Contribution of individual side-chains to the stability of BPTI examined by alanine-scanning mutagenesis.


Abstract

Bovine pancreatic trypsin inhibitor (BPTI) serves as an important model system for the examination of almost all aspects of protein structure. Systematic studies of the effects of mutation on the thermodynamic stability of BPTI, however, have been limited by the extreme stability of the protein. A derivative of BPTI containing only the 5-55 disulfide bond, termed [5-55]Ala, has been shown previously to fold into a structure very similar to that of native BPTI and to be a functional trypsin inhibitor. [5-55]Ala undergoes a reversible thermal unfolding transition with a melting temperature of 39 degrees C, and is therefore well suited for stability studies. Using an alanine-scanning mutagenesis approach, we have examined the contribution to stability of each side-chain in the [5-55]Ala derivative of BPTI. These studies demonstrate the importance of the two hydrophobic cores composed largely of clusters of aromatic residues, as well as the internal hydrogen-bonding network, in stabilizing BPTI. Overall, there is a strong relationship between change in buried surface area and stability for both polar and hydrophobic residues, with proportionality constants of 50 and 20 cal/A2, respectively. None of the alanine substitutions substantially stabilized [5-55]Ala. Nonetheless, approximately 60% (28/46) of the alanine mutants were destabilized by less than 10 degrees C, suggesting that a form of BPTI with up to half of its residues being alanine could fold into a stable structure resembling the native one. Study holds ProTherm entries: 2597, 2598, 2599, 2600, 2601, 2602, 2603, 2604, 2605, 2606, 2607, 2608, 2609, 2610, 2611, 2612, 2613, 2614, 2615, 2616, 2617, 2618, 2619, 2620, 2621, 2622, 2623, 2624, 2625, 2626, 2627, 2628, 2629, 2630, 2631, 2632, 2633, 2634, 2635, 2636, 2637, 2638, 14056, 14057, 14058, 14059, 14060, 14061, 14062, 14063, 14064, 14065, 14066, 14067, 14068, 14069, 14070, 14071, 14072, 14073, 14074, 14075, 14076, 14077, 14078, 14079, 14080, 14081, 14082, 14083, 14084, 14085, 14086, 14087, 14088, 14089, 14090, 14091, 14092, 14093, 14094, 14095, 14096 Extra Details: additive : EDTA(1 mM),wild* [5-55]Ala contains only the 5-55 disulfide bond, with the other,cysteine residues (14, 38, 30 and 51) changed to Alanine BPTI; protein folding; protein stability

