"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
1UUB 2004-01-29 Solution structure of a truncated bovine pancreatic trypsin inhibitor mutant, 3-58 BPTI (K15R, R17A, R42S)
1JV8 2001-09-12 NMR Structure of BPTI Mutant G37A
1PIT 1994-01-31 DETERMINATION OF A HIGH-QUALITY NUCLEAR MAGNETIC RESONANCE SOLUTION STRUCTURE OF THE BOVINE PANCREATIC TRYPSIN INHIBITOR AND COMPARISON WITH THREE CRYSTAL STRUCTURES
1OA5 2003-08-28 The solution structure of bovine pancreatic trypsin inhibitor at high pressure
1K09 2002-07-10 Solution structure of BetaCore, A Designed Water Soluble Four-Stranded Antiparallel b-sheet Protein
1LD5 2002-09-11 STRUCTURE OF BPTI MUTANT A16V
1JV9 2001-09-12 NMR Structure of BPTI Mutant G37A
1UUA 2004-01-29 Solution structure of a truncated bovine pancreatic trypsin inhibitor, 3-58 BPTI.
1LD6 2002-09-11 STRUCTURE OF BPTI_8A MUTANT
3OTJ 2011-01-26 A Crystal Structure of Trypsin Complexed with BPTI (Bovine Pancreatic Trypsin Inhibitor) by X-ray/Neutron Joint Refinement
1OA6 2003-08-28 The solution structure of bovine pancreatic trypsin inhibitor at high pressure
1G6X 2001-05-09 0.86 ULTRA HIGH RESOLUTION STRUCTURE OF BOVINE PANCREATIC TRYPSIN INHIBITOR (BPTI) MUTANT WITH ALTERED BINDING LOOP SEQUENCE
1K6U 2001-12-19 1.0 Crystal Structure of Cyclic Bovine Pancreatic Trypsin Inhibitor
5PTI 1984-10-29 1.0 STRUCTURE OF BOVINE PANCREATIC TRYPSIN INHIBITOR. RESULTS OF JOINT NEUTRON AND X-RAY REFINEMENT OF CRYSTAL FORM II
2ZVX 2009-10-13 1.09 Structure of a BPTI-[5,55] variant containing Gly/Val at the 14/38th positions
2ZJX 2008-10-21 1.09 Bovine pancreatic trypsin inhibitor (BPTI) containing only the [5,55] disulfide bond
1BPI 1995-06-03 1.09 THE STRUCTURE OF BOVINE PANCREATIC TRYPSIN INHIBITOR AT 125K: DEFINITION OF CARBOXYL-TERMINAL RESIDUES GLYCINE-57 AND ALANINE-58
5XX3 2018-07-04 1.12 A BPTI-[5,55] variant with C14GA38G mutations
5XX2 2018-07-04 1.12 A BPTI-[5,55] variant with C14GA38L mutations
4Y0Y 2015-06-24 1.25 Trypsin in complex with with BPTI
5XX8 2018-07-04 1.3 Hetero-micro-seeding: Crystal structure of BPTI-[555]C14GA38I variant using micro-seeds from -C14GA38L variant
3P95 2011-08-31 1.3 Human mesotrypsin complexed with bovine pancreatic trypsin inhibitor variant (BPTI-K15R/R17D)
4Y11 2015-06-24 1.3 Trypsin in complex with with BPTI mutant (2S)-2-amino-4,4,4-trifluorobutanoic acid
3WNY 2014-06-18 1.3 A simplified BPTI variant with poly Lys amino acid tag (C3K) at the C-terminus
5XX6 2018-07-04 1.31 Hetero-micro-seeding: Crystal structure of BPTI-[5,55]C14GA38I variant using micro-seeds from -C14GA38G variant
1P2J 2004-04-20 1.35 Structural consequences of accommodation of four non-cognate amino-acid residues in the S1 pocket of bovine trypsin and chymotrypsin
