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Understanding the mechanism of protein folding requires a detailed knowledge of the structural properties of the barriers separating unfolded from native conformations. The S-peptide from ribonuclease S forms its α-helical structure only upon binding to the folded S-protein. We characterized the transition state for this binding-induced folding reaction at high resolution by determining the effect of site-specific backbone thioxylation and side-chain modifications on the kinetics and thermodynamics of the reaction, which allows us to monitor formation of backbone hydrogen bonds and side-chain interactions in the transition state. The experiments reveal that α-helical structure in the S-peptide is absent in the transition state of binding. Recognition between the unfolded S-peptide and the S-protein is mediated by loosely packed hydrophobic side-chain interactions in two well defined regions on the S-peptide. Close packing and helix formation occurs rapidly after binding. Introducing hydrophobic residues at positions outside the recognition region can drastically slow down association.phi-value analysis | protein-protein interaction | encounter complex formation | thioxo peptide bond C onformational changes in proteins play an important role in many biological processes. A detailed understanding of the mechanism of conformational transitions requires knowledge of the structural and dynamic properties of the initial and final states as well as the characterization of the transition barrier separating them. Site-directed mutagenesis proved a powerful tool to determine the properties of transition states for reactions involving proteins. Comparing the effect of amino acid replacements on kinetics and thermodynamics of a reaction identifies the interactions that are important for the rate-limiting step of a process. This method has been successfully applied to characterize transition states for protein folding (1, 2), for proteinprotein interactions (3-5) and for conformational changes in folded proteins (6). Protein folding transition states were shown to have native-like topology in the whole protein or in major parts of the structure (7-9) and it is commonly assumed that this includes the presence of native-like secondary structure (10, 11). Because site-directed mutagenesis can only modify amino acid side chains, it is still unclear at which stage of a folding reaction backbone hydrogen bonds in secondary structure elements are formed. Several approaches have been applied to test for secondary structure in protein folding transition states. Replacing amino acids by glycyl and prolyl residues leads to changes in backbone conformation, which was used to probe α-helix formation (12), but these replacements introduce additional effects due to altered side-chain interactions. Introducing ester bonds into the polypeptide backbone (13) also has multiple effects because it leads to the loss of a backbone hydrogen bonding donor and strongly increases conformational flexibility of the polypeptide backbone. Deuteration of a...

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psi[CS-NH]4-RNase S, a site specific modified version of RNase S obtained by thioxylation (O/S exchange) at the Ala4-Ala5- peptide bond, was used to evaluate the impact of protein backbone photoswitching on bioactivity. psi[CS-NH](4)-RNase S was yielded by recombination of the S-protein and the respective chemically synthesized thioxylated S-peptide derivative. Comparison with RNase S revealed similar thermodynamic stability of the complex and an unperturbed enzymatic activity toward cytidine 2',3'-cyclic monophosphate (cCMP). Reversible photoisomerization with a highly increased cis/trans isomer ratio of the thioxopeptide bond of psi[CS-NH](4)-RNase S in the photostationary state occurred under UV irradiation conditions (254 nm). The slow thermal reisomerization (t(1/2) = 180 s) permitted us to determine the enzymatic activity of cis psi[CS-NH](4)-RNase S by measurement of initial rates of cCMP hydrolysis. Despite thermodynamic stability of cis psi[CS-NH](4)-RNase S, its enzymatic activity is completely abolished but recovers after reisomerization. We conclude that the thioxopeptide bond modified polypeptide backbone represents a versatile probe for site-directed photoswitching of proteins.