The enzyme peptidyl-prolyl cis-trans isomerase (PPIase) was recently discovered in mammalian tissues and purified from porcine kidney. It catalyses the slow cis-trans isomerization of proline peptide (Xaa-Pro) bonds in oligopeptides and accelerates slow, rate-limiting steps in the folding of several proteins. Here, we report the N-terminal sequence of PPIase together with further chemical and enzymatic properties. The results indicate that this enzyme is probably identical to cyclophilin, a recently discovered mammalian protein which binds tightly to cyclosporin A (CsA). Cyclophilin is thought to be linked to the immunosuppressive action of CsA. The first 38 amino-acid residues of porcine PPIase and of bovine cyclophilin are identical and the two proteins both have a relative molecular mass of about 17,000 (ref. 7). The catalysis of prolyl isomerization in oligopeptides and of protein folding by PPIase are strongly inhibited in the presence of low levels of CsA. The activities of both PPIase and cyclophilin depend on a single sulphydryl group. At present it is unknown whether the inhibition of prolyl isomerase activity is related with the immunosuppressive action of CsA.

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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...

Characterization of the unfolded state is essential for the understanding of the protein folding reaction. We performed time-resolved FRET measurements to gain information on the dimensions and the internal dynamics of unfolded polypeptide chains. Using an approach based on global analysis of data obtained from two different donor–acceptor pairs allowed for the determination of distance distribution functions and diffusion constants between the chromophores. Results on a polypeptide chain consisting of 16 Gly-Ser repeats between the FRET chromophores reveal an increase in the average end-to-end distance from 18.9 to 39.2 Å between 0 and 8 M GdmCl. The increase in chain dimensions is accompanied by an increase in the end-to-end diffusion constant from (3.6 ± 1.0) × 10 −7 cm 2 s −1 in water to (14.8 ± 2.5) × 10 −7 cm 2 s −1 in 8 M GdmCl. This finding suggests that intrachain interactions in water exist even in very flexible chains lacking hydrophobic groups, which indicates intramolecular hydrogen bond formation. The interactions are broken upon denaturant binding, which leads to increased chain flexibility and longer average end-to-end distances. This finding implies that rapid collapse of polypeptide chains during refolding of denaturant-unfolded proteins is an intrinsic property of polypeptide chains and can, at least in part, be ascribed to nonspecific intramolecular hydrogen bonding. Despite decreased intrachain diffusion constants, the conformational search is accelerated in the collapsed state because of shorter diffusion distances. The measured distance distribution functions and diffusion constants in combination with Szabo–Schulten–Schulten theory were able to reproduce experimentally determined rate constants for end-to-end loop formation.