4Y10 2015-06-24 1.37 Trypsin in complex with with BPTI mutant (2S)-2-amino-4,4-difluorobutanoic acid
4Y0Z 2015-06-24 1.37 Trypsin in complex with with BPTI mutant AMINOBUTYRIC ACID
5XX5 2018-07-04 1.38 A BPTI-[5,55] variant with C14GA38I mutations
5XX7 2018-07-04 1.38 Hetero-micro-seeding: Crystal structure of BPTI-[5,55]C14GA38I variant using micro-seeds from -C14GA38I variant
4DG4 2012-09-12 1.4 Human mesotrypsin-S39Y complexed with bovine pancreatic trypsin inhibitor (BPTI)
2R9P 2007-12-11 1.4 Human mesotrypsin complexed with bovine pancreatic trypsin inhibitor(BPTI)
1QLQ 1999-10-05 1.42 Bovine Pancreatic Trypsin Inhibitor (BPTI) Mutant with Altered Binding Loop Sequence
3FP8 2009-02-17 1.46 Anionic trypsin variant S195A in complex with bovine pancreatic trypsin inhibitor (BPTI) determined to the 1.46 A resolution limit
3FP7 2009-02-17 1.46 Anionic trypsin variant S195A in complex with bovine pancreatic trypsin inhibitor (BPTI) cleaved at the scissile bond (LYS15-ALA16) determined to the 1.46 A resolution limit
2RA3 2007-12-11 1.46 Human cationic trypsin complexed with bovine pancreatic trypsin inhibitor (BPTI)
3FP6 2009-02-17 1.49 Anionic trypsin in complex with bovine pancreatic trypsin inhibitor (BPTI) determined to the 1.49 A resolution limit
4PTI 1983-01-18 1.5 THE GEOMETRY OF THE REACTIVE SITE AND OF THE PEPTIDE GROUPS IN TRYPSIN, TRYPSINOGEN AND ITS COMPLEXES WITH INHIBITORS
1F7Z 2001-07-04 1.55 RAT TRYPSINOGEN K15A COMPLEXED WITH BOVINE PANCREATIC TRYPSIN INHIBITOR
2FI5 2006-01-24 1.58 Crystal structure of a BPTI variant (Cys38->Ser) in complex with trypsin
2FI4 2006-01-24 1.58 Crystal structure of a BPTI variant (Cys14->Ser) in complex with trypsin
2FI3 2006-01-24 1.58 Crystal structure of a BPTI variant (Cys14->Ser, Cys38->Ser) in complex with trypsin
3P92 2011-08-31 1.6 Human mesotrypsin complexed with bovine pancreatic trypsin inhibitor variant (BPTI-K15R/R17G)
5JB5 2017-04-19 1.6 A simplified BPTI variant containing 22 alanines out of 58 residues
7PTI 1991-04-15 1.6 STRUCTURAL EFFECTS INDUCED BY REMOVAL OF A DISULFIDE BRIDGE. THE X-RAY STRUCTURE OF THE C30A(SLASH)C51A MUTANT OF BASIC PANCREATIC TRYPSIN INHIBITOR AT 1.6 ANGSTROMS
1AAL 1993-10-31 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
1P2K 2004-04-20 1.6 Structural consequences of accommodation of four non-cognate amino-acid residues in the S1 pocket of bovine trypsin and chymotrypsin
2FTL 2006-02-14 1.62 Crystal structure of trypsin complexed with BPTI at 100K
1D0D 2000-09-09 1.62 CRYSTAL STRUCTURE OF TICK ANTICOAGULANT PROTEIN COMPLEXED WITH BOVINE PANCREATIC TRYPSIN INHIBITOR
1F5R 2001-07-04 1.65 RAT TRYPSINOGEN MUTANT COMPLEXED WITH BOVINE PANCREATIC TRYPSIN INHIBITOR
