Thermodynamics of single peptide bond cleavage in bovine pancreatic trypsin inhibitor (BPTI)

Protein Science : a Publication of the Protein Society
Olga BuczekJ Otlewski

Abstract

A major goal of this paper was to estimate a dynamic range of equilibrium constant for the opening of a single peptide bond in a model protein, bovine pancreatic trypsin inhibitor (BPTI). Ten mutants of BPTI containing a single Xaa-->Met substitution introduced in different parts of the molecule were expressed in Escherichia coli. The mutants were folded, purified to homogeneity, and cleaved with cyanogen bromide to respective cleaved forms. Conformation of the intact mutants was similar to the wildtype, as judged from their circular dichroism spectra. Substantial conformational changes were observed on the chemical cleavage of three single peptide bonds--Met46-Ser, Met49-Cys, and Met53-Thr--located within the C-terminal helix. Cleavage of those peptide bonds caused a significant destabilization of the molecule, with a drop of the denaturation temperature by 56.4 degrees C to 68 degrees C at pH 4.3. Opening of the remaining seven peptide bonds was related to a 10.8 degrees C to 39.4 degrees C decrease in T(den). Free energies of the opening of 10 single peptide bonds in native mutants (Delta G(op,N)) were estimated from the thermodynamic cycle that links denaturation and cleavage free energies. To calculate those values, we ass...Continue Reading

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Citations

May 6, 2008·Protein Engineering, Design & Selection : PEDS·Wenyan WangCharles A Lesburg
Aug 12, 2015·Proceedings of the National Academy of Sciences of the United States of America·Boyuan WangTom W Muir
Apr 21, 2004·Protein Science : a Publication of the Protein Society·Grzegorz BulajDavid P Goldenberg
Jul 24, 2003·Protein Science : a Publication of the Protein Society·Katarzyna Koscielska-Kasprzak, Jacek Otlewski
Aug 15, 2013·BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology·Anna Jean Wirth, Martin Gruebele
Sep 2, 2014·Journal of Biomedical Materials Research. Part B, Applied Biomaterials·Hao-Yang MiLih-Sheng Turng
Apr 10, 2015·Cellular and Molecular Life Sciences : CMLS·Marcela BuchtovaPavel Krejci
Oct 23, 2004·FEBS Letters·D R LivesayD J Jacobs

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