Water contributes actively to the rapid crossing of a protein unfolding barrier.
The cold-shock protein CspB folds rapidly in a N <= => U two-state reaction via a transition state that is about 90% native in its interactions with denaturants and water. This suggested that the energy barrier to unfolding is overcome by processes occurring in the protein itself, rather than in the solvent. Nevertheless, CspB unfolding depends on the solvent viscosity. We determined the activation volumes of unfolding and refolding by pressure-jump and high-pressure stopped-flow techniques in the presence of various denaturants. The results obtained by these methods agree well. The activation volume of unfolding is positive (Delta V(++)(NU)=16(+/-4) ml/mol) and virtually independent of the nature and the concentration of the denaturant. We suggest that in the transition state the protein is expanded and water molecules start to invade the hydrophobic core. They have, however, not yet established favorable interactions to compensate for the loss of intra-protein interactions. The activation volume of refolding is positive as well (Delta V(++)(NU)=53(+/-6) ml/mol) and, above 3 M urea, independent of the concentration of the denaturant. At low concentrations of urea or guanidinium thiocyanate, Delta V(++)(UN) decreases significantly, suggesting that compact unfolded forms become populated under these conditions.
Jacob
Maik H
MH
Laboratorium für Biochemie und Bayreuther Zentrum für Molekulare Biowissenschaften, Universität Bayreuth, D-95440 Bayreuth, Germany. maik.jacob@gmx.de
Saudan
Christophe
C
Holtermann
Georg
G
Martin
Andreas
A
Perl
Dieter
D
Merbach
André E
AE
Schmid
Franz X
FX
D001412:Bacillus subtilis / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry*; D006146:Guanidines; D007700:Kinetics; D011312:Pressure; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry; D013816:Thermodynamics; D013861:Thiocyanates; D014508:Urea; D014867:Water / Q000737:chemistry
D001426:Bacterial Proteins; D006146:Guanidines; D011994:Recombinant Proteins; D013861:Thiocyanates; C077188:cold-shock protein CspB, Bacteria; D014867:Water; C054436:guanidine thiocyanate; D014508:Urea