Impact of point mutations on the structure and thermal stability of ribonuclease T1 in aqueous solution probed by fourier transform infrared spectroscopy

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DOIResolve DOI: http://doi.org/10.1021/bi00201a021
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TypeArticle
Journal titleBiochemistry
ISSN0006-2960
1520-4995
Volume33
Issue35
Pages1072510730; # of pages: 6
AbstractWe 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 thedouble (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 @-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 α-helix and a β-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. In addition, the mutation in position 59 also leads to changes in the microenvironment of (some) tyrosine residues, as revealed by the aromatic ring stretching vibration of tyrosine at 15 16 cm⁻¹, a sensitive local monitor of protein conformation. The infrared results are correlated with X-ray data.
Publication date
LanguageEnglish
AffiliationNRC Institute for Biodiagnostics; National Research Council Canada
Peer reviewedYes
NRC number144
NPARC number9147849
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Record identifiera7d8fb53-fd3b-4102-b374-8f55884b8e04
Record created2009-06-25
Record modified2016-12-06
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