Mutational analysis of hydrophobic domain interactions in gamma B-crystallin from bovine eye lens.


Abstract

gamma B-crystallin is a monomeric member of the beta gamma-superfamily of vertebrate eye lens proteins. It consists of two similar domains with all-beta Greek key topology associating about an approximate two-fold axis. At pH 2, with urea as the denaturant, the domains show independent equilibrium unfolding transitions, suggesting different intrinsic stabilities. Denaturation experiments using recombinant one- or two-domain proteins showed that the N-terminal domain on its own exhibits unaltered intrinsic stability but contributes significantly to the stability of its C-terminal partner. It has been suggested that docking of the domains is determined by a hydrophobic interface that includes phenylalanine at position 56 of the N-terminal domain. In order to test this hypothesis, F56 was substituted by site-directed mutagenesis in both complete gamma B-crystallin and its isolated N-terminal domain. All mutations destabilize the N-terminal domain to about the same extent but affect the C-terminal domain in a different way. Replacement by the small alanine side chain or the charged aspartic acid residue results in a significant destabilization of the C-terminal domain, whereas the more bulky tryptophan residue causes only a moderate decrease in stability. In the mutants F56A and F56D, equilibrium unfolding transitions obtained by circular dichroism and intrinsic fluorescence differ, suggesting a more complex denaturation behavior than the one observed for gamma B wild type. These results confirm how mutations in one crystallin domain can affect the stability of another when they occur at the interface. The results strongly suggest that size, hydrophobicity, and optimal packing of amino acids involved in these interactions are critical for the stability of gamma B-crystallin. Study holds ProTherm entries: 9117, 9118, 9119, 9120, 9121, 9122, 9123, 9124, 9125, 9126, 9127, 9128, 9129 Extra Details: C-domain cyrstallin; protein stability; domain interactions; eye lens proteins;,hydrophobic interactions; protein stability

Submission Details

ID: ZkqjoWvW3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:37 p.m.

Version: 1

Publication Details
Palme S;Slingsby C;Jaenicke R,Protein Sci. (1997) Mutational analysis of hydrophobic domain interactions in gamma B-crystallin from bovine eye lens. PMID:9232654
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
1AMM 1996-11-08 1.2 1.2 ANGSTROM STRUCTURE OF GAMMA-B CRYSTALLIN AT 150K
4W9B 2015-09-09 1.28 Crystal structure of Gamma-B Crystallin expressed in E. coli based on mRNA variant 1
4W9A 2015-09-09 1.38 Crystal structure of Gamma-B Crystallin expressed in E. coli based on mRNA variant 2
4GCR 1993-10-31 1.47 STRUCTURE OF THE BOVINE EYE LENS PROTEIN GAMMA-B (GAMMA-II)-CRYSTALLIN AT 1.47 ANGSTROMS
1DSL 1996-07-11 1.55 GAMMA B CRYSTALLIN C-TERMINAL DOMAIN
1GCS 1994-04-30 2.0 STRUCTURE OF THE BOVINE GAMMA-B CRYSTALLIN AT 150K
1I5I 2001-03-07 2.4 THE C18S MUTANT OF BOVINE (GAMMA-B)-CRYSTALLIN
1GAM 1996-07-11 2.6 GAMMA B CRYSTALLIN TRUNCATED C-TERMINAL DOMAIN

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
92.0 Gamma-crystallin B P10066 CRGB_RAT
94.9 Gamma-crystallin B A3RLE1 CRGB_CANLF
100.0 Gamma-crystallin B P02526 CRGB_BOVIN