Gaining a better understanding of the denatured state ensemble of proteins is important for understanding protein stability and the mechanism of protein folding. We studied the folding kinetics of ribonuclease Sa (RNase Sa) and a charge-reversal variant (D17R). The refolding kinetics are similar, but the unfolding rate constant is 10-fold greater for the variant. This suggests that charge-charge interactions in the denatured state and the transition state ensembles are more favorable in the variant than in RNase Sa, and shows that charge-charge interactions can influence the kinetics and mechanism of protein folding.Keywords: protein folding; protein stability; folding kinetics; denatured state; charge-charge interactions Protein stability is an important consideration in protein engineering. There is considerable interest in developing methods to increase the stability of a protein. Two ways this can be achieved are (1) decrease the free energy of the native state, and/or (2) increase the free energy of the denatured state. In an earlier study , a charge-reversal mutation (D17K) was made to improve electrostatic interactions on the surface of native ribonuclease Sa (RNase Sa) to stabilize the protein. However, the mutant was less stable than the wildtype protein by about 1 kcal/mol. We suggested that this resulted because electrostatic interactions were more favorable in the denatured state of the mutant than in wild-type RNase Sa, and that this might influence the folding kinetics. The results in the present work support this idea, and show that charge-charge interactions in the transition and denatured state ensembles of a protein can exert a substantial effect on the kinetics of protein folding. Recent work on another protein from Raleigh's lab has reached a similar conclusion (Cho et al. 2004;Horng et al. 2005), and thus these findings may be fairly general. ResultsTo monitor the folding and unfolding of WT RNase Sa and the charge-reversal variant, D17R, by fluorescence spectroscopy, a Trp residue was added in place of Tyr 81. The crystal structure of Y81W showed that the Trp is 87% buried, and the variant is 0.4 kcal/mol less stable than WT (Alston et al. 2004). For Y81W RNase Sa, there is a 2.5-fold increase in fluorescence intensity at 319 nm when the protein folds (Alston et al. 2004). Throughout this work, the Y81W variant will be referred to as WT* and the charge-reversal variant as WT*(D17R). Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi

A bivalent recombinant vaccine for human hookworm disease is under development. One of the lead candidate antigens in the vaccine is a glutathione S-transferase cloned from the hookworm Necator americanus (Na-GST-1) which is expressed in the yeast Pichia pastoris. Based on preliminary studies demonstrating that the recombinant protein was not stable in an acetate buffer at pH 6, we undertook an extensive stability analysis of the molecule. To improve and optimize stability we complemented traditional methods employed for macromolecule and vaccine stabilization with biophysical techniques that were incorporated into a systematic process based on an eigenvector approach. Large data sets, obtained from a variety of experimental methods were used to establish a color map ("empirical phase diagram") of the physical stability of the vaccine antigen over a wide range of temperature and pH. The resulting map defined "apparent phase boundaries" that were used to develop high throughput screening assays. These assays were then employed to identify excipients that stabilized the antigen against physical degradation that could otherwise result in losses of physicochemical integrity, immunogenicity, and potency of the vaccine. Based on these evaluations, the recombinant Na-GST-1 antigen was reformulated and ultimately produced under Good Manufacturing Practices and with an acceptable stability profile.

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