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Volume exclusion and soft interaction effects on protein stability under crowded conditions.

ABSTRACT: Most proteins function in nature under crowded conditions, and crowding can change protein properties. Quantification of crowding effects, however, is difficult because solutions containing hundreds of grams of macromolecules per liter often interfere with the observation of the protein being studied. Models for macromolecular crowding tend to focus on the steric effects of crowders, neglecting potential chemical interactions between the crowder and the test protein. Here, we report the first systematic, quantitative, residue-level study of crowding effects on the equilibrium stability of a globular protein. We used a system comprising poly(vinylpyrrolidone)s (PVPs) of varying molecular weights as crowding agents and chymotrypsin inhibitor 2 (CI2) as a small globular test protein. Stability was quantified with NMR-detected amide (1)H exchange. We analyzed the data in terms of hard particle exclusion, confinement, and soft interactions. For all crowded conditions, nearly every observed residue experiences a stabilizing effect. The exceptions are residues for which stabilities are unchanged. At a PVP concentration of 100 g/L, the data are consistent with theories of hard particle exclusion. At higher concentrations, the data are more consistent with confinement. The data show that the crowder also stabilizes the test protein by weakly binding its native state. We conclude that the role of native-state binding and other soft interactions needs to be seriously considered when applying both theory and experiment to studies of macromolecular crowding.

SUBMITTER: Miklos AC

PROVIDER: S-EPMC2927838 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Volume exclusion and soft interaction effects on protein stability under crowded conditions.

Miklos Andrew C AC Li Conggang C Sharaf Naima G NG Pielak Gary J GJ

Biochemistry 20100801 33

Most proteins function in nature under crowded conditions, and crowding can change protein properties. Quantification of crowding effects, however, is difficult because solutions containing hundreds of grams of macromolecules per liter often interfere with the observation of the protein being studied. Models for macromolecular crowding tend to focus on the steric effects of crowders, neglecting potential chemical interactions between the crowder and the test protein. Here, we report the first sy ...[more]

PMID: 20672856

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Project description:BackgroundIn resistance training, the role of training frequency to increase maximal strength is often debated. However, the limited data available does not allow for clear training frequency "optimization" recommendations. The purpose of this study was to investigate the effects of training frequency on maximal muscular strength and rate of perceived exertion (RPE). The total weekly training volume was equally distributed between two and four sessions per muscle group.MethodsTwenty-one experienced resistance-trained male subjects (height: 1.85 ± 0.06 m, body mass: 85.3 ± 12.3 kg, age: 27.6 ± 7.6 years) were tested prior to and after an 8-week training period in one-repetition maximum (1RM) barbell back squat and bench press. Subjects were randomly assigned to a SPLIT group (n = 10), in which there were two training sessions of squats and lower-body exercises and two training sessions of bench press and upper-body exercises, or a FULLBODY group (n = 11), in which four sessions with squats, bench press and supplementary exercises were conducted every session. In each session, the subjects rated their RPE after barbell back squat, bench press, and the full session.ResultsBoth groups significantly increased 1RM strength in barbell back squat (SPLIT group: +13.25 kg; FULLBODY group: +14.31 kg) and bench press (SPLIT group: +7.75 kg; FULLBODY group: +8.86 kg) but training frequency did not affect this increase for squat (p = 0.640) or bench press (p = 0.431). Both groups showed a significant effect for time on RPE on all three measurements. The analyses showed only an interaction effect between groups on time for the RPE after the squat exercise (p = 0.002).ConclusionWe conclude that there are no additional benefits of increasing the training frequency from two to four sessions under volume-equated conditions, but it could be favorable to spread the total training volume into several training bouts through the week to avoid potential increases in RPE, especially after the squat exercise.

Project description:Changes in excluded volume and contact interaction with the surface of a protein have been suggested as mechanisms for the changes in stability induced by cosolvents. The aim of the present paper is to present an analysis that combines both effects in a quantitative manner. The result is that both processes are present in both stabilizing and destabilizing interactions and neither can be ignored. Excluded volume was estimated using accessible surface area calculations of the kind introduced by Lee and Richards. The change in excluded volume on unfolding, deltaX, is quite large. For example, deltaX for ribonuclease is 6.7 L in urea and approximately 16 L in sucrose. The latter number is greater than the molar volume of the protein. Direct interaction with the protein is represented as the solvent exchange mechanism, which differs from ordinary association theory because of the weakness of the interaction and the high concentrations of cosolvents. The balance between the two effects and their contribution to overall stability are most simply presented as bar diagrams as in Fig. 3. Our finding for five proteins is that excluded volume contributes to the stabilization of the native structure and that contact interaction contributes to destabilization. This is true for five proteins and four cosolvents including both denaturants and osmolytes. Whether a substance stabilizes a protein or destabilizes it depends on the relative size of these two contributions. The constant for the cosolvent contact with the protein is remarkably uniform for four of the proteins, indicating a similarity of groups exposed during unfolding. One protein, staphylococcus nuclease, is anomalous in almost all respects. In general, the strength of the interaction with guanidinium is about twice that of urea, which is about twice that of trimethylamine-N-oxide and sucrose. Arguments are presented for the use of volume fractions in equilibrium equations and the ignoring of activity coefficients of the cosolvent. It is shown in the Appendix that both the excluded volume and the direct interaction can be extracted in a unified way from the McMillan-Mayer formula for the second virial coefficient.

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Volume exclusion and soft interaction effects on protein stability under crowded conditions.

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Volume exclusion and soft interaction effects on protein stability under crowded conditions.

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