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Examining solute-induced changes in protein conformational equilibria is a long-standing method for probing the role of water in maintaining protein stability. Interpreting the molecular details governing the solute-induced effects, however, remains controversial. We present experimental and theoretical data for osmolyte-induced changes in the stabilities of the A and N states of yeast iso-1-ferricytochrome c. Using polyol osmolytes of increasing size, we observe that osmolytes alone induce A-state formation from acid-denatured cytochrome c and N state formation from the thermally denatured protein. The stabilities of the A and N states increase linearly with osmolyte concentration. Interestingly, osmolytes stabilize the A state to a greater degree than the N state. To interpret the data, we divide the free energy for the reaction into contributions from nonspecific steric repulsions (excluded volume effects) and from binding interactions. We use scaled particle theory (SPT) to estimate the free energy contributions from steric repulsions, and we estimate the contributions from water–protein and osmolyte–protein binding interactions by comparing the SPT calculations to experimental data. We conclude that excluded volume effects are the primary stabilizing force, with changes in water–protein and solute–protein binding interactions making favorable contributions to stability of the A state and unfavorable contributions to the stability of the N state. The validity of our interpretation is strengthened by analysis of data on osmolyte-induced protein stabilization from the literature, and by comparison with other analyses of solute-induced changes in conformational equilibria. © 2000 John Wiley & Sons, Inc. Biopoly 53: 293–307, 2000

The theta subunit of DNA polymerase III, the main replicative polymerase of Escherichia coli, has been examined by circular dichroism and by NMR spectroscopy. The polymerase core consists of three subunits: alpha, epsilon, and theta, with alpha possessing the polymerase activity, epsilon functioning as a proofreading exonuclease, and theta, a small subunit of 8.9 kD, of undetermined function. The theta subunit has been expressed in E. coli, and a CD analysis of theta indicates the presence of a significant amount of secondary structure: approximately 52% alpha helix, 9% beta sheet, 21% turns, and 18% random coil. However, at higher concentrations, theta yields a poorly-resolved 1D proton NMR spectrum in which both the amide protons and the methyl protons show poor chemical shift dispersion. Subsequent 1H-15N HSQC analysis of uniformly-15N-labeled theta supports the conclusion that approximately half of the protein is reasonably well-structured. Another quarter of the protein, probably including some of the N-terminal region, is highly mobile, exhibiting a chemical shift pattern indicative of random coil structure. The remaining amide resonances exhibit significant broadening, indicative of intermolecular and/or intramolecular exchange processes. Improved chemical shift dispersion and greater uniformity of resonance intensities in the 1H-15N HSQC spectra resulted when [U-15N]-theta was examined in the presence of epsilon186--the N-terminal domain of the epsilon-subunit. Further work is currently in progress to define the solution structure of theta and the theta-epsilon186 complex.