2FTM 2006-02-14 1.65 Crystal structure of trypsin complexed with the BPTI variant (Tyr35->Gly)
1P2I 2004-04-20 1.65 Structural consequences of accommodation of four non-cognate amino-acid residues in the S1 pocket of bovine trypsin and chymotrypsin
5XX4 2018-07-04 1.67 A BPTI-[5,55] variant with C14GA38K mutations
1T8O 2005-03-08 1.7 CRYSTAL STRUCTURE OF THE P1 TRP BPTI MUTANT- BOVINE CHYMOTRYPSIN COMPLEX
1FY8 2000-11-08 1.7 CRYSTAL STRUCTURE OF THE DELTAILE16VAL17 RAT ANIONIC TRYPSINOGEN-BPTI COMPLEX
3LDJ 2010-09-15 1.7 Crystal structure of aprotinin in complex with sucrose octasulfate: unusual interactions and implication for heparin binding
6PTI 1987-10-16 1.7 STRUCTURE OF FORM III CRYSTALS OF BOVINE PANCREATIC TRYPSIN INHIBITOR
4WWY 2015-07-22 1.7 human cationic trypsin G193R mutant in complex with bovine pancreatic trypsin inhibitor
1YLC 2006-04-25 1.7 Trypsin/BPTI complex mutant
1YLD 2006-04-25 1.7 Trypsin/BPTI complex mutant
1YKT 2006-04-25 1.7 Trypsin/Bpti complex mutant
3TGK 2001-07-04 1.7 TRYPSINOGEN MUTANT D194N AND DELETION OF ILE 16-VAL 17 COMPLEXED WITH BOVINE PANCREATIC TRYPSIN INHIBITOR (BPTI)
6F1F 2018-07-18 1.72 The methylene thioacetal BPTI (Bovine Pancreatic Trypsin Inhibitor) mutant structure
1T8L 2005-03-08 1.75 CRYSTAL STRUCTURE OF THE P1 MET BPTI MUTANT- BOVINE CHYMOTRYPSIN COMPLEX
1T8N 2005-03-08 1.75 CRYSTAL STRUCTURE OF THE P1 THR BPTI MUTANT- BOVINE CHYMOTRYPSIN COMPLEX
3BTH 2000-03-13 1.75 THE CRYSTAL STRUCTURES OF THE COMPLEXES BETWEEN BOVINE BETA-TRYPSIN AND TEN P1 VARIANTS OF BPTI
1P2M 2004-04-20 1.75 Structural consequences of accommodation of four non-cognate amino-acid residues in the S1 pocket of bovine trypsin and chymotrypsin
3BTM 2000-03-13 1.8 THE CRYSTAL STRUCTURES OF THE COMPLEXES BETWEEN BOVINE BETA-TRYPSIN AND TEN P1 VARIANTS OF BPTI
3BTF 2000-03-13 1.8 THE CRYSTAL STRUCTURES OF THE COMPLEXES BETWEEN BOVINE BETA-TRYPSIN AND TEN P1 VARIANTS OF BPTI.
8PTI 1991-04-15 1.8 CRYSTAL STRUCTURE OF A Y35G MUTANT OF BOVINE PANCREATIC TRYPSIN INHIBITOR
1P2Q 2004-04-20 1.8 Structural consequences of accommodation of four non-cognate amino-acid residues in the S1 pocket of bovine trypsin and chymotrypsin
1T8M 2005-03-08 1.8 CRYSTAL STRUCTURE OF THE P1 HIS BPTI MUTANT- BOVINE CHYMOTRYPSIN COMPLEX
3U1J 2011-11-09 1.8 Aprotinin bound to Dengue virus protease
1P2N 2004-04-20 1.8 Structural consequences of accommodation of four non-cognate amino-acid residues in the S1 pocket of bovine trypsin and chymotrypsin
3TGI 1998-12-23 1.8 WILD-TYPE RAT ANIONIC TRYPSIN COMPLEXED WITH BOVINE PANCREATIC TRYPSIN INHIBITOR (BPTI)
3BTE 2000-03-15 1.85 The Crystal Structures of the Complexes Between Bovine Beta-Trypsin and Ten P1 Variants of BPTI.
