Pressure-jump-induced kinetics reveals a hydration dependent folding/unfolding mechanism of ribonuclease A.
Pressure-jump (p-jump)-induced relaxation kinetics was used to explore the energy landscape of protein folding/unfolding of Y115W, a fluorescent variant of ribonuclease A. Pressure-jumps of 40 MPa amplitude (5 ms dead-time) were conducted both to higher (unfolding) and to lower (folding) pressure, in the range from 100 to 500 MPa, between 30 and 50 degrees C. Significant deviations from the expected symmetrical protein relaxation kinetics were observed. Whereas downward p-jumps resulted always in single exponential kinetics, the kinetics induced by upward p-jumps were biphasic in the low pressure range and monophasic at higher pressures. The relative amplitude of the slow phase decreased as a function of both pressure and temperature. At 50 degrees C, only the fast phase remained. These results can be interpreted within the framework of a two-dimensional energy surface containing a pressure- and temperature-dependent barrier between two unfolded states differing in the isomeric state of the Asn-113-Pro-114 bond. Analysis of the activation volume of the fast kinetic phase revealed a temperature-dependent shift of the unfolding transition state to a larger volume. The observed compensation of this effect by glycerol offers an explanation for its protein stabilizing effect.
Font
J
J
Laboratori d'Enginyeria de Proteïnes, Departament de Biologia, Facultat de Ciències, Universitat de Girona, Campus de Montilivi s/n, 17071 Girona, Spain.
Torrent
J
J
Ribó
M
M
Laurents
D V
DV
Balny
C
C
Vilanova
M
M
Lange
R
R
D000818:Animals; D005990:Glycerol / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011312:Pressure; D017510:Protein Folding; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000235:genetics; D012997:Solvents; D014867:Water / Q000737:chemistry*
D012997:Solvents; D014867:Water; D012259:Ribonuclease, Pancreatic; D005990:Glycerol