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R Arze

Publications and source records attributed to R Arze.

4 recordsLinked to original sources
PubMed1995

Topochemical catalysis achieved by structure-based ligand design.

Recently, a cyclic peptide ligand, cyclo-Ac-[CHPQG-PPC]-NH2, that binds to streptavidin with high affinity was discovered by screening phage libraries. From the streptavidin-bound crystal structures of cyclo-Ac-[CHPQGPPC]-NH2 and of a related but more weakly binding linear ligand, FSHPQNT, we designed linear thiol-containing streptavidin binding ligands, FCH-PQNT-NH2 and Ac-CHPQNT-NH2, which are dimerized catalytically by the streptavidin crystal lattice of space group I222, as demonstrated by high performance liquid chromatography and mass spectrometry. The catalytic dimerization relies on presentation of the ligand thiols toward one another in the lattice. The streptavidin crystal lattice-mediated catalysis achieved by structure-based design is the first example of catalysis of a chemical reaction by a protein crystal lattice. The spontaneous and crystal catalyzed rates of disulfide formation were determined by high performance liquid chromatography at pH 3.1, 4.0, 5.0, and 6.0. The ratio of the catalyzed to uncatalyzed rate was maximal at pH 3.1 (kcat/kuncat = 3.8), diminishing to 1.2 at pH 6.0. The crystal structures of the streptavidin-bound dimerized peptide ligands, FCHPQNT-NH2 dimer at 1.95 A and Ac-CHPQNT-NH2 dimer at 1.80 A, are described and compared with the structures of streptavidin-bound FSHPQNT monomer and cyclo-Ac-[CHPQGPPC]-NH2 dimer.

Amino Acid Sequence↗
PubMed1995

Screening of cyclic peptide phage libraries identifies ligands that bind streptavidin with high affinities.

The screening of combinatorial peptide libraries has emerged as an important tool in the discovery of novel substrates or ligands for enzyme and receptor targets. For example, screening linear peptide libraries using streptavidin as a model receptor system has previously identified many low-affinity peptide ligands, all of which contain the common motif His-Pro-Gln (HPQ). We reasoned that constraining the conformational freedom of linear peptides by cyclization in a library would yield peptide ligands of increased affinity. Three different cyclic peptide libraries were constructed in an M13 phage display system as N-terminal pIII protein fusions. The random peptide sequences were flanked by two cysteine residues, which allows efficient disulfide bond formation and cyclization during phage assembly. These cyclic peptide libraries were screened with streptavidin as the model receptor system. Many sequences, all of which contained the motif His-Pro-Gln (HPQ), were discovered, and in the preceding paper, the structures of complexes of streptavidin-bound cyclic and linear peptides are described (Katz, 1995). Analysis of binding kinetics and affinities demonstrated that the conformationally constrained cyclic peptides bound streptavidin with affinities up to 3 orders of magnitude higher than linear peptides identified in previous library screens. These results demonstrate the potential of screening conformationally constrained peptide libraries for high-affinity novel receptor ligands or enzyme substrates.

Amino Acid Sequence↗
PubMed1995

Topochemistry for preparing ligands that dimerize receptors.

BACKGROUND: The cyclic, disulfide-containing peptide, cyclo-Ac-[Cys-His-Pro-Gln-Gly-Pro-Pro-Cys]-NH2, binds to streptavidin with high affinity. In streptavidin-peptide cocrystals of space group I222, cyclic peptide monomers are bound on adjacent streptavidin tetramers related by a crystallographic two-fold symmetry axis. We set out to determine whether the disulfide bonds of the peptide, presented close to one another in the crystal, could undergo disulfide interchange to form a dimer. RESULTS: When juxtaposed, the disulfides of neighboring peptides undergo disulfide interchange, breaking and forming covalent disulfide bonds, to produce a peptide dimer adopting the symmetry of the crystal. This is the first example of a chemical transformation mediated by a protein crystal lattice. The structure of the streptavidin-bound monomer, and that of the dimer that was eventually produced from it in the crystal, were both determined from the same single crystal studied at different times. The two-fold symmetric peptide dimer was independently synthesized and shown to form crystals of dimerized streptavidin. CONCLUSIONS: We have shown that formation of a covalently linked peptide dimer can be mediated by a protein crystal lattice. The dimer thus produced dimerizes its target, streptavidin, suggesting that solid-state (or topochemical) reactions of this kind may be broadly useful for the preparation of ligands that can dimerize other protein targets.

Chromatography, High Pressure Liquid↗