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Comment on “Aggregation Interface and Rigid Spots Sustain the Stable Framework of a Thermophilic N-Demethylase”
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  • Daniel M. Wade
    Daniel M. Wade
    Department of Biology, Valdosta State University, Valdosta, Georgia 31698, United States
    More by Daniel M. Wade
  • Walker S. Lewis
    Walker S. Lewis
    Department of Biology, Valdosta State University, Valdosta, Georgia 31698, United States
    More by Walker S. Lewis
  • Jonghoon Kang*
    Jonghoon Kang
    Department of Biology, Valdosta State University, Valdosta, Georgia 31698, United States
    *(229) 333-7140, (229) 245-6585, [email protected]
    More by Jonghoon Kang
    Orcidhttps://orcid.org/0000-0001-5594-6141
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Journal of Agricultural and Food Chemistry

Cite this: J. Agric. Food Chem. 2023, 71, 49, 19900–19902
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https://doi.org/10.1021/acs.jafc.3c07043
Published December 1, 2023

Copyright © 2023 The Authors. Published by American Chemical Society. This publication is licensed under

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Abstract

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The thermal properties of proteins are very important in industrial, agricultural, and food chemistry. A recent article (Li, B., et al. J. Agric. Food Chem. 2023, 71, 5614−5629) examines the thermal denaturation of enzymes TrSOX and BSOX by measuring the enthalpy change and melting temperature in the denaturation. In this work, we report the numerical values of entropy in the denaturation of proteins and show that both proteins TrSOX and BSOX exhibit enthalpy–entropy compensation in thermal denaturation, which results in a limited variation of melting temperature in both proteins. Our analysis may serve to improve our understanding of thermal properties in proteins in food chemistry.

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In a recent publication in the Journal of Agricultural and Food Chemistry, Li et al. (1) used diverse biochemical and biophysical methods to examine the nature of the high thermostability of a N-demethylase from thermophilic Thermomicrobium roseum, compared with that from mesophilic Bacillus subtilis. The N-demethylase they examined in the paper was sarcosine oxidase; the one from T. roseum was denoted TrSOX, while that from B. subtilis was denoted BSOX. (1) The authors examined the molecular basis of the high thermostability of TrSOX using several techniques, including thermodynamic analysis of the thermal denaturation of proteins. This is notably motivating, as evidenced by the increasing prominence of thermodynamics in the field of food science, as reflected in recent scholarly publications. (2−4) One of the main results of the research is that the melting temperature (Tm) for all variants (wild type and mutants) of TrSOX was significantly higher than those of BSOX. They then discussed this phenomenon in terms of denaturation enthalpy (ΔH). Although both ΔH and Tm offer valuable insights into the thermodynamic underpinnings of the thermal stability of proteins, the original paper does not address another essential thermodynamic parameter, the entropy of denaturation (ΔS). ΔS is regarded as being crucial for comprehending the forces involved in protein denaturation. (5−10) In this Correspondence, we present our examination of their findings, aiming to elucidate the ΔS values in the thermal denaturation of the proteins, their correlation with ΔH, and the potential implications of the relationship between ΔH and ΔS within the context of the thermal stability of proteins.

Given that protein denaturation represents a phase transition from the native state to the denatured state, ΔS can be computed using the following equation:

ΔS=ΔHTm
(1)
where Tm is the melting temperature in kelvin. (11) Numerical values of both ΔH and Tm for the 55 denaturation reactions obtained from the original paper, (1) 34 for TrSOX and 21 for BSOX, were used for the calculation of ΔS using eq 1. Figure 1A displays the resulting values of ΔS and the corresponding ΔH values. The plot clearly shows three thermodynamic characteristics in the denaturation of the proteins. First, the denaturation of both TrSOX and BSOX at the melting temperature is an entropy-driven process; in other words, ΔS > 0. Second, site-directed mutagenesis decreases both ΔH and ΔS in the case of TrSOX but increases them in the case of BSOX, in most cases. Third, linear regression using eq 2 indicates a highly significant correlation between ΔH and ΔS in both proteins as the coefficients of determination (R2) are 0.9966 and 0.9992 for TrSOX and BSOX, respectively:
ΔH=TCΔS+β
(2)
where TC, the compensation temperature, is the slope of the fitting line (12) and β is the y-intercept (Figure 1A). The strong correlation between ΔH and ΔS is known as enthalpy–entropy compensation, often observed in a weakly coupled system. (13−16) Figure 1A clearly indicates that the denaturation of TrSOX and BSOX exhibits compensatory behavior, suggesting that the molecular components responsible for the denaturation of those proteins exhibit the property of a weakly coupled system. The compensation means that as ΔH increases the corresponding ΔS also increases so that the resulting differences in Tm are minimized. (10)

Figure 1

Figure 1. Statistical analysis of the thermodynamic parameters of TrSOX and BSOX. (A) Enthalpy–entropy compensation for both TrSOX and BSOX with wild types marked separately. Coefficients of variation of ΔH, ΔS, and Tm for (B) TrSOX and (C) BSOX. Average values and standard deviations of (D) ΔH, (E) ΔS, and (F) Tm for TrSOX and BSOX. SigmaPlot (version 15, Systat Software Inc., San Jose, CA) was used for graph preparation and statistical analysis.

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The values of TC and its standard errors for TrSOX and BSOX are 411.6 ± 4.2 and 331.6 ± 2.1 K, respectively. A Student’s t test indicates that the difference in TC between TrSOX and BSOX is statistically significant (Table 1). In the t test, the degree of freedom (df) (17) was calculated as df = (n1 – 2) + (n2 – 2), where n1 and n2 are the number of data points of TrSOX and BSOX, respectively: n1 = 34, and n2 = 21 (Figure 1A). TC can quantitatively measure the degree of compensation between ΔH and ΔS. (12) The statistical difference in TC between TrSOX and BSOX (Table 1) strongly suggests that denaturation of each protein follows a distinct mechanism.