Submission Details

ID: 98sUUXfU

Submitter: Connie Wang

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

Version: 1

Publication Details
Yu MH;Weissman JS;Kim PS,J. Mol. Biol. (1995) Contribution of individual side-chains to the stability of BPTI examined by alanine-scanning mutagenesis. PMID:7540212
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)
1JV9 2001-09-12 NMR Structure of BPTI Mutant G37A
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
1OA6 2003-08-28 The solution structure of bovine pancreatic trypsin inhibitor at high pressure
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
1LD5 2002-09-11 STRUCTURE OF BPTI MUTANT A16V
1LD6 2002-09-11 STRUCTURE OF BPTI_8A MUTANT
1JV8 2001-09-12 NMR Structure of BPTI Mutant G37A
3OTJ 2011-01-26 A Crystal Structure of Trypsin Complexed with BPTI (Bovine Pancreatic Trypsin Inhibitor) by X-ray/Neutron Joint Refinement
1UUA 2004-01-29 Solution structure of a truncated bovine pancreatic trypsin inhibitor, 3-58 BPTI.
1G6X 2001-05-09 0.86 ULTRA HIGH RESOLUTION STRUCTURE OF BOVINE PANCREATIC TRYPSIN INHIBITOR (BPTI) MUTANT WITH ALTERED BINDING LOOP SEQUENCE
5PTI 1984-10-29 1.0 STRUCTURE OF BOVINE PANCREATIC TRYPSIN INHIBITOR. RESULTS OF JOINT NEUTRON AND X-RAY REFINEMENT OF CRYSTAL FORM II
1K6U 2001-12-19 1.0 Crystal Structure of Cyclic Bovine Pancreatic Trypsin Inhibitor
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
2ZJX 2008-10-21 1.09 Bovine pancreatic trypsin inhibitor (BPTI) containing only the [5,55] disulfide bond
2ZVX 2009-10-13 1.09 Structure of a BPTI-[5,55] variant containing Gly/Val at the 14/38th positions
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
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
3P95 2011-08-31 1.3 Human mesotrypsin complexed with bovine pancreatic trypsin inhibitor variant (BPTI-K15R/R17D)
5XX8 2018-07-04 1.3 Hetero-micro-seeding: Crystal structure of BPTI-[555]C14GA38I variant using micro-seeds from -C14GA38L variant
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
2R9P 2007-12-11 1.4 Human mesotrypsin complexed with bovine pancreatic trypsin inhibitor(BPTI)
4DG4 2012-09-12 1.4 Human mesotrypsin-S39Y complexed with bovine pancreatic trypsin inhibitor (BPTI)
1QLQ 1999-10-05 1.42 Bovine Pancreatic Trypsin Inhibitor (BPTI) Mutant with Altered Binding Loop Sequence
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)
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
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
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
2FI5 2006-01-24 1.58 Crystal structure of a BPTI variant (Cys38->Ser) in complex with trypsin
3P92 2011-08-31 1.6 Human mesotrypsin complexed with bovine pancreatic trypsin inhibitor variant (BPTI-K15R/R17G)
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
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
5JB5 2017-04-19 1.6 A simplified BPTI variant containing 22 alanines out of 58 residues
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
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
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
1F5R 2001-07-04 1.65 RAT TRYPSINOGEN MUTANT COMPLEXED WITH BOVINE PANCREATIC TRYPSIN INHIBITOR
5XX4 2018-07-04 1.67 A BPTI-[5,55] variant with C14GA38K mutations
1FY8 2000-11-08 1.7 CRYSTAL STRUCTURE OF THE DELTAILE16VAL17 RAT ANIONIC TRYPSINOGEN-BPTI COMPLEX
1YLC 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)
1T8O 2005-03-08 1.7 CRYSTAL STRUCTURE OF THE P1 TRP BPTI MUTANT- BOVINE CHYMOTRYPSIN COMPLEX
1YKT 2006-04-25 1.7 Trypsin/Bpti complex mutant
1YLD 2006-04-25 1.7 Trypsin/BPTI complex mutant
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
3LDJ 2010-09-15 1.7 Crystal structure of aprotinin in complex with sucrose octasulfate: unusual interactions and implication for heparin binding
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
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
1T8N 2005-03-08 1.75 CRYSTAL STRUCTURE OF THE P1 THR BPTI MUTANT- BOVINE CHYMOTRYPSIN COMPLEX
1T8M 2005-03-08 1.8 CRYSTAL STRUCTURE OF THE P1 HIS BPTI MUTANT- BOVINE CHYMOTRYPSIN COMPLEX
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
3TGI 1998-12-23 1.8 WILD-TYPE RAT ANIONIC TRYPSIN COMPLEXED WITH BOVINE PANCREATIC TRYPSIN INHIBITOR (BPTI)
8PTI 1991-04-15 1.8 CRYSTAL STRUCTURE OF A Y35G MUTANT OF BOVINE PANCREATIC TRYPSIN INHIBITOR
3BTF 2000-03-13 1.8 THE CRYSTAL STRUCTURES OF THE COMPLEXES BETWEEN BOVINE BETA-TRYPSIN AND TEN P1 VARIANTS OF BPTI.
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
3BTM 2000-03-13 1.8 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
1EJM 2001-03-03 1.85 CRYSTAL STRUCTURE OF THE BPTI ALA16LEU MUTANT IN COMPLEX WITH BOVINE TRYPSIN
1T7C 2005-03-08 1.85 CRYSTAL STRUCTURE OF THE P1 GLU BPTI MUTANT- BOVINE CHYMOTRYPSIN COMPLEX
3BTE 2000-03-15 1.85 The Crystal Structures of the Complexes Between Bovine Beta-Trypsin and Ten P1 Variants of BPTI.
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
3BTT 2000-03-13 1.9 THE CRYSTAL STRUCTURES OF THE COMPLEXES BETWEEN BOVINE BETA-TRYPSIN AND TEN P1 VARIANTS OF BPTI
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
5JB7 2017-04-19 1.9 A simplified BPTI variant containing 24 alanines out of 58 residues
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
3BTG 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
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
3BTD 2000-03-13 1.9 The Crystal Structures of the Complexes Between the Bovine Beta-Trypsin and Ten P1 Variants of BPTI.
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
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
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
1BPT 1993-01-15 2.0 CREVICE-FORMING MUTANTS OF BPTI: 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
1BZX 1998-11-11 2.1 THE CRYSTAL STRUCTURE OF ANIONIC SALMON TRYPSIN IN COMPLEX WITH 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)
2HEX 1999-08-05 2.1 DECAMERS OBSERVED IN THE CRYSTALS OF 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
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)
3LDI 2010-06-23 2.2 Crystal structure of aprotinin in complex with sucrose octasulfate: unusual interactions and implication for heparin binding
2IJO 2007-05-15 2.3 Crystal Structure of the West Nile virus NS2B-NS3 protease complexed with bovine pancreatic trypsin inhibitor
1BTH 1997-12-24 2.3 STRUCTURE OF THROMBIN 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
3GYM 2009-05-05 2.8 Structure of Prostasin in Complex with Aprotinin
1MTN 1996-08-17 2.8 BOVINE ALPHA-CHYMOTRYPSIN:BPTI CRYSTALLIZATION
1B0C 1998-11-11 2.8 EVIDENCE OF A COMMON DECAMER IN THREE CRYSTAL STRUCTURES OF BPTI, CRYSTALLIZED FROM THIOCYANATE, CHLORIDE OR SULFATE
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