We have used potentiometric titrations to measure the pK values of the ionizable groups of proteins in alanine pentapeptides with appropriately blocked termini. These pentapeptides provide an improved model for the pK values of the ionizable groups in proteins. Our pK values determined in 0.1 M KCl at 25°C are: 3.67 6 0.03 (a-carboxyl), 3.67 6 0.04 (Asp), 4.25 6 0.05 (Glu), 6.54 6 0.04 (His), 8.00 6 0.03 (a-amino), 8.55 6 0.03 (Cys), 9.84 6 0.11 (Tyr), and 10.40 6 0.08 (Lys). The pK values of some groups differ from the Nozaki and Tanford (N&T) pK values often used in the literature: Asp (3.67 this work vs. 4.0 N&T); His (6.54 this work vs. 6.3 N&T); a-amino (8.00 this work vs. 7.5 N&T); Cys (8.55 this work vs. 9.5 N&T); and Tyr (9.84 this work vs. 9.6 N&T). Our pK values will be useful to those who study pK perturbations in folded and unfolded proteins, and to those who use theory to gain a better understanding of the factors that determine the pK values of the ionizable groups of proteins.Keywords: pK values; protein ionizable groups; pH titration; peptide model compounds The acid/base properties of proteins have been studied since 1917, when Sorensen, who first defined pH in 1909, showed that egg albumin is an ampholyte (Sorensen et al. 1917). Soon thereafter, Linderstrom-Lang recognized that the net charge on a protein would influence the ionization of individual groups, and incorporated this into the first model developed to understand the acid/base properties of a protein (Linderstrom-Lang 1924). An important contribution by Tanford and Kirkwood triggered an intense interest in the factors that determine the pK values of the ionizable groups of proteins that continues to the present day (Tanford and Kirkwood 1957;Braun-Sand and Warshel 2005). The net charge on a protein varies with pH, and is determined by the content and the pK values of the ionizable groups (Tanford 1962). Thus, the pK values of the ionizable groups are important to biochemists because they influence the structure, stability, solubility, and the many functions of proteins (Tanford 1968;Pace 1975;Fersht 1985;Matthew et al. 1985;Anderson et al. 1990;Ries-Kautt and Ducruix 1997;Shaw et al. 2001;Bartlett et al. 2002).In early studies aimed at interpreting titration curves of proteins, Tanford (1962) introduced the term intrinsic pK (pK int ). He defined the term as the pK an ionizable group would have when the net charge on the molecule is zero. When proteins fold, the perturbations of the pKs of the ionizable groups on the surface of the protein from the pK int values are usually small, and are determined mainly by charge-charge interactions with other ionizable groups (Laurents et al. 2003). However, if these groups are partially or fully buried in the protein interior, large positive and negative perturbations often occur, and it is important that we understand why (Schutz and Warshel 2001). Experimental studies of these perturbations have been reported by several groups (see, for example, Garcia-Moreno et al. 1997;Giletto and Pac...

Our goal was to gain a better understanding of the contribution of the burial of polar groups and their hydrogen bonds to the conformational stability of proteins. We measured the change in stability, D(DG), for a series of hydrogen bonding mutants in four proteins: villin headpiece subdomain (VHP) containing 36 residues, a surface protein from Borrelia burgdorferi (VlsE) containing 341 residues, and two proteins previously studied in our laboratory, ribonucleases Sa (RNase Sa) and T1 (RNase T1). Crystal structures were determined for three of the hydrogen bonding mutants of RNase Sa: S24A, Y51F, and T95A. The structures are very similar to wild type RNase Sa and the hydrogen bonding partners form intermolecular hydrogen bonds to water in all three mutants. We compare our results with previous studies of similar mutants in other proteins and reach the following conclusions. (1) Hydrogen bonds contribute favorably to protein stability. (2) The contribution of hydrogen bonds to protein stability is strongly context dependent. (3) Hydrogen bonds by side chains and peptide groups make similar contributions to protein stability. (4) Polar group burial can make a favorable contribution to protein stability even if the polar groups are not hydrogen bonded. (5) The contribution of hydrogen bonds to protein stability is similar for VHP, a small protein, and VlsE, a large protein.

It is difficult to increase protein stability by adding hydrogen bonds or burying nonpolar surface. The results described here show that reversing the charge on a side chain on the surface of a protein is a useful way of increasing stability. Ribonuclease T1 is an acidic protein with a pI Ϸ 3.5 and a net charge of ϷϪ6 at pH 7. The side chain of Asp49 is hyperexposed, not hydrogen bonded, and 8 Å from the nearest charged group. The stability of Asp49Ala is 0.5 kcal0mol greater than wild-type at pH 7 and 0.4 kcal0mol less at pH 2.5. The stability of Asp49His is 1.1 kcal0mol greater than wild-type at pH 6, where the histidine 49 side chain~pK a ϭ 7.2! is positively charged. Similar results were obtained with ribonuclease Sa where Asp25Lys is 0.9 kcal0mol and Glu74Lys is 1.1 kcal0mol more stable than the wild-type enzyme. These results suggest that protein stability can be increased by improving the coulombic interactions among charged groups on the protein surface. In addition, the stability of RNase T1 decreases as more hydrophobic aromatic residues are substituted for Ala49, indicating a reverse hydrophobic effect.

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