Table 1. Statistical Comparison of the Thermodynamic Parameters between TrSOX and BSOX
 TCΔHΔSTm
df51535353
t14.1–33.3–39.324.2
p3.4 × 10–193.4 × 10–377.3 × 10–412.5 × 10–30

The compensatory tendencies of ΔH and ΔS can be quantitatively described by comparing the coefficient of variation (CV) for each thermodynamic parameter, as determined by eq 3:

CV=sm
(3)
where s and m are the standard deviation and the mean of the samples, respectively. (17) The CVs of ΔH and ΔS are more than 5 or 33 times larger than that of Tm for TrSOX (Figure 1B) or BSOX (Figure 1C), respectively. On the basis of this result, we suggest that variations in ΔH and ΔS are local characteristics for the structural thermodynamics of proteins, while variation in Tm is a global characteristic that stays relatively constant in a weakly coupled system. This thermodynamic explanation is in line with the observations in the original paper (1) that all 20 mutants of BSOX generated with an intention to improve its thermostability showed a large variation in both ΔH and ΔS but a highly limited variation in Tm.

We also compare the thermodynamic parameters of TrSOX and BSOX to elucidate thermodynamic reasons for the high thermal stability of TrSOX. The differences in ΔH (Figure 1D), ΔS (Figure 1E), and Tm (Figure 1F) are shown to be statistically significant on the basis of the p values (Table 1). While ΔH is much smaller in TrSOX suggesting TrSOX must have a smaller value of Tm according to eq 1, ΔS is also much smaller in TrSOX, making it more stable. The much smaller value of ΔS stabilizes TrSOX compared to BSOX. In other words, the high thermal stability of TrSOX can be explained by the small value of ΔS. This is why ΔH is not sufficient in the explanation of the variation of Tm and ΔS should be included in the interpretation. The analysis introduced in this paper can be applied to other thermostable proteins such as TrLipB (18,19) to assess the contribution of small values of denaturation entropy to the thermal stability of proteins. We can conclude that the thermal denaturation of both TrSOX and BSOX exhibits enthalpy–entropy compensation. Statistical analysis suggests that ΔS is responsible for the high thermal stability of TrSOX. It will be interesting to examine whether other thermostable proteins exhibit these phenomena.

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  • Authors
    • Daniel M. Wade - Department of Biology, Valdosta State University, Valdosta, Georgia 31698, United States
    • Walker S. Lewis - Department of Biology, Valdosta State University, Valdosta, Georgia 31698, United States
  • Notes
    The authors declare no competing financial interest.

References

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This article references 19 other publications.

