Individual tyrosine side-chain contributions to circular dichroism of ribonuclease.
Experimental and theoretical studies using site-directed mutants of ribonuclease A (RNase A) offer more extensive information on the tyrosine side-chain contributions to the circular dichroism (CD) of the enzyme. Bovine pancreatic RNase A has three exposed tyrosine residues (Tyr73, Tyr76, and Tyr115) and three buried tyrosine residues (Tyr25, Tyr92 and Tyr97). The difference CD spectra between the wild type and the mutants at pH 7.0 (Deltaepsilon(277,wt) - Deltaepsilon(277,mut)) show bands with more negative DeltaDeltaepsilon(277) values for Y73F and Y115F than those for Y25F and Y92F and bands with positive DeltaDeltaepsilon(277) values for Y76F and Y97F. The theoretical calculations are in good semiquantitative agreement for all the mutants. The pH difference spectrum (pH 11.3-7.0) for the wild type shows a negative band at 295 nm and an enhanced positive band at 245 nm. The three mutants at buried tyrosine sites and one mutant at an exposed tyrosine site (Y76F) exhibit pH-difference spectra that are similar to that of the wild type. In contrast, two mutants at exposed tyrosine sites (Y73F and Y115F) exhibit diminished 295-nm negative bands and, instead of positive bands at 245 nm, negative bands are observed. Our results indicate that Tyr73 and Tyr115, two of the exposed tyrosine residues, are the largest contributors to the 277- and 245-nm CD bands of RNaseA, but the buried tyrosine residues and the one remaining exposed residue also contribute to these bands. Disulfide contributions to the 277- and 240-nm bands and the peptide contribution to the 240-nm band are confirmed theoretically.
Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523, USA.
A-Young Moon
AY
Woody
Robert W
RW
Woody
D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D009154:Mutation; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000235:genetics; D014443:Tyrosine / Q000737:chemistry* / Q000235:genetics
D014443:Tyrosine; D012259:Ribonuclease, Pancreatic