Stabilization of a fibronectin type III domain by the removal of unfavorable electrostatic interactions on the protein surface

It is generally considered that electrostatic interactions on the protein s urface, such as ion pairs, contribute little to protein stability, although they may play important roles in conformational specificity. We found that the tenth fibronectin type III domain of human fibronectin (FNfn 10) is mo re stable at acidic pH than neutral pH, with an apparent midpoint of transi tion near pH 4. Determination of pK(a)'s for all the side chain carboxyl gr oups of Asp and Glu residues revealed that Asp 23 and Glu 9 have an upshift ed pK(a). These residues and Asp 7 form a negatively charged patch on the s urface of FNfn 10, with Asp 7 centrally located between Asp 23 and Glu 9, s uggesting repulsive electrostatic interactions among these residues at neut ral pH. Mutant proteins, D7N and D7K, in which Asp 7 was replaced with Asn and Lys, respectively, exhibited a modest but significant increase in stabi lity at neutral pH, compared to the wild type, and they no longer showed pH dependence of stability. The pKa's of Asp 23 and Glu 9 in these mutant pro teins shifted closer to their respective unperturbed values, indicating tha t the unfavorable electrostatic interactions have been reduced in the mutan t proteins. Interestingly, the wild-type and mutant proteins were all stabi lized to a similar degree by the addition of 1 M sodium chloride at both ne utral and acidic pH, suggesting that the repulsive interactions between the carboxyl groups cannot be effectively shielded by 1 M sodium chloride. The se results indicate that repulsive interactions between like charges on the protein surface can destabilize a protein, and protein stability can be si gnificantly improved by relieving these interactions.