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    Kinetic and thermodynamic compensation. A current and practical review for foods
    Garvin, Alfonso; Ibarz, Raquel; Ibarz, Albert
    Food Research International (2017), 96 (), 132-153CODEN: FORIEU; ISSN:0963-9969. (Elsevier B.V.)
    Kinetic and thermodn. compensations have been reported in many chem., phys., biol. and food processes. Kinetic compensation can be found for any process and when it takes place, it gives information about the reaction mechanism and whether the reaction is controlled by enthalpy or entropy. It consists of the linear relationship between the logarithm of the frequency factor (lnk0) and the activation energy (Ea), both previously obtained from the Arrhenius equation for different values of an environmental variable (e.g. pH, concn. of any substance not involved in the process, pressure, water activity, etc.). A math. consequence of kinetic compensation is the isokinetic temp., this being the temp. at which the kinetic const. should be the same regardless of the environmental variable. Thermodn. compensation can be found for any process involving an equil. and consists of the linear relationship between the variation of enthalpy and entropy, both previously obtained from the Van't Hoff equation for different values of an environmental variable. A math. consequence of thermodn. compensation is the isoequil. temp., this being the temp. at which the equil. const. should be the same regardless of the environmental variable. According to the transition state theory, some kinetic consts. can be related to the equil. const. of the initial equil. stage between the reagents and the transition state. For these cases, it can be concluded that both compensations are related math. and therefore not only does the existence of one kind of compensation imply the existence of the other, but the isokinetic and isoequil. temps. should both be the same, or at least very close to each other. However, there is no reason that forces the linearities that cause either kind of compensation. So, some processes have shown these linear relationships while others have not. Moreover, some authors have reported that, due to the fact that the ests. of the parameters for the couples lnk0-Ea and ΔH≠-ΔS≠ being correlated with each other, there is a statistic compensation that consists of the propagation of exptl. errors, and this effect has to be considered before concluding kinetic and/or thermodn. compensations. This work reviews how to deal with kinetic and thermodn. compensations phys., math. and statistically, prior to a second part that reviews the food processes for which one or both of these compensations have been studied.
  16. 16
    Fox, J. M.; Zhao, M.; Fink, M. J.; Kang, K.; Whitesides, G. M. The molecular origin of enthalpy/entropy compensation in biomolecular recognition. Annu. Rev. Biophys. 2018, 47, 223– 250,  DOI: 10.1146/annurev-biophys-070816-033743
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    The Molecular Origin of Enthalpy/Entropy Compensation in Biomolecular Recognition
    Fox, Jerome M.; Zhao, Mengxia; Fink, Michael J.; Kang, Kyungtae; Whitesides, George M.
    Annual Review of Biophysics (2018), 47 (), 223-250CODEN: ARBNCV; ISSN:1936-122X. (Annual Reviews)
    A review. Biomol. recognition can be stubborn; changes in the structures of assocg. mols., or the environments in which they assoc., often yield compensating changes in enthalpies and entropies of binding and no net change in affinities. This phenomenon-termed enthalpy/entropy (H/S) compensation-hinders efforts in biomol. design, and its incidence-often a surprise to experimentalists-makes interactions between biomols. difficult to predict. Although characterizing H/S compensation requires exptl. care, it is unquestionably a real phenomenon that has, from an engineering perspective, useful phys. origins. Studying H/S compensation can help illuminate the still-murky roles of water and dynamics in biomol. recognition and self-assembly. This review summarizes known sources of H/S compensation (real and perceived) and lays out a conceptual framework for understanding and dissecting-and, perhaps, avoiding or exploiting-this phenomenon in biophys. systems.
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    Fowler, J.; Cohen, L.; Jarvis, P. Practical statistics for field biology; John Wiley & Sons: Chichester, England, 2008.
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    Fang, Y.; Zhou, Y.; Xin, Y.; Shi, Y.; Guo, Z.; Li, Y.; Gu, Z.; Ding, Z.; Shi, G.; Zhang, L. Preparation and characterization of a novel thermostable lipase from Thermomicrobium reseum. Catal. Sci. Technol. 2021, 11, 7386– 7397,  DOI: 10.1039/D1CY01486B
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    18
    Preparation and characterization of a novel thermostable lipase from Thermomicrobium roseum
    Fang, Yakun; Zhou, Yanjie; Xin, Yu; Shi, Yi; Guo, Zitao; Li, Youran; Gu, Zhenghua; Ding, Zhongyang; Shi, Guiyang; Zhang, Liang
    Catalysis Science & Technology (2021), 11 (22), 7386-7397CODEN: CSTAGD; ISSN:2044-4753. (Royal Society of Chemistry)
    In this study, a hypothetical lipase gene from Thermomicrobium roseum DSM 5159 (GenBank: ACM04789.1) was recombinantly expressed and characterized. The TrLIP gene was inserted into two different plasmids (pTIG and pMA5 constructed by our lab.) and further expressed in E. coli BL21 and B. subtilis W600 (TrLIPB/E), resp. After purifn., TrLipE/B showed a single band at approx. 38 kDa on 10% reducing SDS-PAGE gels. The successful expression of TrLipE/B was further confirmed by peptide map fingerprinting (PMF) anal. For both expression systems, the target enzyme revealed marked stability over a wide temp. and pH range. In E. coli BL21, the optimal temp. and pH were 85°C and 8.5, while these were 90°C and 9 in B. subtilis W600. Addnl., the studied TrLipE/B was found to show remarkable tolerance in mixed systems constituted by water and org. solvents. Depending on the different expression systems, TrLipB has better enzymic properties, in particular, thermostability and org. solvent tolerance. Based on the CD (CD) anal., the corresponding helix, β-sheet, β-turn and random coil compns. were slightly different between TrLipE (34.8%, 11.2%, 23.4% and 30.6%) and TrLipB (35.9%, 11.1%, 23.3% and 29.7%). The thermostability of TrLipE/B was further verified with nano-DSC anal. The melting temp. (Tm) and denaturation enthalpy (ΔH) of TrLipE were 97.51°C and 1637 KJ mol-1, 98.53°C and 1463 kJ mol-1 for TrLipB. The substrate specificity and enzymic kinetics were analyzed as well. The studied TrLipE/B's capability to catalyze p-nitrophenol esters with different carbon chain lengths was verified. Enzymic transesterification of immobilized TrLipB was confirmed, with a molar conversion rate of 23.32%. This research therefore provides a candidate that could be applied for biocleanser prodn. and org. synthesis, esp. under environments requiring high temp.
  19. 19
    Fang, Y.; Liu, F.; Shi, Y.; Yang, T.; Liang, C.; Xin, Y.; Gu, Z.; Shi, G.; Zhang, L. Hotspots and mechanisms of action of the thermostable framework of a microbial thermolipase. ACS Synth. Biol. 2022, 11, 3460– 3470,  DOI: 10.1021/acssynbio.2c00360
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    19
    Hotspots and Mechanisms of Action of the Thermostable Framework of a Microbial Thermolipase
    Fang, Yakun; Liu, Fan; Shi, Yi; Yang, Ting; Liang, Chaojuan; Xin, Yu; Gu, Zhenghua; Shi, Guiyang; Zhang, Liang
    ACS Synthetic Biology (2022), 11 (10), 3460-3470CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
    The lipase TrLipB from Thermomicrobium roseum is highly thermostable. However, its thermostable skeleton and mechanism of action should be investigated for industrial applications. Toward this, TrLipB was crystd. using the hanging-drop vapor diffusion method and subjected to X-ray diffraction at 2.0 Å resoln. in this study. The rigid sites, such as the prolines on the relatively flexible loops on the enzyme surface, were scanned. Soft substitutions of these sites were designed using both mol. dynamics (MD) simulation and site-directed mutagenesis. The thermostability of several substitutions decreased markedly, while the catalytic efficiencies of the P9G, P127G, P194G, and P300G mutants reduced substantially; addnl., the thermostable framework of the double mutant, P194G/P300G, was considerably perturbed. However, the substitutions on the lid of the enzyme, including P49G and P48G, promoted the catalytic efficiency to approx. 150% and slightly enhanced the thermostability below 80°C. In MD simulations, the P100G, P194G, P100G/P194G, P194G/P300G, and P100G/P194G/P300G mutants showed high B-factors and RMSD values, whereas the secondary structures, radius of gyration, H-bonds, and solvent accessible surface areas of these mutants were markedly affected. Our observations will assist in understanding the natural framework of a stable lipase, which might contribute to its industrial applications.

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  • Figure 1

    Figure 1. Statistical analysis of the thermodynamic parameters of TrSOX and BSOX. (A) Enthalpy–entropy compensation for both TrSOX and BSOX with wild types marked separately. Coefficients of variation of ΔH, ΔS, and Tm for (B) TrSOX and (C) BSOX. Average values and standard deviations of (D) ΔH, (E) ΔS, and (F) Tm for TrSOX and BSOX. SigmaPlot (version 15, Systat Software Inc., San Jose, CA) was used for graph preparation and statistical analysis.

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  • References

    This article references 19 other publications.

