The burial of nonpolar surface area is known to enhance markedly the conformational stability of proteins. The contribution from the burial of polar surface area is less clear. Here, we report on the tolerance to substitution of Ser75 of bovine pancreatic ribonuclease (RNase A), a residue that has the unusual attributes of being buried, conserved, and polar. To identify variants that retain biological function, we used a genetic selection based on the intrinsic cytotoxicity of ribonucleolytic activity. Cell growth at 30 degrees C, 37 degrees C, and 44 degrees C correlated with residue size, indicating that the primary attribute of Ser75 is its small size. The side-chain hydroxyl group of Ser75 forms a hydrogen bond with a main-chain nitrogen. The conformational stability of the S75A variant, which lacks this hydrogen bond, was diminished by DeltaDeltaG = 2.5 kcal/mol. Threonine, which can reinstate this hydrogen bond, provided a catalytically active RNase A variant at higher temperatures than did some smaller residues (including aspartate), indicating that a secondary attribute of Ser75 is the ability of its uncharged side chain to accept a hydrogen bond. These results provide insight on the imperatives for the conservation of a buried polar residue. Study holds ProTherm entries: 23700, 23701, 23702, 23703, 23704, 23705, 23706 Extra Details: PBS (pH 7.4), was contained in 1.00 L NaCl (8.0 g), KCl (2.0 g), Na2HPO4.7H2O (1.15 g), KH2PO4 (2.0 g), and NaN3 (0.10 g). conformational stability; genetic selection; hydrogen bond; hydrophobic core; molecular evolution; ribonucleases
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:54 p.m.
|Number of data points||16|
|Proteins||Ribonuclease pancreatic ; Ribonuclease pancreatic|
|Assays/Quantities/Protocols||Experimental Assay: ddG ; Experimental Assay: Tm ; Experimental Assay: dHvH ; Derived Quantity: dTm|
|Libraries||Mutations for sequence KETAAAKFERQHMDSSTSAASSSNYCNQMMKSRNLTKDRCKPVNTFVHESLADVQAVCSQKNVACKNGQTNCYQSYSTMSITDCRETGSSKYPNCAYKTTQANKHIIVACEGNPYVPVHFDASV|
|Percent Identity||Matching Chains||Protein||Accession||Entry Name|