Thermal Unfolding Kinetics of Ubiquitin in the Microsecond-to-Second Time Range Probed by Tyr-59 Fluorescence

Abstract

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.

Direct Quantification of the Attempt Frequency Determining the Mechanical Unfolding of Ubiquitin Protein

“…This study complements the extensive work conducted with the protein ubiquitin, the mechanical behavior of which has been vastly characterized in different regions of the folding energy landscape (17,31,(35)(36)(37). Furthermore, the remarkable thermal stability of ubiquitin (above 100°C) makes it an ideal candidate for the thermal studies under force (10). Our experiments demonstrate a significant effect of the temperature on the mechanical rate of unfolding.…”

“…Indeed, the value for the pre-exponential factor obtained for ubiquitin unfolding using bulk techniques using nanosecond laser temperature jumps, where the disruption of interhydrogen bonds between ␤-strands III-V was probed with non-linear infrared spectroscopy as a function of temperature (53-67°C), was measured to be ϳ1.2 ϫ 10 15 s Ϫ1 (43). In similar experiments combining rapid mixing T-jump and laser T-jump with fluorescence detection, the fluorescence changes of Tyr-59 located on the 3 10 -helix of ubiquitin were measured as a function of the temperature, varying within the range 45-65°C (10). These experiments yielded a pre-exponential factor of 8.3 ϫ 10 33 s Ϫ1 .…”

“…Over the last two decades, a great number of studies have reported the effect of temperature on the (un)folding kinetics of a plethora of distinct proteins using different biochemistry bulk techniques. With a few exceptions (5), the apparent consensus (6) is that although protein unfolding usually follows simple Arrhenius kinetics, the folding kinetics exhibit more complex dependences with temperature, resulting in curvature in the ln k f versus 1/T plots (7)(8)(9)(10)(11)(12). The origin of such non-Arrhenius behavior during the folding reaction can be generically explained either in terms of the rate of escape from different minima along a rough energy landscape, thus yielding super-Arrhenius kinetics, or most likely, based on the hydrophobic effect, involving a large change in heat capacity during the folding process (13)(14)(15), with the heat capacity of the transition state placed somewhere in between the folded and unfolded states (16).…”

Identification of two-step chemical mechanisms and determination of thermokinetic parameters using frequency responses to small temperature oscillations

“…[49][50][51] Consequently, it is common for the plots of ln (k ±i ) as a function of 1/T to be nonlinear when data have been collected over several tens of degrees. [49][50][51][52][53][54] The Eyring equation is then more suitable for fitting than Eq. (3).…”

An Outer Membrane Protein Undergoes Enthalpy- and Entropy-Driven Transitions

“…Recently, other groups determined negative activation enthalpies accompanying the kinetics of protein folding (62;63). Noronha and colleagues, using a combination of rapid-mixing temperature jump and laser temperature-jump with fluorescence detection, determined that the activation enthalpy of folding required for a 76-residue ubiquitin-based polypeptide is ~−45 kJ/mol (63). …”