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Article Abstract
A cavity-filling mutation at a hydrophobic cavity is a useful method for increasing protein stability. This method, however, sometimes destabilizes the protein because of the accompanying structural changes by the steric hindrance around the cavity. Thus, detailed knowledge of unfavorable structural changes is important for a comprehensive understanding of the cavity-filling mutation. In the present study, by employing the cavity-filling mutant of RNase HI as a case study, the structural change induced by the substitution of Phe for Ala (Ala52Phe) was analyzed in detail using Raman spectroscopy. In previous studies, the thermodynamic result apparently indicated a small decrease in Δ (destabilization) by the mutation. In the present study, Raman differential spectra show a clear structural difference between wild-type RNase HI and Ala52Phe. Consequently, the direct signature of the conformational strains around the protein cavity is readily acquired, leading to further understanding of the trade-off relationship between the cavity-filling and incidental steric hindrance.
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