Spectroscopic Signature of the Steric Strains in an Escherichia coli RNase HI Cavity-Filling Destabilized Mutant Protein

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 Escherichia coli RNase HI as a case study, the structural change induced by the substitution of Phe for Ala52 (Ala52Phe) was analyzed in detail using Raman spectroscopy. In previous studies, the thermodynamic result apparently indicated a small decrease in ΔG (destabilization) by the mutation. In the present study, Raman differential spectra show a clear structural difference between wild-type E. coli 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.

Spectroscopic Evidence of the Salt-Induced Conformational Change around the Localized Electric Charges on the Protein Surface of Fibronectin Type III

“…Thus, this band is practically useful as a conformational marker band. 16,17,19 The bandwidth of Trp at pH 6.0 immediately increases from 100 to 200 mM. At higher NaCl concentrations, the bandwidth increases almost monotonously with the concentration increase (Figure 4a).…”

“…Figure 2 shows the Raman spectra of FN3 at pH 6.0 (red line) and pH 3.0 (blue line), in the solid state (black line), and their differential spectra (colored filled lines) calculated by the subtraction of the FN3 spectrum at pH 6.0 from each spectrum. Each spectrum shows typical protein Raman bands, such as an amide I band at approximately 1670 cm −1 , 19,20 The detailed band assignment was described previously (Supporting Information 2). 19 Of note, the Tyr bands of FN3 in solution seemed to be detected as triplets.…”

“…Each spectrum shows typical protein Raman bands, such as an amide I band at approximately 1670 cm −1 , 19,20 The detailed band assignment was described previously (Supporting Information 2). 19 Of note, the Tyr bands of FN3 in solution seemed to be detected as triplets. By contrast, many proteins in typical cases have doublets at approximately 850 and 830 cm −1 , which are caused by a Fermi resonance between the symmetric ring-breathing fundamental of Tyr and an overtone of the Tyr out-of-plane ring vibration.…”

“…The experimental setup for the measurement of Raman spectra was previously described in detail. 19 Excitation was accomplished by the 532 nm line with approximately 31 mW at the sample surface. For the salt concentration dependence measurements, a grating having 1800 lines/mm was used to provide a wavenumber resolution of approximately 1.0 cm −1 .…”

“…To obtain detailed conformational knowledge at the molecular scale, Raman spectroscopy and 8-anilino-1-naphthalenesulfonic acid (ANS) fluorescence were employed. Raman spectroscopy can reveal the detailed conformation of each functional group in a protein, especially aromatic acid residues, such as tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe). − ANS is a charge-transfer fluorescence dye and thus compensates the conformational knowledge from the protein surface because the dye typically binds to positively charged residues, such as lysine (Lys) or arginine (Arg) on a protein surface. , These complimentary analyses will be expected to provide detailed molecular knowledge that has been lacking.…”