In vitro unfolding, refolding, and polymerization of human gammaD crystallin, a protein involved in cataract formation.


Abstract

Human gammaD crystallin (HgammaD-Crys), a major protein of the human eye lens, is a primary component of cataracts. This 174-residue primarily beta-sheet protein is made up of four Greek keys separated into two domains. Mutations in the human gene sequence encoding HgammaD-Crys are implicated in early-onset cataracts in children, and the mutant protein expressed in Escherichia coli exhibits properties that reflect the in vivo pathology. We have characterized the unfolding, refolding, and competing aggregation of human wild-type HgammaD-Crys as a function of guanidinium hydrochloride (GuHCl) concentration at neutral pH and 37 degrees C, using intrinsic tryptophan fluorescence to monitor in vitro folding. Wild-type HgammaD-Crys exhibited reversible refolding above 1.0 M GuHCl. The GuHCl unfolded protein was more fluorescent than its native counterpart despite the absence of metal or ion-tryptophan interactions. Aggregation of refolding intermediates of HgammaD-Crys was observed in both equilibrium and kinetic refolding processes. The aggregation pathway competed with productive refolding at denaturant concentrations below 1.0 M GuHCl, beyond the major conformational transition region. Atomic force microscopy of samples under aggregating conditions revealed the sequential appearance of small nuclei, thin protofibrils, and fiber bundles. The HgammaD-Crys fibrous aggregate species bound bisANS appreciably, indicating the presence of exposed hydrophobic pockets. The mechanism of HgammaD-Crys aggregation may provide clues to understanding age-onset cataract formation in vivo. Study holds ProTherm entries: 15990, 15991 Extra Details: 5mM DTT and 1mM EDTA were added in the experiment. Here Cm is the midpoint of the unfolding. human gammaD crystallin; protein folding; aggregation; cataracts; atomic force microscopy; hysteresis

Submission Details

ID: tNgsfxkU

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:47 p.m.

Version: 1

Publication Details
Kosinski-Collins MS;King J,Protein Sci. (2003) In vitro unfolding, refolding, and polymerization of human gammaD crystallin, a protein involved in cataract formation. PMID:12592018
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
2KLJ 2009-10-06 Solution Structure of gammaD-Crystallin with RDC and SAXS
2KFB 2009-07-28 The structure of the cataract causing P23T mutant of human gamma-D crystallin
1H4A 2003-05-08 1.15 Human GammaD Crystallin R58H mutant structure AT 1.15 A resolution
6ETC 2018-11-07 1.2 Crystal Structure of Human gamma-D-crystallin Mutant P23T+R36S at 1.2 Angstroms Resolution
1HK0 2003-05-08 1.25 Human GammaD Crystallin Structure at 1.25 A Resolution
4GR7 2012-11-07 1.7 The human W42R Gamma D-Crystallin Mutant Structure at 1.7A Resolution
6ETA 2018-11-07 2.2 Crystal Structure of Human Gamma-D crystallin Mutant P23T+R36S at Room Temperature
2G98 2006-05-02 2.2 human gamma-D-crystallin
4JGF 2013-05-22 2.5 Crystal Structure of the Cataract-Causing P23T gamma D-Crystallin Mutant

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Gamma-crystallin D P07320 CRGD_HUMAN