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


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 assumed that the free energy of opening of a single peptide bond in the denatured state (Delta G(op,D)) was equal to -2.7 kcal/mole, as reported previously. Calculated Delta G(op,N) values in BPTI were in the range from 0.2 to 10 kcal/mole, which was equivalent to a >1 million-fold difference in equilibrium constants. The values of Delta G(op,N) were the largest for peptide bonds located in the C-terminal helix and significantly lower for peptide bonds in the beta-structure or loop regions. It appears that opening constants for single peptide bonds in various proteins span across 33 orders of magnitude. Typical equilibrium values for a single peptide bond opening in a protein containing secondary structure elements fall into negligibly low values, from 10(-3) to 10(-8), and are efficient to ensure stability against proteolysis. Study holds ProTherm entries: 13114, 13115, 13116, 13117, 13118, 13119, 13120, 13121, 13122, 13123, 13124, 13125, 13126, 13127, 13128, 13129, 13130, 13131, 13132, 13133 Extra Details: bovine pancreatic trypsin inhibitor; calorimetry; denaturation; hydrolysis; peptide bond; thermodynamic stability

Submission Details

ID: SGrEm4k93

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:44 p.m.

Version: 1

Publication Details
Buczek O;Krowarsch D;Otlewski J,Protein Sci. (2002) Thermodynamics of single peptide bond cleavage in bovine pancreatic trypsin inhibitor (BPTI). PMID:11910035
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1AAL 1992-04-09T00:00:00+0000 1.6 STRUCTURAL EFFECTS INDUCED BY MUTAGENESIS AFFECTED BY CRYSTAL PACKING FACTORS: THE STRUCTURE OF A 30-51 DISULFIDE MUTANT OF BASIC PANCREATIC TRYPSIN INHIBITOR
1B0C 1998-11-06T00:00:00+0000 2.8 EVIDENCE OF A COMMON DECAMER IN THREE CRYSTAL STRUCTURES OF BPTI, CRYSTALLIZED FROM THIOCYANATE, CHLORIDE OR SULFATE
1BHC 1998-06-05T00:00:00+0000 2.7 BOVINE PANCREATIC TRYPSIN INHIBITOR CRYSTALLIZED FROM THIOCYANATE
1BPI 1995-02-18T00:00:00+0000 1.09 THE STRUCTURE OF BOVINE PANCREATIC TRYPSIN INHIBITOR AT 125K: DEFINITION OF CARBOXYL-TERMINAL RESIDUES GLYCINE-57 AND ALANINE-58
1BPT 1991-12-11T00:00:00+0000 2.0 CREVICE-FORMING MUTANTS OF BPTI: CRYSTAL STRUCTURES OF F22A, Y23A, N43G, AND F45A
1BRB 1992-12-17T00:00:00+0000 2.1 CRYSTAL STRUCTURES OF RAT ANIONIC TRYPSIN COMPLEXED WITH THE PROTEIN INHIBITORS APPI AND BPTI
1BTH 1996-12-03T00:00:00+0000 2.3 STRUCTURE OF THROMBIN COMPLEXED WITH BOVINE PANCREATIC TRYPSIN INHIBITOR
1BTI 1991-07-11T00:00:00+0000 2.2 CREVICE-FORMING MUTANTS IN THE RIGID CORE OF BOVINE PANCREATIC TRYPSIN INHIBITOR: CRYSTAL STRUCTURES OF F22A, Y23A, N43G, AND F45A
1BZ5 1998-11-05T00:00:00+0000 2.58 EVIDENCE OF A COMMON DECAMER IN THREE CRYSTAL STRUCTURES OF BPTI, CRYSTALLIZE FROM THIOCYANATE, CHLORIDE OR SULFATE
1BZX 1998-11-05T00:00:00+0000 2.1 THE CRYSTAL STRUCTURE OF ANIONIC SALMON TRYPSIN IN COMPLEX WITH BOVINE PANCREATIC TRYPSIN INHIBITOR

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
91.0 Pancreatic trypsin inhibitor P04815 BPT2_BOVIN
100.0 Pancreatic trypsin inhibitor P00974 BPT1_BOVIN
91.4 Pancreatic trypsin inhibitor P00975 IBPS_BOVIN