Genetic selection reveals the role of a buried, conserved polar residue.
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.
Johnson
R Jeremy
RJ
Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706-1544, USA.
Lin
Shawn R
SR
Raines
Ronald T
RT
D000818:Animals; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D006801:Humans; D006860:Hydrogen Bonding; D057927:Hydrophobic and Hydrophilic Interactions; D011487:Protein Conformation; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000235:genetics; D012694:Serine / Q000737:chemistry* / Q000235:genetics; D013696:Temperature; D013816:Thermodynamics
D012694:Serine; D012259:Ribonuclease, Pancreatic