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

Abstract:To understand relationships between protein sequence and stability, we often compare data from proteins that differ by the substitution of one amino acid. Frequently, an amino acid change causes the cooperative denaturation transitions to shift to lower temperatures, diminishing the signal from the native state. Here we show that apparent stability changes, Le., the free energy of denaturation, AG,, can also be caused by a deficiency of points in the low temperature end of the transition. In addition, we suggest a method for overcoming this problem. Keywords: baselines; computer fitting; thermodynamic parametersThere are many techniques, including circular dichroism spectropolarimetry (CD), that can probe protein stability as a function of temperature. Investigators often use computer algorithms to fit these signal-versus-temperature plots to a two-state model (Lumry et al., 1966),(1)The application of this model usually involves defining linear baselines for the native and denatured states:where mN and bN are the slope and y-intercept of the native baseline, mD and bD are these values for the denatured baseline, and T is the absolute temperature. By comparing the signal, AT, at any temperature to the native and denatured baselines, the fraction of protein denatured, CY^,^, can be determined:Reprint requests to: Gary J. Pielak, Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599-3290; e-mail: gar-pielak@ unc.edu. The equilibrium constant for denaturation, KD,T, is obtained via Equation 5, and AGD,T is obtained via Equation 6,where R is the gas constant. The slope of a -R In K,.,-versus-T" plot is the van't Hoff enthalpy, AH,,,. The subscript m means that the slope is evaluated at T,,,, the temperature at which KD,T = I . More often, however, Equations 2 through 6 are inserted into Equation 7, and the data are fit directly.In Equation 7 (Elwell & Schellman, 1977), AC,, is the change in heat capacity upon denaturation.Another simple technique for fitting denaturation curves was developed by Breslauer (1995) through manipulation of the van? Hoff equation to yield:For this method, the baselines are determined manually. A line through the steepest part of the transition ( 8 (~/ 8 T )~, is drawn and its slope is used in conjunction with these baselines and T,, to obtain AH,,, .We have observed that baselines from computer fitting often disagree with those selected by hand. To probe this discrepancy, we chose a large CD thermal denaturation data set (76 points, Fig. l), truncated it at various intervals, and determined the effect of data removal on automated baseline selection,

We have evaluated the use of [1,2-13C2]propionate for the analysis of propionic acid metabolism, based on the ability to distinguish between the methylcitrate and methylmalonate pathways. Studies using propionate-adapted Escherichia coli MG1655 cells were performed. Preservation of the13C-13C-12C carbon skeleton in labeled alanine and alanine-containing peptides involved in cell wall recycling is indicative of the direct formation of pyruvate from propionate via the methylcitrate cycle, the enzymes of which have recently been demonstrated in E. coli. Additionally, formation of 13C-labeled formate from pyruvate by the action of pyruvate-formate lyase is also consistent with the labeling of pyruvate C-1. Carboxylation of the labeled pyruvate leads to formation of [1,2-13C2]oxaloacetate and to multiply labeled glutamate and succinate isotopomers, also consistent with the flux through the methylcitrate pathway, followed by the tricarboxylic acid (TCA) cycle. Additional labeling of TCA intermediates arises due to the formation of [1-13C]acetyl coenzyme A from the labeled pyruvate, formed via pyruvate-formate lyase. Labeling patterns in trehalose and glycine are also interpreted in terms of the above pathways. The information derived from the [1,2-13C2]propionate label is contrasted with information which can be derived from singly or triply labeled propionate and shown to be more useful for distinguishing the different propionate utilization pathways via nuclear magnetic resonance analysis.

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.