title: Protein minimization: characterization of the synthetic cyclic dodecapeptide corresponding to the reactive site region of the oil rape trypsin inhibitor type-III.
abstract: The design of minimal units required for enzyme inhibition is a major field of interest in structural biology and biotechnology. The successful design of the cyclic dodecapeptide corresponding to the Phe17-Val28 reactive site amino acid sequence of the low-molecular-mass trypsin inhibitor RTI-III from Brassica napus (micro-RTI-III) and of the recombinant murine dihydrofolate reductase-(DHFR-)micro-RTI-III fusion protein (DHFR-micro-RTI-III) is reported here. Micro-RTI-III was synthesized using a stepwise solid-phase approach based on the standard Fmoc chemistry, purified by RP-HPLC, and oxidatively refolded. DHFR-micro-RTI-III was expressed in Escherichia coli, purified by metal-chelate affinity chromatography, and oxidatively refolded. The affinity of micro-RTI-III for bovine trypsin (K(d)=1.6x10(-9)M) is similar to that determined for DHFR-micro-RTI-III (K(d)=6.3x10(-10)M) and native RTI-III (K(d)=2.9x10(-10)M), at pH 8.2 and 22.0 degrees C. Remarkably, micro-RTI-III protects the DHFR domain of DHFR-micro-RTI-III from trypsin digestion. Micro-RTI-III is a new minimal trypsin inhibitor and may be regarded as a tool in protein structure-function studies and for developing multifunctional and multidomain proteinase inhibitors.
authors.affiliation: Dipartimento di Genetica e Biologia Molecolare Charles Darwin, Università di Roma La Sapienza, Italy.
authors.forename: Maurizio
authors.initials: M
authors.lastname: Trovato
authors.forename: Elena Caroli
authors.initials: EC
authors.lastname: Casavola
authors.forename: Bruno
authors.initials: B
authors.lastname: Maras
authors.forename: Maria Eugenia
authors.initials: ME
authors.lastname: Schininà
authors.forename: Paolo
authors.initials: P
authors.lastname: Costantino
authors.forename: Paolo
authors.initials: P
authors.lastname: Ascenzi
mesh_terms: D001665:Binding Sites; D029688:Brassica napus / Q000737:chemistry*; D015195:Drug Design; D010456:Peptides, Cyclic / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D010940:Plant Proteins / Q000737:chemistry / Q000494:pharmacology*; D011480:Protease Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D014361:Trypsin Inhibitors / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D010456:Peptides, Cyclic; D010940:Plant Proteins; D011480:Protease Inhibitors; D014361:Trypsin Inhibitors; C499355:trypsin inhibitor type II-S protein, Glycine max; C069599:trypsin inhibitor protein, Brassica napus