The formation of vibrationally excited heme upon photodissociation of carbonmonoxy myoglobin and its subsequent vibrational energy relaxation was monitored by picosecond anti-Stokes resonance Raman spectroscopy. The anti-Stokes intensity of the nu4 band showed immediate generation of vibrationally excited hemes and biphasic decay of the excited populations. The best fit to double exponentials gave time constants of 1.9 +/- 0.6 and 16 +/- 9 picoseconds for vibrational population decay and 3.0 +/- 1.0 and 25 +/- 14 picoseconds for temperature relaxation of the photolyzed heme when a Boltzmann distribution was assumed. The decay of the nu4 anti-Stokes intensity was accompanied by narrowing and frequency upshift of the Stokes counterpart. This direct monitoring of the cooling dynamics of the heme cofactor within the globin matrix allows the characterization of the vibrational energy flow through the protein moiety and to the water bath.

A temperature jump (T-jump) method capable of initiating thermally induced processes on the picosecond time scale in aqueous solutions is introduced. Protein solutions are heated by energy from a laser pulse that is absorbed by homogeneously dispersed molecules of the dye crystal violet. These act as transducers by releasing the energy as heat to cause a T-jump of up to 10 K with a time resolution of 70 ps. The method was applied to the unfolding of RNase A. At pH 5.7 and 59°C, a T-jump of 3-6 K induced unfolding which was detected by picosecond transient infrared spectroscopy of the amide I region between 1600 and 1700 cm-'.The difference spectral profile at 3.5 ns closely resembled that found for the equilibrium (native -unfolded) states. The signal at 1633 cm-1, corresponding to the fl-sheet structure, achieved 15 ± 2% of the decrease found at equilibrium, within 5.5 ns. However, no decrease in absorbance was detected until 1 ns after the T-jump. The disruption of fl-sheet therefore appears to be subject to a delay of -1 ns. Prior to 1 ns after the T-jump, water might be accessing the intact hydrophobic regions.Questions concerning the physical and chemical nature of protein folding are among the most challenging in biological research (1-4). Folding and unfolding events have seldom been studied on time scales shorter than milliseconds. Internal motions of macromolecules such as rotations about single bonds, chemical exchange reactions, diffusion over molecular dimensions, and barrier crossing processes can occur on nanosecond or even picosecond time scales, so protein structure reorganization might be expected to involve ultrafast intermediate steps. An example is the recent report of tens-ofmicroseconds folding in cytochrome c (5). Some of the faster processes in protein folding might involve relatively small alterations in electronic structure. Therefore the probes used to examine them must be sensitive to subtle changes in, for example, nonbonded interactions, weaker chemical bonds, charge distributions, and motions of pieces of the structure. For this reason we decided to use transient infrared (IR) spectroscopy (6-8), which is structure sensitive at a chemicalbond resolution, to identify any ultrafast folding steps.The IR spectra of proteins in the region of the amide vibrations of the polypeptide structures are well known to be sensitive to the state of the protein. For example, there are distinct differences between the IR spectra of random coil, a-helical, }3-sheet, 13'-sheet, and turn structures of polypeptides (9). These differences arise from the dependence of interactions between the various amide groups on the local polypeptide structures. One can therefore conceive of carrying out time-resolved IR capable of following the kinetics of structure change as it affects these different spatial regions of the polypeptide backbone. Experiments on the kinetics of folding also require that the system be triggered to suddenly change. For this purpose we have developed an ultrafast temperature jump...

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