Amino-acid substitutions at the fully exposed P1 site of bovine pancreatic trypsin inhibitor affect its stability.


Abstract

It is widely accepted that solvent-exposed sites in proteins play only a negligible role in determining protein energetics. In this paper we show that amino acid substitutions at the fully exposed Lys15 in bovine pancreatic trypsin inhibitor (BPTI) influenced the CD- and DSC-monitored stability: The T(den) difference between the least (P1 Trp) and the most stable (P1 His) mutant is 11.2 degrees C at pH 2.0. The DeltaH(den) versus T(den) plot for all the variants at three pH values (2.0, 2.5, 3.0) is linear (DeltaC(p,den) = 0.41 kcal* mole(-1) * K(-1); 1 cal = 4.18 J) leading to a DeltaG(den) difference of 2.1 kcal*mole(-1). Thermal denaturation of the variants monitored by CD signal at pH 2.0 in the presence of 6 M GdmCl again showed differences in their stability, albeit somewhat smaller (DeltaT(den) =7.1 degrees C). Selective reduction of the Cys14-Cys 38 disulfide bond, which is located in the vicinity of the P1 position did not eliminate the stability differences. A correlation analysis of the P1 stability with different properties of amino acids suggests that two mechanisms may be responsible for the observed stability differences: the reverse hydrophobic effect and amino acid propensities to occur in nonoptimal dihedral angles adopted by the P1 position. The former effect operates at the denatured state level and causes a drop in protein stability for hydrophobic side chains, due to their decreased exposure upon denaturation. The latter factor influences the native state energetics and results from intrinsic properties of amino acids in a way similar to those observed for secondary structure propensities. In conclusion, our results suggest that the protein-stability-derived secondary structure propensity scales should be taken with more caution. Study holds ProTherm entries: 10396, 10397, 10398, 10399, 10400, 10401, 10402, 10403, 10404, 10405, 10406, 10407, 10408, 10409, 10410, 10411, 10412, 10413, 10414, 10415, 10416, 10417, 10418, 10419, 10420, 10421, 10422, 10423, 10424, 10425, 10426, 10427, 10428, 10429, 10430, 10431, 10432, 10433, 10434, 10435, 10436, 10437, 10438, 10439, 10440, 10441, 10442, 10443, 10444, 10445, 10446, 10447, 10448, 10449, 10450, 10451, 10452, 10453, 10454, 10455, 10456, 10457, 10458, 10459, 10460, 10461, 10462, 10463, 10464, 10465, 10466, 10467, 10468, 10469, 10470, 10471, 10472, 10473, 10474, 10475, 10476, 10477, 10478, 10479, 10480, 10481, 10482, 10483, 10484, 10485, 10486, 10487, 10488, 10489, 10490, 10491, 10492, 10493, 10494, 10495, 10496, 10497, 10498, 10499 Extra Details: thermodynamic stability; solvent-exposed residue; reverse hydrophobic effect;,bovine pancreatic trypsin inhibitor

Submission Details

ID: DwwzwSaW

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:40 p.m.

Version: 1

Publication Details
Krowarsch D;Otlewski J,Protein Sci. (2001) Amino-acid substitutions at the fully exposed P1 site of bovine pancreatic trypsin inhibitor affect its stability. PMID:11274462
Additional Information

