Folding, stability and aggregation of the long-lived eye lens protein human gamma D crystallin

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2006.Vita.Includes bibliographical references (p. 179-194).Human [gamma]D crystallin (H[gamma]D-Crys) is a monomeric, two domain, primarily P-sheet protein found in high concentrations in the human eye lens. H[gamma]D-Crys and other crystallins are found in insoluble protein inclusions associated with the eye disease cataract. H[gamma]D-Crys is expressed in utero and does not regenerate during life, thus necessitating high stability and solubility. Covalent damage, including glutamine deamidation, of the lens crystallins increases with age and as a result of exposure to environmental insults. Such covalent damage may cause partial-unfolding into aggregation-prone confomations that cause cataract. The in vitro stability of H[gamma]D-Crys was analyzed in the denaturant guanidine h[gamma]Drochloride at pH 7.0 and 370C. An off-pathway aggregation reaction that competed with refolding was previously reported when H[gamma]D-Crys was refolded to less than 1 M GuHC1. Equilibrium transitions of H[gamma]D-Crys were best fit to a three-state model suggesting the presence of a partially-folded intermediate that likely had a structured C-terminal domain (C-td) and unstructured N-terminal domain (N-td). Similarly, previous analyses revealed a sequential domain refolding pathway where the C-td refolds first followed by the N-td.(cont.) These findings suggest that the inter-domain interface of H[gamma]D-Crys is important in both folding and stability. The domain interface of H[gamma]D-Crys contains a central h[gamma]Drophobic cluster of six residues and two pairs of peripheral interacting residues. To test this importance of these residues in folding and stability, site-directed alanine mutants were constructed at all ten positions and properties of the mutant proteins were analyzed. Single mutations of h[gamma]Drophobic domain interface residues caused a decrease in stability of the N-td, but did not affect stability of the C-td. Similarly, stability of the N-td but not the C-td was reduced as a result of single and double mutations of peripheral interface residues. Minimal to no interaction energy was observed for the peripheral residues suggesting they contribute to stability indirectly, perhaps by shielding the central h[gamma]Drophobic cluster from solvent. Both the h[gamma]Drophobic and peripheral domain interface alanine mutants also had reduced rates of productive refolding for the N-td while refolding rates for the C-td were unchanged. These results suggest a productive folding pathway where the C-td refolds first and domain interface residues of the structured C-td act as a nucleating center for refolding of the N-td.(cont.) Effects on N-td refolding rates were most prominent for the h[gamma]Drophobic residues indicating the importance of proper h[gamma]Drophobic burial during refolding. The peripheral domain interface residues of H[gamma]D-Crys include a pair of two glutamines that are targets for covalent damage during aging. Deamidation mimics at these sites were constructed by site directed mutagenesis of glutamine to glutamate. Properties of the mutants were analyzed to assess the affects of deamidation on stability and folding. Similar to the alanine mutants at these sites, the deamidation mutants had a destabilized N-td but not C-td at pH 7.0. In contrast, stabilities of the mutants were indistinguishable from wild type at pH 3.0. The N-td of the deamidation mutants also unfolded faster than that of wild type during kinetic unfolding. These results indicate that deamidation of domain interface glutamines destabilizes H[gamma]D-Crys and lowers the kinetic barrier to unfolding. A reduction in the thermodynamic and kinetic stability as a result of domain interface deamidation could result in the population of partially-unfolded conformations in the lens that may aggregate through mechanisms such as domain swapping or loop-sheet insertion.by Shannon L. Flaugh.Ph.D

Contributions of hydrophobic domain interface interactions to the folding and stability of human γD-crystallin

Human γD-crystallin (HγD-Crys) is a monomeric eye lens protein composed of two highly homologous β-sheet domains. The domains interact through interdomain side chain contacts forming two structurally distinct regions, a central hydrophobic cluster and peripheral residues. The hydrophobic cluster contains Met43, Phe56, and Ile81 from the N-terminal domain (N-td) and Val132, Leu145, and Val170 from the C-terminal domain (C-td). Equilibrium unfolding/refolding of wild-type HγD-Crys in guanidine hydrochloride (GuHCl) was best fit to a three-state model with transition midpoints of 2.2 and 2.8 M GuHCl. The two transitions likely corresponded to sequential unfolding/refolding of the N-td and the C-td. Previous kinetic experiments revealed that the C-td refolds more rapidly than the N-td. We constructed alanine substitutions of the hydrophobic interface residues to analyze their roles in folding and stability. After purification from E. coli, all mutant proteins adopted a native-like structure similar to wild type. The mutants F56A, I81A, V132A, and L145A had a destabilized N-td, causing greater population of the single folded domain intermediate. