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Amyloid-like Fibrils from an α-Helical Transmembrane Protein

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† Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, U.K.
‡ Department of Chemistry, King’s College London, London SE1 1DB, U.K.
§ Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, U.K.
*E-mail: [email protected]. Phone: +44 1223 763873.
Cite this: Biochemistry 2017, 56, 25, 3225–3233
Publication Date (Web):May 11, 2017
https://doi.org/10.1021/acs.biochem.7b00157

Copyright © 2022 American Chemical Society. This publication is licensed under CC-BY.

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Abstract

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The propensity to misfold and self-assemble into stable aggregates is increasingly being recognized as a common feature of protein molecules. Our understanding of this phenomenon and of its links with human disease has improved substantially over the past two decades. Studies thus far, however, have been almost exclusively focused on cytosolic proteins, resulting in a lack of detailed information about the misfolding and aggregation of membrane proteins. As a consequence, although such proteins make up approximately 30% of the human proteome and have high propensities to aggregate, relatively little is known about the biophysical nature of their assemblies. To shed light on this issue, we have studied as a model system an archetypical representative of the ubiquitous major facilitator superfamily, the Escherichia coli lactose permease (LacY). By using a combination of established indicators of cross-β structure and morphology, including the amyloid diagnostic dye thioflavin-T, circular dichroism spectroscopy, Fourier transform infrared spectroscopy, X-ray fiber diffraction, and transmission electron microscopy, we show that LacY can form amyloid-like fibrils under destabilizing conditions. These results indicate that transmembrane α-helical proteins, similarly to cytosolic proteins, have the ability to adopt this generic state.

A wide range of medical conditions, including neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases, are associated with the misfolding and aggregation of specific proteins into amyloid fibrils. (1-4)In vivo and in vitro studies of these disease-related aggregates have led to the recognition of their common structural features, despite the diversity of the amino acid sequences and native state structures of the proteins by which they are formed. (4) In particular, it has been shown that amyloid fibrils possess a cross-β architecture, in which the β-strands that make up the β-sheets are perpendicular to the fibril axis. (2, 4, 5) As this fibrillar architecture can be adopted by a wide variety of different proteins, (6-9) it has been suggested that polypeptide chains have a generic ability to form amyloid fibrils. (4, 10)
Despite a growing body of evidence of the widespread nature of the amyloid phenomenon, (3, 4, 6) little evidence has been obtained about this type of behavior in the case of membrane proteins. This absence of information is particularly remarkable, as it is well-known that the highly hydrophobic nature of membrane proteins makes them prone to self-association and aggregation under many conditions. (11) Indeed, hundreds of membrane proteins have been observed to aggregate upon aging in Caenorhabditis elegans, (12) and a set of genes found to be associated with human neurodegenerative disorders has been linked with membrane proteins involved in the oxidative phosphorylation pathway. (13) In addition, predictive models of protein homeostasis indicate that the aggregation propensity of membrane proteins is very high. (14) Moreover, the amyloid-β peptide (Aβ), which is associated with Alzheimer’s disease, is a proteolytic fragment of the membrane protein APP, (15) and the aggregation and formation of fibrils by fragments of luminal membrane protein domains have been reported. (16)
Several reasons lie behind the current lack of detailed structural information about membrane protein aggregation, many of which are related to the challenges inherent in their recombinant production and purification. These steps remain among the major bottlenecks in many structural and functional analyses of these important biological components. (17) In contrast to the situation for cytosolic proteins, membrane proteins often require expression and purification procedures to be performed in lipid or detergent environments. (18) Despite these difficulties, a more detailed knowledge of this group of proteins is absolutely essential for the understanding of normal and aberrant biological processes, as they represent ∼30% of the human proteome (19) and comprise a substantial fraction of currently known drug targets. (20)
Moreover, an increased level of β-sheet structure has sparsely been reported for disease-related membrane proteins. Perhaps the best studied of these proteins is cystic fibrosis transmembrane conductance regulator (CFTR), an ABC transporter containing 12 transmembrane helices. Several mutations of this membrane protein result in a variety of cystic fibrosis phenotypes, (21) spanning defects in synthesis, processing, trafficking, and functional deficits. Although most studies report a fast degradation of dysfunctional CFTR rather than formation of deposits, (22) it has been indicated that misfolded aggregates with highly organized arrays of β-strands can form for the P205 variant. (23) Retinitis pigmentosa, a progressive retinal degenerative disease, has been shown to be associated with mutations in rhodopsin, an α-helical membrane protein of the G-coupled protein receptor (GCPR) family. (24) Indeed, because a majority of these mutations result in the incorrect folding of the apoprotein opsin, retinitis pigmentosa has been classified as a protein misfolding disease. (25, 26) Despite the fact that misfolded opsin was initially presumed to form disordered aggregates in the endoplasmic reticulum (ER), (27) it has recently been shown that aggregates of the opsin variants G188R and P23H, formed in HEK293 cells, exhibit a high β-structure content. (28) These aggregates appear to be distinct from amyloid fibrils because they do not enhance the fluorescence of thioflavin-T (ThT). (28) A stronger tendency to form ordered β-sheet structure has also been reported for a model peptide with the partial sequence of a transmembrane domain of peripheral myelin protein 22 (PMP22). (29) PMP22 is an α-helical transmembrane glycoprotein primarily expressed in Schwann cells, and its mutations are known to cause the peripheral neuropathy Charcot-Marie-Tooth disease. (30) Aggresome formation by this protein has been reported upon its overexpression in mice, although the structure of the protein within these aggregate-rich inclusions is not yet clear. (31) In addition, there is recent evidence of the formation of ThT-positive aggregates of the β-barrel transmembrane domain of outer membrane protein A (OmpA) formed in the absence of lipid or lipid-mimetic membranes and in the presence of high concentrations of urea. (32)
Overall, although increases in the level of β-sheet structure have been reported in a number of cases, (23, 28, 31, 32) it remains unclear whether membrane proteins have an inherent propensity to convert into amyloid fibrils similar to that of cytosolic proteins. To address this problem, we have studied LacY, a representative of the major facilitator superfamily of transporters that are prevalent across prokaryotes and eukaryotes. This 417-residue protein is composed of 12 transmembrane α-helices arranged in two domains, with a substrate binding site at the domain interface. (33) Its crystal structure has been determined to 3.6 Å resolution, (34) and its function has been studied in great detail. (35) The structure and in vitro folding of LacY have been studied within both micelles and liposomes. (36-38) These simple lipid-mimetic systems represent an ideal starting point for biophysical studies, and the relevance of their use is supported by extensive studies showing the tolerance of membrane proteins to changing lipid environments. (39)
We should mention that, in vivo, LacY is translated directly into lipid membranes in Escherichia coli, in the same way that most membrane proteins are directly translated into the ER membrane where they then reside. (40) Furthermore, a network of quality-control mechanisms has co-evolved with the biosynthesis and trafficking of membrane proteins in the ER, with the specific purpose of preventing their aggregation. (41) By contrast, in our in vitro study, we observe aggregation reactions in a convenient detergent system. Although these conditions are nonphysiological, and our in vitro assays also lack the protein homeostasis controls present in the cellular environment, they allow us to explore the range of structural states accessible to LacY. Consistently, a number of experimental studies have already provided evidence of membrane protein aggregation within the cellular environment. For example, even if in many proteomics protocols membrane proteins tend to be discarded prior to analysis, (42) several studies have reported the presence of membrane proteins in aggregates found in disease tissues. In particular, lysosomal ATPase and mitochondrial ATPase components have been found to co-aggregate in post-mortem amyloid plaques (43) and neurofibrillary tangles, (44) respectively, and the accumulation of the Parkin-associated transmembrane receptor Pael-R in Lewy bodies has been reported. (45) These examples indicate that membrane proteins under stress can aggregate in vivo outside their native environments.
We report here the conversion of LacY into fibrillar aggregates with the characteristic features of cross-β structure and therefore demonstrate that an α-helical membrane protein can adopt a stable amyloid-like state analogous to those observed for cytosolic proteins.

Materials and Methods

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Generation of the Aggregation Propensity Profile of LacY

The CamSol method (46) was used to calculate the sequence-dependent intrinsic solubility of LacY, which we adopted as an indicator of its aggregation propensity, as both solubility and aggregation propensity describe the tendency of proteins to convert from the native state to insoluble aggregates. The CamSol method was derived by considering the physicochemical properties of the amino acid sequences that are most responsible for their aggregation behavior, including in particular the hydrophobic patterns, which tend to drive the aggregation process, and the presence of “gatekeeper” residues (charged residues and prolines), which tend to prevent it. (46) The indication of α-helices and coil in the figure was based on the native structure of LacY. (34, 47)

LacY Expression and Purification

LacY was purified as previously described (37) with modifications. LacY was expressed in One Shot BL21-AI chemically competent E. coli (Thermo Fisher Scientific, Paisley, U.K.), using the kanamycin resistant plasmid pET-28a. The cultures were grown at 37 °C in LB medium and induced with 0.1% arabinose and 1 mM isopropyl β-d-1-thiogalactopyranoside at an OD600 of 0.7–0.8 AU, and the cells were harvested by centrifugation (5000g for 45 min). Following growth and induction, the cells were lysed by three cycles in a high-pressure homogenizer, and the membranes were sedimented by centrifugation (100000g for 4 °C at 30 min) (ultracentrifuge, rotor 70Ti, Beckman Coulter, High Wycombe, U.K.). The pellets were resuspended and incubated (4 °C for 2 h) in 25 mL of solubilization buffer [200 mM NaCl, 20 mM imidazole, 10 mM β-mercaptoethanol, 40 mM DDM, and an EDTA free protease inhibitor cocktail tablet (Roche Diagnostics Ltd., Burgess Hill, U.K.)]. The solubilized membranes were centrifuged (100000g and 4 °C for 30 min), and the supernatant was retained for purification. For all steps of purification, the buffer contained 50 mM sodium phosphate (pH 7.4), 10% (v/v) glycerol, 10 mM β-mercaptoethanol, and 1 mM DDM, with additional components indicated in brackets. Initial purification was performed with a 1 mL HisTrap HP Ni2+ affinity column (GE Healthcare Ltd., Little Chalfont, U.K.), previously equilibrated with 10 column volumes (CV) of purification buffer (20 mM imidazole). Following loading of the sample, the column was washed with 50 CV of purification buffer (20 mM imidazole) and LacY was eluted with 5 CV of elution buffer (75 mM imidazole). The eluted protein was transferred directly to a gel filtration column (SUP200 Superdex, GE Healthcare) to remove imidazole as well as multimeric protein species. The resulting protein fractions were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) using NuPAGE 4 to 12% gels with MES buffer (Thermo Fisher Scientific) and UV–vis spectroscopy to verify the purity of the protein samples and determine their concentrations, respectively. The LacY concentration in the supernatant was determined from the absorbance at 280 nm, using an εLacY of 76391 M–1 cm–1. After purification, aliquoted samples were frozen in liquid N2 and stored at −80 °C until they were used.

LacY Aggregation Procedure

LacY in 50 mM sodium phosphate and 0.05% DDM (pH 7.4) was used directly as obtained from the purification (2 μM, 4 μM, and occasionally 8 μM samples) or concentrated (to 8, 16, or 32 μM), and aggregation was initiated by increasing the temperature of the sample to 37 °C. When concentration of the protein was required, Vivaspin20 concentrators with a 100 kDa cutoff (Millipore, Watford, U.K.) were used as these devices were reported to cause insignificant changes in the DDM concentration. (48, 49) The final DDM concentration was determined via colorimetric determination of the glycoside content. (49) The samples were continuously kept on ice or at 4 °C to minimize aggregate formation prior to any measurement. Concentrating the samples took approximately 35 min from defrosting. Samples were used immediately after being concentrated and were not refrozen.

ThT Fluorescence Assay

LacY (2, 4, and 8 μM as obtained after purification) in 50 mM sodium phosphate and 0.05% DDM (pH 7.4) was incubated at 37 °C in the presence of 4 μM ultrapure ThT (Sigma-Aldrich, Dorset, U.K.). The change in the ThT fluorescence intensity (excitation at 440 nm, emission at 480 nm) was monitored over 48 h using a FLUOstar Optima plate reader (BMG Labtech, Aylesbury, U.K.) under quiescent conditions. Corning 96-well plates with half-area (black/clear bottom polystyrene) nonbinding surfaces (Fisher Scientific, Loughborough, U.K.) were used.

Analysis of LacY in the Supernatant

At the end of an aggregation experiment, the samples were centrifuged (15000g for 15 min), and the concentration of the free monomer was calculated by measuring the UV–vis spectrum of the supernatant. The same samples were analyzed via SDS–PAGE. An aliquot of the LacY supernatant solution (15 μL) was mixed with NuPAGE LDS sample buffer (ThermoFisher Scientific), and 10 μL of the solution was run on a NuPAGE 4 to 12% Bis-Tris gel (ThermoFisher Scientific). A tricolor protein marker (Stratech Scientific, Suffolk, U.K.) was run on the same gel. The gel was stained with InstantBlue (Expedeon, Swavesey, U.K.).

Circular Dichroism Spectroscopy

A LacY solution (2 μM) in 50 mM phosphate buffer and 0.05% DDM (pH 7.4), before and after incubation at 37 °C for 48 h, was deposited in a circular cell. Far-UV CD spectra were recorded on a model 410 Aviv circular dichroism spectrophotometer (Biomedical Inc., Lakewood, NJ), with specially adapted sample detection (the detector is placed closer to the sample) to minimize scattering artifacts. Quartz circular cells were used, and CD spectra were obtained by averaging two to five individual spectra recorded between 260 and 190 nm at a 0.5 nm interval with an averaging time of 2 s. For each protein sample, the CD signal of the buffer without protein was recorded and subtracted from the CD signal of each protein sample. The multiple scans were averaged, and the buffer background was subtracted. The data were then converted from millidegrees to mean residue ellipticity. (50)

Fourier Transform Infrared Spectroscopy

A LacY solution (concentrated to 16 μM) in 50 mM phosphate buffer and 0.05% DDM (pH 7.4), before and after incubation at 37 °C for 48 h, was deposited (10 μL) into a BioATR cell (Bruker Optics, Coventry, U.K.). Fourier transform infrared (FT-IR) absorption spectra were collected in absorbance mode on an FT-IR Vertex 70 (Bruker) spectrometer fitted with a BioATR cell (Bruker Optics, Coventry, U.K.). Spectra were corrected for the presence of 50 mM sodium phosphate (pH 7.4) and 0.05% DDM. The secondary derivative for each spectrum was calculated using OriginPRO. To reduce the noise, a FFT filter (10 pt) was initially applied; the curves were successively normalized and smoothed by a Savitski–Golay filter (9 points).

Transmission Electron Microscopy

LacY solutions (concentrated to 16 μM) in 50 mM phosphate buffer and 0.05% DDM (pH 7.4) were incubated at 37 °C for 48 h, and 5 μL of the sample was applied to carbon-coated 400 mesh copper grids (EM Resolutions, Saffron Walden, U.K.). Samples were stained with 2% (w/v) uranyl acetate and imaged using a FEI Tecnai G2 transmission electron microscope (Cambridge Advanced Imaging Centre (CAIC), University of Cambridge). Images were analyzed using the SIS Megaview II Image Capture system (Olympus).

X-ray Fiber Diffraction Pattern Analysis

LacY solutions (concentrated to 16 and 32 μM) in 50 mM phosphate buffer and 0.05% DDM (pH 7.4) were incubated at 37 °C for 48 h. The samples for fiber diffraction were dialyzed against 50 mM sodium phosphate (pH 7.4) and 0.05% DDM, and a 5 μL aliquot was pipetted between wax stalks and left to dry. The experiments were performed at the Crystallographic X-ray Facility of the Department of Biochemistry of the University of Cambridge. X-rays with a wavelength of 1.5418 Å were produced by a rotating copper anode MISCROSTAR generator (Bruker-AXS, Ltd.), collimated, and focused by HELIOS-MX high-brightness multilayer X-ray optics. Images were acquired on a PLATINUM135 CCD area detector (Bruker-AXS, Ltd., Rigaku-MSC). The exposure time for the diffraction images was 60 s in each case. Viewing and analysis of the diffraction pattern were performed using the PORTEUM2 software package (PROTEUM 2 User Manual, Bruker AXS, 2010).

Results and Discussion

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LacY Has a Strong Intrinsic Propensity to Aggregate

In a preliminary assessment, we used the CamSol method (46) to calculate the sequence-dependent solubility of LacY (Figure 1), which is inversely related to the aggregation propensity score (see Materials and Methods). As the native state of LacY comprises 12 transmembrane α-helices, the sequence may be expected to include highly hydrophobic and aggregation-prone regions outside its natural lipid environment, which are indeed present (red regions in the sequence profile in Figure 1).

Figure 1

Figure 1. CamSol profile of LacY, as an indicator of its aggregation propensity. (A) Native structure of LacY (front and bottom view), which is shown for reference. The Protein Data Bank structure 2V8N (34) was used to draw the cartoons, with cylinders representing α-helices and ribbons representing disordered coils. (B) CamSol (46) profile of LacY (see Materials and Methods). Regions colored red have a low intrinsic solubility and hence a high aggregation propensity. The positions of α-helices (as derived from ref 47) are indicated by horizontal gray bars.

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LacY Readily Forms Aggregates at 37 °C and pH 7.4

LacY was expressed and purified into n-dodecyl β-d-maltoside (DDM) micelles according to standard procedures. (37) The detergent was used at a concentration of 1 mM (or 0.05%) in all experiments, which is well above its critical micelle concentration (CMC) of 0.16 mM. (51) Long chain maltoside detergents are generally used for LacY purification as they are well-known to effectively maintain the protein in solution. (52) In addition, it was established that LacY is stabilized by low temperature and glycerol, (52) which was added in all purification buffers (Materials and Methods). Despite these considerations, purification of LacY is challenging as the protein tends to aggregate readily at room temperature and even when kept on ice for a few hours. Furthermore, as several biophysical techniques utilized in our studies require protein concentrations (in the range of at least 15–30 μM) higher than those initially obtained from the purification (typically in the range of 2–6 μM), the samples often required concentrating, thereby placing the protein under conditions where it is particularly vulnerable to aggregation prior to the start of any experiment. This aspect of the system made it difficult to characterize the precise starting point of the aggregation reaction and to avoid consistently the presence of a small fraction of aggregated species at the initiation of the experiments.
In the experimental protocol described here, LacY [in 50 mM sodium phosphate (pH 7.4) and 0.05% DDM] was used as obtained from the purification procedure (2, 4, and 8 μM samples) or concentrated to 8, 16, or 32 μM. Amyloid-like structures often are observed under conditions that destabilize the protein under study, (7) and as an increase in temperature in particular is known to destabilize LacY, (52) aggregation of the protein was initiated by increasing the temperature of the sample from 4 to 37 °C. Visible precipitation of LacY in the micellar environment was observed after approximately 4 h for the 16 μM samples to approximately 8 h for the 2 μM samples, with increasing turbidity of the solutions at longer incubation times of ≤48 h. Aliquots taken at different time points during the incubation were centrifuged, and the supernatant at each time point was analyzed. The concentration of LacY in the supernatant at each time point was obtained from UV–vis spectra at 280 nm (Table 1) and observed to decrease gradually over time, resulting in <20% of the initial protein remaining in solution after incubation for 48 h. This ratio is consistent for the samples at higher concentrations. The same samples were analyzed by SDS–PAGE (Figure 2), and the increasingly faint bands observed for the samples after increasing incubation times confirm the nearly complete conversion of the monomer into precipitating species after 48 h.
Table 1. LacY Concentrations in the Supernatant over Timea
time (h)A280c (μM)
00.1481.94
20.1281.68
40.0891.15
60.0851.11
240.0520.68
480.0310.40
a

LacY solutions (2 μM) incubated in 50 mM phosphate buffer (pH 7.4) with 0.05% DDM (37 °C for 48 h) were centrifuged after 0, 2, 4, 6, 24, and 48 h to precipitate the aggregated fraction, and the UV–vis signal at 280 nm of the remaining supernatant was monitored. The LacY concentration in the supernatant was determined from the absorbance at 280 nm, using an εLacY of 76391 M–1 cm–1. An average of two measurements from separate samples is shown (standard deviation ± 0.005 for A280).

Figure 2

Figure 2. Fraction of LacY in the supernatant over time. LacY solutions (2 μM) incubated in 50 mM phosphate buffer (pH 7.4) with 0.05% DDM (37 °C for 48 h) were centrifuged to precipitate the aggregated fraction, and the remaining supernatant was analyzed by SDS–PAGE after 0, 1, 2, 3, 4, 5, 6, 7, 8, 24, and 48 h (as indicated). LacY appears at ∼28 kDa because it does not unfold in SDS and thus runs in its folded state (its expected MW is 48 kDa). The experiment was repeated twice; a representative data set is shown.

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In the following sections, we describe a range of biophysical techniques that we have used to define the structural nature of the observed aggregates and to characterize their properties. Because nearly complete precipitation of LacY from solution was observed after 48 h, we did not extend experiments beyond this initial time frame.

LacY Aggregates Are ThT-Positive

Amyloid-specific fluorescent dyes have been used for many years to identify amyloid assemblies, (1) and in vitro assays involving these compounds remain a powerful approach to characterizing amyloid-like fibrils and to monitoring the kinetics of their formation. One of the most widely used dyes is ThT, a positively charged benzothiazole salt that shows increased fluorescence upon interaction with amyloid fibrils. (53) Membrane proteins are highly hydrophobic in nature and could therefore adopt a mode of binding to ThT different from that of cytosolic proteins. Although some studies have suggested that binding of ThT to amyloid fibrils involves the formation of ThT micelles as a result of hydrophobic interactions, (54) a model in which ThT specifically interacts with the cross-β strands that characterize the unique amyloid architecture is more widely accepted. (53)
LacY solutions at concentrations of 2, 4, and 8 μM were incubated at 37 °C in the presence of 4 μM ThT, and the fluorescence signal was monitored over the course of 48 h. Within minutes, an increase in the fluorescence intensity at 480 nm was observed, and sigmoidal aggregation curves were obtained by plotting the relative fluorescence intensities as a function of time (Figure 3). The initial high intensities, which were seen in all samples, are often observed in this type of kinetic experiment and can be attributed to an equilibration at the higher temperature (from 4 to 37 °C in this case). Normalization of the data shows an increase in the aggregation rate with an increasing initial protein concentration (≤8 μM, as these samples could be obtained directly after purification without the need for concentration) (Figure 3B). The ThT-positive character of the LacY aggregates provides a first indication that they may have a β-sheet-rich amyloid-like nature. The enhanced fluorescence of ThT inside micelles, however, could be a result of electrostatic interactions with the charged detergent molecules. (55) Indeed, significant effects of this type have been observed for ThT simply in the presence of the anionic surfactant sodium dodecyl sulfate (SDS), whereas relatively insignificant intensity changes were observed with neutral detergents such as Tween 20 and Triton X-100. (55) Because DDM belongs to the latter category of detergents, only minor intensity changes were expected, and none were observed, in its presence (Figure S1). All spectra were corrected for potential buffer effects by monitoring the ThT fluorescence in sodium phosphate buffer containing DDM concentrations equal to those of the sample under investigation, showing that the increase in ThT fluorescence correlates with the presence of LacY aggregates as determined by centrifugation.

Figure 3

Figure 3. Enhancement of ThT fluorescence by LacY aggregates. (A) ThT fluorescence changes in monomer solutions of 2 μM (yellow), 4 μM (red), and 8 μM (blue) LacY (in duplicate) in 50 mM sodium phosphate (pH 7.4) with 0.05% DDM. The solutions were incubated (37 °C for 48 h) in the presence of 4 μM ThT, and the ThT fluorescence was continuously monitored. The fluorescence spectra were corrected for the signal in 50 mM sodium phosphate (pH 7.4) with 0.05% DDM. (B) The spectra shown in panel A were normalized by subtracting the minimum and dividing by the maximal fluorescence intensity for the respective data set. This procedure allows a straightforward comparison of the slopes of the intensity profiles for different concentrations. The experiment was repeated five times; a representative data set is presented.

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LacY Is Converted from α-Helical to Parallel β-Sheet Structure

In addition to monitoring dye binding properties, direct detection of their characteristic β-sheet architecture is a key parameter in the identification of amyloid-like fibrils. Circular dichroism (CD) and FT-IR spectroscopies are powerful techniques used to characterize protein secondary structure content and were used to monitor the changes in the LacY secondary structure upon aggregation. (56) The native LacY monomer has been studied extensively; an α-helical structure content of ∼85% has been reported from CD measurements and is consistent with the crystal structure. (57)
Aliquots from a LacY solution at a concentration of 2 μM were taken before and after incubation at 37 °C for 48 h, and CD spectra were recorded for each sample (Figure 4). The spectrum of LacY as obtained from the purification and before incubation possesses characteristic minima at 208 and 223 nm, indicative of a highly α-helical structure, in accord with the LacY native fold. (57) The signal intensity was reduced significantly after incubation of the sample at 37 °C, indicating a loss of soluble protein. This result indicates that only soluble protein is being detected, while the aggregated fraction crashes out of solution and does not result in a detectable signal. Further secondary structure determination therefore was performed using FT-IR spectroscopy. This technique furthermore has a higher sensitivity and accuracy for resolving β-sheet compositions. (58-60) For example, whereas only changes in α-helical content could be detected by CD spectroscopy in the study of the secondary structure changes of opsin variants, changes in both α-helical and β-sheet content were observed for the same system using FT-IR spectroscopy. (28) As FT-IR requires protein concentrations higher than those obtained from the purification procedure, a concentrated sample was used for this analysis. The DDM concentration of the concentrated samples was verified via colorimetric determination of the glycoside content, (49) and only small increases [from 0.050 to 0.057% (Table S1)] were detected. Aliquots from the LacY solution at a concentration of 16 μM were taken before and after incubation at 37 °C for 48 h, and FT-IR spectra were recorded for each sample (Figure 5A). The amide I/II regions of the spectra at both time points are shown, as well as the second-derivative spectra (Figure 5B).

Figure 4

Figure 4. CD intensity decrease upon aggregation of LacY. LacY (2 μM) was incubated in 50 mM phosphate buffer (pH 7.4) with 0.05% DDM at 37 °C. CD spectra were recorded before (blue) and after (red) incubation for 48 h. The spectra were corrected for 50 mM sodium phosphate (pH 7.4) with 0.05% DDM. A minimum of three scans was averaged per time point. The experiment was repeated twice; a representative data set is presented.

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Figure 5

Figure 5. Secondary structure in LacY aggregates detected using FT-IR. (A) LacY (16 μM) was incubated in 50 mM phosphate buffer (pH 7.4) with 0.05–0.06% DDM at 37 °C. FT-IR spectra were recorded before (blue) and after (red) incubation for 48 h and corrected by subtracting the spectrum of the buffer solution. The dotted lines indicate the peak maxima, and the arrows illustrate the peak shifts. A spectrum of LacY prior to concentration is not shown, because the protein directly obtained from the purification was not concentrated enough to result in a detectable spectrum. (B) Second derivative of the spectra before (blue) and after (red) incubation for 48 h showing the appearance of peak positions in the amide I region at 1653 and 1623 cm–1, respectively. The dotted lines indicate the main peak minima. The experiment was repeated twice; a representative data set is presented.

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The amide I band obtained for the LacY sample before incubation has its maximum at 1653 cm–1, which is the characteristic region for α-helical/disordered structure content. (61, 62) This preincubation sample, although predominantly α-helical in nature, may contain some aggregated species because of its high protein concentration. After 48 h, the main peak is shifted to 1623 cm–1, where intermolecular β-sheet structure has its characteristic maximum. (61) The differences between β-sheet structure in amyloid-like fibrils compared to that in native states of proteins is evident from FT-IR data, with amyloid-like fibrils specifically populating the amide I region between 1610 and 1630 cm–1. (63) A shift of the amide II band to lower wavelengths further reflects the formation of intermolecular hydrogen bonds upon incubation of the sample at 37 °C for 48 h. Deconvolution of the FT-IR data in Figure 5A indicates the conversion of mainly α-helical structure to a 35% parallel β-sheet conformation after incubation at 37 °C for 48 h. In addition, the β-turn conformation (1682 cm–1) and antiparallel β-sheet (1695 cm–1) contents slightly increase upon incubation of the sample, and these changes are intimately related to formation of cross-β sheet structure. (63) The FT-IR data thus indicate that the nature of the β-sheet fraction is typical of amyloid-like fibrils. (63)

Denaturant Resistant LacY Aggregates Have a Fibrillar Architecture

In light of the fibrillar nature of the aggregated samples indicated by the ThT assay and FT-IR data, we set out to obtain the corresponding fiber diffraction patterns. The diffraction patterns of the LacY sample after incubation at 37 °C for 48 h (Figure 6A) show a diffuse ring at a spacing between 4.5 and 4.8 Å and a very weak reflection at ∼10 Å. While the observation of these reflections does confirm that fibrillar species are present, the weakness of the signal is a consequence of the low concentrations of fibrils and/or of a dilution effect due to the presence of less ordered species. To gain more insights into the morphology of the species present in the LacY aggregates, transmission electron microscopy (TEM) images were recorded for the incubated (37 °C for 48 h) LacY sample (concentration of 16–32 μM). These measurements, although not sufficiently accurate to reveal quantitative parameters such as width and height, showed that the aggregates have a fibrillar morphology but are coated with less ordered material (Figure 6C), suggesting that the low resolution of the diffraction pattern is at least in part a consequence of dilution by disordered species.

Figure 6

Figure 6. TEM and X-ray fiber diffraction data of LacY aggregates. (A) LacY (16–32 μM) was incubated in 50 mM phosphate buffer (pH 7.4) with 0.05–0.06% DDM at 37 °C. Fiber diffraction patterns from air-dried stalks of the samples were measured for the samples incubated for 48 h and (B) samples treated with guanidinium hydrochloride. The arrowheads indicate the reflections at ∼4.6 Å, while the arrows indicate the reflections at ∼10.8 Å. TEM grids were prepared for the same samples (C) after incubation for 48 h and (D) after incubation for 48 h followed by washing with 3 M guanidinium hydrochloride. These experiments were repeated three times, and representative diffraction patterns and TEM images are shown.

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To obtain more detailed images of the fibrillar species in the samples, we set out to separate the fibrillar and disordered components of the aggregates. Amyloid-like fibrils are typically extremely stable and considerably more resistant to relatively high concentrations of denaturant than disordered aggregates are. (64) We therefore incubated the aggregates in urea (8 M) or guanidinium hydrochloride (3 M) for 1 h, centrifuged the remaining insoluble material, and re-examined it via TEM. The samples prepared in this manner show clearly the presence of substantially more homogeneous structures (Figure 6D), indicating that a denaturant resistant fibrillar fraction can be isolated. Following treatment of the samples in urea (8 M) or guanidinium hydrochloride (3 M), the diffraction pattern was seen to be much more highly resolved as well (Figure 6B), showing reflections at 4.6 Å, characteristic of interstrand spacing, and at 10.8 Å, corresponding to the intersheet spacing as observed in amyloid fibrils. (2) The combination of these reflections is consistent with the cross-β pattern indicative of a β-sheet core structure, in which the constituent β-strands lie at right angles to the axis of the fibril. The characterized interstrand and intersheet spacing, in combination with an extended degree of β-sheet content, enhanced ThT fluorescence emission, and evidence of a fibrillar morphology indicates the ability of LacY to convert into amyloid-like fibrils in vitro.

Conclusions

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The generality of the amyloid phenomenon has been firmly established for cytosolic proteins through a large number of case studies. (1-8, 10) By contrast, its extent remains rather unclear for membrane proteins, for which the conversion into β-sheet-rich aggregates has been previously suggested, (16, 23, 28, 31, 32) but only preliminary evidence of fibril formation so far has been provided. (16, 23, 32) In this work, we have reported an extensive characterization of aggregates of LacY as amyloid-like fibrils. As many membrane proteins have sequences and overall architectures similar in nature to those of LacY, we anticipate that amyloid-like structures will be described in detail for a wider range of membrane proteins, and under a variety of conditions, thus allowing the biological relevance of these aggregates to be progressively defined.

Supporting Information

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.7b00157.

  • DDM concentrations of LacY samples after concentration (Table S1) and ThT intensity profiles for the DDM-containing buffer in the absence of protein (Figure S1) (PDF)

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Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

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  • Corresponding Author
  • Authors
    • Karen Stroobants - Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, U.K.
    • Janet R. Kumita - Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, U.K.;  Orcidhttp://orcid.org/0000-0002-3887-4964
    • Nicola J. Harris - Department of Chemistry, King’s College London, London SE1 1DB, U.K.
    • Dimitri Y. Chirgadze - Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, U.K.
    • Christopher M. Dobson - Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, U.K.
    • Paula J. Booth - Department of Chemistry, King’s College London, London SE1 1DB, U.K.
  • Funding

    We are grateful for the award of the Marie Curie Career Development Fellowship (K.S.) and for support of this work by a Wellcome Trust Programme Grant 094425/Z/10/Z (C.M.D. and M.V.) and by an ERC Advanced Grant (294342) (N.J.H., P.J.B.).

  • Notes
    The authors declare no competing financial interest.

Acknowledgment

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The authors thank Prof. Kathryn Lilley and Dr. Daniel Nightingale of the Cambridge Centre for Proteomics for the use of the ultracentrifuge, Dr. Ewa Klimont for the help with setting up the membrane protein purification procedures in Cambridge, Dr. Francesco Simone Ruggeri for the help with the FT-IR deconvolution, and Dr. Lyn Carter and Dr. Jeremy Skepper of the Cambridge Advanced Imaging Centre (CAIC), University of Cambridge, for the help with acquiring the TEM images. The X-ray fiber diffraction experiments were performed in the Crystallographic X-ray Facility (CXF) at the Department of Biochemistry of the University of Cambridge (DYC).

References

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    A review. Transmembrane transporters are responsible for maintaining a correct internal cellular environment. The inherent flexibility of transporters together with their hydrophobic environment means that they are challenging to study in vitro, but recently significant progress been made. Here, the authors focus on in vitro stability and folding studies of transmembrane α-helical transporters, including reversible folding systems and thermal denaturation. The successful re-assembly of a small no. of ATP-binding cassette transporters is also described as this is a significant step forward in terms of understanding the folding and assembly of these more complex, multi-subunit proteins. The studies on transporters discussed here represent substantial advances for membrane protein studies as well as for research into protein folding. The work demonstrates that large flexible hydrophobic proteins are within reach of in vitro folding studies, thus holding promise for furthering knowledge on the structure, function and biogenesis of ubiquitous membrane transporter families.
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  19. 19
    Krogh, A., Larsson, B., von Heijne, G., and Sonnhammer, E. L. L. (2001) Predicting transmembrane protein topology with a hidden Markov model: Application to complete genomes J. Mol. Biol. 305, 567– 580 DOI: 10.1006/jmbi.2000.4315
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    19
    Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes
    Krogh, Anders; Larsson, Bjorn; von Heijne, Gunnar; Sonnhammer, Erik L. L.
    Journal of Molecular Biology (2001), 305 (3), 567-580CODEN: JMOBAK; ISSN:0022-2836. (Academic Press)
    We describe and validate a new membrane protein topol. prediction method, TMHMM, based on a hidden Markov model. We present a detailed anal. of TMHMM's performance, and show that it correctly predicts 97-98 % of the transmembrane helixes. Addnl., TMHMM can discriminate between sol. and membrane proteins with both specificity and sensitivity better than 99 %, although the accuracy drops when signal peptides are present. This high degree of accuracy allowed us to predict reliably integral membrane proteins in a large collection of genomes. Based on these predictions, we est. that 20-30 % of all genes in most genomes encode membrane proteins, which is in agreement with previous ests. We further discovered that proteins with Nin-Cin topologies are strongly preferred in all examd. organisms, except Caenorhabditis elegans, where the large no. of 7TM receptors increases the counts for Nout-Cin topologies. We discuss the possible relevance of this finding for our understanding of membrane protein assembly mechanisms. A TMHMM prediction service is available at http://www.cbs.dtu.dk/services/TMHMM/. (c) 2001 Academic Press.
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    Overington, J. P., Al-Lazikani, B., and Hopkins, A. L. (2006) Opinion - How many drug targets are there? Nat. Rev. Drug Discovery 5, 993– 996 DOI: 10.1038/nrd2199
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    How many drug targets are there?
    Overington, John P.; Al-Lazikani, Bissan; Hopkins, Andrew L.
    Nature Reviews Drug Discovery (2006), 5 (12), 993-996CODEN: NRDDAG; ISSN:1474-1776. (Nature Publishing Group)
    A review. For the past decade, the no. of mol. targets for approved drugs has been debated. Here, we reconcile apparently contradictory previous reports into a comprehensive survey, and propose a consensus no. of current drug targets for all classes of approved therapeutic drugs. One striking feature is the relatively const. historical rate of target innovation (the rate at which drugs against new targets are launched); however, the rate of developing drugs against new families is significantly lower. The recent approval of drugs that target protein kinases highlights two addnl. trends: an emerging realization of the importance of polypharmacol., and also the power of a gene-family-led approach in generating novel and important therapies.
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    Welsh, M. J. and Smith, A. E. (1993) Molecular mechanisms of CFTR chloride channel dysfunction in cystic fibrosis Cell 73, 1251– 1254 DOI: 10.1016/0092-8674(93)90353-R
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    Molecular mechanisms of CFTR chloride channel dysfunction in cystic fibrosis
    Welsh, Michael J.; Smith, Alan E.
    Cell (Cambridge, MA, United States) (1993), 73 (7), 1251-4CODEN: CELLB5; ISSN:0092-8674.
    A review with 24 refs. on mol. mechanisms by which cystic fibrosis-assocd. mutations cause a loss of CFTR chloride channel function.
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  22. 22
    Ward, C. L., Omura, S., and Kopito, R. R. (1995) Degradation of CFTR by the ubiquitin-proteasome pathway Cell 83, 121– 127 DOI: 10.1016/0092-8674(95)90240-6
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    Wigley, W. C., Corboy, M. J., Cutler, T. D., Thibodeau, P. H., Oldan, J., Lee, M. G., Rizo, J., Hunt, J. F., and Thomas, P. J. (2002) A protein sequence that can encode native structure by disfavoring alternate conformations Nat. Struct. Biol. 9, 381– 388 DOI: 10.1038/nsb784
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    A protein sequence that can encode native structure by disfavoring alternate conformations
    Wigley, W. C.; Corboy, M. J.; Cutler, T. D.; Thibodeau, P. H.; Oldan, J.; Lee, M. G.; Rizo, J.; Hunt, J. F.; Thomas, P. J.
    Nature Structural Biology (2002), 9 (5), 381-388CODEN: NSBIEW; ISSN:1072-8368. (Nature America Inc.)
    The linear sequence of amino acids contains all the necessary information for a protein to fold into its unique three-dimensional structure. Native protein sequences are known to accomplish this by promoting the formation of stable, kinetically accessible structures. Here we describe a Pro residue in the center of the third transmembrane helix of the cystic fibrosis transmembrane conductance regulator that promotes folding by a distinct mechanism: disfavoring the formation of a misfolded structure. The generality of this mechanism is supported by genome-wide transmembrane sequence analyses. Furthermore, the results provide an explanation for the increased frequency of Pro residues in transmembrane α-helixes. Incorporation by nature of such 'neg. folding determinants', aimed at preventing the formation of off-pathway structures, represents an addnl. mechanism by which folding information is encoded within the evolved sequences of proteins.
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    Hartong, D. T., Berson, E. L., and Dryja, T. P. (2006) Retinitis pigmentosa Lancet 368, 1795– 1809 DOI: 10.1016/S0140-6736(06)69740-7
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    Retinitis pigmentosa
    Hartong, Dyonne T.; Berson, Eliot L.; Dryja, Thaddeus P.
    Lancet (2006), 368 (9549), 1795-1809CODEN: LANCAO; ISSN:0140-6736. (Elsevier Ltd.)
    A review. Hereditary degenerations of the human retina are genetically heterogeneous, with well over 100 genes implicated so far. This Seminar focuses on the subset of diseases called retinitis pigmentosa, in which patients typically lose night vision in adolescence, side vision in young adulthood, and central vision in later life because of progressive loss of rod and cone photoreceptor cells. Measures of retinal function, such as the electroretinogram, show that photoreceptor function is diminished generally many years before symptomic night blindness, visual-field scotomas, or decreased visual acuity arise. More than 45 genes for retinitis pigmentosa have been identified. These genes account for only about 60% of all patients; the remainder have defects in as yet unidentified genes. Findings of controlled trials indicate that nutritional interventions, including vitamin A palmitate and omega-3-rich fish, slow progression of disease in many patients. Imminent treatments for retinitis pigmentosa are greatly anticipated, esp. for genetically defined subsets of patients, because of newly identified genes, growing knowledge of affected biochem. pathways, and development of animal models.
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    Krebs, M. P., Holden, D. C., Joshi, P., Clark, C. L., III, Lee, A. H., and Kaushal, S. (2010) Molecular mechanisms of rhodopsin retinitis pigmentosa and the efficacy of pharmacological rescue J. Mol. Biol. 395, 1063– 1078 DOI: 10.1016/j.jmb.2009.11.015
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    25
    Molecular Mechanisms of Rhodopsin Retinitis Pigmentosa and the Efficacy of Pharmacological Rescue
    Krebs, Mark P.; Holden, David C.; Joshi, Parth; Clark, Charles L.; Lee, Andrew H.; Kaushal, Shalesh
    Journal of Molecular Biology (2010), 395 (5), 1063-1078CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
    Variants of rhodopsin, a complex of 11-cis retinal and opsin, cause retinitis pigmentosa (RP), a degenerative disease of the retina. Trafficking defects due to rhodopsin misfolding have been proposed as the most likely basis of the disease, but other potentially overlapping mechanisms may also apply. Pharmacol. therapies for RP must target the major disease mechanism and contend with overlap, if it occurs. To this end, we have explored the mol. basis of rhodopsin RP in the context of pharmacol. rescue with 11-cis retinal. Stable inducible cell lines were constructed to express wild-type opsin; the pathogenic variants T4R, T17M, P23A, P23H, P23L, and C110Y; or the nonpathogenic variants F220L and A299S. Pharmacol. rescue was measured as the fold increase in rhodopsin or opsin levels upon addn. of 11-cis retinal during opsin expression. Only Pro23 and T17M variants were rescued significantly. C110Y opsin was produced at low levels and did not yield rhodopsin, whereas the T4R, F220L, and A299S proteins reached near-wild-type levels and changed little with 11-cis retinal. All of the mutant rhodopsins exhibited misfolding, which increased over a broad range in the order F220L, A299S, T4R, T17M, P23A, P23H, P23L, as detd. by decreased thermal stability in the dark and increased hydroxylamine sensitivity. Pharmacol. rescue increased as misfolding decreased, but was limited for the least misfolded variants. Significantly, pathogenic variants also showed abnormal photobleaching behavior, including an increased ratio of metarhodopsin-I-like species to metarhodopsin-II-like species and aberrant photoproduct accumulation with prolonged illumination. These results, combined with an anal. of published biochem. and clin. studies, suggest that many rhodopsin variants cause disease by affecting both biosynthesis and photoactivity. We conclude that pharmacol. rescue is promising as a broadly effective therapy for rhodopsin RP, particularly if implemented in a way that minimizes the photoactivity of the mutant proteins.
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  26. 26
    Saliba, R. S., Munro, P. M. G., Luthert, P. J., and Cheetham, M. E. (2002) The cellular fate of mutant rhodopsin: quality control, degradation and aggresome formation J. Cell Sci. 115, 2907– 2918
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    26
    The cellular fate of mutant rhodopsin: quality control, degradation and aggresome formation
    Saliba, Richard S.; Munro, Peter M. G.; Luthert, Philip J.; Cheetham, Michael E.
    Journal of Cell Science (2002), 115 (14), 2907-2918CODEN: JNCSAI; ISSN:0021-9533. (Company of Biologists Ltd.)
    Mutations in the photopigment rhodopsin are the major cause of autosomal dominant retinitis pigmentosa. The majority of mutations in rhodopsin lead to misfolding of the protein. Through the detailed examn. of P23H and K296E mutant opsin processing in COS-7 cells, we have shown that the mutant protein does not accumulate in the Golgi, as previously thought, instead it forms aggregates that have many of the characteristic features of an aggresome. The aggregates form close to the centrosome and lead to the dispersal of the Golgi app. Furthermore, these aggregates are ubiquitinated, recruit cellular chaperones and disrupt the intermediate filament network. Mutant opsin expression can disrupt the processing of normal opsin, as co-transfection revealed that the wild-type protein is recruited to mutant opsin aggregates. The degrdn. of mutant opsin is dependent on the proteasome machinery. Unlike the situation with ΔF508-CFTR, proteasome inhibition does not lead to a marked increase in aggresome formation but increases the retention of the protein within the ER, suggesting that the proteasome is required for the efficient retro-translocation of the mutant protein. Inhibition of N-linked glycosylation with tunicamycin leads to the selective retention of the mutant protein within the ER and increases the steady state level of mutant opsin. Glycosylation, however, has no influence on the biogenesis and targeting of wild-type opsin in cultured cells. This demonstrates that N-linked glycosylation is required for ER-assocd. degrdn. of the mutant protein but is not essential for the quality control of opsin folding. The addn. of 9-cis-retinal to the media increased the amt. of P23H, but not K296E, that was sol. and reached the plasma membrane. These data show that rhodopsin autosomal dominant retinitis pigmentosa is similar to many other neurodegenerative diseases in which the formation of intracellular protein aggregates is central to disease pathogenesis, and they suggest a mechanism for disease dominance.
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  27. 27
    Surguchev, A. and Surguchov, A. (2010) Conformational diseases: Looking into the eyes Brain Res. Bull. 81, 12– 24 DOI: 10.1016/j.brainresbull.2009.09.015
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    Conformational diseases: Looking into the eyes
    Surguchev, Alexei; Surguchov, Andrei
    Brain Research Bulletin (2010), 81 (1), 12-24CODEN: BRBUDU; ISSN:0361-9230. (Elsevier Inc.)
    A review. Conformational diseases, a general term comprising more than 40 disorders are caused by the accumulation of unfolded or misfolded proteins. Improper protein folding (misfolding) as well as accrual of unfolded proteins can lead to the formation of disordered (amorphous) or ordered (amyloid fibril) aggregates. The gradual accumulation of protein aggregates and the acceleration of their formation by stress explain the characteristic late or episodic onset of the diseases. The best studied in this group are neurodegenerative diseases and amyloidosis accompanied by the deposition of a specific aggregation-prone proteins or protein fragments and formation of insol. fibrils. Amyloidogenic protein accumulation often occurs in the brain tissues, e.g. in Alzheimer's disease with the deposition of amyloid-β and Tau, in scrapie and bovine spongiform encephalopathy with the accumulation of prion protein, in Parkinson's disease with the deposition of α-synuclein. Other examples of amyloid proteins are transthyretin, Ig light chain, gelsolin, etc. In addn. to the brain, the accumulation of unfolded or misfolded proteins leading to pathol. takes place in a wide variety of organs and tissues, including different parts of the eye. The best studied ocular conformational diseases are cataract in the lens and retinitis pigmentosa in the retina, but accumulation of misfolded proteins also occurs in other parts of the eye causing various disorders. Furthermore, ocular manifestation of systemic amyloidosis often causes the deposition of amyloidogenic proteins in different ocular tissues. Here we present the data regarding naturally unfolded and misfolded proteins in eye tissues, their structure-function relationships, and mol. mechanisms underlying their involvement in diseases. We also summarize the etiol. of ocular conformational diseases and discuss approaches to their treatment.
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  28. 28
    Miller, L. M., Gragg, M., Kim, T. G., and Park, P. S. H. (2015) Misfolded opsin mutants display elevated beta-sheet structure FEBS Lett. 589, 3119– 3125 DOI: 10.1016/j.febslet.2015.08.042
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    28
    Misfolded opsin mutants display elevated β-sheet structure
    Miller, Lisa M.; Gragg, Megan; Kim, Tae Gyun; Park, Paul S.-H.
    FEBS Letters (2015), 589 (20_Part_B), 3119-3125CODEN: FEBLAL; ISSN:0014-5793. (Elsevier B.V.)
    Mutations in rhodopsin can cause misfolding and aggregation of the receptor, which leads to retinitis pigmentosa, a progressive retinal degenerative disease. The structure adopted by misfolded opsin mutants and the assocd. cell toxicity is poorly understood. Forster resonance energy transfer (FRET) and Fourier transform IR (FTIR) microspectroscopy were utilized to probe within cells the structures formed by G188R and P23H opsins, which are misfolding mutants that cause autosomal dominant retinitis pigmentosa. Both mutants formed aggregates in the endoplasmic reticulum and exhibited altered secondary structure with elevated β-sheet and reduced α-helical content. The newly formed β-sheet structure may facilitate the aggregation of misfolded opsin mutants. The effects obsd. for the mutants were unrelated to retention of opsin mols. in the endoplasmic reticulum itself.
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    Yamada, K., Sato, J., Oku, H., and Katakai, R. (2003) Conformation of the transmembrane domains in peripheral myelin protein 22. Part 1. Solution-phase synthesis and circular dichroism study of protected 17-residue partial peptides in the first putative transmembrane domain J. Pept. Res. 62, 78– 87 DOI: 10.1034/j.1399-3011.2003.00073.x
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    29
    Conformation of the transmembrane domains in peripheral myelin protein 22. Part 1. Solution-phase synthesis and circular dichroism study of protected 17-residue partial peptides in the first putative transmembrane domain
    Yamada, K.; Sato, J.; Oku, H.; Katakai, R.
    Journal of Peptide Research (2003), 62 (2), 78-87CODEN: JPERFA; ISSN:1397-002X. (Blackwell Munksgaard)
    Charcot-Marie-Tooth disease (CMT) is the most commonly inherited peripheral neuropathy. DNA duplication and point mutation of the gene encoding peripheral myelin protein 22 (PMP22) have been found in CMT type 1A dominants. To investigate the influence of the point mutation of PMP22 on the secondary structure, protected partial peptides in the putative first transmembrane domain, wild type Boc-IVLH(Bom)VAVLVLLFVSTIV-OMe (1; Bom = benzyloxymethyl) and its Pro16 mutant Boc-IVLH(Bom)VAVPVLLFVSTIV-OMe (2) were synthesized. CD anal. suggested that peptide 1 adopts a stable α-helical conformation in membrane-mimetic solvent, 1-BuOH/1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) system. On the contrary, the mutant 2 favors β-sheet conformation in the same solvent system. Interestingly, α-helix to β-sheet transition of 2 was obsd. at higher contents of 1-BuOH than 70%.
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  30. 30
    Naef, R. and Suter, U. (1998) Many facets of the peripheral myelin protein PMP22 in myelination and disease Microsc. Res. Tech. 41, 359– 371 DOI: 10.1002/(SICI)1097-0029(19980601)41:5<359::AID-JEMT3>3.0.CO;2-L
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    30
    Many facets of the peripheral myelin protein PMP22 in myelination and disease
    Naef, Roland; Suter, Ueli
    Microscopy Research and Technique (1998), 41 (5), 359-371CODEN: MRTEEO; ISSN:1059-910X. (Wiley-Liss, Inc.)
    A review with many refs. Peripheral myelin protein 22 (PMP22) is a small, hydrophobic glycoprotein, which is most prominently expressed by Schwann cells as a component of compact myelin of the peripheral nervous system (PNS). Recent progress in mol. genetics revealed that mutations affecting the PMP22 gene including duplications, deletions, and point mutations are responsible for the most common forms of hereditary peripheral neuropathies including Charcot-Marie-Tooth disease type 1A (CMT1A), hereditary neuropathy with liability to pressure palsies (HNPP), and a subtype of Dejerine-Sottas Syndrome (DSS). Functionally, PMP22 is involved in correct myelination during development of peripheral nerves, the stability of myelin, and the maintenance of axons. While most of these functions relate to a role of PMP22 as a structural component of myelin, PMP22 has also been proposed as a regulator of Schwann cell proliferation and differentiation. In this review, the authors will discuss their current knowledge of PMP22 and its related proteins in the normal organism as well as in disease. In particular, the authors will focus on how the function of PMP22 and its regulation may be relevant to particular disease mechanisms.
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    Ryan, M. C., Shooter, E. M., and Notterpek, L. (2002) Aggresome formation in neuropathy models based on peripheral myelin protein 22 mutations Neurobiol. Dis. 10, 109– 118 DOI: 10.1006/nbdi.2002.0500
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    Aggresome Formation in Neuropathy Models Based on Peripheral Myelin Protein 22 Mutations
    Ryan, Mary C.; Shooter, Eric M.; Notterpek, Lucia
    Neurobiology of Disease (2002), 10 (2), 109-118CODEN: NUDIEM; ISSN:0969-9961. (Elsevier Science)
    Alterations in peripheral myelin protein 22 (PMP22) gene expression are assocd. with demyelinating peripheral neuropathies. Overexpression of wild type (wt) PMP22 or inhibition of proteasomal degrdn. lead to the formation of aggresomes, intracellular ubiquitinated PMP22 aggregates. Aggresome formation has now been obsd. with two mutant PMP22s, the Tr- and TrJ-PMP22 when the proteasome is inhibited. The formation of these aggresomes required intact microtubules and involved the recruitment of chaperones, including Hsp40, Hsp70, and αB-crystallin. Spontaneously formed ubiquitinated PMP22 aggregates were also obsd. in Schwann cells of homozygous TrJ mice. Significant upregulation of both the ubiquitin-proteasomal and lysosomal pathways occurred in affected nerves suggesting that two pathways of PMP22 degrdn. are present. Thus, the presence of aggresomes appears to be a common finding in neuropathy models of PMP22 overexpression and of some point mutations known to cause neuropathy in mice and humans.
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  32. 32
    Danoff, E. J. and Fleming, K. G. (2015) Aqueous, unfolded OmpA forms amyloid-like fibrils upon self-association PLoS One 10, e0132301 DOI: 10.1371/journal.pone.0132301
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    Aqueous, unfolded OmpA forms amyloid- like fibrils upon self-association
    Danoff, Emily J.; Fleming, Karen G.
    PLoS One (2015), 10 (7), e0132301/1-e0132301/10CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
    Unfolded outer membrane beta-barrel proteins have been shown to self-assoc. in the absence of lipid bilayers. We previously investigated the formation of high mol. wt. species by OmpA, with both the transmembrane domain alone and the full-length protein, and discovered that the oligomeric form contains non-native β -sheet structure. We have further probed the conformation of self-assocd. OmpA by monitoring binding to Thioflavin T, a dye that is known to bind the cross- β a structure inherent in amyloid fibrils, and by observing the species by electron microscopy. The significant increase in fluorescence indicative of Thioflavin T binding and the appearance of fibrillar species by electron microscopy verify that the protein forms amyloid-like fibril structures upon oligomerization. These results are also consistent with our previous kinetic anal. of OmpA self-assocn. that revealed a nucleated growth polymn. mechanism, which is frequently obsd. in amyloid formation. The discovery of OmpA's ability to form amyloid-like fibrils provides a new model protein with which to study fibrillization, and implicates periplasmic chaperone proteins as capable of inhibiting fibril formation.
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    Abramson, J., Smirnova, I., Kasho, V., Verner, G., Kaback, H. R., and Iwata, S. (2003) Structure and mechanism of the lactose permease of Escherichia coli Science 301, 610– 615 DOI: 10.1126/science.1088196
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    Structure and Mechanism of the Lactose Permease of Escherichia coli
    Abramson, Jeff; Smirnova, Irina; Kasho, Vladimir; Verner, Gillian; Kaback, H. Ronald; Iwata, So
    Science (Washington, DC, United States) (2003), 301 (5633), 610-616CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    Membrane transport proteins that transduce free energy stored in electrochem. ion gradients into a concn. gradient are a major class of membrane proteins. We report the crystal structure at 3.5 angstroms of the Escherichia coli lactose permease, an intensively studied member of the major facilitator superfamily of transporters. The mol. is composed of N- and C-terminal domains, each with six transmembrane helixes, sym. positioned within the permease. A large internal hydrophilic cavity open to the cytoplasmic side represents the inward-facing conformation of the transporter. The structure with a bound lactose homolog, β-D-galactopyranosyl-1-thio-β-D-galactopyranoside, reveals the sugar-binding site in the cavity, and residues that play major roles in substrate recognition and proton translocation are identified. We propose a possible mechanism for lactose/proton symport (cotransport) consistent with both the structure and a large body of exptl. data.
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    Guan, L., Mirza, O., Verner, G., Iwata, S., and Kaback, H. R. (2007) Structural determination of wild-type lactose permease Proc. Natl. Acad. Sci. U. S. A. 104, 15294– 15298 DOI: 10.1073/pnas.0707688104
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    34
    Structural determination of wild-type lactose permease
    Guan, Lan; Mirza, Osman; Verner, Gillian; Iwata, So; Kaback, H. Ronald
    Proceedings of the National Academy of Sciences of the United States of America (2007), 104 (39), 15294-15298CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Here we describe an X-ray structure of wild-type lactose permease (LacY) from Escherichia coli detd. by manipulating phospholipid content during crystn. The structure exhibits the same global fold as the previous X-ray structures of a mutant that binds sugar but cannot catalyze translocation across the membrane. LacY is organized into two six-helix bundles with twofold pseudosymmetry sepd. by a large interior hydrophilic cavity open only to the cytoplasmic side and contg. the side chains important for sugar and H+ binding. To initiate transport, binding of sugar and/or an H+ electrochem. gradient increases the probability of opening on the periplasmic side. Because the inward-facing conformation represents the lowest free-energy state, the rate-limiting step for transport may be the conformational change leading to the outward-facing conformation.
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    Guan, L. and Kaback, H. R. (2006) Lessons from lactose permease Annu. Rev. Biophys. Biomol. Struct. 35, 67– 91 DOI: 10.1146/annurev.biophys.35.040405.102005
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    Lessons from lactose permease
    Guan, Lan; Kaback, H. Ronald
    Annual Review of Biophysics and Biomolecular Structure (2006), 35 (), 67-91CODEN: ABBSE4; ISSN:1056-8700. (Annual Reviews Inc.)
    A review. The x-ray crystal structure of the lactose permease of Escherichia coli (LacY) in an inward-facing conformation has been solved. LacY contains N- and C-terminal domains, each with 6 transmembrane helixes, positioned pseudosym. The ligand is bound at the apex of a hydrophilic cavity in approx. the middle of the mol. Residues involved in substrate binding and H+ translocation are aligned parallel to the membrane at the same level and may be exposed to a water-filled cavity in both the inward- and outward-facing conformations, thereby allowing both sugar and H+ release directly into either cavity. These structural features may explain why LacY catalyzes galactoside/H+ symport in both directions utilizing the same residues. A working model for the mechanism is presented that involves alternating access of both the sugar- and H+-binding sites to either side of the membrane.
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    Madej, M. G. (2014) Function, structure, and evolution of the Major Facilitator Superfamily: the LacY manifesto Adv. Biol. 2014, 1– 20 DOI: 10.1155/2014/523591
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    Harris, N. J., Findlay, H. E., Simms, J., Liu, X., and Booth, P. J. (2014) Relative domain folding and stability of a membrane transport protein J. Mol. Biol. 426, 1812– 1825 DOI: 10.1016/j.jmb.2014.01.012
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    Findlay, H. E. and Booth, P. J. (2013) Folding alpha-helical membrane proteins into liposomes in vitro and determination of secondary structure Methods Mol. Biol. 1063, 117– 124 DOI: 10.1007/978-1-62703-583-5_6
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    Folding Alpha-Helical Membrane Proteins into Liposomes In Vitro and Determination of Secondary Structure
    Findlay, Heather E.; Booth, Paula J.
    Methods in Molecular Biology (New York, NY, United States) (2013), 1063 (Membrane Proteins), 117-124CODEN: MMBIED; ISSN:1064-3745. (Springer)
    The native environment of integral membrane proteins is a highly complex lipid bilayer composed of many different types of lipids, the phys. characteristics of which can profoundly influence protein stability, folding, and function. Secondary transporters are a class of protein where changes to both structure and activity have been obsd. in different bilayer environments. In order to study these interactions in vitro, it is necessary to ext. and purify the protein and exchange it into an artificial lipid system that can be manipulated to control protein behavior. Liposomes are a commonly used model system that is particularly suitable for studying transporters. GalP and LacY can be reconstituted or refolded into vesicles with a high degree of efficiency for further structural anal. CD spectroscopy is an important technique in monitoring protein folding, which allows the decompn. of spectra into secondary structural components.
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    Sanders, C. R. and Mittendorf, K. F. (2011) Tolerance to changes in membrane lipid composition as a selected trait of membrane proteins Biochemistry 50, 7858– 7867 DOI: 10.1021/bi2011527
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    Tolerance to Changes in Membrane Lipid Composition as a Selected Trait of Membrane Proteins
    Sanders, Charles R.; Mittendorf, Kathleen F.
    Biochemistry (2011), 50 (37), 7858-7867CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    A review. Membrane lipid compn. can vary dramatically across the three domains of life and even within single organisms. Here evidence that the lipid-exposed surfaces of membrane proteins have generally evolved to maintain correct structure and function in the face of major changes in lipid compn. is reviewed. Such tolerance has allowed evolution to extensively remodel membrane lipid compns. during the emergence of new species without having to extensively remodel the assocd. membrane proteins. The tolerance of membrane proteins also permits single-cell organisms to vary their membrane lipid compn. in response to their changing environments and allows dynamic and organelle-specific variations in the lipid compns. of eukaryotic cells. Membrane protein structural biol. has greatly benefited from this seemingly intrinsic property of membrane proteins: the majority of structures detd. to date have been characterized under model membrane conditions that little resemble those of native membranes. Nevertheless, with a few notable exceptions, most exptl. detd. membrane protein structures appear, to a good approxn., to faithfully report on native structure.
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    Alder, N. N. and Johnson, A. E. (2004) Cotranslational membrane protein biogenesis at the endoplasmic reticulum J. Biol. Chem. 279, 22787– 22790 DOI: 10.1074/jbc.R400002200
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    Vembar, S. S. and Brodsky, J. L. (2008) One step at a time: endoplasmic reticulum-associated degradation Nat. Rev. Mol. Cell Biol. 9, 944– 957 DOI: 10.1038/nrm2546
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    One step at a time: endoplasmic reticulum-associated degradation
    Vembar, Shruthi S.; Brodsky, Jeffrey L.
    Nature Reviews Molecular Cell Biology (2008), 9 (12), 944-957CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)
    A review. Protein folding in the endoplasmic reticulum (ER) is monitored by ER quality control (ERQC) mechanisms. Proteins that pass ERQC criteria traffic to their final destinations through the secretory pathway, whereas non-native and unassembled subunits of multimeric proteins are degraded by the ER-assocd. degrdn. (ERAD) pathway. During ERAD, mol. chaperones and assocd. factors recognize and target substrates for retrotranslocation to the cytoplasm, where they are degraded by the ubiquitin-proteasome machinery. The discovery of diseases that are assocd. with ERAD substrates highlights the importance of this pathway. Here, the authors summarize their current understanding of each step during ERAD, with emphasis on the factors that catalyze distinct activities.
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    Ibstedt, S., Sideri, T. C., Grant, C. M., and Tamas, M. J. (2014) Global analysis of protein aggregation in yeast during physiological conditions and arsenite stress Biol. Open 3, 913– 923 DOI: 10.1242/bio.20148938
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    Global analysis of protein aggregation in yeast during physiological conditions and arsenite stress
    Ibstedt, Sebastian; Sideri, Theodora C.; Grant, Chris M.; Tamas, Markus J.
    Biology Open (2014), 3 (10), 913-923, 11 pp.CODEN: BOIPBR; ISSN:2046-6390. (Company of Biologists Ltd.)
    Protein aggregation is a widespread phenomenon in cells and assocd. with pathol. conditions. Yet, little is known about the rules that govern protein aggregation in living cells. In this study, we biochem. isolated aggregation-prone proteins and used computational analyses to identify characteristics that are linked to physiol. and arsenite-induced aggregation in living yeast cells. High protein abundance, extensive phys. interactions, and certain structural properties are pos. correlated with an increased aggregation propensity. The aggregated proteins have high translation rates and are substrates of ribosome-assocd. Hsp70 chaperones, indicating that they are susceptible for aggregation primarily during translation/folding. The aggregation-prone proteins are enriched for multiple chaperone interactions, thus high protein abundance is probably counterbalanced by mol. chaperones to allow sol. expression in vivo. Our data support the notion that arsenite interferes with chaperone activity and indicate that arsenite-aggregated proteins might engage in extensive aberrant protein-protein interactions. Expression of aggregation-prone proteins is down-regulated during arsenite stress, possibly to prevent their toxic accumulation. Several aggregation-prone yeast proteins have human homologues that are implicated in misfolding diseases, suggesting that similar mechanisms may apply in disease- and non-disease settings.
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    Liao, L., Cheng, D., Wang, J., Duong, D. M., Losik, T. G., Gearing, M., Rees, H. D., Lah, J. J., Levey, A. I., and Peng, J. (2004) Proteomic characterization of postmortem amyloid plaques isolated by laser capture microdissection J. Biol. Chem. 279, 37061– 37068 DOI: 10.1074/jbc.M403672200
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    Proteomic Characterization of Postmortem Amyloid Plaques Isolated by Laser Capture Microdissection
    Liao, Lujian; Cheng, Dongmei; Wang, Jian; Duong, Duc M.; Losik, Tatyana G.; Gearing, Marla; Rees, Howard D.; Lah, James J.; Levey, Allan I.; Peng, Junmin
    Journal of Biological Chemistry (2004), 279 (35), 37061-37068CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    The presence of amyloid plaques in the brain is one of the pathol. hallmarks of Alzheimer's disease (AD). We report here a comprehensive proteomic anal. of senile plaques from postmortem AD brain tissues. Senile plaques labeled with thioflavin-S were procured by laser capture microdissection, and their protein components were analyzed by liq. chromatog. coupled with tandem mass spectrometry. We identified a total of 488 proteins coisolated with the plaques, and we found multiple phosphorylation sites on the neurofilament intermediate chain, implicating the complexity and diversity of cellular processes involved in the plaque formation. More significantly, we identified 26 proteins enriched in the plaques of two AD cases by quant. comparison with surrounding non-plaque tissues. The localization of several proteins in the plaques was further confirmed by the approach of immunohistochem. In addn. to previously identified plaque constituents, we discovered novel assocn. of dynein heavy chain with the plaques in human postmortem brain and in a double transgenic AD mouse model, suggesting that neuronal transport may play a role in neuritic degeneration. Overall, our results revealed for the first time the sub-proteome of amyloid plaques that is important for further studies on disease biomarker identification and mol. mechanisms of AD pathogenesis.
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    Sergeant, N., Wattez, A., Galvan-Valencia, M., Ghestem, A., David, J. P., Lemoine, J., Sautiere, P.-E., Dachary, J., Mazat, J. P., Michalski, J. C., Velours, J., Mena-Lopez, R., and Delacourte, A. (2003) Association of ATP synthase alpha-chain with neurofibrillary degeneration in Alzheimer’s disease Neuroscience 117, 293– 303 DOI: 10.1016/S0306-4522(02)00747-9
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    Murakami, T., Shoji, M., Imai, Y., Inoue, H., Kawarabayashi, T., Matsubara, E., Harigaya, Y., Sasaki, A., Takahashi, R., and Abe, K. (2004) Pael-R is accumulated in Lewy bodies of Parkinson’s disease Ann. Neurol. 55, 439– 442 DOI: 10.1002/ana.20064
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    Pael-R is accumulated in Lewy bodies of Parkinson's disease
    Murakami, Tetsuro; Shoji, Mikio; Imai, Yuzuru; Inoue, Haruhisa; Kawarabayashi, Takeshi; Matsubara, Etsuro; Harigaya, Yasuo; Sasaki, Atsushi; Takahashi, Ryosuke; Abe, Koji
    Annals of Neurology (2004), 55 (3), 439-442CODEN: ANNED3; ISSN:0364-5134. (Wiley-Liss, Inc.)
    The authors examd. the distribution of Pael-R, a newly identified substrate for Parkin, in Parkinson's disease (PD) and multiple system atrophy (MSA). Pael-R, Parkin, α-synuclein, and ubiquitin accumulated in Lewy bodies (LBs) and neurites. Pael-R was localized in the care of LBs. Parkin and α-synuclein accumulated in the halo, neuronal cell bodies, and processes. These findings potentially suggest the involvement of Pael-R in LB formation, and protection role of Parkin in Pael-R-mediated neurotoxicity in PD. The absence of Pael-R and Parkin in glial cytoplasmic inclusions (GCIs) in MSA implies a distinct pathway involved in the formation of LBs and GCIs.
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    Sormanni, P., Aprile, F. A., and Vendruscolo, M. (2015) The CamSol method of rational design of protein mutants with enhanced solubility J. Mol. Biol. 427, 478– 490 DOI: 10.1016/j.jmb.2014.09.026
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    The CamSol Method of Rational Design of Protein Mutants with Enhanced Solubility
    Sormanni, Pietro; Aprile, Francesco A.; Vendruscolo, Michele
    Journal of Molecular Biology (2015), 427 (2), 478-490CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
    Protein soly. is often an essential requirement in biotechnol. and biomedical applications. Great advances in understanding the principles that det. this specific property of proteins have been made during the past decade, in particular concerning the physicochem. characteristics of their constituent amino acids. By exploiting these advances, we present the CamSol method for the rational design of protein variants with enhanced soly. The method works by performing a rapid computational screening of tens of thousand of mutations to identify those with the greatest impact on the soly. of the target protein while maintaining its native state and biol. activity. The application to a single-domain antibody that targets the Alzheimer's Aβ peptide demonstrates that the method predicts with great accuracy soly. changes upon mutation, thus offering a cost-effective strategy to help the prodn. of sol. proteins for academic and industrial purposes.
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    Guan, L., Murphy, F. D., and Kaback, H. R. (2002) Surface-exposed positions in the transmembrane helices of the lactose permease of Escherichia coli determined by intermolecular thiol cross-linking Proc. Natl. Acad. Sci. U. S. A. 99, 3475– 3480 DOI: 10.1073/pnas.052703699
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    Strop, P. and Brunger, A. T. (2005) Refractive index-based determination of detergent concentration and its application to the study of membrane proteins Protein Sci. 14, 2207– 2211 DOI: 10.1110/ps.051543805
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    Refractive index-based determination of detergent concentration and its application to the study of membrane proteins
    Strop, Pavel; Brunger, Axel T.
    Protein Science (2005), 14 (8), 2207-2211CODEN: PRCIEI; ISSN:0961-8368. (Cold Spring Harbor Laboratory Press)
    The concn. of detergent in membrane protein prepns. can have a crit. role on protein stability, function, and the potential for crystn. Unfortunately, dialysis or protein concn. can lead to an unknown amt. of detergent in the final membrane protein prepns. Here we present a method for the detn. of detergent concn. based on refractive index of the detergent soln. This method was applied to quantitate the amt. of detergent remaining in soln. after concn. in various concentrators. We found that the ability of the tested detergents to pass through the mol. wt. cutoff membrane correlates well with detergent micelle size. Therefore, the micelle size can be used as a rough guide to est. the retention of a given detergent in various mol. wt. cutoff concentrators. The refractive index method is exceptionally informative when coupled with size exclusion chromatog. and light scattering, and can be used to det. the oligomeric state of the membrane protein, the size of a protein-assocd. micelle, as well as the amt. and size of the unbound detergent micelle.
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    Urbani, A. and Warne, T. (2005) A colorimetric determination for glycosidic and bile salt-based detergents: applications in membrane protein research Anal. Biochem. 336, 117– 124 DOI: 10.1016/j.ab.2004.09.040
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    A colorimetric determination for glycosidic and bile salt-based detergents: applications in membrane protein research
    Urbani, Andrea; Warne, Tony
    Analytical Biochemistry (2005), 336 (1), 117-124CODEN: ANBCA2; ISSN:0003-2697. (Elsevier)
    Detergents are crucial to the isolation of integral membrane proteins. During membrane protein purifn., it is useful to accurately quantify detergent, esp. if concn. steps have been used. Previously, this has been difficult and time-consuming. We present a simple, rapid, and sensitive procedure for the quantification of glycosidic and bile salt-based detergents such as dodecylmaltoside, octylglucoside, and CHAPS. The method directly quantifies sugar or cholate moieties via colorimetric reactions with phenol and sulfuric acid. A no. of detergents have been screened, and the assay has been validated in the presence of commonly used reagents. In addn. to detg. the overall detergent concn. in soln., the procedure allows accurate quantification of specific binding of glycosidic or bile salt-based detergents to purified membrane proteins. Both the colorimetric method and the radiometric 14C method were used to det. detergent binding to two integral membrane proteins: the cytochrome cbb3 oxidase from Pseudomonas stutzeri and the turkey β-adrenergic receptor. Both methods gave similar results. After sepg. monomeric glycosidic detergent from micellar solns. by ultrafiltration, we used the colorimetric method to det. the concn. of monomeric detergent present. We obsd. that values obtained are in close agreement with previously detd. crit. micelle concns.
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    Kelly, S. M., Jess, T. J., and Price, N. C. (2005) How to study proteins by circular dichroism Biochim. Biophys. Acta, Proteins Proteomics 1751, 119– 139 DOI: 10.1016/j.bbapap.2005.06.005
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    How to study proteins by circular dichroism
    Kelly, Sharon M.; Jess, Thomas J.; Price, Nicholas C.
    Biochimica et Biophysica Acta, Proteins and Proteomics (2005), 1751 (2), 119-139CODEN: BBAPBW; ISSN:1570-9639. (Elsevier B.V.)
    A review. CD is being increasingly recognized as a valuable technique for examg. the structure of proteins in soln. However, the value of many studies using CD is compromised either by inappropriate exptl. design or by lack of attention to key aspects of instrument calibration or sample characterization. In this article, we summarize the basis of the CD approach and its application to the study of proteins, and then present clear guidelines on how reliable data can be obtained and analyzed.
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    Kuhlbrandt, W. (1988) 3-Dimensional cystallization of membrane-proteins Q. Rev. Biophys. 21, 429– 477 DOI: 10.1017/S0033583500004625
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    Three-dimensional crystallization of membrane proteins
    Kuhlbrandt W
    Quarterly reviews of biophysics (1988), 21 (4), 429-77 ISSN:0033-5835.
    There is no expanded citation for this reference.
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    Engel, C. K., Chen, L., and Privé, G. G. (2002) Stability of the lactose permease in detergent solutions Biochim. Biophys. Acta, Biomembr. 1564, 47– 56 DOI: 10.1016/S0005-2736(02)00397-8
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    Stability of the lactose permease in detergent solutions
    Engel, Christian K.; Chen, Lu; Prive, Gilbert G.
    Biochimica et Biophysica Acta, Biomembranes (2002), 1564 (1), 47-56CODEN: BBBMBS; ISSN:0005-2736. (Elsevier B.V.)
    Protein stability, as measured by irreversible protein aggregation, is one of the central difficulties in the handling of detergent-solubilized membrane proteins. We present a quant. anal. of the stability of the Escherichia coli lactose (lac) permease and a series of lac permease fusion proteins contg. an insertion of cytochromeb562, T4 lysozyme or β-lactamase in the central hydrophilic loop of the permease. The stability of the proteins was evaluated under a variety of storage conditions by both a qual. SDS-PAGE assay and by a quant. HPLC assay. Long-chain maltoside detergents were more effective at maintaining purified protein in soln. than detergents with smaller head groups and/or shorter alkyl tails. A full factorial expt. established that the proteins were insensitive to sodium chloride concns., but greatly stabilized by glycerol, low temp. and the combination of glycerol and low temp. The accurate quantitation of the protein by absorbance spectroscopy required exclusion of all contact with clarified polypropylene or polyvinyl chloride (PVC) materials. Although some of the fusion proteins were more prone to aggregation than the wild-type permease, the stability of a fusion protein contg. a cytochromeb562 insertion was indistinguishable from that of native lac permease.
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    Biancalana, M. and Koide, S. (2010) Molecular mechanism of Thioflavin-T binding to amyloid fibrils Biochim. Biophys. Acta, Proteins Proteomics 1804, 1405– 1412 DOI: 10.1016/j.bbapap.2010.04.001
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    Molecular mechanism of thioflavin-T binding to amyloid fibrils
    Biancalana, Matthew; Koide, Shohei
    Biochimica et Biophysica Acta, Proteins and Proteomics (2010), 1804 (7), 1405-1412CODEN: BBAPBW; ISSN:1570-9639. (Elsevier B. V.)
    A review. Intense efforts to detect, diagnose, and analyze the kinetic and structural properties of amyloid fibrils have generated a powerful toolkit of amyloid-specific mol. probes. Since its 1st description in 1959, the fluorescent dye, thioflavin-T (ThT), has become among the most widely used "gold stds." for selectively staining and identifying amyloid fibrils both in vivo and in vitro. The large enhancement of its fluorescence emission upon its binding to fibrils makes ThT a particularly powerful and convenient tool. Despite its widespread use in clin. and basic science applications, the mol. mechanism for the ability of ThT to recognize diverse types of amyloid fibrils and for the dye's characteristic fluorescence has only begun to be elucidated. Here, the authors review recent progress in the understanding of ThT-fibril interactions at at. resoln. These studies have yielded important insights into amyloid structures and the processes of fibril formation, and they also offer guidance for designing the next generation of amyloid assembly diagnostics, inhibitors, and therapeutics.
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    Khurana, R., Coleman, C., Ionescu-Zanetti, C., Carter, S. A., Krishna, V., Grover, R. K., Roy, R., and Singh, S. (2005) Mechanism of thioflavin T binding to amyloid fibrils J. Struct. Biol. 151, 229– 238 DOI: 10.1016/j.jsb.2005.06.006
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    Mechanism of thioflavin T binding to amyloid fibrils
    Khurana, Ritu; Coleman, Chris; Ionescu-Zanetti, Cristian; Carter, Sue A.; Krishna, Vinay; Grover, Rajesh K.; Roy, Raja; Singh, Shashi
    Journal of Structural Biology (2005), 151 (3), 229-238CODEN: JSBIEM; ISSN:1047-8477. (Elsevier)
    Thioflavin T is a benzothiazole dye that exhibits enhanced fluorescence upon binding to amyloid fibrils and is commonly used to diagnose amyloid fibrils, both ex vivo and in vitro. In aq. solns., thioflavin T was found to exist as micelles at concns. commonly used to monitor fibrils by fluorescence assay (∼10-20 μM). Specific cond. changes were measured at varying concn. of thioflavin T and the crit. micellar concn. was calcd. to be 4.0±0.5 μM. Interestingly, changes in the fluorescence excitation and emission of thioflavin T were also dependent on the micelle formation. The thioflavin T micelles of 3 nm diam. were directly visualized using at. force microscopy, and bound thioflavin T micelles were obsd. along the fibril length for representative fibrils. Increasing concn. of thioflavin T above the crit. micellar concn. shows increased nos. of micelles bound along the length of the amyloid fibrils. Thioflavin T micelles were disrupted at low pH as obsd. by at. force microscopy and fluorescence enhancement upon binding of thioflavin T to amyloid fibrils also reduced by several-fold upon decreasing the pH to below 3. This suggests that pos. charge on the thioflavin T mol. has a role in its micelle formation that then bind the amyloid fibrils. The authors' data suggests that the micelles of thioflavin T bind amyloid fibrils leading to enhancement of fluorescence emission.
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  • Abstract

