Protein stabilization by removal of unsatisfied polar groups: Computational approaches and experimental tests

The role of polar and charged side chains in partially buried protein environments has been probed in a variant of Are repressor (MYL) in which hydrophobic interactions between Met31, Tyr36, and Leu40 replace the wild-type salt-bridge interactions between Arg31, Glu36, and Arg40. In the absence of this salt-bridge triad, three additional side chains were identified by continuum electrostatic calculations as incurring larger desolvation penalties during folding than were recovered in favorable electrostatic interactions in the folded state. These side chains (Asn29, Ser44, and Glu48) were mutated singly and collectively to alanine in the Mn background, and the thermodynamic stabilities of the resulting mutant proteins were found to be increased by 0.1 to 1.3 kcal/mol of dimer. All of the mutants displayed cooperative thermal melts and appeared to have well-packed hydrophobic cores by near-UV circular dichroism spectroscopy, indicating that conformational specificity is maintained. The Are variant (MYL-NA29/SA44/EA48) in which the entire six-residue polar network is replaced by nonpolar groups is 5.1 kcal/mol of dimer more stable than wild type, indicating that the strategy of replacing buried or partially buried charged and polar side chains with hydrophobic residues can lead to substantial stabilization.