Context dependence of protein secondary structure formation: the three-dimensional structure and stability of a hybrid between chymotrypsin inhibitor 2 and helix E from subtilisin Carlsberg.


Abstract

The loop region of chymotrypsin inhibitor 2 from barley has been employed as a scaffold for testing the intrinsic propensity of a peptide fragment to form a secondary structure. The helix formation of the nine amino acid residue segment Lys-Gln-Ala-Val-Asp-Asn-Ala-Tyr-Ala of helix E from subtilisin Carlsberg has been studied by the construction of a hybrid consisting of chymotrypsin inhibitor 2 (CI2) where part of the active loop has been replaced by the nonapeptide. An expression system for a truncated form of CI2 where the 19 structureless residues of the N-terminus have been removed and Leu20 replaced by methionyl was constructed from the entire 83-residue wild-type CI2 gene by polymerase chain reaction methodology. The gene encoding the hybrid was constructed from the truncated inhibitor gene. The stability of the truncated inhibitor and of the hybrid toward guanidinium chloride denaturation was examined. From these measurements, the energy of unfolding in pure water was extrapolated to 30.5 +/- 1.0 kJ/mol for the truncated inhibitor and 10.9 +/- 0.3 kJ/mol for the hybrid. These energies show that the stability of CI2 is unaffected by the N-terminal truncation but severely decreased by the loop mutations. The three-dimensional structure of the hybrid protein has been determined in solution by nuclear magnetic resonance spectroscopy using 893 distance restraints and 84 torsional angle restraints. The average root-mean-square deviation (rmsd) of 15 structures compared to their geometrical average was 0.8 +/- 0.2 A for heavy backbone atoms and 1.3 +/- 0.2 A for all heavy atoms.(ABSTRACT TRUNCATED AT 250 WORDS) Study holds ProTherm entries: 4621, 4622 Extra Details: M20: without 19 N-terminal residues and with mutation L 20 M secondary structure; helix formation; hybrid; loop mutations;,CI2 scaffold

Submission Details

ID: tD4jaQsn

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:26 p.m.

Version: 1

Publication Details
Osmark P;Sørensen P;Poulsen FM,Biochemistry (1993) Context dependence of protein secondary structure formation: the three-dimensional structure and stability of a hybrid between chymotrypsin inhibitor 2 and helix E from subtilisin Carlsberg. PMID:8218165
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
1CIQ 1995-10-02T00:00:00+0000 2.2 COMPLEX OF TWO FRAGMENTS OF CI2, RESIDUES 1-40 AND 41-64
1CIQ 1995-10-02T00:00:00+0000 2.2 COMPLEX OF TWO FRAGMENTS OF CI2, RESIDUES 1-40 AND 41-64
1CIR 1995-10-02T00:00:00+0000 0 COMPLEX OF TWO FRAGMENTS OF CI2 [(1-40)(DOT)(41-64)]
1CIR 1995-10-02T00:00:00+0000 0 COMPLEX OF TWO FRAGMENTS OF CI2 [(1-40)(DOT)(41-64)]
1CIS 1993-04-23T00:00:00+0000 0 CONTEXT DEPENDENCE OF PROTEIN SECONDARY STRUCTURE FORMATION. THE THREE-DIMENSIONAL STRUCTURE AND STABILITY OF A HYBRID BETWEEN CHYMOTRYPSIN INHIBITOR 2 AND HELIX E FROM SUBTILISIN CARLSBERG
1COA 1993-05-14T00:00:00+0000 2.2 THE EFFECT OF CAVITY CREATING MUTATIONS IN THE HYDROPHOBIC CORE OF CHYMOTRYPSIN INHIBITOR 2
1CQ4 1998-11-17T00:00:00+0000 1.8 CI2 MUTANT WITH TETRAGLUTAMINE (MGQQQQGM) REPLACING MET59
1CQ4 1998-11-17T00:00:00+0000 1.8 CI2 MUTANT WITH TETRAGLUTAMINE (MGQQQQGM) REPLACING MET59
1LW6 2002-05-30T00:00:00+0000 1.5 Crystal Structure of the Complex of Subtilisin BPN' with Chymotrypsin Inhibitor 2 at 1.5 Angstrom Resolution
1YPA 1993-01-10T00:00:00+0000 2.0 DIRECT OBSERVATION OF BETTER HYDRATION AT THE N-TERMINUS OF AN ALPHA-HELIX WITH GLYCINE RATHER THAN ALANINE AS N-CAP

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
92.6 Subtilisin-chymotrypsin inhibitor-2A P08626 ICI3_HORVU
100.0 Subtilisin-chymotrypsin inhibitor-2A P01053 ICI2_HORVU