Thermal unfolding kinetics of ubiquitin in the microsecond-to-second time range probed by Tyr-59 fluorescence.
Thermal folding/unfolding kinetics of wild-type ubiquitin (wt-UBQ) was studied in a wide time range, from microseconds to seconds, by combining rapid-mixing T-jump and laser T-jump with fluorescence detection (MTJ-F and LTJ-F, respectively) to monitor the fluorescence changes of Tyr-59 located on the 310-helix. The kinetics occurs exclusively in the millisecond to second time range, and the decays are strictly single exponential. From global analysis of folding and unfolding decays, the kf and ku values were determined, without use of the equilibrium constant Ku. The activation enthalpy of folding is negative (DeltaHf(#)(Tm) = -10.8 kcal/mol), but the free energy of the transition state is substantially larger than that of the unfolded state (DeltaGf(#)(Tm) = 7.6 kcal/mol RTm). Thus, wt-UBQ behaves as a two-state folder, when folding is monitored by the fluorescence of Tyr-59. The observation of kinetics on the microsecond time scale, when folding is monitored by the disruption of hydrogen bonds between beta-strands, using nonlinear infrared spectroscopy of the amide I vibrations (LTJ-DVE) [Chung, H. S.; Tokmakoff, A. Proteins: Struct., Funct., Bioinf. 2008, 72, 474-487], seems to result from the fact that MTJ-F monitors the effective unfolding (backbone exposure to water) of the thermally excited protein alone, while LTJ-DVE also monitors preliminary events (hydrogen-bond breaking) and thermal re-equilibration of the thermally excited protein.
Noronha
Melinda
M
Centro de Química Estrutural, Instituto Superior Técnico, Technical University of Lisbon, 1049-001 Lisboa, Portugal. mnoronha@ist.utl.pt
Gerbelová
Hana
H
Faria
Tiago Q
TQ
Lund
Daniel N
DN
Smith
D Alastair
DA
Santos
Helena
H
Maçanita
António L
AL
D000818:Animals; D002152:Calorimetry, Differential Scanning; D002417:Cattle; D005453:Fluorescence; D007700:Kinetics; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry*; D025801:Ubiquitin / Q000737:chemistry*
D025801:Ubiquitin; D014443:Tyrosine