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 of 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 because they are known to affect the activity of the enzyme. The structural differences were evaluated by using peptide backbone and side-chain "marker" bands as conformation-sensitive monitors. All mutations lead to a decrease of the thermal transition temperature, though the mutation Tyr-45-->Trp affects the Tm to a lesser degree than the replacement 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 point mutations, could be detected. In particular, we found subtle differences in the hydrogen bonding pattern of the beta-strands in the mutants 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 were obtained for the double mutant Y45W/W59Y and the single mutant W59Y, 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) which affects the overall protein conformation. The mutation Tyr to Trp in position 45, on the other hand, has practically no impact on the polypeptide backbone conformation.(ABSTRACT TRUNCATED AT 250 WORDS)
Fabian
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H
Institute for Biochemistry, Humboldt-University Berlin, FRG.
Schultz
C
C
Backmann
J
J
Hahn
U
U
Saenger
W
W
Mantsch
H H
HH
Naumann
D
D
D001236:Aspergillus oryzae / Q000201:enzymology; D005656:Fungal Proteins / Q000737:chemistry; D006358:Hot Temperature; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011489:Protein Denaturation; D006163:Ribonuclease T1 / Q000737:chemistry*; D012996:Solutions; D017550:Spectroscopy, Fourier Transform Infrared; D013329:Structure-Activity Relationship
D005656:Fungal Proteins; D012996:Solutions; D006163:Ribonuclease T1