Wuyuan Lu

A novel method to synthesize cyclic peptides

Abstract Successful synthesis of cyclic peptides resembling the binding loop of Eglin c, a serine protease inhibitor, provides extended native chemical ligation as a novel method to synthesize cyclic peptides.

Total chemical synthesis of bovine pancreatic trypsin inhibitor by native chemical ligation

Bovine pancreatic trypsin inhibitor (BPTI) is an important model for the study of protein folding. Herein we describe a robust approach to the total chemical synthesis of BPTI using native chemical ligation of unprotected peptide segments in aqueous solution. After refolding and oxidative formation of disulfides, the target protein was purified by affinity chromatography. The synthetic BPTI was characterized by mass spectrometry, inhibition assay, thermal denaturation and 2D NMR spectroscopy, and was shown to be structurally and functionally identical to natural BPTI. The synthetic strategy presented in this paper has enabled us to establish rapid access to novel analogues of BPTI.

Functional evolution within a protein superfamily

The ability to predict and characterize distributions of reactivities over families and even superfamilies of proteins opens the door to an array of analyses regarding functional evolution. In this article, insights into functional evolution in the Kazal inhibitor superfamily are gained by analyzing and comparing predicted association free energy distributions against six serine proteinases, over a number of groups of inhibitors: all possible Kazal inhibitors, natural avian ovomucoid first and third domains, and sets of Kazal inhibitors with statistically weighted combinations of residues. The results indicate that, despite the great hypervariability of residues in the 10 proteinase‐binding positions, avian ovomucoid third domains evolved to inhibit enzymes similar to the six enzymes selected, whereas the orthologous first domains are not inhibitors of these enzymes on purpose. Hypervariability arises because of similarity in energetic contribution from multiple residue types; conservation is in terms of functionality, with “good” residues, which make positive or less deleterious contributions to the binding, selected more frequently, and yielding overall the same distributional characteristics. Further analysis of the distributions indicates that while nature did optimize inhibitor strength, the objective may not have been the strongest possible inhibitor against one enzyme but rather an inhibitor that is relatively strong against a number of enzymes. Proteins 2006.

Additivity-based design of the strongest possible turkey ovomucoid third domain inhibitors for porcine pancreatic elastase (PPE) and Streptomyces griseus protease B (SGPB)

We describe here successful designs of strong inhibitors for porcine pancreatic elastase (PPE) and Streptomyces griseus protease B (SGPB). For each enzyme two inhibitor variants were designed. In one, the reactive site residue (position 18) was retained and the best residues were substituted at contact positions 13, 14, and 15. In the other variant the best residues were substituted at all contact positions except the reactive site where a Gly was substituted. The four designed variants were: for PPE, T13E14Y15‐OMTKY3 and T13E14Y15G18M21P32V36‐OMTKY3, and for SGPB, S13D14Y15‐OMTKY3 and S13D14Y15G18I19K21‐OMTKY3. The free energies of association (ΔG 0) of expressed variants have been measured with the proteases for which they were designed as well as with five other serine proteases and the results are discussed.