Irreversible thermal denaturation of elongation factor Ts from Thermus thermophilus effect of the residual structure and intermonomer disulfide bond.
The homodimeric wild-type elongation factor Ts, EF-Ts(wt), and its C190A mutant, EF-Ts(C190A), from Thermus thermophilus goes through thermal denaturation in a way consistent with a two state irreversible model with a relatively high activation energy, approximately 530 kJ/mol (Supplemental materials provides a list of 98 activation energies from 54 proteins in various solvent conditions). Removing the intermonomeric disulfide bond by substituting alanine for cysteine 190 affects the rate constant of the irreversible thermal transition. At physiological temperatures, the half-life of the native conformations was estimated to be approximately 21 days for wt and 1.3 days for C190A. Thermally denatured EF-Ts refolds into a molten-globule-like state as indicated by its native-like circular dichroism spectrum in the far UV region and the enhanced fluorescence of the hydrophobic probe, 1-anilinonaphtalene-8-sulphonate. The residual secondary structure observed in the thermally denatured state of EF-Ts at high temperatures affects its apparent temperature of thermal transition, T(trs), independent of the presence or absence of the intermonomeric disulfide bond. The effect of the GdmHCl concentration on the activation energy, E(a), and the temperature, T*, i.e., the temperature at which the rate of the irreversible step is 1 min(-1), indicates that the intermonomeric disulfide bond contributes to the irreversibility of thermal transition of EF-Ts.
Zoldák
Gabriel
G
Department of Biochemistry, Faculty of Sciences, P. J. Safárik University, Moyzesova 11, 041 54 Kosice, Slovakia.
Sedlák
Erik
E
Valusová
Eva
E
Wolfrum
Alexandra
A
Marek
Jozef
J
Antalík
Marián
M
Sprinzl
Mathias
M
D000465:Algorithms; D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D003553:Cystine / Q000737:chemistry* / Q000235:genetics; D019791:Guanidine / Q000737:chemistry; D006358:Hot Temperature; D009154:Mutation / Q000235:genetics; D010445:Peptide Elongation Factors / Q000737:chemistry* / Q000235:genetics; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D016963:Thermus thermophilus / Q000737:chemistry* / Q000235:genetics
D000817:Anilino Naphthalenesulfonates; D010445:Peptide Elongation Factors; D011994:Recombinant Proteins; C023512:elongation factor Ts; D003553:Cystine; C027132:1-anilino-8-naphthalenesulfonate; D019791:Guanidine