The speed limit for protein folding measured by triplet–triplet energy transfer

Abstract

A direct measure of intramolecular chain diffusion is obtained by the determination of triplet-triplet energytransfer rates between a donor and an acceptor chromophore attached at defined points on a polypeptide chain. Single exponential kinetics of contact formation are observed on the nanosecond time scale for polypeptides in which donor and acceptor are linked by repeating units of glycine and serine residues. The rates depend on the number of peptide bonds (N) separating donor and acceptor and show a maximum for the shortest peptides (N ‫؍‬ 3) with a time constant ( ‫؍‬ 1/k) of 20 ns. This sets an upper limit for the speed of formation of the first side-chain contacts during protein folding.Protein folding starts from an ensemble of random coil conformations to finally reach the native state with well defined side-chain contacts. For many proteins, rapid chain collapse precedes formation of the native structure (1-3). In a number of small proteins, in contrast, collapse and formation of the native interactions occur simultaneously (4-8). In both scenarios, the search for energetically favorable conformations requires formation of interactions between parts of the polypeptide chain, which is limited by chain dynamics. Intrachain diffusion can thus be regarded as the elementary process that determines the maximum rate at which a protein can fold. Models for the rate-limiting steps and for the distance dependence of intrachain diffusion have been proposed in a number of theoretical studies (9-13), but to date no direct experimental data are available for the rates of contact formation between two defined points on a polypeptide chain.Triplet-triplet energy transfer provides an excellent tool to measure such rates of contact formation. Energy transfer between an electronically excited triplet donor (sensitizer) and an acceptor proceeds by an electron-exchange mechanism (Dexter mechanism), which requires van der Waals contact between the donor and acceptor. Rates of intermolecular exothermic triplet energy transfer approach, but do not exceed, the diffusion-controlled limit (14). The process is readily monitored by triplet-triplet absorption by using laser-flash photolysis. The fast formation and the long lifetimes of many triplet states allow measurements of processes in the range from nanoseconds to microseconds. MATERIALS AND METHODSThioxanthone (E T ϭ 265 kJ⅐mol Ϫ1) was used as a triplet donor, which can be excited selectively with an excimer laser pulse at 351 nm in the presence of naphthalene (E T ϭ 253 kJ⅐mol Ϫ1) as the acceptor (15). The amount of thioxanthone triplets was monitored by their strong triplet-triplet absorbance at max ϭ 620 nm. Fig. 1a shows the transient absorbance of triplet thioxanthone after excitation by a 351-nm laser flash of 20-ns duration. In the absence of acceptor, the thioxanthone triplets decay with a half-life of 30 s. The decay rate of the thioxanthone triplets increases on addition of naphthalene, because of the formation of triplet naphthalene (Fig. 1b). The secondor...

Structure and Dynamics of End-to-End Loop Formation of the Penta-Peptide Cys-Ala-Gly-Gln-Trp in Implicit Solvents

“…The end-to-end contact formation of amino acid residues is one of the elementary processes in protein folding and has been extensively studied both experimentally [22][23][24][25][26] and theoretically. 25,[27][28][29][30][31][32][33][34][35][36] The end-to-end contact formation dynamics of the penta-peptide Cys-Ala-Gly-Gln-Trp (CAGQW) has been experimentally studied with triplet quenching.…”

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.…”

Mechanical Properties of Bovine Rhodopsin and Bacteriorhodopsin: Possible Roles in Folding and Function

“…23 For protein dynamics, τ A has a value of 10 −9 s. 48 The ΔG u ‡ values for all structural segments were estimated using this value. The errors in ΔG u ‡ were estimated by propagating the errors of k u .…”