3BTK 2000-03-13 1.85 THE CRYSTAL STRUCTURES OF THE COMPLEXES BETWEEN BOVINE BETA-TRYPSIN AND TEN P1 VARIANTS OF BPTI
1T7C 2005-03-08 1.85 CRYSTAL STRUCTURE OF THE P1 GLU BPTI MUTANT- BOVINE CHYMOTRYPSIN COMPLEX
1EJM 2001-03-03 1.85 CRYSTAL STRUCTURE OF THE BPTI ALA16LEU MUTANT IN COMPLEX WITH BOVINE TRYPSIN
2TGP 1983-01-18 1.9 THE GEOMETRY OF THE REACTIVE SITE AND OF THE PEPTIDE GROUPS IN TRYPSIN, TRYPSINOGEN AND ITS COMPLEXES WITH INHIBITORS
1TPA 1983-01-18 1.9 THE GEOMETRY OF THE REACTIVE SITE AND OF THE PEPTIDE GROUPS IN TRYPSIN, TRYPSINOGEN AND ITS COMPLEXES WITH INHIBITORS
3BTQ 2000-03-15 1.9 THE CRYSTAL STRUCTURES OF THE COMPLEXES BETWEEN BOVINE BETA-TRYPSIN AND TEN P1 VARIANTS OF BPTI
1NAG 1993-10-31 1.9 CREVICE-FORMING MUTANTS IN THE RIGID CORE OF BOVINE PANCREATIC TRYPSIN INHIBITOR: CRYSTAL STRUCTURES OF F22A, Y23A, N43G, AND F45A
3BTT 2000-03-13 1.9 THE CRYSTAL STRUCTURES OF THE COMPLEXES BETWEEN BOVINE BETA-TRYPSIN AND TEN P1 VARIANTS OF BPTI
5JB6 2017-04-19 1.9 A simplified BPTI variant containing 23 alanines out of 58 residues
3BTD 2000-03-13 1.9 The Crystal Structures of the Complexes Between the Bovine Beta-Trypsin and Ten P1 Variants of BPTI.
5JB7 2017-04-19 1.9 A simplified BPTI variant containing 24 alanines out of 58 residues
2PTC 1983-01-18 1.9 THE GEOMETRY OF THE REACTIVE SITE AND OF THE PEPTIDE GROUPS IN TRYPSIN, TRYPSINOGEN AND ITS COMPLEXES WITH INHIBITORS
3TPI 1983-01-18 1.9 THE GEOMETRY OF THE REACTIVE SITE AND OF THE PEPTIDE GROUPS IN TRYPSIN, TRYPSINOGEN AND ITS COMPLEXES WITH INHIBITORS
3BTG 2000-03-13 1.9 THE CRYSTAL STRUCTURES OF THE COMPLEXES BETWEEN BOVINE BETA-TRYPSIN AND TEN P1 VARIANTS OF BPTI
5JB4 2017-04-19 1.99 A simplified BPTI variant containing 21 alanines out 58 of residues
4BNR 2013-09-04 2.0 Extremely stable complex of crayfish trypsin with bovine trypsin inhibitor
1P2O 2004-04-20 2.0 Structural consequences of accommodation of four non-cognate amino-acid residues in the S1 pocket of bovine trypsin and chymotrypsin
1BPT 1993-01-15 2.0 CREVICE-FORMING MUTANTS OF BPTI: CRYSTAL STRUCTURES OF F22A, Y23A, N43G, AND F45A
1FAN 1993-10-31 2.0 CREVICE-FORMING MUTANTS IN THE RIGID CORE OF BOVINE PANCREATIC TRYPSIN INHIBITOR: CRYSTAL STRUCTURES OF F22A, Y23A, N43G, AND F45A
3BTW 2000-03-13 2.05 THE CRYSTAL STRUCTURES OF THE COMPLEXES BETWEEN BOVINE BETA-TRYPSIN AND TEN P1 VARIANTS OF BPTI
1BRB 1994-07-31 2.1 CRYSTAL STRUCTURES OF RAT ANIONIC TRYPSIN COMPLEXED WITH THE PROTEIN INHIBITORS APPI AND BPTI
2HEX 1999-08-05 2.1 DECAMERS OBSERVED IN THE CRYSTALS OF BOVINE PANCREATIC TRYPSIN INHIBITOR
2TPI 1982-03-04 2.1 ON THE DISORDERED ACTIVATION DOMAIN IN TRYPSINOGEN. CHEMICAL LABELLING AND LOW-TEMPERATURE CRYSTALLOGRAPHY
