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Interrogating Detergent Desolvation of Nanopore-Forming Proteins by Fluorescence Polarization Spectroscopy

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Department of Physics, Syracuse University, 201 Physics Building, Syracuse, New York 13244-1130, United States
Structural Biology, Biochemistry, and Biophysics Program, Syracuse University, 111 College Place, Syracuse, New York 13244-4100, United States
§ Department of Biochemistry and Molecular Biology, State University of New York, Upstate Medical University, 4249 Weiskotten Hall, 766 Irving Avenue, Syracuse, New York 13210, United States
Department of Chemistry, University of Massachusetts, Amherst, 710 North Pleasant Street, Amherst, Massachusetts 01003-9336, United States
Department of Biomedical and Chemical Engineering, Syracuse University, 329 Link Hall, Syracuse, New York 13244, United States
*Phone: 315-443-8078. Fax: 315-443-9103. E-mail: [email protected]
Cite this: Anal. Chem. 2017, 89, 15, 8013–8020
Publication Date (Web):June 26, 2017
https://doi.org/10.1021/acs.analchem.7b01339
Copyright © 2017 American Chemical Society

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    Abstract

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    Understanding how membrane proteins interact with detergents is of fundamental and practical significance in structural and chemical biology as well as in nanobiotechnology. Current methods for inspecting protein–detergent complex (PDC) interfaces require high concentrations of protein and are of low throughput. Here, we describe a scalable, spectroscopic approach that uses nanomolar protein concentrations in native solutions. This approach, which is based on steady-state fluorescence polarization (FP) spectroscopy, kinetically resolves the dissociation of detergents from membrane proteins and protein unfolding. For satisfactorily solubilizing detergents, at concentrations much greater than the critical micelle concentration (CMC), the fluorescence anisotropy was independent of detergent concentration. In contrast, at detergent concentrations comparable with or below the CMC, the anisotropy readout underwent a time-dependent decrease, showing a specific and sensitive protein unfolding signature. Functionally reconstituted membrane proteins into a bilayer membrane confirmed predictions made by these FP-based determinations with respect to varying refolding conditions. From a practical point of view, this 96-well analytical approach will facilitate a massively parallel assessment of the PDC interfacial interactions under a fairly broad range of micellar and environmental conditions. We expect that these studies will potentially accelerate research in membrane proteins pertaining to their extraction, solubilization, stabilization, and crystallization, as well as reconstitution into bilayer membranes.

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    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.7b01339.

    • (i) Protein expression and purification under denaturing condition; (ii) protein labeling of FhuA derivatives; (iii) expression, purification, and labeling of OmpG D224C proteins; (iv) contributions of anisotropy values to the Langmuir–Hill isothermal binding curves; (v) secondary structure determination of the refolded FhuA ΔC/Δ5L protein in solution using circular dichroism; (vi) single-channel and macroscopic electrical recordings using planar lipid bilayers; (vii) acquiring equilibrium steady-state end points of the FP anisotropy at different concentrations of detergents of varying chemistry; (viii) rotational motions of the protein nanopores under detergent solvation and desolvation conditions; (ix) fluorescence anisotropy readout acquired with LPPG-refolded FhuA ΔC/Δ5L at final refolding detergent concentration of 25 mM; (x) detailed time- and concentration-dependent anisotropy traces acquired with anionic and zwitterionic detergents; (xi) steroidal group-containing detergents are weakly binding to FhuA ΔC/Δ5L; (xii) dissociation of maltoside-containing detergents from FhuA ΔC/Δ5L; (xiii) dependence of time-dependent, steady-state fluorescence anisotropy on proteins of closely similar structure, but varying isoelectric point; (xiv) current–voltage relationship of FhuA ΔC/Δ5L refolded in detergents of varying chemistry; (xv) stability of the open-state current of the refolded FhuA ΔC/Δ5L proteins at higher applied transmembrane potentials. (PDF)

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    This article is cited by 11 publications.

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    4. Aaron J. Wolfe, Jack F. Gugel, Min Chen, Liviu Movileanu. Kinetics of Membrane Protein–Detergent Interactions Depend on Protein Electrostatics. The Journal of Physical Chemistry B 2018, 122 (41) , 9471-9481. https://doi.org/10.1021/acs.jpcb.8b07889
    5. Aaron J. Wolfe, Jack F. Gugel, Min Chen, Liviu Movileanu. Detergent Desorption of Membrane Proteins Exhibits Two Kinetic Phases. The Journal of Physical Chemistry Letters 2018, 9 (8) , 1913-1919. https://doi.org/10.1021/acs.jpclett.8b00549
    6. Aaron J. Wolfe, Wei Si, Zhengqi Zhang, Adam R. Blanden, Yi-Ching Hsueh, Jack F. Gugel, Bach Pham, Min Chen, Stewart N. Loh, Sharon Rozovsky, Aleksei Aksimentiev, and Liviu Movileanu . Quantification of Membrane Protein-Detergent Complex Interactions. The Journal of Physical Chemistry B 2017, 121 (44) , 10228-10241. https://doi.org/10.1021/acs.jpcb.7b08045
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    9. Ali Imran, Brandon S. Moyer, Ashley J. Canning, Dan Kalina, Thomas M. Duncan, Kelsey J. Moody, Aaron J. Wolfe, Michael S. Cosgrove, Liviu Movileanu. Kinetics of the multitasking high-affinity Win binding site of WDR5 in restricted and unrestricted conditions. Biochemical Journal 2021, 478 (11) , 2145-2161. https://doi.org/10.1042/BCJ20210253
    10. Olga D. Hendrickson, Nadezhda A. Taranova, Anatoly V. Zherdev, Boris B. Dzantiev, Sergei A. Eremin. Fluorescence Polarization-Based Bioassays: New Horizons. Sensors 2020, 20 (24) , 7132. https://doi.org/10.3390/s20247132
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