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) Study holds ProTherm entries: 2573, 2574, 2575, 2576 Extra Details: Ribonuclease T1; point mutations; hydrogen bonding; structure;,thermal stability; beta-strands; Fourier transform infrared spectroscopy
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:19 p.m.
|Number of data points||8|
|Proteins||Guanyl-specific ribonuclease T1 ; Guanyl-specific ribonuclease T1 ; Guanyl-specific ribonuclease T1 ; Guanyl-specific ribonuclease T1 ; Guanyl-specific ribonuclease T1|
|Assays/Quantities/Protocols||Experimental Assay: Tm ; Derived Quantity: dTm|
|Libraries||Mutations for sequence ACDYTCGSNCYSSSDVSTAQAAGYQLHEDGETVGSNSYPHKYNNYEGFDFSVSSPYYEWPILSSGDVYSGGSPGADRVVFNENNQLAGVITHTGASGNNFVECT|