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      Li, B.; Sun, Y.; Zhu, X.; Qian, S.; Pu, J.; Guo, Y.; Wu, H.; Zhang, L.; Xin, Y. Aggregation interface and rigid spots sustain the stable framework of a thermophilic N-demethylase. J. Agric. Food Chem. 2023, 71, 5614– 5629,  DOI: 10.1021/acs.jafc.3c00877
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      Aggregation Interface and Rigid Spots Sustain the Stable Framework of a Thermophilic N-Demethylase
      Li, Bingjie; Sun, Yuqian; Zhu, Xinyi; Qian, Siyu; Pu, Jiayang; Guo, Yuwen; Wu, Haobo; Zhang, Liang; Xin, Yu
      Journal of Agricultural and Food Chemistry (2023), 71 (14), 5614-5629CODEN: JAFCAU; ISSN:0021-8561. (American Chemical Society)
      Enzymes from thermophilic microorganisms usually show high thermostability, which is of great potential in industrial application; to understand the structural logic of these enzymes is helpful for the construction of robust biocatalysts. In this study, based on the crystal structure of an N-demethylase-TrSOX-with outstanding thermostability from Thermomicrobium roseum, substitutions were introduced on the aggregation interface and rigid spots to reduce the aggregation ratio and the rigidity. Four substitutions on the aggregation interface-V162S, M308S, F170S, and V306S-considerably reduced the thermostability and slightly enhanced the catalytic efficiency. In addn., the thermostable framework was considerably disrupted in several multiple P → G substitutions in several local motifs (P129G/P134G, P237G/P259G, and P259G/P276G). These structural fluctuations were in good accordance with whole-structure or partial root-mean-square deviation, radius of gyration H-bonds, and solvent-accessible surface area values in mol. dynamics simulation. Furthermore, these key spots were introduced into an unstable homolog from Bacillus sp., resulting in a dramatical increase in the half-life at 60°C from <10 to 1440 min. These results could help understand the natural stable framework of thermophilic enzymes, which could be refs. for the construction of robust enzymes in industrial applications.
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      Arroyo-Maya, I. J.; McClements, D. J. Application of ITC in foods: A powerful tool for understanding the gastrointestinal fate of lipophilic compounds. Biochim. Biophys. Acta, Gen. Subj. 2016, 1860, 1026– 1035,  DOI: 10.1016/j.bbagen.2015.10.001
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      Application of ITC in foods: A powerful tool for understanding the gastrointestinal fate of lipophilic compounds
      Arroyo-Maya, Izlia J.; McClements, David Julian
      Biochimica et Biophysica Acta, General Subjects (2016), 1860 (5), 1026-1035CODEN: BBGSB3; ISSN:0304-4165. (Elsevier B.V.)
      A review. Isothermal titrn. calorimetry (ITC) is a biophys. technique widely used to study mol. interactions in biol. and non-biol. systems. It can provide important information about mol. interactions (such as binding const., no. of binding sites, free energy, enthalpy, and entropy) simply by measuring the heat absorbed or released during an interaction between two liq. solns. In this review, we present an overview of ITC applications in food science, with particular focus on understanding the fate of lipids within the human gastrointestinal tract. In this area, ITC can be used to study micellization of bile salts, inclusion complex formation, the interaction of surface-active mols. with proteins, carbohydrates and lipids, and the interactions of lipid droplets. ITC is an extremely powerful tool for measuring mol. interactions in food systems, and can provide valuable information about many types of interactions involving food components such as proteins, carbohydrates, lipids, surfactants, and minerals. For systems at equil., ITC can provide fundamental thermodn. parameters that can be used to establish the physiochem. origin of mol. interactions. It is expected that ITC will continue to be utilized as a means of providing fundamental information about complex materials such as those found in foods. This knowledge may be used to create functional foods designed to behave in the gastrointestinal tract in a manner that will improve human health and well-being.
    3. 3
      Garvín, A.; Ibarz, R.; Ibarz, A. Kinetic and thermodynamic compensation. A current and practical review for foods. Food Res. Int. 2017, 96, 132– 153,  DOI: 10.1016/j.foodres.2017.03.004
      3
      Kinetic and thermodynamic compensation. A current and practical review for foods
      Garvin, Alfonso; Ibarz, Raquel; Ibarz, Albert
      Food Research International (2017), 96 (), 132-153CODEN: FORIEU; ISSN:0963-9969. (Elsevier B.V.)
      Kinetic and thermodn. compensations have been reported in many chem., phys., biol. and food processes. Kinetic compensation can be found for any process and when it takes place, it gives information about the reaction mechanism and whether the reaction is controlled by enthalpy or entropy. It consists of the linear relationship between the logarithm of the frequency factor (lnk0) and the activation energy (Ea), both previously obtained from the Arrhenius equation for different values of an environmental variable (e.g. pH, concn. of any substance not involved in the process, pressure, water activity, etc.). A math. consequence of kinetic compensation is the isokinetic temp., this being the temp. at which the kinetic const. should be the same regardless of the environmental variable. Thermodn. compensation can be found for any process involving an equil. and consists of the linear relationship between the variation of enthalpy and entropy, both previously obtained from the Van't Hoff equation for different values of an environmental variable. A math. consequence of thermodn. compensation is the isoequil. temp., this being the temp. at which the equil. const. should be the same regardless of the environmental variable. According to the transition state theory, some kinetic consts. can be related to the equil. const. of the initial equil. stage between the reagents and the transition state. For these cases, it can be concluded that both compensations are related math. and therefore not only does the existence of one kind of compensation imply the existence of the other, but the isokinetic and isoequil. temps. should both be the same, or at least very close to each other. However, there is no reason that forces the linearities that cause either kind of compensation. So, some processes have shown these linear relationships while others have not. Moreover, some authors have reported that, due to the fact that the ests. of the parameters for the couples lnk0-Ea and ΔH≠-ΔS≠ being correlated with each other, there is a statistic compensation that consists of the propagation of exptl. errors, and this effect has to be considered before concluding kinetic and/or thermodn. compensations. This work reviews how to deal with kinetic and thermodn. compensations phys., math. and statistically, prior to a second part that reviews the food processes for which one or both of these compensations have been studied.
