IMPACT OF POINT MUTATIONS ON THE STRUCTURE AND THERMAL-STABILITY OF RIBONUCLEASE-T1 IN AQUEOUS-SOLUTION PROBED BY FOURIER-TRANSFORM INFRARED-SPECTROSCOPY

We undertook a detailed comparative analysis of the infrared spectra o f wild-type ribonuclease T1 and three mutants: two single mutants, Tyr -45 --> Trp (Y45W) and Trp-59 --> Tyr (W59Y), and a double mutant, Tyr -45 --> Trp/Trp-59 --> Tyr (Y45W/W59Y). These mutants were selected be cause they are known to affect the activity of the enzyme. The structu ral differences were evaluated by using peptide backbone and side-chai n ''marker'' bands as conformation-sensitive monitors. All mutations l ead to a decrease of the thermal transition temperature, though the mu tation Tyr-45 --> Trp affects the T-m to a lesser degree than the repl acement of Trp-59 by Tyr, both in the single (W59Y) and in the double (Y45W/W59Y) mutant. Small changes in the protein backbone conformation and in the microenvironment of certain amino acids, induced by the po int mutations, could be detected. In particular, we found subtle diffe rences in the hydrogen bonding pattern of the beta-strands in the muta nts W59Y and Y45W/W59Y, compared to that in wild-type RNase T1 and in the mutant Y45W. Practically identical spectra in the amide I region w ere obtained for the double mutant Y45W/W59Y and the single mutant W59 Y, demonstrating that it is the change from Trp to Tyr in position 59 (located at the interface between the alpha-helix and a beta-strand) w hich affects the overall protein conformation. The mutation Tyr to Trp in position 45, on the other hand, has practically no impact on the p olypeptide backbone conformation. In addition, the mutation in positio n 59 also leads to changes in the microenvironment of (some) tyrosine residues, as revealed by the aromatic ring stretching vibration of tyr osine at 1516 cm(-1), a sensitive local monitor of protein conformatio n. The infrared results are correlated with X-ray data.