Highly nonexponential folding kinetics in aqueous solution have been observed during temperature jump-induced refolding of two proteins, yeast phosphoglycerate kinase and a ubiquitin mutant. The observations are most easily interpreted in terms of downhill folding, which posits a heterogeneous ensemble of structures en route to the folded state. The data are also reconciled with exponential kinetics measured under different experimental conditions and with titration experiments indicating cooperative folding.

The rapid refolding dynamics of apomyoglobin are followed by a new temperature-jump fluorescence technique on a 15-ns to 0. EXPERIMENTAL METHODSOutline. The heart of the experiment is shown in Fig. 1 Heating and Sample Cell. The sample is held in a custom 0.4-mm path length-fused silica cell (Fig. 1) cooled by two thermoelectric devices. The temperature is held constant to <0.2°C by a thermistor feedback loop. Two -120-mJ, 1.54-,um infrared beams are generated by Raman shifting a 700-mJ neodymium:yttrium/aluminum-garnet laser in a modeoptimized high-efficiency methane cell, resulting in uniform, near-gaussian heating profiles of 2-mm diameter. The counterpropagating beams are delayed by 8 ns from one another to avoid transient grating formation, and heating (by OH overtone relaxation in water) is completed within the pulse duration due to picosecond vibrational equilibration. The two mirror-image exponential absorption profiles add up to a longitudinal temperature uniformity of ±3% over the length of the cell, and the large uniform pump profile minimizes thermal lensing and diffusion effects. The T-jump is measured by transmission of a 1.5-,Lm diode laser focused to <400 tLm Abbreviations: T-jump, temperature jump; Mb, myoglobin; apoMb, apomyoglobin; h-apoMb, horse apoMb.

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The dynamics of protein-refolding experiments initiated by a temperature jump depend critically on the nature of the initial cold-denatured ensemble. The cold-denatured state of equine apomyoglobin has been investigated in aqueous buffers by near- and far-UV circular dichroism, fluorescence, infrared, and NMR spectroscopies at temperatures ranging from −20 to 98 °C. Cold denaturation of apomyoglobin is well described by a cooperative transition below 3 °C and differs in many aspects from acid-induced unfolding. As a reference system, the N-terminal A-peptide fragment of equine apomyoglobin has also been studied in aqueous and trifluoroethanol solutions. The A-peptide has a low helix-forming propensity in the absence of any stabilizing tertiary interactions. The results show that cold denaturation breaks the AGH-hydrophobic interface of equine apomyoglobin. Furthermore, at least some GH-helical structure appears to be preserved at the expense of the less stable A-helix.

We describe a fast temperature-jump (T-jump) apparatus capable of acquiring kinetic relaxation transients via real-time fluorescence detection over a time interval from nanoseconds to milliseconds in a single sweep. The method is suitable for aqueous solutions, relying upon the direct absorption of laser light by the bulk water. This obviates the need for additives (serving as optical or conductive heaters) that may interact with the sample under investigation. The longitudinal temperature profile is made uniform by counterpropagating heating pulses. Dead time is limited to one period of the probe laser (16 ns). The apparatus response is tested with aqueous tryptophan and the diffusion-controlled dimerization of proflavine.