1FAK 1999-12-03 2.1 HUMAN TISSUE FACTOR COMPLEXED WITH COAGULATION FACTOR VIIA INHIBITED WITH A BPTI-MUTANT
4WXV 2015-07-22 2.1 Human cationic trypsin K97D mutant in complex with bovine pancreatic trypsin inhibitor (BPTI)
1BZX 1998-11-11 2.1 THE CRYSTAL STRUCTURE OF ANIONIC SALMON TRYPSIN IN COMPLEX WITH BOVINE PANCREATIC TRYPSIN INHIBITOR
1BTI 1993-10-31 2.2 CREVICE-FORMING MUTANTS IN THE RIGID CORE OF BOVINE PANCREATIC TRYPSIN INHIBITOR: CRYSTAL STRUCTURES OF F22A, Y23A, N43G, AND F45A
3LDI 2010-06-23 2.2 Crystal structure of aprotinin in complex with sucrose octasulfate: unusual interactions and implication for heparin binding
4TPI 1985-11-08 2.2 THE REFINED 2.2-ANGSTROMS (0.22-NM) X-RAY CRYSTAL STRUCTURE OF THE TERNARY COMPLEX FORMED BY BOVINE TRYPSINOGEN, VALINE-VALINE AND THE ARG15 ANALOGUE OF BOVINE PANCREATIC TRYPSIN INHIBITOR
3TGJ 1998-12-23 2.2 S195A TRYPSINOGEN COMPLEXED WITH BOVINE PANCREATIC TRYPSIN INHIBITOR (BPTI)
1BTH 1997-12-24 2.3 STRUCTURE OF THROMBIN COMPLEXED WITH BOVINE PANCREATIC TRYPSIN INHIBITOR
2IJO 2007-05-15 2.3 Crystal Structure of the West Nile virus NS2B-NS3 protease complexed with bovine pancreatic trypsin inhibitor
5YVU 2018-11-28 2.49 Crystal structures of unlinked full length NS3 from Dengue virus provide insights into dynamics of protease domain
2KAI 1984-07-19 2.5 REFINED 2.5 ANGSTROMS X-RAY CRYSTAL STRUCTURE OF THE COMPLEX FORMED BY PORCINE KALLIKREIN A AND THE BOVINE PANCREATIC TRYPSIN INHIBITOR. CRYSTALLIZATION, PATTERSON SEARCH, STRUCTURE DETERMINATION, REFINEMENT, STRUCTURE AND COMPARISON WITH ITS COMPONENTS AND WITH THE BOVINE TRYPSIN-PANCREATIC TRYPSIN INHIBITOR COMPLEX
1BZ5 1998-11-11 2.58 EVIDENCE OF A COMMON DECAMER IN THREE CRYSTAL STRUCTURES OF BPTI, CRYSTALLIZE FROM THIOCYANATE, CHLORIDE OR SULFATE
1CBW 1997-07-23 2.6 BOVINE CHYMOTRYPSIN COMPLEXED TO BPTI
5YW1 2019-01-02 2.6 Crystal structure of full length NS3 protein (eD4NS2BNS3) in complex with Bovine Pancreatic Trypsin Inhibitor
3LDM 2010-06-23 2.6 Crystal structure of aprotinin in complex with sucrose octasulfate: unusual interactions and implication for heparin binding
1BHC 1998-09-16 2.7 BOVINE PANCREATIC TRYPSIN INHIBITOR CRYSTALLIZED FROM THIOCYANATE
1B0C 1998-11-11 2.8 EVIDENCE OF A COMMON DECAMER IN THREE CRYSTAL STRUCTURES OF BPTI, CRYSTALLIZED FROM THIOCYANATE, CHLORIDE OR SULFATE
3GYM 2009-05-05 2.8 Structure of Prostasin in Complex with Aprotinin
1MTN 1996-08-17 2.8 BOVINE ALPHA-CHYMOTRYPSIN:BPTI CRYSTALLIZATION
1EAW 2002-01-28 2.93 Crystal structure of the MTSP1 (matriptase)-BPTI (aprotinin) complex

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