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    Figure 1

    Figure 1. CamSol profile of LacY, as an indicator of its aggregation propensity. (A) Native structure of LacY (front and bottom view), which is shown for reference. The Protein Data Bank structure 2V8N (34) was used to draw the cartoons, with cylinders representing α-helices and ribbons representing disordered coils. (B) CamSol (46) profile of LacY (see Materials and Methods). Regions colored red have a low intrinsic solubility and hence a high aggregation propensity. The positions of α-helices (as derived from ref 47) are indicated by horizontal gray bars.

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    Figure 2

    Figure 2. Fraction of LacY in the supernatant over time. LacY solutions (2 μM) incubated in 50 mM phosphate buffer (pH 7.4) with 0.05% DDM (37 °C for 48 h) were centrifuged to precipitate the aggregated fraction, and the remaining supernatant was analyzed by SDS–PAGE after 0, 1, 2, 3, 4, 5, 6, 7, 8, 24, and 48 h (as indicated). LacY appears at ∼28 kDa because it does not unfold in SDS and thus runs in its folded state (its expected MW is 48 kDa). The experiment was repeated twice; a representative data set is shown.

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    Figure 3

    Figure 3. Enhancement of ThT fluorescence by LacY aggregates. (A) ThT fluorescence changes in monomer solutions of 2 μM (yellow), 4 μM (red), and 8 μM (blue) LacY (in duplicate) in 50 mM sodium phosphate (pH 7.4) with 0.05% DDM. The solutions were incubated (37 °C for 48 h) in the presence of 4 μM ThT, and the ThT fluorescence was continuously monitored. The fluorescence spectra were corrected for the signal in 50 mM sodium phosphate (pH 7.4) with 0.05% DDM. (B) The spectra shown in panel A were normalized by subtracting the minimum and dividing by the maximal fluorescence intensity for the respective data set. This procedure allows a straightforward comparison of the slopes of the intensity profiles for different concentrations. The experiment was repeated five times; a representative data set is presented.

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    Figure 4

    Figure 4. CD intensity decrease upon aggregation of LacY. LacY (2 μM) was incubated in 50 mM phosphate buffer (pH 7.4) with 0.05% DDM at 37 °C. CD spectra were recorded before (blue) and after (red) incubation for 48 h. The spectra were corrected for 50 mM sodium phosphate (pH 7.4) with 0.05% DDM. A minimum of three scans was averaged per time point. The experiment was repeated twice; a representative data set is presented.

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    Figure 5

    Figure 5. Secondary structure in LacY aggregates detected using FT-IR. (A) LacY (16 μM) was incubated in 50 mM phosphate buffer (pH 7.4) with 0.05–0.06% DDM at 37 °C. FT-IR spectra were recorded before (blue) and after (red) incubation for 48 h and corrected by subtracting the spectrum of the buffer solution. The dotted lines indicate the peak maxima, and the arrows illustrate the peak shifts. A spectrum of LacY prior to concentration is not shown, because the protein directly obtained from the purification was not concentrated enough to result in a detectable spectrum. (B) Second derivative of the spectra before (blue) and after (red) incubation for 48 h showing the appearance of peak positions in the amide I region at 1653 and 1623 cm–1, respectively. The dotted lines indicate the main peak minima. The experiment was repeated twice; a representative data set is presented.

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    Figure 6

    Figure 6. TEM and X-ray fiber diffraction data of LacY aggregates. (A) LacY (16–32 μM) was incubated in 50 mM phosphate buffer (pH 7.4) with 0.05–0.06% DDM at 37 °C. Fiber diffraction patterns from air-dried stalks of the samples were measured for the samples incubated for 48 h and (B) samples treated with guanidinium hydrochloride. The arrowheads indicate the reflections at ∼4.6 Å, while the arrows indicate the reflections at ∼10.8 Å. TEM grids were prepared for the same samples (C) after incubation for 48 h and (D) after incubation for 48 h followed by washing with 3 M guanidinium hydrochloride. These experiments were repeated three times, and representative diffraction patterns and TEM images are shown.

