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
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