Rational Modification of Protein Stability by the Mutation of Charged Surface Residues ² Shari Spector, ‡,§ Minghui Wang, | Stefan A. Carp, ⊥ James Robblee, O Zachary S. Hendsch, ⊥ Robert Fairman, O Bruce Tidor,* ,⊥ and Daniel P. Raleigh* ,|,X Department of Physiology and Biophysics, State UniVersity of New York at Stony Brook, Stony Brook, New York 11794-8661, Department of Chemistry, State UniVersity of New York at Stony Brook, Stony Brook, New York 11794-3400, Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139-4307, Department of Molecular, Cellular and DeVelopmental Biology, HaVerford College, HaVerford PennsylVania 19041, and Graduate Programs in Biophysics and in Molecular and Cellular Biology, State UniVersity of New York at Stony Brook, Stony Brook, New York 11794 ReceiVed September 7, 1999; ReVised Manuscript ReceiVed NoVember 3, 1999 ABSTRACT: Continuum methods were used to calculate the electrostatic contributions of charged and polar side chains to the overall stability of a small 41-residue helical protein, the peripheral subunit-binding domain. The results of these calculations suggest several residues that are destabilizing, relative to hydrophobic isosteres. One position was chosen to test the results of these calculations. Arg8 is located on the surface of the protein in a region of positive electrostatic potential. The calculations suggest that Arg8 makes a significant, unfavorable electrostatic contribution to the overall stability. The experiments described in this paper represent the first direct experimental test of the theoretical methods, taking advantage of solid-phase peptide synthesis to incorporate approximately isosteric amino acid substitutions. Arg8 was replaced with norleucine (Nle), an amino acid that is hydrophobic and approximately isosteric, or with R-amino adipic acid (Aad), which is also approximately isosteric but oppositely charged. In this manner, it is possible to isolate electrostatic interactions from the effects of hydrophobic and van der Waals interactions. Both Arg8Nle and Arg8Aad are more thermostable than the wild-type sequence, testifying to the validity of the calculations. These replacements led to stability increases at 52.6 °C, the T m of the wild-type, of 0.86 and 1.08 kcal mol -1 , respectively. The stability of Arg8Nle is particularly interesting as a rare case in which replacement of a surface charge with a hydrophobic residue leads to an increase in the stability of the protein. The amino acid sequences of proteins include a wide variety of different residue types, including several acidic and basic groups. The charges on the surface of a protein are certainly important for its solubility, but what effect do electrostatic interactions have on the overall stability of the molecule? A number of recent experimental and theoretical studies have suggested that partially or completely buried salt bridges function at least in part to provide specificity to the fold, although they do not generally provide added stability beyond that of a hydrophobic bridge of similar geometry (1, 2). This conclusion is based on results showing that the favorable electrostatic interactions from the salt bridge are often insufficient to overcome the electrostatic desolvation penalty (3-8). Surface salt bridges appear to make only small contributions to protein stability (9-12). However, in T4 lysozyme, a partially exposed salt bridge appears to contribute 3-5 kcal mol -1 to the stability of the protein (13). Experimental studies have also been performed to examine the contribution of a single charged residue to the stability of a protein. The binding face of barstar, the inhibitor of the ribonuclease barnase, has four acidic residues. Replacement of any of these with alanine leads to an increase in the stability of the protein. On the basis of the ionic strength dependence, the increased stability of the barstar mutants is ascribed to the removal of unfavorable electrostatic interactions (14). Comparison of these results is complicated by the choice of different reference states. In the T4 lysozyme study, the salt bridge in the wild-type protein is only partially exposed, and the mutation cycle involves changing each member of the salt bridge pair to asparagine, together and individually (13). In one barnase study investigating an existing, solvent- exposed salt bridge triad, a triple mutant cycle is used in which each residue is substituted with alanine (10). In another ² This research was supported by NIH grant GM 54233 to DPR who is a Pew Scholar in the Biomedical Sciences, and by NIH grants GM 55758 and GM 56552 to BT. SS was supported in part by a Graduate Council Fellowship from the State University of New York. SAC is a Beckman Scholar. * To whom correspondence should be addressed. Bruce Tidor: telephone, 617-253-7258; fax, 617-252-1816; e-mail, tidor@mit.edu. Daniel Raleigh: telephone, 516-632-9547; fax, 516-632-7960; e-mail, draleigh@notes.cc.sunysb.edu. ‡ Department of Physiology and Biophysics, State University of New York at Stony Brook. § Current address: Departments of Chemistry and Biology, Mas- sachusetts Institute of Technology 68-565, 77 Massachusetts Avenue, Cambridge, MA 02139-4307. | Department of Chemistry, State University of New York at Stony Brook. ⊥ Department of Chemistry, Massachusetts Institute of Technology. O Department of Molecular, Cellular and Developmental Biology, Haverford College. X Graduate Programs in Biophysics and in Molecular and Cellular Biology, State University of New York at Stony Brook. 872 Biochemistry 2000, 39, 872-879 10.1021/bi992091m CCC: $19.00 © 2000 American Chemical Society Published on Web 01/13/2000