Comparison of heat- and pressure-induced unfolding of ribonuclease a: the critical role of Phe46 which appears to belong to a new hydrophobic chain-folding initiation site

Biochemistry
Eri ChataniR Lange

Abstract

To clarify the structural role of Phe46 inside the hydrophobic core of bovine pancreatic ribonuclease A (RNase A), thermal and pressure unfolding of wild-type RNase A and three mutant forms (F46V, F46E, and F46K) were analyzed by fourth-derivative UV absorbance spectroscopy. All the mutants, as well as the wild type, exhibited a two-state transition during both thermal and pressure unfolding, and both T(m) and P(m) decreased markedly when Phe46 was replaced with valine, glutamic acid, or lysine. The strongest effect was on the F46K mutant and the weakest on F46V. Both unfolding processes produced identical blue shifts in the fourth-derivative spectra, indicating that the tyrosine residues are similarly exposed in the temperature- and pressure-induced unfolded states. A comparison of Gibbs free energies determined from the pressure and temperature unfoldings, however, gave DeltaG(p)/DeltaG(t) ratios (r) of 1.7 for the wild type and 0.92 +/- 0.03 for the mutants. Furthermore, the DeltaV value for each mutant was larger than that for the wild type. CD spectra and activity measurements showed no obvious major structural differences in the folded state, indicating that the structures of the Phe46 mutants and wild type differ in the ...Continue Reading

References

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Citations

Jan 6, 2005·Macromolecular Bioscience·Alexander Wittemann, Matthias Ballauff
Dec 21, 2002·European Journal of Biochemistry·Marie TrovasletMarie Dominique Legoy
Oct 14, 2004·European Journal of Biochemistry·Jens KöditzUlrich Arnold
Apr 18, 2009·Biopolymers·Roger VilàMaria Vilanova
Nov 11, 2011·Journal of Chromatography. a·Pete Gagnon
Aug 8, 2007·Protein Expression and Purification·M Walid QoronflehMatthew B Seefeldt
Sep 25, 2004·Protein Science : a Publication of the Protein Society·Matthew B SeefeldtTheodore W Randolph
Jan 15, 2008·Biophysical Journal·Marta BruixMaria Vilanova
Jul 18, 2006·Biophysical Journal·Jason K CheungThomas M Truskett
Aug 22, 2006·Journal of Molecular Biology·Bryan D Smith, Ronald T Raines
Nov 9, 2012·The Journal of Biological Chemistry·Donald GagnéNicolas Doucet

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