title: Internally quenched fluorescent peptide libraries with randomized sequences designed to detect endopeptidases.
abstract: Identification of synthetic peptide substrates for novel peptidases is an essential step for their study. With this purpose we synthesized fluorescence resonance energy transfer (FRET) peptide libraries Abz (or MCA)-GXXXXXQ-EDDnp and Abz (or MCA)-GXXZXXQ-EDDnp, where X consists of an equimolar mixture of all amino acids, the Z position is fixed with one of the proteinogenic amino acids (cysteine was excluded), Abz (ortho-aminobenzoic acid) or MCA ([7-amino-4-methyl]coumarin) is the fluorescence donor and Q-EDDnp (glutamine-[N-(2,4-dinitrophenyl)-ethylenediamine]) is the fluorescence acceptor. The peptide libraries MCA-GXXX↓XXQ-EDDnp and MCA-GXXZ↓XXQ-EDDnp were cleaved as indicated (↓) by trypsin, chymotrypsin, cathepsin L, pepsin A, and Eqolisin as confirmed by Edman degradation of the products derived from the digestion of these libraries. The best hydrolyzed Abz-GXXZXXQ-EDDnp sublibraries by these proteases, including Dengue 2 virus NS2B-NS3 protease, contained amino acids at the Z position that are reported to be well accepted by their S(1) subsite. The pH profiles of the hydrolytic activities of these canonical proteases on the libraries were similar to those reported for typical substrates. The FRET peptide libraries provide an efficient and simple approach for detecting nanomolar concentrations of endopeptidases and are useful for initial specificity characterization as performed for two proteases secreted by a Bacillus subtilis.
authors: Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Três de Maio 100, São Paulo 04044-020, Brazil.
authors: Lilian C G
authors: LC
authors: Oliveira
authors: Vinícius O
authors: VO
authors: Silva
authors: Debora N
authors: DN
authors: Okamoto
authors: Marcia Y
authors: MY
authors: Kondo
authors: Saara M B
authors: SM
authors: Santos
authors: Isaura Y
authors: IY
authors: Hirata
authors: Marcelo A
authors: MA
authors: Vallim
authors: Renata C
authors: RC
authors: Pascon
authors: Iuri E
authors: IE
authors: Gouvea
authors: Maria A
authors: MA
authors: Juliano
authors: Luiz
authors: L
authors: Juliano
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D010450:Endopeptidases / Q000032:analysis*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics; D019151:Peptide Library
chemical_list: D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D019151:Peptide Library; D010450:Endopeptidases