Fast events in protein folding initiated by nanosecond laser photolysis.

Abstract

Initiation of protein folding by light can dramaticafly improve the time resolution of kinetic studies. Here we present an example ofan optically triggered folding reaction by using nanosecond photodissociation of the heme-carbon monoxide complex of reduced cytochrome c. The optical trigger is based on the observation that under destabilizing conditions cytochrome c can be unfolded by preferential binding of carbon monoxide to the covalently attached heme group in the unfolded state. Photodissociation of the carbon monoxide thus triggers the folding reaction. We used time-resolved absorption spectroscopy to monitor binding at the heme. Before folding begins we observe transient binding of both nonnative and native ligands from the unfolded polypeptide on a microsecond time scale. Kinetic modeling suggests that the intramolecular binding of methionine-65 and -80 is faster than that of histidine-26 and -33, even though the histidines are doser to the heme. This optical trigger should provide a powerful method for studying chain collapse and secondary structure formation in cytochrome c without any limitations in time resolution.Protein folding generally occurs in two phases, one rapid and one slow. The rapid phase is the collapse of the unfolded polypeptide into a compact structure and the formation of secondary structure. The slow phase is the subsequent, often multistep rearrangement to the native conformation (1-7). Because of the limited time resolution (milliseconds) of conventional stopped-flow mixing experiments, the complete time course of protein folding has not yet been observed. A dramatic improvement in time resolution would result if protein folding could be initiated by light.Here we present an example of an optically triggered folding reaction. We take advantage of the fact that under destabilizing conditions cytochrome c can be unfolded by preferential binding of carbon monoxide (CO) to the covalently attached heme group in the unfolded state. The folding reaction can thus be triggered by photodissociating the CO complex. In this study, we used time-resolved absorption spectroscopy with nanosecond lasers to monitor binding events at the heme (8-11). Although rebinding of CO prevents the complete formation of the native conformation, the rapid recovery of the sample permits repetitive photolysis and therefore the acquisition of high signal/noise transient spectra for investigating submillisecond events. Simulations of the multiwavelength data with kinetic models were carried out to generate the spectra of intermediates, as well as the rate constants connecting them. We find that before folding begins there is transient binding of both nonnative and native ligands from the unfolded polypeptide chain on a microsecond time scale. MATERIALS AND METHODSTye VI horse heart cytochrome c from Sigma was purified by ion-exchange chromatography using carboxymethyl-cellulose (Whatman) (12), reduced with sodium dithionite anaerobically, and separated anaerobically from the sodium dithionite by gel filtrat...

Multi-basin dynamics of a protein in a crystal environment

“…On longer timescales (milliseconds to seconds) many proteins seem to follow an energy-like funnel to the folded state [9,10]. Fast folding (submillisecond regime) proteins have also been observed [11][12][13][14]. Studies of the protein folding kinetics and stability analyses based on 3D lattice and off-lattice models of polymers with binary interactions (i.e., polar and hydrophobic) that mimic proteins [15][16][17][18][19][20][21] also agree with this picture.…”

Molecular dynamics simulations of hydrophobic collapse of ubiquitin

“…Furthermore, simple collapse to any one of many collapsed states is an easier search problem than a protein folding to one native and precisely-packed structure. Also, as shorter time scales become experimentally accessible through fast spectroscopic techniques (Jones et al, 1993;Ballew et al, 1996;Williams et al, 1996;and reviewed by Plaxco & Dobson, 1996;Wolynes et al, 1996;Eaton et al, 1997), it is becoming clear that both secondary and tertiary structure can form on the nanosecond-microsecond time scale even at room temperature, or lower. Thirdly, ubiquitin is small and contains a welldefined hydrophobic core.…”

Minimal Models for Proteins and RNA: From Folding to Function

“…(5)). When t ≈ 10 −1 τ f the non-native single disulfide intermediates rearrange to form the more stable native [9][10][11][12][13][14][15][16][17][18][19][20][21][22] and [2][3][4][5][6][7][8][9][10][11][12][13][14][15] form early in the folding process when the ordering is determined by entropic considerations. The current experiments on BPTI are far too slow to detect these intermediates.…”

“…[5][6][7][8][9] In the last two decades, considerable progress has been made in attaining a global understanding of the mechanisms by which proteins fold thanks to breakthroughs in experiments, 10-12 theory, [13][14][15] and computations. [16][17][18][19] Fast folding experiments 4,11,[20][21][22] and single molecule methods [23][24][25] have begun to provide a direct glimpse into the initial stages of protein folding. These experiments show that there is a great diversity in the routes explored during the transitions from unfolded states to the folded state that were unanticipated in ensemble experiments.…”