title: Proteolytic excision and in situ cyclization of a bioactive loop from an REI-VL presentation scaffold.
abstract: Covalent cyclization of peptides is an important tool in structure-function analysis of bioactive peptides, because it constrains the molecule to enrich or exclude the receptor-bound conformation. Previously we described a 2-step procedure for cyclizing purified, native peptides in aqueous solution by reacting a Met or Lys side chain with an iodoacetylated N-terminus (Wood SJ, Wetzel R, 1992a, Int J Pept Protein Res 39:533-539). We show here that the cyclization reaction scheme can be extended to peptides excised from proteins by endo-LysC proteolysis, which generates fragments terminating with Lys. To illustrate the method, we used an immunoglobulin VL domain (REI-VL) with an RGD-containing sequence engineered into its CDR3 and flanked by Lys residues. This REI-VL/RGD hybrid displayed an IC50 of 24 nM for ligand competition at the platelet fibrinogen receptor alpha IIb beta 3. The RGD-containing peptide excised by endo-LysC from the REI-VL presentation scaffold exhibited an IC50 of about 50 nM, and the corresponding cyclized peptide, and IC50 of about 10 nM. Significantly, both the N alpha-acylation and the cyclization reactions occur efficiently even in the context of the other endo-LysC fragments of REI-VL, which suggests that the reaction may prove useful in converting mixtures of endo-LysC products of many proteins into the corresponding cyclic peptides in situ.
authors.affiliation: Macromolecular Sciences Department, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406-0939.
authors.forename: L R
authors.initials: LR
authors.lastname: Helms
authors.forename: R
authors.initials: R
authors.lastname: Wetzel
mesh_terms: D000107:Acetylation; D000595:Amino Acid Sequence; D001536:Bence Jones Protein / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D003500:Cyclization; D005340:Fibrinogen / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000737:chemistry; D007461:Iodoacetates; D019807:Iodoacetic Acid; D008666:Metalloendopeptidases / Q000378:metabolism; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009842:Oligopeptides / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism
chemical_list: D007135:Immunoglobulin Variable Region; D007461:Iodoacetates; D009842:Oligopeptides; D010446:Peptide Fragments; D010980:Platelet Membrane Glycoproteins; C047981:arginyl-glycyl-aspartic acid; D005340:Fibrinogen; D001536:Bence Jones Protein; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase; D019807:Iodoacetic Acid