Study Summary

Number of data points 256
Proteins Pancreatic trypsin inhibitor ; Pancreatic trypsin inhibitor
Unique complexes 18
Assays/Quantities/Protocols Experimental Assay: Tm prot_conc:- ; Experimental Assay: dHvH prot_conc:- ; Experimental Assay: dG temp:89.0 C, pH:3.0, prot_conc:180 micro g/mL ; Experimental Assay: dG pH:2.5, prot_conc:150 micro g/mL, temp:83.0 C ; Experimental Assay: dG temp:89.0 C, prot_conc:300 micro g/mL, pH:3.0 ; Experimental Assay: dG prot_conc:270 micro g/mL, temp:81.0 C, pH:2.0 ; Experimental Assay: dG prot_conc:110 micro g/mL, temp:81.0 C, pH:2.0 ; Experimental Assay: dG pH:2.5, prot_conc:200 micro g/mL, temp:83.0 C ; Experimental Assay: dG temp:81.0 C, pH:2.0, prot_conc:230 micro g/mL ; Experimental Assay: dG temp:89.0 C, prot_conc:220 micro g/mL, pH:3.0 ; Experimental Assay: dG pH:2.5, prot_conc:280 micro g/mL, temp:83.0 C ; Experimental Assay: dG temp:81.0 C, pH:2.0, prot_conc:260 micro g/mL ; Experimental Assay: dG temp:81.0 C, pH:2.0, prot_conc:210 micro g/mL ; Experimental Assay: dG prot_conc:220 micro g/mL, pH:2.5, temp:83.0 C ; Experimental Assay: dG prot_conc:100 micro g/mL, temp:81.0 C, pH:2.0 ; Experimental Assay: dG temp:89.0 C, pH:3.0, prot_conc:250 micro g/mL ; Experimental Assay: dG pH:2.5, prot_conc:230 micro g/mL, temp:83.0 C ; Experimental Assay: dG temp:81.0 C, pH:2.0, prot_conc:250 micro g/mL ; Experimental Assay: dG temp:89.0 C, pH:3.0, prot_conc:90 micro g/mL ; Experimental Assay: dG prot_conc:60 micro g/mL, pH:2.5, temp:83.0 C ; Experimental Assay: dG temp:81.0 C, pH:2.0, prot_conc:130 micro g/mL ; Experimental Assay: dG temp:89.0 C, pH:3.0, prot_conc:140 micro g/mL ; Experimental Assay: dG prot_conc:180 micro g/mL, temp:81.0 C, pH:2.0 ; Experimental Assay: dG temp:89.0 C, prot_conc:60 micro g/mL, pH:3.0 ; Experimental Assay: dG prot_conc:50 micro g/mL, pH:2.5, temp:83.0 C ; Experimental Assay: dG temp:81.0 C, pH:2.0, prot_conc:80 micro g/mL ; Experimental Assay: dG pH:2.5, temp:83.0 C, prot_conc:130 micro g/mL ; Experimental Assay: dG prot_conc:300 micro g/mL, temp:81.0 C, pH:2.0 ; Experimental Assay: dG temp:89.0 C, pH:3.0, prot_conc:150 micro g/mL ; Experimental Assay: dG pH:2.5, prot_conc:90 micro g/mL, temp:83.0 C ; Experimental Assay: dG temp:89.0 C, prot_conc:120 micro g/mL, pH:3.0 ; Experimental Assay: dG pH:2.5, prot_conc:250 micro g/mL, temp:83.0 C ; Experimental Assay: dG temp:81.0 C, pH:2.0, prot_conc:140 micro g/mL ; Experimental Assay: dG prot_conc:120 micro g/mL, pH:2.5, temp:83.0 C ; Experimental Assay: dG prot_conc:200 micro g/mL, temp:81.0 C, pH:2.0 ; Experimental Assay: dCp pH:3.0, prot_conc:180 micro g/mL ; Experimental Assay: dHcal pH:3.0, prot_conc:180 micro g/mL ; Experimental Assay: Tm pH:3.0, prot_conc:180 micro g/mL ; Experimental Assay: dHvH pH:3.0, prot_conc:180 micro g/mL ; Experimental Assay: dCp pH:2.5, prot_conc:150 micro g/mL ; Experimental Assay: dHcal pH:2.5, prot_conc:150 micro g/mL ; Experimental Assay: Tm prot_conc:150 micro g/mL, pH:2.5 ; Experimental Assay: dHvH prot_conc:150 micro g/mL, pH:2.5 ; Experimental Assay: dCp prot_conc:300 micro g/mL, pH:3.0 ; Experimental Assay: dHcal prot_conc:300 micro g/mL, pH:3.0 ; Experimental Assay: Tm prot_conc:300 micro g/mL, pH:3.0 ; Experimental Assay: dHvH prot_conc:300 micro g/mL, pH:3.0 ; Experimental Assay: dCp prot_conc:270 micro g/mL, pH:2.0 ; Experimental Assay: dHcal prot_conc:270 micro g/mL, pH:2.0 ; Experimental Assay: Tm prot_conc:270 micro g/mL ; Experimental Assay: dHvH prot_conc:270 micro g/mL ; Experimental