    4. 4
      Lin, S. Y.; Lin, C. C. One-step real-time food quality analysis by simultaneous DSC-FTIR microspectroscopy. Crit. Rev. Food Sci. Nutr. 2016, 56, 292– 305,  DOI: 10.1080/10408398.2012.740523
      4
      One-step Real-time Food Quality Analysis by Simultaneous DSC-FTIR Microspectroscopy
      Lin, Shan-Yang; Lin, Chih-Cheng
      Critical Reviews in Food Science and Nutrition (2016), 56 (2), 292-305CODEN: CRFND6; ISSN:1040-8398. (Taylor & Francis, Inc.)
      This review discusses an anal. technique that combines differential scanning calorimetry and Fourier-transform IR (DSC-FTIR) microspectroscopy, which simulates the accelerated stability test and detects decompn. products simultaneously in real time. We show that the DSC-FTIR technique is a fast, simple and powerful anal. tool with applications in food sciences. This technique has been applied successfully to the simultaneous investigation of: encapsulated squid oil stability; the dehydration and intramol. condensation of sweetener (aspartame); the dehydration, rehydration and solidification of trehalose; and online monitoring of the Maillard reaction for glucose (Glc)/asparagine (Asn) in the solid state. This technique delivers rapid and appropriate interpretations with food science applications.
    5. 5
      Khechinashvili, N. N.; Janin, J.; Rodier, F. Thermodynamics of the temperature-induced unfolding of globular proteins. Protein Sci. 1995, 4, 1315– 1324,  DOI: 10.1002/pro.5560040707
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      Thermodynamics of the temperature-induced unfolding of globular proteins
      Khechinashvili, Nikolay N.; Janin, Joeel; Rodier, Francis
      Protein Science (1995), 4 (7), 1315-24CODEN: PRCIEI; ISSN:0961-8368. (Cambridge University Press)
      The heat capacity, enthalpy, entropy, and Gibbs free energy changes for the temp.-induced unfolding of 11 globular proteins (including several enzymes) of known 3-dimensional structure were obtained by microcalorimetric measurements. Their exptl. values were compared to those calcd. from the change in solvent-accessible surface area between the native proteins and the extended polypeptide chain. Proportionality coeffs. were used for the transfer (hydration) of aliph., arom., and polar groups from the gas phase to aq. soln., vibrational effects were estd., and the temp. dependence of each constituent of the thermodn. functions was discussed. At 25°, stabilization of the native state of a globular protein is largely due to 2 favorable terms: the entropy of nonpolar group hydration and the enthalpy of interactions within the protein. They compensate the unfavorable entropy change assocd. with these interactions (conformational entropy) and with vibrational effects. Due to the large heat capacity of nonpolar group hydration, its stabilizing contribution decreases quickly at higher temps., and the 2 unfavorable entropy terms take over, leading to temp.-induced unfolding.
    6. 6
      Tamoliu Nas, K.; Galamba, N. Protein denaturation, zero entropy temperature, and the structure of water around hydrophobic and amphiphilic solutes. J. Phys. Chem. B 2020, 124, 10994– 11006,  DOI: 10.1021/acs.jpcb.0c08055
      6
      Protein Denaturation, Zero Entropy Temperature, and the Structure of Water around Hydrophobic and Amphiphilic Solutes
      Tamoliu Nas Kazimieras; Galamba Nuno
      The journal of physical chemistry. B (2020), 124 (48), 10994-11006 ISSN:.
      The hydrophobic effect plays a key role in many chemical and biological processes, including protein folding. Nonetheless, a comprehensive picture of the effect of temperature on hydrophobic hydration and protein denaturation remains elusive. Here, we study the effect of temperature on the hydration of model hydrophobic and amphiphilic solutes, through molecular dynamics, aiming at getting insight on the singular behavior of water, concerning the zero-entropy temperature, TS, and entropy convergence, TS(*), also observed for some proteins, upon denaturation. We show that, similar to hydrocarbons, polar amphiphilic solutes exhibit a TS, although strongly dependent on solute-water interactions, opposite to hydrocarbons. Further, the temperature dependence of the hydration entropy, normalized by the solvent accessible surface area, is shown to be nearly solute size independent for hydrophobic but not for amphiphilic solutes, for similar reasons. These results are further discussed in the light of information theory (IT) and the structure of water around hydrophobic groups. The latter shows that the tetrahedral enhancement of some water molecules around hydrophobic groups, associated with the reduction of water defects, leads to the strengthening of the weakest hydrogen bonds, relative to bulk water. In addition, a larger tetrahedrality is found in low density water populations, demonstrating that pure water has encoded structural information, similar to that associated with hydrophobic hydration. The reversal of the hydration entropy dependence on the solute size, above TS(*), is also analyzed and shown to be associated with a greater loss of water molecules exhibiting enhanced orientational order, in the coordination sphere of large solutes. Finally, the source of the differences between Kauzmann's "hydrocarbon model" on protein denaturation and hydrophobic hydration is discussed, with relatively large amphiphilic hydrocarbons seemingly displaying a more similar behavior to some globular proteins than aliphatic hydrocarbons.
    7. 7