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  • References

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      A review. The phenomenon of protein aggregation and amyloid formation has become the subject of rapidly increasing research activities across a wide range of scientific disciplines. Such activities have been stimulated by the assocn. of amyloid deposition with a range of debilitating medical disorders, from Alzheimer's disease to type II diabetes, many of which are major threats to human health and welfare in the modern world. It has become clear, however, that the ability to form the amyloid state is more general than previously imagined, and that its study can provide unique insights into the nature of the functional forms of peptides and proteins, as well as understanding the means by which protein homeostasis can be maintained and protein metastasis avoided.
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    4. 4
      Chiti, F. and Dobson, C. M. (2006) Protein misfolding, functional amyloid, and human disease Annu. Rev. Biochem. 75, 333– 366 DOI: 10.1146/annurev.biochem.75.101304.123901
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      4
      Protein misfolding, functional amyloid, and human disease
      Chiti, Fabrizio; Dobson, Christopher M.
      Annual Review of Biochemistry (2006), 75 (), 333-366CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews Inc.)
      A review. Peptides or proteins convert under some conditions from their sol. forms into highly ordered fibrillar aggregates. Such transitions can give rise to pathol. conditions ranging from neurodegenerative disorders to systemic amyloidoses. In this review, we identify the diseases known to be assocd. with formation of fibrillar aggregates and the specific peptides and proteins involved in each case. We describe, in addn., that living organisms can take advantage of the inherent ability of proteins to form such structures to generate novel and diverse biol. functions. We review recent advances toward the elucidation of the structures of amyloid fibrils and the mechanisms of their formation at a mol. level. Finally, we discuss the relative importance of the common main-chain and side-chain interactions in detg. the propensities of proteins to aggregate and describe some of the evidence that the oligomeric fibril precursors are the primary origins of pathol. behavior.
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    5. 5
      Sunde, M., Serpell, L. C., Bartlam, M., Fraser, P. E., Pepys, M. B., and Blake, C. C. F. (1997) Common core structure of amyloid fibrils by synchrotron X-ray diffraction J. Mol. Biol. 273, 729– 739 DOI: 10.1006/jmbi.1997.1348
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      Common core structure of amyloid fibrils by synchrotron X-ray diffraction
      Sunde, Margaret; Serpell, Louise C.; Bartlam, Mark; Fraser, Paul E.; Pepys, Mark B.; Blake, Colin C. F.
      Journal of Molecular Biology (1997), 273 (3), 729-739CODEN: JMOBAK; ISSN:0022-2836. (Academic Press Ltd.)
      Tissue deposition of normally sol. proteins as insol. amyloid fibrils is assocd. with serious diseases including the systemic amyloidoses, maturity onset diabetes, Alzheimer's disease, and transmissible spongiform encephalopathy. Although the precursor proteins in different diseases do not share sequence homol. or related native structure, the morphol. and properties of all amyloid fibrils are remarkably similar. Using intense synchrotron sources, we obsd. that six different ex vivo amyloid fibrils and two synthetic fibril prepns. all gave similar high-resoln. X-ray fiber diffraction patterns, consistent with a helical array of β-sheets parallel to the fiber long axis, with the strands perpendicular to this axis. This confirms that amyloid fibrils comprise a structural superfamily and share a common protofilament substructure, irresp. of the nature of their precursor proteins.
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    6. 6
      Glenner, G. G., Eanes, E. D., Bladen, H. A., Linke, R. P., and Termine, J. D. (1974) Beta-pleated sheet fibrils - comparison of native amyloid with synthetic protein fibrils J. Histochem. Cytochem. 22, 1141– 1158 DOI: 10.1177/22.12.1141
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      β-Pleated sheet fibrils. Comparison of native amyloid with synthetic protein fibrils
      Glenner, G. G.; Eanes, E. D.; Bladen, H. A.; Linke, R. P.; Termine, J. D.
      Journal of Histochemistry and Cytochemistry (1974), 22 (12), 1141-58CODEN: JHCYAS; ISSN:0022-1554.
      The title materials were compared by staining, electron microscopy, x-ray diffraction, and ir spectroscopy. These properties were analyzed and an attempt was made to define amyloids further and to clarify the terminol. related to the β-conformation. The term amyloidosis should be reserved for the lethal systemic disease and perhaps some benign hereditary varieties.
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    7. 7
      Chiti, F., Webster, P., Taddei, N., Clark, A., Stefani, M., Ramponi, G., and Dobson, C. M. (1999) Designing conditions for in vitro formation of amyloid protofilaments and fibrils Proc. Natl. Acad. Sci. U. S. A. 96, 3590– 3594 DOI: 10.1073/pnas.96.7.3590
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      7
      Designing conditions for in vitro formation of amyloid protofilaments and fibrils
      Chiti, Fabrizio; Webster, Paul; Taddei, Niccolo; Clark, Anne; Stefani, Massimo; Ramponi, Giampietro; Dobson, Christopher M.
      Proceedings of the National Academy of Sciences of the United States of America (1999), 96 (7), 3590-3594CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      We have been able to convert a small α/β protein, acylphosphatase, from its sol. and native form into insol. amyloid fibrils of the type obsd. in a range of pathol. conditions. This was achieved by allowing slow growth in a soln. contg. moderate concns. of trifluoroethanol. When analyzed with electron microscopy, the protein aggregate present in the sample after long incubation times consisted of extended, unbranched filaments of 30-50 Å in width that assemble subsequently into higher order structures. This fibrillar material possesses extensive β-sheet structure as revealed by far-UV CD and IR spectroscopy. Furthermore, the fibrils exhibit Congo red birefringence, increased fluorescence with thioflavine T and cause a red shift of the Congo red absorption spectrum. All of these characteristics are typical of amyloid fibrils. The results indicate that formation of amyloid occurs when the native fold of a protein is destabilized under conditions in which noncovalent interactions, and in particular hydrogen bonding, within the polypeptide chain remain favorable. We suggest that amyloid formation is not restricted to a small no. of protein sequences but is a property common to many, if not all, natural polypeptide chains under appropriate conditions.
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    8. 8
      Fandrich, M., Fletcher, M. A., and Dobson, C. M. (2001) Amyloid fibrils from muscle myoglobin - Even an ordinary globular protein can assume a rogue guise if conditions are right Nature 410, 165– 166 DOI: 10.1038/35065514
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      8
      Amyloid fibrils from muscle myoglobin
      Fandrich, Marcus; Fletcher, Matthew A.; Dobson, Christopher M.
      Nature (London, United Kingdom) (2001), 410 (6825), 165-166CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      A review, with 12 refs. The authors show here that myoglobin, the archetypal globular protein, can convert into an alternative and radically different structure that closely resembles the amyloid and prion aggregates seen in pathol. conditions such as Alzheimer's and Creutzfeldt-Jakob diseases". This most striking example of such a structural transition provides compelling evidence for the idea that amyloid represents a generic form of polypeptide conformation, but one that evolution has ensured is not normally formed in living systems.
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    9. 9
      Guijarro, J. I., Sunde, M., Jones, J. A., Campbell, I. D., and Dobson, C. M. (1998) Amyloid fibril formation by an SH3 domain Proc. Natl. Acad. Sci. U. S. A. 95, 4224– 4228 DOI: 10.1073/pnas.95.8.4224
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      9
      Amyloid fibril formation by an SH3 domain
      Guijarro, J. Inaki; Sunde, Margaret; Jones, Jonathan A.; Campbell, Iain D.; Dobson, Christopher M.
      Proceedings of the National Academy of Sciences of the United States of America (1998), 95 (8), 4224-4228CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      The SH3 domain is a well-characterized small protein module with a simple fold found in many proteins. At acid pH, the SH3 domain (PI3-SH3) of the p85α subunit of bovine phosphatidylinositol 3-kinase slowly forms a gel that consists of typical amyloid fibrils, as assessed by electron microscopy, a Congo red binding assay, and x-ray fiber diffraction. The sol. form of PI3-SH3 at acid pH (the A state by a variety of techniques) from which fibrils are generated has been characterized. CD in the far- and near-UV regions and 1H NMR indicate that the A state is unfolded relative to the native protein at neutral pH. NMR diffusion measurements indicate, however, that the effective hydrodynamic radius of the A state is only 23% higher than that of the native protein and is 20% lower than that of the protein denatured in 3.5 M guanidinium chloride. The A state binds the hydrophobic dye 1-anilinonaphthalene-8-sulfonic acid, which suggests that SH3 in this state has a partially formed hydrophobic core. These results indicate that the A state is partially folded and support the hypothesis that partially-folded states formed in soln. are precursors of amyloid deposition. Moreover, that this domain aggregates into amyloid fibrils suggests that the potential for amyloid deposition may be a common property of proteins, and not only of a few proteins assocd. with disease.
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    10. 10
      Dobson, C. M. (1999) Protein misfolding, evolution and disease Trends Biochem. Sci. 24, 329– 332 DOI: 10.1016/S0968-0004(99)01445-0
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      10
      Protein misfolding, evolution and disease
      Dobson, C. M.
      Trends in Biochemical Sciences (1999), 24 (9), 329-332CODEN: TBSCDB; ISSN:0968-0004. (Elsevier Science Ltd.)
      A review with 35 refs. discussing the linkage of protein misfolding to disease, aggregation of sol. proteins under denaturing conditions, amyloid as a generic structural form of proteins, amyloid formation in living systems, and new insights into evolutionary biol.
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      Rath, A. and Deber, C. M. (2007) Membrane protein assembly patterns reflect selection for non-proliferative structures FEBS Lett. 581, 1335– 1341 DOI: 10.1016/j.febslet.2007.02.050
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      Walther, D. M., Kasturi, P., Zheng, M., Pinkert, S., Vecchi, G., Ciryam, P., Morimoto, R. I., Dobson, C. M., Vendruscolo, M., Mann, M., and Hartl, F. U. (2015) Widespread proteome remodeling and aggregation in aging C-elegans Cell 161, 919– 932 DOI: 10.1016/j.cell.2015.03.032
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      Ciryam, P., Kundra, R., Freer, R., Morimoto, R. I., Dobson, C. M., and Vendruscolo, M. (2016) A transcriptional signature of Alzheimer’s disease is associated with a metastable subproteome at risk for aggregation Proc. Natl. Acad. Sci. U. S. A. 113, 4753– 4758 DOI: 10.1073/pnas.1516604113
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      Ciryam, P., Tartaglia, G. G., Morimoto, R. I., Dobson, C. M., and Vendruscolo, M. (2013) Widespread aggregation and neurodegenerative diseases are associated with supersaturated proteins Cell Rep. 5, 781– 790 DOI: 10.1016/j.celrep.2013.09.043
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      14
      Widespread Aggregation and Neurodegenerative Diseases Are Associated with Supersaturated Proteins
      Ciryam, Prajwal; Tartaglia, Gian Gaetano; Morimoto, Richard I.; Dobson, Christopher M.; Vendruscolo, Michele
      Cell Reports (2013), 5 (3), 781-790CODEN: CREED8; ISSN:2211-1247. (Cell Press)
      The maintenance of protein soly. is a fundamental aspect of cellular homeostasis because protein aggregation is assocd. with a wide variety of human diseases. Numerous proteins unrelated in sequence and structure, however, can misfold and aggregate, and widespread aggregation can occur in living systems under stress or aging. A crucial question in this context is why only certain proteins appear to aggregate readily in vivo, whereas others do not. We identify here the proteins most vulnerable to aggregation as those whose cellular concns. are high relative to their solubilities. We find that these supersatd. proteins represent a metastable subproteome involved in pathol. aggregation during stress and aging and are overrepresented in biochem. processes assocd. with neurodegenerative disorders. Consequently, such cellular processes become dysfunctional when the ability to keep intrinsically supersatd. proteins sol. is compromised. Thus, the simultaneous anal. of abundance and soly. can rationalize the diverse cellular pathologies linked to neurodegenerative diseases and aging.
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    15. 15
      Kang, J., Lemaire, H. G., Unterbeck, A., Salbaum, J. M., Masters, C. L., Grzeschik, K. H., Multhaup, G., Beyreuther, K., and Mullerhill, B. (1987) The precursor of Alzheimers disease amyloid-a4 protein resembles a cell-surface receptor Nature 325, 733– 736 DOI: 10.1038/325733a0
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      Berson, J. F., Theos, A. C., Harper, D. C., Tenza, D., Raposo, G., and Marks, M. S. (2003) Proprotein convertase cleavage liberates a fibrillogenic fragment of a resident glycoprotein to initiate melanosome biogenesis J. Cell Biol. 161, 521– 533 DOI: 10.1083/jcb.200302072
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      16
      Proprotein convertase cleavage liberates a fibrillogenic fragment of a resident glycoprotein to initiate melanosome biogenesis
      Berson, Joanne F.; Theos, Alexander C.; Harper, Dawn C.; Tenza, Danielle; Raposo, Graca; Marks, Michael S.
      Journal of Cell Biology (2003), 161 (3), 521-533CODEN: JCLBA3; ISSN:0021-9525. (Rockefeller University Press)
      Lysosome-related organelles are cell type-specific intracellular compartments with distinct morphologies and functions. The mol. mechanisms governing the formation of their unique structural features are not known. Melanosomes and their precursors are lysosome-related organelles that are characterized morphol. by intralumenal fibrous striations upon which melanins are polymd. The integral membrane protein Pmel17 is a component of the fibrils and can nucleate their formation in the absence of other pigment cell-specific proteins. Here, we show that formation of intralumenal fibrils requires cleavage of Pmel17 by a furin-like proprotein convertase (PC). As in the generation of amyloid, proper cleavage of Pmel17 liberates a lumenal domain fragment that becomes incorporated into the fibrils; longer Pmel17 fragments generated in the absence of PC activity are unable to form organized fibrils. Our results demonstrate that PC-dependent cleavage regulates melanosome biogenesis by controlling the fibrillogenic activity of a resident protein. Like the pathol. process of amyloidogenesis, the formation of other tissue-specific organelle structures may be similarly dependent on proteolytic activation of physiol. fibrillogenic substrates.
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    17. 17
      Wagner, S., Baars, L., Ytterberg, A. J., Klussmeier, A., Wagner, C. S., Nord, O., Nygren, P.-A., van Wijk, K. J., and de Gier, J.-W. (2007) Consequences of membrane protein overexpression in Escherichia coli Mol. Cell. Proteomics 6, 1527– 1550 DOI: 10.1074/mcp.M600431-MCP200
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      Consequences of membrane protein overexpression in Escherichia coli
      Wagner, Samuel; Baars, Louise; Ytterberg, A. Jimmy; Klussmeier, Anja; Wagner, Claudia S.; Nord, Olof; Nygren, Per-Aake; van Wijk, Klaas J.; de Gier, Jan-Willem
      Molecular and Cellular Proteomics (2007), 6 (9), 1527-1550CODEN: MCPOBS; ISSN:1535-9476. (American Society for Biochemistry and Molecular Biology)
      Overexpression of membrane proteins is often essential for structural and functional studies, but yields are frequently too low. An understanding of the physiol. response to overexpression is needed to improve such yields. Therefore, the authors analyzed the consequences of overexpression of three different membrane proteins (YidC, YedZ, and LepI) fused to green fluorescent protein (GFP) in the bacterium Escherichia coli and compared this with overexpression of a sol. protein, GST-GFP. Proteomes of total lysates, purified aggregates, and cytoplasmic membranes were analyzed by one- and two-dimensional gel electrophoresis and mass spectrometry complemented with flow cytometry, microscopy, Western blotting, and pulse labeling expts. Compn. and accumulation levels of protein complexes in the cytoplasmic membrane were analyzed with improved two-dimensional blue native PAGE. Overexpression of the three membrane proteins, but not sol. GST-GFP, resulted in accumulation of cytoplasmic aggregates contg. the overexpressed proteins, chaperones (DnaK/J and GroEL/S), and sol. proteases (HslUV and ClpXP) as well as many precursors of periplasmic and outer membrane proteins. This was consistent with lowered accumulation levels of secreted proteins in the three membrane protein overexpressors and is likely to be a direct consequence of satn. of the cytoplasmic membrane protein translocation machinery. Importantly, accumulation levels of respiratory chain complexes in the cytoplasmic membrane were strongly reduced. Induction of the acetate-phosphotransacetylase pathway for ATP prodn. and a downregulated tricarboxylic acid cycle indicated the activation of the Arc two-component system, which mediates adaptive responses to changing respiratory states. This study provides a basis for designing rational strategies to improve yields of membrane protein overexpression in E. coli.
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    18. 18
      Harris, N. J. and Booth, P. J. (2012) Folding and stability of membrane transport proteins in vitro Biochim. Biophys. Acta, Biomembr. 1818, 1055– 1066 DOI: 10.1016/j.bbamem.2011.11.006
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      18
      Folding and stability of membrane transport proteins in vitro
      Harris, Nicola J.; Booth, Paula J.
      Biochimica et Biophysica Acta, Biomembranes (2012), 1818 (4), 1055-1066CODEN: BBBMBS; ISSN:0005-2736. (Elsevier B.V.)
      A review. Transmembrane transporters are responsible for maintaining a correct internal cellular environment. The inherent flexibility of transporters together with their hydrophobic environment means that they are challenging to study in vitro, but recently significant progress been made. Here, the authors focus on in vitro stability and folding studies of transmembrane α-helical transporters, including reversible folding systems and thermal denaturation. The successful re-assembly of a small no. of ATP-binding cassette transporters is also described as this is a significant step forward in terms of understanding the folding and assembly of these more complex, multi-subunit proteins. The studies on transporters discussed here represent substantial advances for membrane protein studies as well as for research into protein folding. The work demonstrates that large flexible hydrophobic proteins are within reach of in vitro folding studies, thus holding promise for furthering knowledge on the structure, function and biogenesis of ubiquitous membrane transporter families.
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    19. 19
      Krogh, A., Larsson, B., von Heijne, G., and Sonnhammer, E. L. L. (2001) Predicting transmembrane protein topology with a hidden Markov model: Application to complete genomes J. Mol. Biol. 305, 567– 580 DOI: 10.1006/jmbi.2000.4315
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      19
      Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes
      Krogh, Anders; Larsson, Bjorn; von Heijne, Gunnar; Sonnhammer, Erik L. L.
      Journal of Molecular Biology (2001), 305 (3), 567-580CODEN: JMOBAK; ISSN:0022-2836. (Academic Press)
      We describe and validate a new membrane protein topol. prediction method, TMHMM, based on a hidden Markov model. We present a detailed anal. of TMHMM's performance, and show that it correctly predicts 97-98 % of the transmembrane helixes. Addnl., TMHMM can discriminate between sol. and membrane proteins with both specificity and sensitivity better than 99 %, although the accuracy drops when signal peptides are present. This high degree of accuracy allowed us to predict reliably integral membrane proteins in a large collection of genomes. Based on these predictions, we est. that 20-30 % of all genes in most genomes encode membrane proteins, which is in agreement with previous ests. We further discovered that proteins with Nin-Cin topologies are strongly preferred in all examd. organisms, except Caenorhabditis elegans, where the large no. of 7TM receptors increases the counts for Nout-Cin topologies. We discuss the possible relevance of this finding for our understanding of membrane protein assembly mechanisms. A TMHMM prediction service is available at http://www.cbs.dtu.dk/services/TMHMM/. (c) 2001 Academic Press.
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    20. 20
      Overington, J. P., Al-Lazikani, B., and Hopkins, A. L. (2006) Opinion - How many drug targets are there? Nat. Rev. Drug Discovery 5, 993– 996 DOI: 10.1038/nrd2199
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      20
      How many drug targets are there?
      Overington, John P.; Al-Lazikani, Bissan; Hopkins, Andrew L.
      Nature Reviews Drug Discovery (2006), 5 (12), 993-996CODEN: NRDDAG; ISSN:1474-1776. (Nature Publishing Group)
      A review. For the past decade, the no. of mol. targets for approved drugs has been debated. Here, we reconcile apparently contradictory previous reports into a comprehensive survey, and propose a consensus no. of current drug targets for all classes of approved therapeutic drugs. One striking feature is the relatively const. historical rate of target innovation (the rate at which drugs against new targets are launched); however, the rate of developing drugs against new families is significantly lower. The recent approval of drugs that target protein kinases highlights two addnl. trends: an emerging realization of the importance of polypharmacol., and also the power of a gene-family-led approach in generating novel and important therapies.
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      Welsh, M. J. and Smith, A. E. (1993) Molecular mechanisms of CFTR chloride channel dysfunction in cystic fibrosis Cell 73, 1251– 1254 DOI: 10.1016/0092-8674(93)90353-R
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      21
      Molecular mechanisms of CFTR chloride channel dysfunction in cystic fibrosis
      Welsh, Michael J.; Smith, Alan E.
      Cell (Cambridge, MA, United States) (1993), 73 (7), 1251-4CODEN: CELLB5; ISSN:0092-8674.
      A review with 24 refs. on mol. mechanisms by which cystic fibrosis-assocd. mutations cause a loss of CFTR chloride channel function.
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    22. 22
      Ward, C. L., Omura, S., and Kopito, R. R. (1995) Degradation of CFTR by the ubiquitin-proteasome pathway Cell 83, 121– 127 DOI: 10.1016/0092-8674(95)90240-6
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      Wigley, W. C., Corboy, M. J., Cutler, T. D., Thibodeau, P. H., Oldan, J., Lee, M. G., Rizo, J., Hunt, J. F., and Thomas, P. J. (2002) A protein sequence that can encode native structure by disfavoring alternate conformations Nat. Struct. Biol. 9, 381– 388 DOI: 10.1038/nsb784
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      A protein sequence that can encode native structure by disfavoring alternate conformations
      Wigley, W. C.; Corboy, M. J.; Cutler, T. D.; Thibodeau, P. H.; Oldan, J.; Lee, M. G.; Rizo, J.; Hunt, J. F.; Thomas, P. J.
      Nature Structural Biology (2002), 9 (5), 381-388CODEN: NSBIEW; ISSN:1072-8368. (Nature America Inc.)
      The linear sequence of amino acids contains all the necessary information for a protein to fold into its unique three-dimensional structure. Native protein sequences are known to accomplish this by promoting the formation of stable, kinetically accessible structures. Here we describe a Pro residue in the center of the third transmembrane helix of the cystic fibrosis transmembrane conductance regulator that promotes folding by a distinct mechanism: disfavoring the formation of a misfolded structure. The generality of this mechanism is supported by genome-wide transmembrane sequence analyses. Furthermore, the results provide an explanation for the increased frequency of Pro residues in transmembrane α-helixes. Incorporation by nature of such 'neg. folding determinants', aimed at preventing the formation of off-pathway structures, represents an addnl. mechanism by which folding information is encoded within the evolved sequences of proteins.
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      Hartong, D. T., Berson, E. L., and Dryja, T. P. (2006) Retinitis pigmentosa Lancet 368, 1795– 1809 DOI: 10.1016/S0140-6736(06)69740-7
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      24
      Retinitis pigmentosa
      Hartong, Dyonne T.; Berson, Eliot L.; Dryja, Thaddeus P.
      Lancet (2006), 368 (9549), 1795-1809CODEN: LANCAO; ISSN:0140-6736. (Elsevier Ltd.)
      A review. Hereditary degenerations of the human retina are genetically heterogeneous, with well over 100 genes implicated so far. This Seminar focuses on the subset of diseases called retinitis pigmentosa, in which patients typically lose night vision in adolescence, side vision in young adulthood, and central vision in later life because of progressive loss of rod and cone photoreceptor cells. Measures of retinal function, such as the electroretinogram, show that photoreceptor function is diminished generally many years before symptomic night blindness, visual-field scotomas, or decreased visual acuity arise. More than 45 genes for retinitis pigmentosa have been identified. These genes account for only about 60% of all patients; the remainder have defects in as yet unidentified genes. Findings of controlled trials indicate that nutritional interventions, including vitamin A palmitate and omega-3-rich fish, slow progression of disease in many patients. Imminent treatments for retinitis pigmentosa are greatly anticipated, esp. for genetically defined subsets of patients, because of newly identified genes, growing knowledge of affected biochem. pathways, and development of animal models.