Assay: dCp prot_conc:110 micro g/mL, pH:2.0 ; Experimental Assay: dHcal prot_conc:110 micro g/mL, pH:2.0 ; Experimental Assay: Tm prot_conc:110 micro g/mL ; Experimental Assay: dHvH prot_conc:110 micro g/mL ; Experimental Assay: dCp pH:2.5, prot_conc:200 micro g/mL ; Experimental Assay: dHcal pH:2.5, prot_conc:200 micro g/mL ; Experimental Assay: Tm prot_conc:200 micro g/mL, pH:2.5 ; Experimental Assay: dHvH prot_conc:200 micro g/mL, pH:2.5 ; Experimental Assay: dCp pH:2.0, prot_conc:230 micro g/mL ; Experimental Assay: dHcal pH:2.0, prot_conc:230 micro g/mL ; Experimental Assay: Tm prot_conc:230 micro g/mL ; Experimental Assay: dHvH prot_conc:230 micro g/mL ; Experimental Assay: dCp prot_conc:220 micro g/mL, pH:3.0 ; Experimental Assay: dHcal prot_conc:220 micro g/mL, pH:3.0 ; Experimental Assay: Tm prot_conc:220 micro g/mL, pH:3.0 ; Experimental Assay: dHvH prot_conc:220 micro g/mL, pH:3.0 ; Experimental Assay: dCp pH:2.5, prot_conc:280 micro g/mL ; Experimental Assay: dHvH prot_conc:210 micro g/mL ; Experimental Assay: dCp prot_conc:220 micro g/mL, pH:2.5 ; Experimental Assay: dHcal prot_conc:220 micro g/mL, pH:2.5 ; Experimental Assay: Tm prot_conc:220 micro g/mL, pH:2.5 ; Experimental Assay: dHvH prot_conc:220 micro g/mL, pH:2.5 ; Experimental Assay: dCp prot_conc:100 micro g/mL, pH:2.0 ; Experimental Assay: dHcal prot_conc:100 micro g/mL, pH:2.0 ; Experimental Assay: Tm prot_conc:100 micro g/mL ; Experimental Assay: dHvH prot_conc:100 micro g/mL ; Experimental Assay: dCp pH:3.0, prot_conc:250 micro g/mL ; Experimental Assay: dHcal pH:3.0, prot_conc:250 micro g/mL ; Experimental Assay: Tm pH:3.0, prot_conc:250 micro g/mL ; Experimental Assay: dHvH pH:3.0, prot_conc:250 micro g/mL ; Experimental Assay: dCp pH:2.5, prot_conc:230 micro g/mL ; Experimental Assay: dHcal pH:2.5, prot_conc:230 micro g/mL ; Experimental Assay: Tm pH:2.5, prot_conc:230 micro g/mL ; Experimental Assay: dHvH pH:2.5, prot_conc:230 micro g/mL ; Experimental Assay: dCp pH:2.0, prot_conc:200 micro M ; Experimental Assay: dHcal pH:2.0, prot_conc:200 micro M ; Experimental Assay: Tm prot_conc:200 micro M ; Experimental Assay: dHvH prot_conc:200 micro M ; Experimental Assay: dCp prot_conc:250 micro g/mL, pH:2.0 ; Experimental Assay: dHcal prot_conc:250 micro g/mL, pH:2.0 ; Experimental Assay: Tm prot_conc:250 micro g/mL ; Experimental Assay: dHvH prot_conc:250 micro g/mL ; Experimental Assay: dCp pH:3.0, prot_conc:90 micro g/mL ; Experimental Assay: dHcal pH:3.0, prot_conc:90 micro g/mL ; Experimental Assay: Tm prot_conc:90 micro g/mL, pH:3.0 ; Experimental Assay: dHvH prot_conc:90 micro g/mL, pH:3.0 ; Experimental Assay: dCp prot_conc:60 micro g/mL, pH:2.5 ; Experimental Assay: dHcal prot_conc:60 micro g/mL, pH:2.5 ; Experimental Assay: Tm prot_conc:60 micro g/mL, pH:2.5 ; Experimental Assay: dHvH prot_conc:60 micro g/mL, pH:2.5 ; Experimental Assay: dCp pH:2.0, prot_conc:130 micro g/mL ; Experimental Assay: dHcal pH:2.0, prot_conc:130 micro g/mL ; Experimental Assay: Tm prot_conc:130 micro g/mL ; Experimental Assay: dHvH prot_conc:130 micro g/mL ; Experimental Assay: dCp pH:3.0, prot_conc:140 micro g/mL ; Experimental Assay: dHcal pH:3.0, prot_conc:140 micro g/mL ; Experimental Assay: Tm pH:3.0, prot_conc:140 micro g/mL ; Experimental Assay: dHvH pH:3.0, prot_conc:140 micro g/mL ; Experimental Assay: dCp prot_conc:180 micro g/mL, pH:2.0 ; Experimental Assay: dHcal prot_conc:180 micro g/mL, pH:2.0 ; Experimental Assay: Tm prot_conc:180 micro g/mL ; Experimental Assay: dHvH