      Pereira, R. N.; Teixeira, J. A.; Vicente, A. A. Exploring the denaturation of whey proteins upon application of moderate electric fields: a kinetic and thermodynamic study. J. Agric. Food Chem. 2011, 59, 11589– 11597,  DOI: 10.1021/jf201727s
      7
      Exploring the Denaturation of Whey Proteins upon Application of Moderate Electric Fields: A Kinetic and Thermodynamic Study
      Pereira, Ricardo N.; Teixeira, Jose A.; Vicente, Antonio A.
      Journal of Agricultural and Food Chemistry (2011), 59 (21), 11589-11597CODEN: JAFCAU; ISSN:0021-8561. (American Chemical Society)
      Thermal processing often results in disruption of the native conformation of whey proteins, thus affecting functional properties. The aim of this work was to evaluate the effects of moderate elec. fields on denaturation kinetics and thermodn. properties of whey protein dispersions at temps. ranging from 75 to 90 °C. Application of elec. fields led to a lower denaturation of whey proteins, kinetically traduced by lower values of reaction order (n) and rate const. (k) (p < 0.05), when compared to those from conventional heating under equiv. heating rates and holding times. Furthermore, the application of elec. fields combined with short come-up times has reduced considerably the denaturation of proteins during early stages of heating (>30% of native sol. protein than conventional heating) and has detd. also considerable changes in calcd. thermodn. properties (such as Ea, ΔH‡, ΔS‡). In general, denaturation reactions during moderate elec. fields processing were less dependent on temp. increase.
    8. 8
      Helmick, H.; Turasan, H.; Yildirim, M.; Bhunia, A.; Liceaga, A.; Kokini, J. L. Cold denaturation of proteins: Where bioinformatics meets thermodynamics to offer a mechanistic understanding: Pea protein as a case study. J. Agric. Food Chem. 2021, 69, 6339– 6350,  DOI: 10.1021/acs.jafc.0c06558
      8
      Cold Denaturation of Proteins: Where Bioinformatics Meets Thermodynamics to Offer a Mechanistic Understanding: Pea Protein As a Case Study
      Helmick, Harrison; Turasan, Hazal; Yildirim, Merve; Bhunia, Arun; Liceaga, Andrea; Kokini, Jozef L.
      Journal of Agricultural and Food Chemistry (2021), 69 (22), 6339-6350CODEN: JAFCAU; ISSN:0021-8561. (American Chemical Society)
      Protein structure can be altered with heat, but models which predict denaturation show that globular proteins also spontaneously unfold at low temps. through cold denaturation. By an anal. of the primary structure of pea protein using bioinformatic modeling, a mechanism of pea protein cold denaturation is proposed. Pea protein is then fractionated into partially purified legumin and vicilin components, suspended in ethanol, and subjected to low temps. (-10 to -20°). The structural characterizations of the purified fractions are conducted through FTIR, ζ potential, dynamic light scattering, and oil binding, and these are compared to the results of com. protein isolates. The obsd. structural changes suggest that pea protein undergoes changes in structure as the result of low-temp. treatments, which could lead to innovative industrial processing techniques for functionalization by low-temp. processing.
    9. 9
      Montserrat, M.; Mayayo, C.; Sánchez, L.; Calvo, M.; Pérez, M. D. Study of the thermoresistance of the allergenic Ara h1 protein from peanut (Arachis hypogaea). J. Agric. Food Chem. 2013, 61, 3335– 3340,  DOI: 10.1021/jf305450s
      9
      Study of the thermoresistance of the allergenic Ara h1 protein from peanut (Arachis hypogaea)
      Montserrat, Mercedes; Mayayo, Cristina; Sanchez, Lourdes; Calvo, Miguel; Perez, Maria D.
      Journal of Agricultural and Food Chemistry (2013), 61 (13), 3335-3340CODEN: JAFCAU; ISSN:0021-8561. (American Chemical Society)
      The effect of heat treatment on denaturation of Ara h1 protein, a major allergen from peanut, was studied using several techniques. Previously, Ara h1 protein was isolated from raw peanut using ammonium sulfate pptn. and chromatograpic techniques. Antibodies against Ara h1 protein were obtained in rabbits, conjugated with horseradish peroxidase, and used to develop a sandwich ELISA. Denaturation of Ara h1 protein was estd. by the loss of reactivity with its specific antibodies by ELISA. Kinetic and thermodn. parameters of the denaturation process of Ara h1 protein were detd. over a temp. range of 82-90 °C. Denaturation of Ara h1 was best described assuming a reaction order of 1.5. Thermal denaturation of Ara h1 protein was also studied by differential scanning calorimetry using several heating rates. The max. peak temp. and the enthalpy of denaturation obtained by extrapolation to a scan rate of 0 °C/min were 90.22 °C and 1574 kJ/mol, resp. The hydrophobicity of Ara h1 protein increased with the intensity of heat treatment, and aggregates were formed when the protein was treated at 90 °C for 10 min.
    10. 10
      Kang, J.; Solis Rueda, K. A. Enthalpy–entropy compensation in the denaturation of proteins of bovine masseter and cutaneous trunci. Meat Sci. 2022, 184, 108688  DOI: 10.1016/j.meatsci.2021.108688
      10
      Enthalpy-entropy compensation in the denaturation of proteins of bovine masseter and cutaneous trunci
      Kang, Jonghoon; Solis Rueda, Karla A.
      Meat Science (2022), 184 (), 108688CODEN: MESCDN; ISSN:0309-1740. (Elsevier Ltd.)
      Thermal properties of muscles are of great interest in meat science. A recent article (Vaskoska et al., 2021) examines the thermal denaturation of proteins in bovine muscles, masseter and cutaneous trunci, by measuring the enthalpy and melting temp. of the denaturation. In the present article, we report the numerical values of entropy in the denaturation of proteins. Furthermore, we describe a characteristic phenomenon, enthalpy-entropy compensation in the denaturation of bovine proteins. To our knowledge, this is the first observation of the compensation in situ condition. The anal. shown in this paper may develop a new approach in meat science by introducing a new parameter, entropy, that is rarely reported in relation to differential scanning calorimetry results in this field of research. Therefore, it may enhance our understanding of the thermal properties of meat from a phys. chem. perspective.
    11. 11