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    25. 25
      Krebs, M. P., Holden, D. C., Joshi, P., Clark, C. L., III, Lee, A. H., and Kaushal, S. (2010) Molecular mechanisms of rhodopsin retinitis pigmentosa and the efficacy of pharmacological rescue J. Mol. Biol. 395, 1063– 1078 DOI: 10.1016/j.jmb.2009.11.015
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      25
      Molecular Mechanisms of Rhodopsin Retinitis Pigmentosa and the Efficacy of Pharmacological Rescue
      Krebs, Mark P.; Holden, David C.; Joshi, Parth; Clark, Charles L.; Lee, Andrew H.; Kaushal, Shalesh
      Journal of Molecular Biology (2010), 395 (5), 1063-1078CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
      Variants of rhodopsin, a complex of 11-cis retinal and opsin, cause retinitis pigmentosa (RP), a degenerative disease of the retina. Trafficking defects due to rhodopsin misfolding have been proposed as the most likely basis of the disease, but other potentially overlapping mechanisms may also apply. Pharmacol. therapies for RP must target the major disease mechanism and contend with overlap, if it occurs. To this end, we have explored the mol. basis of rhodopsin RP in the context of pharmacol. rescue with 11-cis retinal. Stable inducible cell lines were constructed to express wild-type opsin; the pathogenic variants T4R, T17M, P23A, P23H, P23L, and C110Y; or the nonpathogenic variants F220L and A299S. Pharmacol. rescue was measured as the fold increase in rhodopsin or opsin levels upon addn. of 11-cis retinal during opsin expression. Only Pro23 and T17M variants were rescued significantly. C110Y opsin was produced at low levels and did not yield rhodopsin, whereas the T4R, F220L, and A299S proteins reached near-wild-type levels and changed little with 11-cis retinal. All of the mutant rhodopsins exhibited misfolding, which increased over a broad range in the order F220L, A299S, T4R, T17M, P23A, P23H, P23L, as detd. by decreased thermal stability in the dark and increased hydroxylamine sensitivity. Pharmacol. rescue increased as misfolding decreased, but was limited for the least misfolded variants. Significantly, pathogenic variants also showed abnormal photobleaching behavior, including an increased ratio of metarhodopsin-I-like species to metarhodopsin-II-like species and aberrant photoproduct accumulation with prolonged illumination. These results, combined with an anal. of published biochem. and clin. studies, suggest that many rhodopsin variants cause disease by affecting both biosynthesis and photoactivity. We conclude that pharmacol. rescue is promising as a broadly effective therapy for rhodopsin RP, particularly if implemented in a way that minimizes the photoactivity of the mutant proteins.
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    26. 26
      Saliba, R. S., Munro, P. M. G., Luthert, P. J., and Cheetham, M. E. (2002) The cellular fate of mutant rhodopsin: quality control, degradation and aggresome formation J. Cell Sci. 115, 2907– 2918
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      26
      The cellular fate of mutant rhodopsin: quality control, degradation and aggresome formation
      Saliba, Richard S.; Munro, Peter M. G.; Luthert, Philip J.; Cheetham, Michael E.
      Journal of Cell Science (2002), 115 (14), 2907-2918CODEN: JNCSAI; ISSN:0021-9533. (Company of Biologists Ltd.)
      Mutations in the photopigment rhodopsin are the major cause of autosomal dominant retinitis pigmentosa. The majority of mutations in rhodopsin lead to misfolding of the protein. Through the detailed examn. of P23H and K296E mutant opsin processing in COS-7 cells, we have shown that the mutant protein does not accumulate in the Golgi, as previously thought, instead it forms aggregates that have many of the characteristic features of an aggresome. The aggregates form close to the centrosome and lead to the dispersal of the Golgi app. Furthermore, these aggregates are ubiquitinated, recruit cellular chaperones and disrupt the intermediate filament network. Mutant opsin expression can disrupt the processing of normal opsin, as co-transfection revealed that the wild-type protein is recruited to mutant opsin aggregates. The degrdn. of mutant opsin is dependent on the proteasome machinery. Unlike the situation with ΔF508-CFTR, proteasome inhibition does not lead to a marked increase in aggresome formation but increases the retention of the protein within the ER, suggesting that the proteasome is required for the efficient retro-translocation of the mutant protein. Inhibition of N-linked glycosylation with tunicamycin leads to the selective retention of the mutant protein within the ER and increases the steady state level of mutant opsin. Glycosylation, however, has no influence on the biogenesis and targeting of wild-type opsin in cultured cells. This demonstrates that N-linked glycosylation is required for ER-assocd. degrdn. of the mutant protein but is not essential for the quality control of opsin folding. The addn. of 9-cis-retinal to the media increased the amt. of P23H, but not K296E, that was sol. and reached the plasma membrane. These data show that rhodopsin autosomal dominant retinitis pigmentosa is similar to many other neurodegenerative diseases in which the formation of intracellular protein aggregates is central to disease pathogenesis, and they suggest a mechanism for disease dominance.
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    27. 27
      Surguchev, A. and Surguchov, A. (2010) Conformational diseases: Looking into the eyes Brain Res. Bull. 81, 12– 24 DOI: 10.1016/j.brainresbull.2009.09.015
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      27
      Conformational diseases: Looking into the eyes
      Surguchev, Alexei; Surguchov, Andrei
      Brain Research Bulletin (2010), 81 (1), 12-24CODEN: BRBUDU; ISSN:0361-9230. (Elsevier Inc.)
      A review. Conformational diseases, a general term comprising more than 40 disorders are caused by the accumulation of unfolded or misfolded proteins. Improper protein folding (misfolding) as well as accrual of unfolded proteins can lead to the formation of disordered (amorphous) or ordered (amyloid fibril) aggregates. The gradual accumulation of protein aggregates and the acceleration of their formation by stress explain the characteristic late or episodic onset of the diseases. The best studied in this group are neurodegenerative diseases and amyloidosis accompanied by the deposition of a specific aggregation-prone proteins or protein fragments and formation of insol. fibrils. Amyloidogenic protein accumulation often occurs in the brain tissues, e.g. in Alzheimer's disease with the deposition of amyloid-β and Tau, in scrapie and bovine spongiform encephalopathy with the accumulation of prion protein, in Parkinson's disease with the deposition of α-synuclein. Other examples of amyloid proteins are transthyretin, Ig light chain, gelsolin, etc. In addn. to the brain, the accumulation of unfolded or misfolded proteins leading to pathol. takes place in a wide variety of organs and tissues, including different parts of the eye. The best studied ocular conformational diseases are cataract in the lens and retinitis pigmentosa in the retina, but accumulation of misfolded proteins also occurs in other parts of the eye causing various disorders. Furthermore, ocular manifestation of systemic amyloidosis often causes the deposition of amyloidogenic proteins in different ocular tissues. Here we present the data regarding naturally unfolded and misfolded proteins in eye tissues, their structure-function relationships, and mol. mechanisms underlying their involvement in diseases. We also summarize the etiol. of ocular conformational diseases and discuss approaches to their treatment.
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    28. 28
      Miller, L. M., Gragg, M., Kim, T. G., and Park, P. S. H. (2015) Misfolded opsin mutants display elevated beta-sheet structure FEBS Lett. 589, 3119– 3125 DOI: 10.1016/j.febslet.2015.08.042
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      28
      Misfolded opsin mutants display elevated β-sheet structure
      Miller, Lisa M.; Gragg, Megan; Kim, Tae Gyun; Park, Paul S.-H.
      FEBS Letters (2015), 589 (20_Part_B), 3119-3125CODEN: FEBLAL; ISSN:0014-5793. (Elsevier B.V.)
      Mutations in rhodopsin can cause misfolding and aggregation of the receptor, which leads to retinitis pigmentosa, a progressive retinal degenerative disease. The structure adopted by misfolded opsin mutants and the assocd. cell toxicity is poorly understood. Forster resonance energy transfer (FRET) and Fourier transform IR (FTIR) microspectroscopy were utilized to probe within cells the structures formed by G188R and P23H opsins, which are misfolding mutants that cause autosomal dominant retinitis pigmentosa. Both mutants formed aggregates in the endoplasmic reticulum and exhibited altered secondary structure with elevated β-sheet and reduced α-helical content. The newly formed β-sheet structure may facilitate the aggregation of misfolded opsin mutants. The effects obsd. for the mutants were unrelated to retention of opsin mols. in the endoplasmic reticulum itself.
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    29. 29
      Yamada, K., Sato, J., Oku, H., and Katakai, R. (2003) Conformation of the transmembrane domains in peripheral myelin protein 22. Part 1. Solution-phase synthesis and circular dichroism study of protected 17-residue partial peptides in the first putative transmembrane domain J. Pept. Res. 62, 78– 87 DOI: 10.1034/j.1399-3011.2003.00073.x
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      29
      Conformation of the transmembrane domains in peripheral myelin protein 22. Part 1. Solution-phase synthesis and circular dichroism study of protected 17-residue partial peptides in the first putative transmembrane domain
      Yamada, K.; Sato, J.; Oku, H.; Katakai, R.
      Journal of Peptide Research (2003), 62 (2), 78-87CODEN: JPERFA; ISSN:1397-002X. (Blackwell Munksgaard)
      Charcot-Marie-Tooth disease (CMT) is the most commonly inherited peripheral neuropathy. DNA duplication and point mutation of the gene encoding peripheral myelin protein 22 (PMP22) have been found in CMT type 1A dominants. To investigate the influence of the point mutation of PMP22 on the secondary structure, protected partial peptides in the putative first transmembrane domain, wild type Boc-IVLH(Bom)VAVLVLLFVSTIV-OMe (1; Bom = benzyloxymethyl) and its Pro16 mutant Boc-IVLH(Bom)VAVPVLLFVSTIV-OMe (2) were synthesized. CD anal. suggested that peptide 1 adopts a stable α-helical conformation in membrane-mimetic solvent, 1-BuOH/1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) system. On the contrary, the mutant 2 favors β-sheet conformation in the same solvent system. Interestingly, α-helix to β-sheet transition of 2 was obsd. at higher contents of 1-BuOH than 70%.
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    30. 30
      Naef, R. and Suter, U. (1998) Many facets of the peripheral myelin protein PMP22 in myelination and disease Microsc. Res. Tech. 41, 359– 371 DOI: 10.1002/(SICI)1097-0029(19980601)41:5<359::AID-JEMT3>3.0.CO;2-L
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      30
      Many facets of the peripheral myelin protein PMP22 in myelination and disease
      Naef, Roland; Suter, Ueli
      Microscopy Research and Technique (1998), 41 (5), 359-371CODEN: MRTEEO; ISSN:1059-910X. (Wiley-Liss, Inc.)
      A review with many refs. Peripheral myelin protein 22 (PMP22) is a small, hydrophobic glycoprotein, which is most prominently expressed by Schwann cells as a component of compact myelin of the peripheral nervous system (PNS). Recent progress in mol. genetics revealed that mutations affecting the PMP22 gene including duplications, deletions, and point mutations are responsible for the most common forms of hereditary peripheral neuropathies including Charcot-Marie-Tooth disease type 1A (CMT1A), hereditary neuropathy with liability to pressure palsies (HNPP), and a subtype of Dejerine-Sottas Syndrome (DSS). Functionally, PMP22 is involved in correct myelination during development of peripheral nerves, the stability of myelin, and the maintenance of axons. While most of these functions relate to a role of PMP22 as a structural component of myelin, PMP22 has also been proposed as a regulator of Schwann cell proliferation and differentiation. In this review, the authors will discuss their current knowledge of PMP22 and its related proteins in the normal organism as well as in disease. In particular, the authors will focus on how the function of PMP22 and its regulation may be relevant to particular disease mechanisms.
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    31. 31
      Ryan, M. C., Shooter, E. M., and Notterpek, L. (2002) Aggresome formation in neuropathy models based on peripheral myelin protein 22 mutations Neurobiol. Dis. 10, 109– 118 DOI: 10.1006/nbdi.2002.0500
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      Aggresome Formation in Neuropathy Models Based on Peripheral Myelin Protein 22 Mutations
      Ryan, Mary C.; Shooter, Eric M.; Notterpek, Lucia
      Neurobiology of Disease (2002), 10 (2), 109-118CODEN: NUDIEM; ISSN:0969-9961. (Elsevier Science)
      Alterations in peripheral myelin protein 22 (PMP22) gene expression are assocd. with demyelinating peripheral neuropathies. Overexpression of wild type (wt) PMP22 or inhibition of proteasomal degrdn. lead to the formation of aggresomes, intracellular ubiquitinated PMP22 aggregates. Aggresome formation has now been obsd. with two mutant PMP22s, the Tr- and TrJ-PMP22 when the proteasome is inhibited. The formation of these aggresomes required intact microtubules and involved the recruitment of chaperones, including Hsp40, Hsp70, and αB-crystallin. Spontaneously formed ubiquitinated PMP22 aggregates were also obsd. in Schwann cells of homozygous TrJ mice. Significant upregulation of both the ubiquitin-proteasomal and lysosomal pathways occurred in affected nerves suggesting that two pathways of PMP22 degrdn. are present. Thus, the presence of aggresomes appears to be a common finding in neuropathy models of PMP22 overexpression and of some point mutations known to cause neuropathy in mice and humans.
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    32. 32
      Danoff, E. J. and Fleming, K. G. (2015) Aqueous, unfolded OmpA forms amyloid-like fibrils upon self-association PLoS One 10, e0132301 DOI: 10.1371/journal.pone.0132301
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      Aqueous, unfolded OmpA forms amyloid- like fibrils upon self-association
      Danoff, Emily J.; Fleming, Karen G.
      PLoS One (2015), 10 (7), e0132301/1-e0132301/10CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
      Unfolded outer membrane beta-barrel proteins have been shown to self-assoc. in the absence of lipid bilayers. We previously investigated the formation of high mol. wt. species by OmpA, with both the transmembrane domain alone and the full-length protein, and discovered that the oligomeric form contains non-native β -sheet structure. We have further probed the conformation of self-assocd. OmpA by monitoring binding to Thioflavin T, a dye that is known to bind the cross- β a structure inherent in amyloid fibrils, and by observing the species by electron microscopy. The significant increase in fluorescence indicative of Thioflavin T binding and the appearance of fibrillar species by electron microscopy verify that the protein forms amyloid-like fibril structures upon oligomerization. These results are also consistent with our previous kinetic anal. of OmpA self-assocn. that revealed a nucleated growth polymn. mechanism, which is frequently obsd. in amyloid formation. The discovery of OmpA's ability to form amyloid-like fibrils provides a new model protein with which to study fibrillization, and implicates periplasmic chaperone proteins as capable of inhibiting fibril formation.
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      Abramson, J., Smirnova, I., Kasho, V., Verner, G., Kaback, H. R., and Iwata, S. (2003) Structure and mechanism of the lactose permease of Escherichia coli Science 301, 610– 615 DOI: 10.1126/science.1088196
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      Structure and Mechanism of the Lactose Permease of Escherichia coli
      Abramson, Jeff; Smirnova, Irina; Kasho, Vladimir; Verner, Gillian; Kaback, H. Ronald; Iwata, So
      Science (Washington, DC, United States) (2003), 301 (5633), 610-616CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      Membrane transport proteins that transduce free energy stored in electrochem. ion gradients into a concn. gradient are a major class of membrane proteins. We report the crystal structure at 3.5 angstroms of the Escherichia coli lactose permease, an intensively studied member of the major facilitator superfamily of transporters. The mol. is composed of N- and C-terminal domains, each with six transmembrane helixes, sym. positioned within the permease. A large internal hydrophilic cavity open to the cytoplasmic side represents the inward-facing conformation of the transporter. The structure with a bound lactose homolog, β-D-galactopyranosyl-1-thio-β-D-galactopyranoside, reveals the sugar-binding site in the cavity, and residues that play major roles in substrate recognition and proton translocation are identified. We propose a possible mechanism for lactose/proton symport (cotransport) consistent with both the structure and a large body of exptl. data.
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      Guan, L., Mirza, O., Verner, G., Iwata, S., and Kaback, H. R. (2007) Structural determination of wild-type lactose permease Proc. Natl. Acad. Sci. U. S. A. 104, 15294– 15298 DOI: 10.1073/pnas.0707688104
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      Structural determination of wild-type lactose permease
      Guan, Lan; Mirza, Osman; Verner, Gillian; Iwata, So; Kaback, H. Ronald
      Proceedings of the National Academy of Sciences of the United States of America (2007), 104 (39), 15294-15298CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Here we describe an X-ray structure of wild-type lactose permease (LacY) from Escherichia coli detd. by manipulating phospholipid content during crystn. The structure exhibits the same global fold as the previous X-ray structures of a mutant that binds sugar but cannot catalyze translocation across the membrane. LacY is organized into two six-helix bundles with twofold pseudosymmetry sepd. by a large interior hydrophilic cavity open only to the cytoplasmic side and contg. the side chains important for sugar and H+ binding. To initiate transport, binding of sugar and/or an H+ electrochem. gradient increases the probability of opening on the periplasmic side. Because the inward-facing conformation represents the lowest free-energy state, the rate-limiting step for transport may be the conformational change leading to the outward-facing conformation.
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      Guan, L. and Kaback, H. R. (2006) Lessons from lactose permease Annu. Rev. Biophys. Biomol. Struct. 35, 67– 91 DOI: 10.1146/annurev.biophys.35.040405.102005
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      Lessons from lactose permease
      Guan, Lan; Kaback, H. Ronald
      Annual Review of Biophysics and Biomolecular Structure (2006), 35 (), 67-91CODEN: ABBSE4; ISSN:1056-8700. (Annual Reviews Inc.)
      A review. The x-ray crystal structure of the lactose permease of Escherichia coli (LacY) in an inward-facing conformation has been solved. LacY contains N- and C-terminal domains, each with 6 transmembrane helixes, positioned pseudosym. The ligand is bound at the apex of a hydrophilic cavity in approx. the middle of the mol. Residues involved in substrate binding and H+ translocation are aligned parallel to the membrane at the same level and may be exposed to a water-filled cavity in both the inward- and outward-facing conformations, thereby allowing both sugar and H+ release directly into either cavity. These structural features may explain why LacY catalyzes galactoside/H+ symport in both directions utilizing the same residues. A working model for the mechanism is presented that involves alternating access of both the sugar- and H+-binding sites to either side of the membrane.
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      Madej, M. G. (2014) Function, structure, and evolution of the Major Facilitator Superfamily: the LacY manifesto Adv. Biol. 2014, 1– 20 DOI: 10.1155/2014/523591
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      Harris, N. J., Findlay, H. E., Simms, J., Liu, X., and Booth, P. J. (2014) Relative domain folding and stability of a membrane transport protein J. Mol. Biol. 426, 1812– 1825 DOI: 10.1016/j.jmb.2014.01.012
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      Findlay, H. E. and Booth, P. J. (2013) Folding alpha-helical membrane proteins into liposomes in vitro and determination of secondary structure Methods Mol. Biol. 1063, 117– 124 DOI: 10.1007/978-1-62703-583-5_6
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      Folding Alpha-Helical Membrane Proteins into Liposomes In Vitro and Determination of Secondary Structure
      Findlay, Heather E.; Booth, Paula J.
      Methods in Molecular Biology (New York, NY, United States) (2013), 1063 (Membrane Proteins), 117-124CODEN: MMBIED; ISSN:1064-3745. (Springer)
      The native environment of integral membrane proteins is a highly complex lipid bilayer composed of many different types of lipids, the phys. characteristics of which can profoundly influence protein stability, folding, and function. Secondary transporters are a class of protein where changes to both structure and activity have been obsd. in different bilayer environments. In order to study these interactions in vitro, it is necessary to ext. and purify the protein and exchange it into an artificial lipid system that can be manipulated to control protein behavior. Liposomes are a commonly used model system that is particularly suitable for studying transporters. GalP and LacY can be reconstituted or refolded into vesicles with a high degree of efficiency for further structural anal. CD spectroscopy is an important technique in monitoring protein folding, which allows the decompn. of spectra into secondary structural components.
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      Sanders, C. R. and Mittendorf, K. F. (2011) Tolerance to changes in membrane lipid composition as a selected trait of membrane proteins Biochemistry 50, 7858– 7867 DOI: 10.1021/bi2011527
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      Tolerance to Changes in Membrane Lipid Composition as a Selected Trait of Membrane Proteins
      Sanders, Charles R.; Mittendorf, Kathleen F.
      Biochemistry (2011), 50 (37), 7858-7867CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      A review. Membrane lipid compn. can vary dramatically across the three domains of life and even within single organisms. Here evidence that the lipid-exposed surfaces of membrane proteins have generally evolved to maintain correct structure and function in the face of major changes in lipid compn. is reviewed. Such tolerance has allowed evolution to extensively remodel membrane lipid compns. during the emergence of new species without having to extensively remodel the assocd. membrane proteins. The tolerance of membrane proteins also permits single-cell organisms to vary their membrane lipid compn. in response to their changing environments and allows dynamic and organelle-specific variations in the lipid compns. of eukaryotic cells. Membrane protein structural biol. has greatly benefited from this seemingly intrinsic property of membrane proteins: the majority of structures detd. to date have been characterized under model membrane conditions that little resemble those of native membranes. Nevertheless, with a few notable exceptions, most exptl. detd. membrane protein structures appear, to a good approxn., to faithfully report on native structure.
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      Alder, N. N. and Johnson, A. E. (2004) Cotranslational membrane protein biogenesis at the endoplasmic reticulum J. Biol. Chem. 279, 22787– 22790 DOI: 10.1074/jbc.R400002200
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      Vembar, S. S. and Brodsky, J. L. (2008) One step at a time: endoplasmic reticulum-associated degradation Nat. Rev. Mol. Cell Biol. 9, 944– 957 DOI: 10.1038/nrm2546
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      One step at a time: endoplasmic reticulum-associated degradation
      Vembar, Shruthi S.; Brodsky, Jeffrey L.
      Nature Reviews Molecular Cell Biology (2008), 9 (12), 944-957CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)
      A review. Protein folding in the endoplasmic reticulum (ER) is monitored by ER quality control (ERQC) mechanisms. Proteins that pass ERQC criteria traffic to their final destinations through the secretory pathway, whereas non-native and unassembled subunits of multimeric proteins are degraded by the ER-assocd. degrdn. (ERAD) pathway. During ERAD, mol. chaperones and assocd. factors recognize and target substrates for retrotranslocation to the cytoplasm, where they are degraded by the ubiquitin-proteasome machinery. The discovery of diseases that are assocd. with ERAD substrates highlights the importance of this pathway. Here, the authors summarize their current understanding of each step during ERAD, with emphasis on the factors that catalyze distinct activities.
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      Ibstedt, S., Sideri, T. C., Grant, C. M., and Tamas, M. J. (2014) Global analysis of protein aggregation in yeast during physiological conditions and arsenite stress Biol. Open 3, 913– 923 DOI: 10.1242/bio.20148938
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      Global analysis of protein aggregation in yeast during physiological conditions and arsenite stress
      Ibstedt, Sebastian; Sideri, Theodora C.; Grant, Chris M.; Tamas, Markus J.
      Biology Open (2014), 3 (10), 913-923, 11 pp.CODEN: BOIPBR; ISSN:2046-6390. (Company of Biologists Ltd.)
      Protein aggregation is a widespread phenomenon in cells and assocd. with pathol. conditions. Yet, little is known about the rules that govern protein aggregation in living cells. In this study, we biochem. isolated aggregation-prone proteins and used computational analyses to identify characteristics that are linked to physiol. and arsenite-induced aggregation in living yeast cells. High protein abundance, extensive phys. interactions, and certain structural properties are pos. correlated with an increased aggregation propensity. The aggregated proteins have high translation rates and are substrates of ribosome-assocd. Hsp70 chaperones, indicating that they are susceptible for aggregation primarily during translation/folding. The aggregation-prone proteins are enriched for multiple chaperone interactions, thus high protein abundance is probably counterbalanced by mol. chaperones to allow sol. expression in vivo. Our data support the notion that arsenite interferes with chaperone activity and indicate that arsenite-aggregated proteins might engage in extensive aberrant protein-protein interactions. Expression of aggregation-prone proteins is down-regulated during arsenite stress, possibly to prevent their toxic accumulation. Several aggregation-prone yeast proteins have human homologues that are implicated in misfolding diseases, suggesting that similar mechanisms may apply in disease- and non-disease settings.
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    43. 43
      Liao, L., Cheng, D., Wang, J., Duong, D. M., Losik, T. G., Gearing, M., Rees, H. D., Lah, J. J., Levey, A. I., and Peng, J. (2004) Proteomic characterization of postmortem amyloid plaques isolated by laser capture microdissection J. Biol. Chem. 279, 37061– 37068 DOI: 10.1074/jbc.M403672200
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      Proteomic Characterization of Postmortem Amyloid Plaques Isolated by Laser Capture Microdissection
      Liao, Lujian; Cheng, Dongmei; Wang, Jian; Duong, Duc M.; Losik, Tatyana G.; Gearing, Marla; Rees, Howard D.; Lah, James J.; Levey, Allan I.; Peng, Junmin
      Journal of Biological Chemistry (2004), 279 (35), 37061-37068CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      The presence of amyloid plaques in the brain is one of the pathol. hallmarks of Alzheimer's disease (AD). We report here a comprehensive proteomic anal. of senile plaques from postmortem AD brain tissues. Senile plaques labeled with thioflavin-S were procured by laser capture microdissection, and their protein components were analyzed by liq. chromatog. coupled with tandem mass spectrometry. We identified a total of 488 proteins coisolated with the plaques, and we found multiple phosphorylation sites on the neurofilament intermediate chain, implicating the complexity and diversity of cellular processes involved in the plaque formation. More significantly, we identified 26 proteins enriched in the plaques of two AD cases by quant. comparison with surrounding non-plaque tissues. The localization of several proteins in the plaques was further confirmed by the approach of immunohistochem. In addn. to previously identified plaque constituents, we discovered novel assocn. of dynein heavy chain with the plaques in human postmortem brain and in a double transgenic AD mouse model, suggesting that neuronal transport may play a role in neuritic degeneration. Overall, our results revealed for the first time the sub-proteome of amyloid plaques that is important for further studies on disease biomarker identification and mol. mechanisms of AD pathogenesis.
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      Sergeant, N., Wattez, A., Galvan-Valencia, M., Ghestem, A., David, J. P., Lemoine, J., Sautiere, P.-E., Dachary, J., Mazat, J. P., Michalski, J. C., Velours, J., Mena-Lopez, R., and Delacourte, A. (2003) Association of ATP synthase alpha-chain with neurofibrillary degeneration in Alzheimer’s disease Neuroscience 117, 293– 303 DOI: 10.1016/S0306-4522(02)00747-9
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      Murakami, T., Shoji, M., Imai, Y., Inoue, H., Kawarabayashi, T., Matsubara, E., Harigaya, Y., Sasaki, A., Takahashi, R., and Abe, K. (2004) Pael-R is accumulated in Lewy bodies of Parkinson’s disease Ann. Neurol. 55, 439– 442 DOI: 10.1002/ana.20064
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      Pael-R is accumulated in Lewy bodies of Parkinson's disease
      Murakami, Tetsuro; Shoji, Mikio; Imai, Yuzuru; Inoue, Haruhisa; Kawarabayashi, Takeshi; Matsubara, Etsuro; Harigaya, Yasuo; Sasaki, Atsushi; Takahashi, Ryosuke; Abe, Koji
      Annals of Neurology (2004), 55 (3), 439-442CODEN: ANNED3; ISSN:0364-5134. (Wiley-Liss, Inc.)
      The authors examd. the distribution of Pael-R, a newly identified substrate for Parkin, in Parkinson's disease (PD) and multiple system atrophy (MSA). Pael-R, Parkin, α-synuclein, and ubiquitin accumulated in Lewy bodies (LBs) and neurites. Pael-R was localized in the care of LBs. Parkin and α-synuclein accumulated in the halo, neuronal cell bodies, and processes. These findings potentially suggest the involvement of Pael-R in LB formation, and protection role of Parkin in Pael-R-mediated neurotoxicity in PD. The absence of Pael-R and Parkin in glial cytoplasmic inclusions (GCIs) in MSA implies a distinct pathway involved in the formation of LBs and GCIs.
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      Sormanni, P., Aprile, F. A., and Vendruscolo, M. (2015) The CamSol method of rational design of protein mutants with enhanced solubility J. Mol. Biol. 427, 478– 490 DOI: 10.1016/j.jmb.2014.09.026
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      The CamSol Method of Rational Design of Protein Mutants with Enhanced Solubility
      Sormanni, Pietro; Aprile, Francesco A.; Vendruscolo, Michele
      Journal of Molecular Biology (2015), 427 (2), 478-490CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
      Protein soly. is often an essential requirement in biotechnol. and biomedical applications. Great advances in understanding the principles that det. this specific property of proteins have been made during the past decade, in particular concerning the physicochem. characteristics of their constituent amino acids. By exploiting these advances, we present the CamSol method for the rational design of protein variants with enhanced soly. The method works by performing a rapid computational screening of tens of thousand of mutations to identify those with the greatest impact on the soly. of the target protein while maintaining its native state and biol. activity. The application to a single-domain antibody that targets the Alzheimer's Aβ peptide demonstrates that the method predicts with great accuracy soly. changes upon mutation, thus offering a cost-effective strategy to help the prodn. of sol. proteins for academic and industrial purposes.
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      Guan, L., Murphy, F. D., and Kaback, H. R. (2002) Surface-exposed positions in the transmembrane helices of the lactose permease of Escherichia coli determined by intermolecular thiol cross-linking Proc. Natl. Acad. Sci. U. S. A. 99, 3475– 3480 DOI: 10.1073/pnas.052703699
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      Strop, P. and Brunger, A. T. (2005) Refractive index-based determination of detergent concentration and its application to the study of membrane proteins Protein Sci. 14, 2207– 2211 DOI: 10.1110/ps.051543805
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      Refractive index-based determination of detergent concentration and its application to the study of membrane proteins
      Strop, Pavel; Brunger, Axel T.
      Protein Science (2005), 14 (8), 2207-2211CODEN: PRCIEI; ISSN:0961-8368. (Cold Spring Harbor Laboratory Press)
      The concn. of detergent in membrane protein prepns. can have a crit. role on protein stability, function, and the potential for crystn. Unfortunately, dialysis or protein concn. can lead to an unknown amt. of detergent in the final membrane protein prepns. Here we present a method for the detn. of detergent concn. based on refractive index of the detergent soln. This method was applied to quantitate the amt. of detergent remaining in soln. after concn. in various concentrators. We found that the ability of the tested detergents to pass through the mol. wt. cutoff membrane correlates well with detergent micelle size. Therefore, the micelle size can be used as a rough guide to est. the retention of a given detergent in various mol. wt. cutoff concentrators. The refractive index method is exceptionally informative when coupled with size exclusion chromatog. and light scattering, and can be used to det. the oligomeric state of the membrane protein, the size of a protein-assocd. micelle, as well as the amt. and size of the unbound detergent micelle.
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      Urbani, A. and Warne, T. (2005) A colorimetric determination for glycosidic and bile salt-based detergents: applications in membrane protein research Anal. Biochem. 336, 117– 124 DOI: 10.1016/j.ab.2004.09.040
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      A colorimetric determination for glycosidic and bile salt-based detergents: applications in membrane protein research
      Urbani, Andrea; Warne, Tony
      Analytical Biochemistry (2005), 336 (1), 117-124CODEN: ANBCA2; ISSN:0003-2697. (Elsevier)
      Detergents are crucial to the isolation of integral membrane proteins. During membrane protein purifn., it is useful to accurately quantify detergent, esp. if concn. steps have been used. Previously, this has been difficult and time-consuming. We present a simple, rapid, and sensitive procedure for the quantification of glycosidic and bile salt-based detergents such as dodecylmaltoside, octylglucoside, and CHAPS. The method directly quantifies sugar or cholate moieties via colorimetric reactions with phenol and sulfuric acid. A no. of detergents have been screened, and the assay has been validated in the presence of commonly used reagents. In addn. to detg. the overall detergent concn. in soln., the procedure allows accurate quantification of specific binding of glycosidic or bile salt-based detergents to purified membrane proteins. Both the colorimetric method and the radiometric 14C method were used to det. detergent binding to two integral membrane proteins: the cytochrome cbb3 oxidase from Pseudomonas stutzeri and the turkey β-adrenergic receptor. Both methods gave similar results. After sepg. monomeric glycosidic detergent from micellar solns. by ultrafiltration, we used the colorimetric method to det. the concn. of monomeric detergent present. We obsd. that values obtained are in close agreement with previously detd. crit. micelle concns.
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      Kelly, S. M., Jess, T. J., and Price, N. C. (2005) How to study proteins by circular dichroism Biochim. Biophys. Acta, Proteins Proteomics 1751, 119– 139 DOI: 10.1016/j.bbapap.2005.06.005
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      How to study proteins by circular dichroism
      Kelly, Sharon M.; Jess, Thomas J.; Price, Nicholas C.
      Biochimica et Biophysica Acta, Proteins and Proteomics (2005), 1751 (2), 119-139CODEN: BBAPBW; ISSN:1570-9639. (Elsevier B.V.)
      A review. CD is being increasingly recognized as a valuable technique for examg. the structure of proteins in soln. However, the value of many studies using CD is compromised either by inappropriate exptl. design or by lack of attention to key aspects of instrument calibration or sample characterization. In this article, we summarize the basis of the CD approach and its application to the study of proteins, and then present clear guidelines on how reliable data can be obtained and analyzed.
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      Kuhlbrandt, W. (1988) 3-Dimensional cystallization of membrane-proteins Q. Rev. Biophys. 21, 429– 477 DOI: 10.1017/S0033583500004625
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      Three-dimensional crystallization of membrane proteins
      Kuhlbrandt W
      Quarterly reviews of biophysics (1988), 21 (4), 429-77 ISSN:0033-5835.
      There is no expanded citation for this reference.
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      Engel, C. K., Chen, L., and Privé, G. G. (2002) Stability of the lactose permease in detergent solutions Biochim. Biophys. Acta, Biomembr. 1564, 47– 56 DOI: 10.1016/S0005-2736(02)00397-8
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      Stability of the lactose permease in detergent solutions
      Engel, Christian K.; Chen, Lu; Prive, Gilbert G.
      Biochimica et Biophysica Acta, Biomembranes (2002), 1564 (1), 47-56CODEN: BBBMBS; ISSN:0005-2736. (Elsevier B.V.)
      Protein stability, as measured by irreversible protein aggregation, is one of the central difficulties in the handling of detergent-solubilized membrane proteins. We present a quant. anal. of the stability of the Escherichia coli lactose (lac) permease and a series of lac permease fusion proteins contg. an insertion of cytochromeb562, T4 lysozyme or β-lactamase in the central hydrophilic loop of the permease. The stability of the proteins was evaluated under a variety of storage conditions by both a qual. SDS-PAGE assay and by a quant. HPLC assay. Long-chain maltoside detergents were more effective at maintaining purified protein in soln. than detergents with smaller head groups and/or shorter alkyl tails. A full factorial expt. established that the proteins were insensitive to sodium chloride concns., but greatly stabilized by glycerol, low temp. and the combination of glycerol and low temp. The accurate quantitation of the protein by absorbance spectroscopy required exclusion of all contact with clarified polypropylene or polyvinyl chloride (PVC) materials. Although some of the fusion proteins were more prone to aggregation than the wild-type permease, the stability of a fusion protein contg. a cytochromeb562 insertion was indistinguishable from that of native lac permease.
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    53. 53
      Biancalana, M. and Koide, S. (2010) Molecular mechanism of Thioflavin-T binding to amyloid fibrils Biochim. Biophys. Acta, Proteins Proteomics 1804, 1405– 1412 DOI: 10.1016/j.bbapap.2010.04.001
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      Molecular mechanism of thioflavin-T binding to amyloid fibrils
      Biancalana, Matthew; Koide, Shohei
      Biochimica et Biophysica Acta, Proteins and Proteomics (2010), 1804 (7), 1405-1412CODEN: BBAPBW; ISSN:1570-9639. (Elsevier B. V.)
      A review. Intense efforts to detect, diagnose, and analyze the kinetic and structural properties of amyloid fibrils have generated a powerful toolkit of amyloid-specific mol. probes. Since its 1st description in 1959, the fluorescent dye, thioflavin-T (ThT), has become among the most widely used "gold stds." for selectively staining and identifying amyloid fibrils both in vivo and in vitro. The large enhancement of its fluorescence emission upon its binding to fibrils makes ThT a particularly powerful and convenient tool. Despite its widespread use in clin. and basic science applications, the mol. mechanism for the ability of ThT to recognize diverse types of amyloid fibrils and for the dye's characteristic fluorescence has only begun to be elucidated. Here, the authors review recent progress in the understanding of ThT-fibril interactions at at. resoln. These studies have yielded important insights into amyloid structures and the processes of fibril formation, and they also offer guidance for designing the next generation of amyloid assembly diagnostics, inhibitors, and therapeutics.
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    54. 54
      Khurana, R., Coleman, C., Ionescu-Zanetti, C., Carter, S. A., Krishna, V., Grover, R. K., Roy, R., and Singh, S. (2005) Mechanism of thioflavin T binding to amyloid fibrils J. Struct. Biol. 151, 229– 238 DOI: 10.1016/j.jsb.2005.06.006
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      Mechanism of thioflavin T binding to amyloid fibrils
      Khurana, Ritu; Coleman, Chris; Ionescu-Zanetti, Cristian; Carter, Sue A.; Krishna, Vinay; Grover, Rajesh K.; Roy, Raja; Singh, Shashi
      Journal of Structural Biology (2005), 151 (3), 229-238CODEN: JSBIEM; ISSN:1047-8477. (Elsevier)
      Thioflavin T is a benzothiazole dye that exhibits enhanced fluorescence upon binding to amyloid fibrils and is commonly used to diagnose amyloid fibrils, both ex vivo and in vitro. In aq. solns., thioflavin T was found to exist as micelles at concns. commonly used to monitor fibrils by fluorescence assay (∼10-20 μM). Specific cond. changes were measured at varying concn. of thioflavin T and the crit. micellar concn. was calcd. to be 4.0±0.5 μM. Interestingly, changes in the fluorescence excitation and emission of thioflavin T were also dependent on the micelle formation. The thioflavin T micelles of 3 nm diam. were directly visualized using at. force microscopy, and bound thioflavin T micelles were obsd. along the fibril length for representative fibrils. Increasing concn. of thioflavin T above the crit. micellar concn. shows increased nos. of micelles bound along the length of the amyloid fibrils. Thioflavin T micelles were disrupted at low pH as obsd. by at. force microscopy and fluorescence enhancement upon binding of thioflavin T to amyloid fibrils also reduced by several-fold upon decreasing the pH to below 3. This suggests that pos. charge on the thioflavin T mol. has a role in its micelle formation that then bind the amyloid fibrils. The authors' data suggests that the micelles of thioflavin T bind amyloid fibrils leading to enhancement of fluorescence emission.
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    55. 55
      Kumar, S., Singh, A. K., Krishnamoorthy, G., and Swaminathan, R. (2008) Thioflavin T displays enhanced fluorescence selectively inside anionic micelles and mammalian cells J. Fluoresc. 18, 1199– 1205 DOI: 10.1007/s10895-008-0378-2
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      Thioflavin T displays enhanced fluorescence selectively inside anionic micelles and mammalian cells
      Kumar, Satish; Singh, Atul K.; Krishnamoorthy, G.; Swaminathan, Rajaram
      Journal of Fluorescence (2008), 18 (6), 1199-1205CODEN: JOFLEN; ISSN:1053-0509. (Springer)
      Thioflavin T (ThT) has been widely employed to detect amyloid fibrils in tissues and recently in presence of SDS micelles. However, the contribution of membranes or micelles to ThT fluorescence has never been investigated. In this paper, we show for the first time that the anionic micellar microenvironment of SDS has a profound impact on the absorption and fluorescence spectra of ThT in sharp contrast to cationic (CTAB) and neutral micelles (Triton X-100 & Tween 20). Unlike CTAB or Triton X-100 or Tween 20 micelles, formation of SDS micelles shifts the λmax for ThT absorption from 412 nm in buffer to 428 nm inside the micelle, with a 28% increase in the peak molar absorptivity and a ∼13 fold increase in ThT fluorescence (λmax = 489 nm). Extending these observations to cell plasma membranes, we show that ThT can quickly enter and appear selectively fluorescent inside mammalian cells like BHK21 and HT29, against a dark background owing to negligible fluorescence from free ThT in aq. medium. The above results suggest that ThT can be a useful probe for live cell imaging and for selectively labeling micelles on the basis of the charge in the polar headgroup.
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    56. 56
      Bouchard, M., Zurdo, J., Nettleton, E. J., Dobson, C. M., and Robinson, C. V. (2000) Formation of insulin amyloid fibrils followed by FTIR simultaneously with CD and electron microscopy Protein Sci. 9, 1960– 1967 DOI: 10.1110/ps.9.10.1960
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      Formation of insulin amyloid fibrils followed by FTIR simultaneously with CD and electron microscopy
      Bouchard, Mario; Zurdo, Jesus; Nettleton, Ewan J.; Dobson, Christopher M.; Robinson, Carol V.
      Protein Science (2000), 9 (10), 1960-1967CODEN: PRCIEI; ISSN:0961-8368. (Cambridge University Press)
      Fourier transform IR spectroscopy (FTIR), CD, and electron microscopy (EM) have been used simultaneously to follow the temp.-induced formation of amyloid fibrils by bovine insulin at acidic pH. The FTIR and CD data confirm that, before heating, insulin mols. in soln. at pH 2.3 have a predominantly native-like α-helical structure. On heating to 70°, partial unfolding occurs and results initially in aggregates that are shown by CD and FT-IR spectra to retain a predominantly helical structure. Following this step, changes in the CD and FTIR spectra occur that are indicative of the extensive conversion of the mol. conformation from α-helical to β-sheet structure. At later stages, EM shows the development of fibrils with well-defined repetitive morphologies including structures with a periodic helical twist of ∼450 Å. The results indicate that formation of fibrils by insulin requires substantial unfolding of the native protein, and that the most highly ordered structures result from a slow evolution of the morphol. of the initially formed fibrillar species.
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      Foster, D. L., Boublik, M., and Kaback, H. R. (1983) Structure of the lac carrier protein of Escherichia-coli J. Biol. Chem. 258, 31– 34
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      Uversky, V. N., Li, J., and Fink, A. L. (2001) Evidence for a partially folded intermediate in alpha-synuclein fibril formation J. Biol. Chem. 276, 10737– 10744 DOI: 10.1074/jbc.M010907200
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      Evidence for a partially folded intermediate in α-synuclein fibril formation
      Uversky, Vladimir N.; Li, Jie; Fink, Anthony L.
      Journal of Biological Chemistry (2001), 276 (14), 10737-10744CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Intracellular proteinaceous aggregates (Lewy bodies and Lewy neurites) of α-synuclein are hallmarks of neurodegenerative diseases such as Parkinson's disease, dementia with Lewy bodies, and multiple systemic atrophy. However, the mol. mechanisms underlying α-synuclein aggregation into such filamentous inclusions remain unknown. An intriguing aspect of this problem is that α-synuclein is a natively unfolded protein, with little or no ordered structure under physiol. conditions. This raises the question of how an essentially disordered protein is transformed into highly organized fibrils. In the search for an answer to this question, we have investigated the effects of pH and temp. on the structural properties and fibrillation kinetics of human recombinant α-synuclein. Either a decrease in pH or an increase in temp. transformed α-synuclein into a partially folded conformation. The presence of this intermediate is strongly correlated with the enhanced formation of α-synuclein fibrils. We propose a model for the fibrillation of α-synuclein in which the first step is the conformational transformation of the natively unfolded protein into the aggregation-competent partially folded intermediate.
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      Barth, A. and Zscherp, C. (2002) What vibrations tell us about proteins Q. Rev. Biophys. 35, 369– 430 DOI: 10.1017/S0033583502003815
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      What vibrations tell about proteins
      Barth, Andreas; Zscherp, Christian
      Quarterly Reviews of Biophysics (2002), 35 (4), 369-430CODEN: QURBAW; ISSN:0033-5835. (Cambridge University Press)
      This review deals with current concepts of vibrational spectroscopy for the investigation of protein structure and function. While the focus is on IR (IR) spectroscopy, some of the general aspects also apply to Raman spectroscopy. Special emphasis is on the amide I vibration of the polypeptide backbone that is used for secondary-structure anal. Theor. as well as exptl. aspects are covered including transition dipole coupling. Further topics are discussed, namely the absorption of amino-acid side-chains, 1H/2H exchange to study the conformational flexibility and reaction-induced difference spectroscopy for the investigation of reaction mechanisms with a focus on interpretation tools.
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      Calero, M. and Gasset, M. (2012) Featuring amyloids with fourier transform infrared and circular dichroism spectroscopies Methods Mol. Biol. 849, 53– 68 DOI: 10.1007/978-1-61779-551-0_5
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      Featuring amyloids with Fourier transform infrared and circular dichroism spectroscopies
      Calero, Miguel; Gasset, Maria
      Methods in Molecular Biology (New York, NY, United States) (2012), 849 (Amyloid Proteins), 53-68CODEN: MMBIED; ISSN:1064-3745. (Springer)
      Amyloids are fibrillar aggregates of proteins characterized by a basic scaffold consisting of cross β-sheet structure that can exert physiol. or pathol. effects. Both far-UV CD and Fourier transform IR (FTIR) spectroscopies are techniques used for the fast anal. of protein secondary structure. Both techniques are complementary and preferentially used depending on the phys. state of the analyte, the major secondary structure element and the relative abundance of given amino acids. Although there are special setups for working with films, CD is best suited for ideal dild. solns. of polypeptides exhibiting α-helix as major structural element and low content of arom. residues. During the last decade, a related technique, linear dichroism, has been applied to study the orientation of protein subunits within amyloid oligomers or fibrils in soln. Alternatively, FTIR works best with concd. solns., solids and films, and resolves with accuracy the β-sheet compn., but it is affected by contributions of amide groups. The advent of new IR techniques based on correlation anal. of time-dependent variations induced by external perturbations that generates two-dimensional IR maps has enabled to greatly increase spectral resoln. and to extend its applicability to protein secondary structure characterization in a variety of phys. environments. Within the amyloid field, conjunction of both spectroscopies has provided the first filter step for amyloid detection and has contributed to decipher the structural aspects of the amyloid formation mechanism.
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    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.7b00157.

    • DDM concentrations of LacY samples after concentration (Table S1) and ThT intensity profiles for the DDM-containing buffer in the absence of protein (Figure S1) (PDF)


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