prot_conc:180 micro g/mL ; Experimental Assay: dCp prot_conc:60 micro g/mL, pH:3.0 ; Experimental Assay: dHcal prot_conc:60 micro g/mL, pH:3.0 ; Experimental Assay: Tm prot_conc:60 micro g/mL, pH:3.0 ; Experimental Assay: dHvH prot_conc:60 micro g/mL, pH:3.0 ; Experimental Assay: dCp prot_conc:50 micro g/mL, pH:2.5 ; Experimental Assay: dHcal prot_conc:50 micro g/mL, pH:2.5 ; Experimental Assay: Tm prot_conc:50 micro g/mL, pH:2.5 ; Experimental Assay: dHvH prot_conc:50 micro g/mL, pH:2.5 ; Experimental Assay: dCp pH:2.0, prot_conc:80 micro g/mL ; Experimental Assay: dHcal pH:2.5, prot_conc:280 micro g/mL ; Experimental Assay: Tm prot_conc:280 micro g/mL, pH:2.5 ; Experimental Assay: dHvH prot_conc:280 micro g/mL, pH:2.5 ; Experimental Assay: dCp pH:2.0, prot_conc:260 micro g/mL ; Experimental Assay: dHcal pH:2.0, prot_conc:260 micro g/mL ; Experimental Assay: Tm prot_conc:260 micro g/mL ; Experimental Assay: dHvH prot_conc:260 micro g/mL ; Experimental Assay: dCp pH:2.0, prot_conc:210 micro g/mL ; Experimental Assay: dHcal pH:2.0, prot_conc:210 micro g/mL ; Experimental Assay: Tm prot_conc:210 micro g/mL ; Experimental Assay: dHcal pH:2.0, prot_conc:80 micro g/mL ; Experimental Assay: Tm prot_conc:80 micro g/mL ; Experimental Assay: dHvH prot_conc:80 micro g/mL ; Experimental Assay: dCp pH:2.5, prot_conc:130 micro g/mL ; Experimental Assay: dHcal pH:2.5, prot_conc:130 micro g/mL ; Experimental Assay: Tm pH:2.5, prot_conc:130 micro g/mL ; Experimental Assay: dHvH pH:2.5, prot_conc:130 micro g/mL ; Experimental Assay: dCp prot_conc:300 micro g/mL, pH:2.0 ; Experimental Assay: dHcal prot_conc:300 micro g/mL, pH:2.0 ; Experimental Assay: Tm prot_conc:300 micro g/mL ; Experimental Assay: dHvH prot_conc:300 micro g/mL ; Experimental Assay: dCp pH:3.0, prot_conc:150 micro g/mL ; Experimental Assay: dHcal pH:3.0, prot_conc:150 micro g/mL ; Experimental Assay: Tm prot_conc:150 micro g/mL, pH:3.0 ; Experimental Assay: dHvH prot_conc:150 micro g/mL, pH:3.0 ; Experimental Assay: dCp pH:2.5, prot_conc:90 micro g/mL ; Experimental Assay: dHcal pH:2.5, prot_conc:90 micro g/mL ; Experimental Assay: Tm prot_conc:90 micro g/mL, pH:2.5 ; Experimental Assay: dHvH prot_conc:90 micro g/mL, pH:2.5 ; Experimental Assay: dCp prot_conc:120 micro g/mL, pH:3.0 ; Experimental Assay: dHcal prot_conc:120 micro g/mL, pH:3.0 ; Experimental Assay: Tm prot_conc:120 micro g/mL, pH:3.0 ; Experimental Assay: dHvH prot_conc:120 micro g/mL, pH:3.0 ; Experimental Assay: dCp pH:2.5, prot_conc:250 micro g/mL ; Experimental Assay: dHcal pH:2.5, prot_conc:250 micro g/mL ; Experimental Assay: Tm pH:2.5, prot_conc:250 micro g/mL ; Experimental Assay: dHvH pH:2.5, prot_conc:250 micro g/mL ; Experimental Assay: dCp pH:2.0, prot_conc:140 micro g/mL ; Experimental Assay: dHcal pH:2.0, prot_conc:140 micro g/mL ; Experimental Assay: Tm prot_conc:140 micro g/mL ; Experimental Assay: dHvH prot_conc:140 micro g/mL ; Experimental Assay: dCp prot_conc:120 micro g/mL, pH:2.5 ; Experimental Assay: dHcal prot_conc:120 micro g/mL, pH:2.5 ; Experimental Assay: Tm prot_conc:120 micro g/mL, pH:2.5 ; Experimental Assay: dHvH prot_conc:120 micro g/mL, pH:2.5 ; Experimental Assay: dCp prot_conc:200 micro g/mL, pH:2.0 ; Experimental Assay: dHcal pH:2.0, prot_conc:200 micro g/mL ; Experimental Assay: Tm prot_conc:200 micro g/mL ; Experimental Assay: dHvH prot_conc:200 micro g/mL
Libraries Mutations for sequence RPDFCLEPPYTGPCKARIIRYFYNAKAGLCQTFVYGGCRAKRNNFKSAEDCMRTCGGA

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