Abstract
Site-directed mutagenesis was used to examine the contribution of buried and exposed aromatic amino acids to the spectral properties and conformational stability of ribonuclease T, (RNase T,). The side chain of Trp59 is completely buried in the hydrophobic core of RNase T,. Mutants were made in which Trp59 was replaced by tyrosine and phenylalanine. Analysis of these mutants indicated that tryptophan in the wild-type protein made a major contribution to the fluorescence and near-UV CD spectra. Aromatic amino acids at this position contributed to the far-UV CD as well. Differential scanning calorimetry (DSC) and urea denaturation curves were used to determine the conformational stability of the mutant and wild-type proteins. The conformational stability of Trp59Phe was 0.5 kcal/mol greater than wild-type RNase TI, but the stability of Trp59Tyr was 0.8 kcal/mole less. An analysis suggests that these mutations may relieve steric strain between Pro39 and Trp59 present in the wild-type protein as suggested previously [Keifhaber Grunert, T., H.-P., Hahn, U., & Schmid, F.X. (1992) Proteins 12, 171-179], and that this steric strain may destabilize RNase T, by approximately 1 kcal/mol. Aspartate 49 has the most hyperexposed side chain in RNase T,. Mutants were constructed in which Asp 49 was replaced by tryptophan, tyrosine, phenylalanine, and alanine. Only the fluorescence spectrum of Asp49Trp differed from that of wild type; the difference spectrum was consistent with a solvent-exposed tryptophan at position 49. Near-UV CD and far-UV CD both showed increases in the intensity of bands when aspartate 49 was mutated to an aromatic amino acid. Both urea denaturation and DSC results showed that the stability of the Asp49AIa mutant increased by 0.6 kcal/mole. The stability decreased as the more hydrophobic aromatic residues were substituted for Ala 49, showing a reverse hydrophobic effect of the type observed by Pakula and Sauer [Pakula, A.A. & Sauer R.T. (1990) Nature 344, 363-364].
Fee, Lanette Renee (1995). The contribution of aromatic amino acids to the spectral properties and conformational stability of ribonuclease T1. Texas A&M University. Texas A&M University. Libraries. Available electronically from
https : / /hdl .handle .net /1969 .1 /DISSERTATIONS -1559802.