      Chang, R. Physical Chemistry for the Chemical and Biological Sciences; University Science Books: Sausalito, CA, 2000.
      There is no corresponding record for this reference.
    12. 12
      Griessen, R.; Dam, B. Simple accurate verification of enthalpy–entropy compensation and isoequilibrium relationship. Chemphyschem. 2021, 22, 1774– 1784,  DOI: 10.1002/cphc.202100431
      12
      Simple Accurate Verification of Enthalpy-Entropy Compensation and Isoequilibrium Relationship
      Griessen, Ronald; Dam, Bernard
      ChemPhysChem (2021), 22 (17), 1774-1784CODEN: CPCHFT; ISSN:1439-4235. (Wiley-VCH Verlag GmbH & Co. KGaA)
      In many exptl. investigations of thermodn. equil. or kinetic properties of series of similar reactions it is found that the enthalpies and entropies derived from Van 't Hoff or Arrhenius plots exhibit a strong linear correlation. The origin of this Enthalpy-Entropy compensation, which is strongly related to the coalescence tendency of Van't Hoff or Arrhenius plots, is not necessarily due to a phys./chem./biol. process. It can also be a merely statistical artifact. A new method, called Combined K-CQF makes it possible both to quantify the degree of coalescence of exptl. Van't Hoff lines and to verify whether or not the Enthalpy-Entropy Compensation is of a statistical origin at a desired confidence level. The method is universal and can handle data sets with any degree of coalescence of Van't Hoff (or Arrhenius) plots. The new method requires only a std. least square fit of the enthalpy ΔH vs. entropy ΔS plot to det. the two essential dimensionless parameters K and CQF. The parameter K indicates the position (in inverse temp.) of the coalescence region of Van't Hoff plots and CQF is a quant. measure of the smallest spread of the Van't Hoff plots. The position of the (K, CQF) couple with respect to universal confidence contours detd. from a large no. of simulations of random Van't Hoff plots indicates straightforwardly whether or not the ΔH-ΔS compensation is a statistical artifact.
    13. 13
      Haynie, D. T. Biological thermodynamics, 1st ed.; Cambridge University Press: Cambridge, U.K., 2001.
      There is no corresponding record for this reference.
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      Kang, J.; Auerbach, J. D. Thermodynamic characterization of dissociation rate variations of human leukocyte antigen and peptide complexes. Mol. Immunol. 2009, 46, 2873– 2875,  DOI: 10.1016/j.molimm.2009.05.184
      14
      Thermodynamic characterization of dissociation rate variations of human leukocyte antigen and peptide complexes
      Kang, Jonghoon; Auerbach, Jeremy D.
      Molecular Immunology (2009), 46 (15), 2873-2875CODEN: MOIMD5; ISSN:0161-5890. (Elsevier Ltd.)
      Stability of minor histocompatibility antigen-MHC mol. complexes is a major requirement for the successful presentation of the antigen to T cell receptors. In this letter the authors show thermodn. features of the complexes made of a peptide antigen and its three variants to explain mol. basis of variable stability of the complexes. The anal. suggests that enthalpy is a major factor in detg. the stability of the complexes. The authors also found that the dissocn. of the peptides from the complexes exhibits enthalpy-entropy compensation. Two structural features of the complexes, noncovalent chem. bondings and flexibility of the peptides in the complexes, are in a good agreement with the thermodn. anal. The authors expect thermodn. investigation of peptide antigen-MHC protein complexes will provide valuable information on the stability.
    15. 15
      Garvín, A.; Ibarz, R.; Ibarz, A. Kinetic and thermodynamic compensation. A current and practical review for foods. Food Res. Int. 2017, 96, 132– 153,  DOI: 10.1016/j.foodres.2017.03.004
      15
      Kinetic and thermodynamic compensation. A current and practical review for foods
      Garvin, Alfonso; Ibarz, Raquel; Ibarz, Albert
      Food Research International (2017), 96 (), 132-153CODEN: FORIEU; ISSN:0963-9969. (Elsevier B.V.)
      Kinetic and thermodn. compensations have been reported in many chem., phys., biol. and food processes. Kinetic compensation can be found for any process and when it takes place, it gives information about the reaction mechanism and whether the reaction is controlled by enthalpy or entropy. It consists of the linear relationship between the logarithm of the frequency factor (lnk0) and the activation energy (Ea), both previously obtained from the Arrhenius equation for different values of an environmental variable (e.g. pH, concn. of any substance not involved in the process, pressure, water activity, etc.). A math. consequence of kinetic compensation is the isokinetic temp., this being the temp. at which the kinetic const. should be the same regardless of the environmental variable. Thermodn. compensation can be found for any process involving an equil. and consists of the linear relationship between the variation of enthalpy and entropy, both previously obtained from the Van't Hoff equation for different values of an environmental variable. A math. consequence of thermodn. compensation is the isoequil. temp., this being the temp. at which the equil. const. should be the same regardless of the environmental variable. According to the transition state theory, some kinetic consts. can be related to the equil. const. of the initial equil. stage between the reagents and the transition state. For these cases, it can be concluded that both compensations are related math. and therefore not only does the existence of one kind of compensation imply the existence of the other, but the isokinetic and isoequil. temps. should both be the same, or at least very close to each other. However, there is no reason that forces the linearities that cause either kind of compensation. So, some processes have shown these linear relationships while others have not. Moreover, some authors have reported that, due to the fact that the ests. of the parameters for the couples lnk0-Ea and ΔH≠-ΔS≠ being correlated with each other, there is a statistic compensation that consists of the propagation of exptl. errors, and this effect has to be considered before concluding kinetic and/or thermodn. compensations. This work reviews how to deal with kinetic and thermodn. compensations phys., math. and statistically, prior to a second part that reviews the food processes for which one or both of these compensations have been studied.
