"Designing out" disulfide bonds: thermodynamic properties of 30-51 cystine substitution mutants of bovine pancreatic trypsin inhibitor.


Abstract

We have used a combination of spectroscopic and calorimetric techniques to assess the thermodynamic and extrathermodynamic consequences of paired amino acid substitutions at positions 30 and 51 in bovine pancreatic trypsin inhibitor (BPTI). Correctly folded, wild type BPTI contains a disulfide at the 30-51 positions, with the nonbackbone atoms of this cystine being relatively solvent inaccessible. Mutants missing this buried 30-51 disulfide adopt a conformation very similar to that of the native state of wild type BPTI (Eigenbrot et al., 1990, 1992), although they are severely destabilized relative to the wild type molecule (Hurle et al., 1990). We have conducted a systematic effort to find the energetically most favorable substitution for this buried 30-51 disulfide in BPTI. To this end, we have studied and characterized the thermally induced and guanidine hydrochloride-induced denaturation transitions for a family of mutants in which the amino acid residue(s) at positions 30 and/or 51 have been systematically altered. Specifically, we studied the unfolding transitions of the following series of residue 30/residue 51 paired substitution mutants: C30A/C51A, C30V/C51A, C30G/C51A, C30S/C51A, C30T/C51A, C30A/C51S, C30S/C51S, and C30G/C51M. For this series of mutants, comparisons between the relative stabilization free energies, derived from analysis of the denaturation profiles, allow us to reach the following conclusions: (a) side chains containing polar moieties (Ser and Thr) are destabilizing, with this effect being position dependent (i.e., a serine substitution is more destabilizing at position 51 than at position 30); (b) the destabilizing effects of two serine substitutions are approximately additive, suggesting that side chain-side chain hydrogen bonds between the two serine hydroxyl groups probably are weak or nonexistent; (c) the thermodynamic impact of a Gly30 substitution is consistent with a glycine-induced increase in the configurational entropy of the unfolded state; (d) the C30G/C51M mutant is highly destabilized relative to the C30A/C51A mutant despite the fact that, based on considerations of hydrophobicity and steric fit, substitution of a buried disulfide by Gly30 and Met51 would be expected to be optimal. Methionine may be particularly ill-suited as a buried disulfide substitute due to the large loss of side chain conformational entropy it undergoes during the transition from the unfolded to the native state. In the aggregate, our data provide insight into the residue-, position-, and context-dependent consequences on protein stability of "designing out" the buried 30-51 disulfide bond in the BPTI molecule. These data also suggest that a previously unrecognized component of disulfide bridge stabilization of proteins is the relatively minor penalty in side chain conformational entropy incurred by cystine residues during folding due to their severely restricted rotation even in the unfolded state. Study holds ProTherm entries: 2767, 2768, 2769, 2770, 2771, 2772, 2773, 14098, 14099, 14100, 14101, 14102, 14103 Extra Details: additive : EDTA(0.2 mM), BPTI; disulfide bonds; cystine; thermodynamic properties;,amino acid substitutions; configurational entropy; protein stability

Submission Details

ID: LKKZ3NZp3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:19 p.m.

Version: 1

Publication Details
Liu Y;Breslauer K;Anderson S,Biochemistry (1997) "Designing out" disulfide bonds: thermodynamic properties of 30-51 cystine substitution mutants of bovine pancreatic trypsin inhibitor. PMID:9154914
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
100.0 Pancreatic trypsin inhibitor P00974 BPT1_BOVIN
91.0 Pancreatic trypsin inhibitor P04815 BPT2_BOVIN
91.4 Pancreatic trypsin inhibitor P00975 IBPS_BOVIN