Ultrafast thermally induced unfolding of RNase A.

Abstract

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...

Making connections between ultrafast protein folding kinetics and molecular dynamics simulations

“…is p F Ã ðtÞ þ p U Ã ðtÞ p F Ã ð0Þ þ p U Ã ð0Þ ¼ AðtÞ∕Að0Þ ¼ a expð−λ þ tÞ þ ð1 − aÞ expð−λ − tÞ; [3] where…”

“…Polynomial extrapolation of all the data to zero denaturant yields a folding time of 220 ðþ100, − 70Þ ns at 283 K, suggesting that under these conditions the barrier between folded and unfolded states has effectively disappeared-the so-called "downhill scenario." tryptophan triplet lifetime | villin headpiece subdomain | downhill protein folding | laser temperature jump T he introduction of a variety of experimental methods to study protein folding with much improved time resolution has resulted in the discovery of proteins that fold on a microsecond timescale (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). The investigation of these so-called ultrafast folding proteins is important for several reasons.…”

Triggered infrared spectroscopy for investigating metalloprotein chemistry

“…In addition to studies of CO photolysis from proteins, lighttriggered time-resolved IR methods have been applied to a range of small-molecule systems including transition metal carbonyl complexes (Butler et al 2007) and photodamaged DNA bases (Kuimova et al 2006). A related temperature-jump method makes use of a laser-induced temperature jump in the sample, followed at variable time intervals by an IR probe pulse, to obtain time-resolved IR data on temperature-triggered spectral changes, such as protein unfolding studied by Phillips et al (1995). Another related suite of methods is building up around the concept of two-dimensional IR in which both the pump and probe pulses are in the IR region, and coupling between vibrational modes in a protein can be deduced from cross-peaks in the two-dimensional plot, analogous to two-dimensional nuclear magnetic resonance methods.…”

“…Similar IR methods were used recently alongside molecular dynamics simulations to address ligand binding and movement in another haem protein, neuroglobin (Lutz et al 2009). Temperature triggers have also been used elegantly in time-resolved laser temperature jump studies to study protein folding or unfolding: in a study of RNase unfolding, Phillips et al (1995) achieved an increase of about 10 K in around 100 ps. This approach should also be able to provide time-resolved information on thermally induced ligand release, rebinding or movement within metalloproteins.…”

Conformational changes during the nanosecond-to-millisecond unfolding of ubiquitin

“…IR spectroscopy on amide I transitions (primarily CO stretching; 1,600-1,700 cm Ϫ1 ) has been widely used for folding studies of proteins (7,(14)(15)(16)(17) and peptides (9,(18)(19)(20), primarily because amide I spectra have peak positions that depend on secondary structure. The frequency-structure correlation is a result of through-space electrostatic couplings that depend on the distance and orientation between the many amide I vibrations of each of the protein's peptide units (21,22).…”