Folding and stability of the isolated Greek key domains of the long-lived human lens proteins gammaD-crystallin and gammaS-crystallin.


Abstract

The transparency of the eye lens depends on the high solubility and stability of the lens crystallin proteins. The monomeric gamma-crystallins and oligomeric beta-crystallins have paired homologous double Greek key domains, presumably evolved through gene duplication and fusion. Prior investigation of the refolding of human gammaD-crystallin revealed that the C-terminal domain folds first and nucleates the folding of the N-terminal domain. This result suggested that the human N-terminal domain might not be able to fold on its own. We constructed and expressed polypeptide chains corresponding to the isolated N- and C-terminal domains of human gammaD-crystallin, as well as the isolated domains of human gammaS-crystallin. Both circular dichroism and fluorescence spectroscopy indicated that the isolated domains purified from Escherichia coli were folded into native-like monomers. After denaturation, the isolated domains refolded efficiently at pH 7 and 37 degrees C into native-like structures. The in vitro refolding of all four domains revealed two kinetic phases, identifying partially folded intermediates for the Greek key motifs. When subjected to thermal denaturation, the isolated N-terminal domains were less stable than the full-length proteins and less stable than the C-terminal domains, and this was confirmed in equilibrium unfolding/refolding experiments. The decrease in stability of the N-terminal domain of human gammaD-crystallin with respect to the complete protein indicated that the interdomain interface contributes of 4.2 kcal/mol to the overall stability of this very long-lived protein. Study holds ProTherm entries: 23707, 23708, 23709, 23710, 23711, 23712 Extra Details: human gammaD-crystallin; human gammaS-crystallin; domain interface; cataract; equilibrium unfolding/refolding transitions; refolding kinetic intermediates; protein stability

Submission Details

ID: raFiftU23

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:54 p.m.

Version: 1

Publication Details
Mills IA;Flaugh SL;Kosinski-Collins MS;King JA,Protein Sci. (2007) Folding and stability of the isolated Greek key domains of the long-lived human lens proteins gammaD-crystallin and gammaS-crystallin. PMID:17905830
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
1ZWO 2005-07-05 NMR structure of murine gamma-S crystallin
6IF9 2019-04-10 Solution-state NMR structure of G57W human gammaS crystallin
2M3T 2013-11-13 Solution-state NMR structure of wild-type human gamma(S)-crystallin
2M3U 2013-11-13 Solution-state NMR structure of cataract-related human gamma(S)-crystallin point variant G18V
2A5M 2005-07-19 NMR structure of murine gamma-S crystallin from joint refinement with SAXS data
1ZWM 2005-07-05 NMR structure of murine gamma-S crystallin
6FD8 2018-12-26 2.1 Gamma-s crystallin dimer
1HA4 2001-11-20 2.4 GammaS crystallin C terminal domain from Homo Sapiens
1A7H 1998-05-27 2.56 GAMMA S CRYSTALLIN C-TERMINAL DOMAIN
6MYH 2018-11-14 2.9 Mouse Gamma S Crystallin L16 Octamer
6MYG 2018-11-14 2.92 Mouse Gamma S Crystallin L16 Octamer

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
92.0 Beta-crystallin S P0C5E9 CRYGS_RAT
90.8 Beta-crystallin S O35486 CRYGS_MOUSE
95.4 Beta-crystallin S A2IBY7 CRYGS_CANLF
93.1 Beta-crystallin S P06504 CRYGS_BOVIN
94.3 Beta-crystallin S A4L9I8 CRYGS_RABIT
100.0 Beta-crystallin S P22914 CRYGS_HUMAN