It is generally considered that electrostatic interactions on the protein surface, 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 (FNfn10) is more stable at acidic pH than neutral pH, with an apparent midpoint of transition near pH 4. Determination of pK(a)'s for all the side chain carboxyl groups of Asp and Glu residues revealed that Asp 23 and Glu 9 have an upshifted pK(a). These residues and Asp 7 form a negatively charged patch on the surface of FNfn10, with Asp 7 centrally located between Asp 23 and Glu 9, suggesting repulsive electrostatic interactions among these residues at neutral pH. Mutant proteins, D7N and D7K, in which Asp 7 was replaced with Asn and Lys, respectively, exhibited a modest but significant increase in stability at neutral pH, compared to the wild type, and they no longer showed pH dependence of stability. The pK(a)'s of Asp 23 and Glu 9 in these mutant proteins shifted closer to their respective unperturbed values, indicating that the unfavorable electrostatic interactions have been reduced in the mutant proteins. Interestingly, the wild-type and mutant proteins were all stabilized to a similar degree by the addition of 1 M sodium chloride at both neutral and acidic pH, suggesting that the repulsive interactions between the carboxyl groups cannot be effectively shielded by 1 M sodium chloride. These results indicate that repulsive interactions between like charges on the protein surface can destabilize a protein, and protein stability can be significantly improved by relieving these interactions. Study holds ProTherm entries: 11625, 11626, 11627, 11628, 11629, 11630, 11631, 11632, 11633, 11634, 11635, 11636 Extra Details: in the presence of 6.3 M Urea electrostatic interactions; protein surface; ion pairs; negatively charged patch
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:42 p.m.
|Number of data points||12|
|Proteins||Fibronectin ; Fibronectin|
|Assays/Quantities/Protocols||Experimental Assay: Tm ionic:NaCl: 1 M, buffers:sodium phosphate: 20 mM, pH:7.0 ; Experimental Assay: Tm ionic:NaCl: 0.1 M, buffers:sodium phosphate: 20 mM, pH:7.0 ; Experimental Assay: Tm pH:2.4, ionic:NaCl: 1 M, buffers:glycine hydrochloride: 20 mM ; Experimental Assay: Tm pH:2.4, ionic:NaCl: 0.1 M, buffers:glycine hydrochloride: 20 mM|
|Libraries||Mutations for sequence VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT|