Proline is unique among the natural amino acids in the similar propensity of its peptide bond to be in the cis or trans conformation. This attribute affects many processes, including the rate at which proteins fold, their structures, and their activities. Other aliphatic amino acids can serve as mimics for proline residues with trans peptide bonds. In contrast, chemical synthesis is needed to create surrogates for cis prolyl peptide bonds. Here, 1,5-disubstituted-[1,2,3]-triazoles were assessed as cispeptide bond surrogates. Huisgen's 1,3-dipolar cycloaddition reaction of amino alkynes and azido acids and a Ru(II) catalyst were used to synthesize a variety of Xaa-1,5-triazole-Ala modules in moderate-to-high yields. Two of these modules, along with their 1,4-triazole regioisomers, were installed in a turn region of bovine pancreatic ribonuclease by using expressed protein ligation. The resulting semisynthetic enzymes displayed full enzymatic activity, indicating the maintenance of native structure. The 1,5-triazole surrogates instilled conformational stability that was comparable to that of Xaa-cis-Pro segments, whereas the 1,4-triazoles conferred markedly less stability. The stability conferred by both surrogates was independent of the Xaa residue, eliminating an uncertainty in protein design. We conclude that Xaa-1,5-triazole-Ala modules can serve as viable mimics of Xaa-cis-Pro segments. The possibility of synthesizing this surrogate by the ligation of fragments in situ and the emergence of biocompatible catalysts for that process portends its widespread use.Proline is unique among the natural amino acids in the similar propensity of its peptide bond to be in the cis or trans conformation. 1 This attribute affects many processes, including the rate at which proteins fold, 2 their structures, 3 and their activities. 4 Other aliphatic amino acids can serve as mimics for proline residues with trans peptide bonds. In contrast, chemical synthesis is needed to create surrogates for cis prolyl peptide bonds. 1,5-7

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The thermal stabilities of ribonuclease A (RNase A) and ribonuclease B (RNase B), which possess identical protein structures but differ by the presence of a carbohydrate chain attached to Asn34 in RNase B, were studied by proteolysis and UV spectroscopy at pH 8.0. Proteolysis was quantified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and densitometry. Increasing protease concentrations led to a hyperbolic increase of the rate constants of proteolysis. With thermolysin, which attacks the unfolded molecules only, the thermal unfolding constants were determined by extrapolating the rate constants of proteolysis to infinite concentration of protease. With trypsin, the unfolding constants of RNase A could be confirmed. Subtilisin attacked even the native RNases, where RNase B was more stable toward proteolytic degradation. Kinetic stabilities (deltaG++) calculated from the unfolding constants for temperatures between 52.5 and 65 degrees C revealed a higher kinetic stability of RNase B, which results from enthalpic effects only, whereas entropic effects counteract stabilization. delta deltaG++ at the transition temperature of RNase A (60.4 degrees C) was 2.2 +/- 0.3 kJ mol(-1). Thermodynamic stabilities (deltaG) were estimated from the thermal transition curves at 287 nm for the temperature range from 55 to 70 degrees C. For 17.5-25 degrees C, deltaG values were determined from transition curves of unfolding induced by guanidine hydrochloride and extrapolation of the free energy values to those in the absence of denaturant. At all temperatures, RNase B proved to be more stable than RNase A with essentially the same enthalpy and entropy of unfolding. delta deltaG was 2.5 +/- 0.2 kJ mol(-1) at 60.4 degrees C and 2.3 kJ mol(-1) at 25 degrees C.