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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...

Oligopeptides derived from the gag polyprotein (Pr55 gag) of human immunodeficiency virus type 1 (HIV-1) segment were used to evaluate the extension of the putative binding region for the complex of Pr55 gag and the human cytosolic peptidyl prolyl cis/trans isomerase (PPIase) 18 kDa cyclophilin (Cypl8). Five N-terminally acetylated, C-terminally amidated oligopeptides containing one (HIV-1 Gag218-224; 1), two (HIV-1 Gag 21s-226 and HIV-1 Gag217-224; 2 and 3, respectively), three (HIV-I Gag217-226; 4) or four (HIV-1 Gag213-237; 5) proline residues were synthesized. Using competition experiments with a standard substrate the binding affinities to Cypl8 of the synthesized peptides were determined. The ICs0 value of 184 ~M for the 25-mer peptide 5 was fivefold or more lower than those of the peptides 1--4 lacking one or more prolines. Failure of competition in assays containing enzymes of other PPlase families by millimolar concentrations of 5 revealed a Cyp18 specific interaction involving the active site of the enzyme. In its far UV circular dichroism, aqueous solutions of 5 display properties of random coil conformation, but spectra were also consistent with a small contribution of proline specific secondary structures. However, a proline-rich peptide typical of forming left-handed polyproline II helices did not compete for the active site of Cypl8. The results demonstrate that the putative binding region of HIV-1 gag polyprotein has a certain degree of binding affinity to the PPIase site of Cypl8, and may add a previously unrecognized topological component to the known subsite specificity of cyciophilins.