Rational modification of protein stability by the mutation of charged surface residues

Continuum methods were used to calculate the electrostatic contributions of charged and polar side chains to the overall stability of a small 41-resid ue helical protein, the peripheral subunit-binding domain. The results of t hese calculations suggest several residues that are destabilizing, relative to hydrophobic isosteres. One position was chosen to test the results of t hese calculations. Arg8 is located on the surface of the protein in a regio n of positive electrostatic potential. The calculations suggest that Arg8 m akes a significant, unfavorable electrostatic contribution to the overall s tability, The experiments described in this paper represent the first direc t experimental test of the theoretical methods, taking advantage of solid-p hase peptide synthesis to incorporate approximately isosteric amino acid su bstitutions. Arg8 was replaced with norleucine (Nle), an amino acid that is hydrophobic and approximately isosteric, or with alpha-amino adipic acid ( Aad), which is also approximately isosteric but oppositely charged. In this manner, it is possible to isolate electrostatic interactions from the effe cts of hydrophobic and van der Waals interactions. Both Arg8Nle and Arg8Aad are more thermostable than the wild-type sequence, testifying to the valid ity of the calculations. These replacements led to stability increases at 5 2.6 degrees C, the T-m of the wild-type, of 0.86 and 1.08 kcal mol(-1), res pectively. The stability of Arg8Nle is particularly interesting as a rare c ase in which replacement of a surface charge with a hydrophobic residue lea ds to an increase in the stability of the protein.