    16. 16
      Fox, J. M.; Zhao, M.; Fink, M. J.; Kang, K.; Whitesides, G. M. The molecular origin of enthalpy/entropy compensation in biomolecular recognition. Annu. Rev. Biophys. 2018, 47, 223– 250,  DOI: 10.1146/annurev-biophys-070816-033743
      16
      The Molecular Origin of Enthalpy/Entropy Compensation in Biomolecular Recognition
      Fox, Jerome M.; Zhao, Mengxia; Fink, Michael J.; Kang, Kyungtae; Whitesides, George M.
      Annual Review of Biophysics (2018), 47 (), 223-250CODEN: ARBNCV; ISSN:1936-122X. (Annual Reviews)
      A review. Biomol. recognition can be stubborn; changes in the structures of assocg. mols., or the environments in which they assoc., often yield compensating changes in enthalpies and entropies of binding and no net change in affinities. This phenomenon-termed enthalpy/entropy (H/S) compensation-hinders efforts in biomol. design, and its incidence-often a surprise to experimentalists-makes interactions between biomols. difficult to predict. Although characterizing H/S compensation requires exptl. care, it is unquestionably a real phenomenon that has, from an engineering perspective, useful phys. origins. Studying H/S compensation can help illuminate the still-murky roles of water and dynamics in biomol. recognition and self-assembly. This review summarizes known sources of H/S compensation (real and perceived) and lays out a conceptual framework for understanding and dissecting-and, perhaps, avoiding or exploiting-this phenomenon in biophys. systems.
    17. 17
      Fowler, J.; Cohen, L.; Jarvis, P. Practical statistics for field biology; John Wiley & Sons: Chichester, England, 2008.
      There is no corresponding record for this reference.
    18. 18
      Fang, Y.; Zhou, Y.; Xin, Y.; Shi, Y.; Guo, Z.; Li, Y.; Gu, Z.; Ding, Z.; Shi, G.; Zhang, L. Preparation and characterization of a novel thermostable lipase from Thermomicrobium reseum. Catal. Sci. Technol. 2021, 11, 7386– 7397,  DOI: 10.1039/D1CY01486B
      18
      Preparation and characterization of a novel thermostable lipase from Thermomicrobium roseum
      Fang, Yakun; Zhou, Yanjie; Xin, Yu; Shi, Yi; Guo, Zitao; Li, Youran; Gu, Zhenghua; Ding, Zhongyang; Shi, Guiyang; Zhang, Liang
      Catalysis Science & Technology (2021), 11 (22), 7386-7397CODEN: CSTAGD; ISSN:2044-4753. (Royal Society of Chemistry)
      In this study, a hypothetical lipase gene from Thermomicrobium roseum DSM 5159 (GenBank: ACM04789.1) was recombinantly expressed and characterized. The TrLIP gene was inserted into two different plasmids (pTIG and pMA5 constructed by our lab.) and further expressed in E. coli BL21 and B. subtilis W600 (TrLIPB/E), resp. After purifn., TrLipE/B showed a single band at approx. 38 kDa on 10% reducing SDS-PAGE gels. The successful expression of TrLipE/B was further confirmed by peptide map fingerprinting (PMF) anal. For both expression systems, the target enzyme revealed marked stability over a wide temp. and pH range. In E. coli BL21, the optimal temp. and pH were 85°C and 8.5, while these were 90°C and 9 in B. subtilis W600. Addnl., the studied TrLipE/B was found to show remarkable tolerance in mixed systems constituted by water and org. solvents. Depending on the different expression systems, TrLipB has better enzymic properties, in particular, thermostability and org. solvent tolerance. Based on the CD (CD) anal., the corresponding helix, β-sheet, β-turn and random coil compns. were slightly different between TrLipE (34.8%, 11.2%, 23.4% and 30.6%) and TrLipB (35.9%, 11.1%, 23.3% and 29.7%). The thermostability of TrLipE/B was further verified with nano-DSC anal. The melting temp. (Tm) and denaturation enthalpy (ΔH) of TrLipE were 97.51°C and 1637 KJ mol-1, 98.53°C and 1463 kJ mol-1 for TrLipB. The substrate specificity and enzymic kinetics were analyzed as well. The studied TrLipE/B's capability to catalyze p-nitrophenol esters with different carbon chain lengths was verified. Enzymic transesterification of immobilized TrLipB was confirmed, with a molar conversion rate of 23.32%. This research therefore provides a candidate that could be applied for biocleanser prodn. and org. synthesis, esp. under environments requiring high temp.
    19. 19
      Fang, Y.; Liu, F.; Shi, Y.; Yang, T.; Liang, C.; Xin, Y.; Gu, Z.; Shi, G.; Zhang, L. Hotspots and mechanisms of action of the thermostable framework of a microbial thermolipase. ACS Synth. Biol. 2022, 11, 3460– 3470,  DOI: 10.1021/acssynbio.2c00360
      19
      Hotspots and Mechanisms of Action of the Thermostable Framework of a Microbial Thermolipase
      Fang, Yakun; Liu, Fan; Shi, Yi; Yang, Ting; Liang, Chaojuan; Xin, Yu; Gu, Zhenghua; Shi, Guiyang; Zhang, Liang
      ACS Synthetic Biology (2022), 11 (10), 3460-3470CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
      The lipase TrLipB from Thermomicrobium roseum is highly thermostable. However, its thermostable skeleton and mechanism of action should be investigated for industrial applications. Toward this, TrLipB was crystd. using the hanging-drop vapor diffusion method and subjected to X-ray diffraction at 2.0 Å resoln. in this study. The rigid sites, such as the prolines on the relatively flexible loops on the enzyme surface, were scanned. Soft substitutions of these sites were designed using both mol. dynamics (MD) simulation and site-directed mutagenesis. The thermostability of several substitutions decreased markedly, while the catalytic efficiencies of the P9G, P127G, P194G, and P300G mutants reduced substantially; addnl., the thermostable framework of the double mutant, P194G/P300G, was considerably perturbed. However, the substitutions on the lid of the enzyme, including P49G and P48G, promoted the catalytic efficiency to approx. 150% and slightly enhanced the thermostability below 80°C. In MD simulations, the P100G, P194G, P100G/P194G, P194G/P300G, and P100G/P194G/P300G mutants showed high B-factors and RMSD values, whereas the secondary structures, radius of gyration, H-bonds, and solvent accessible surface areas of these mutants were markedly affected. Our observations will assist in understanding the natural framework of a stable lipase, which might contribute to its industrial applications.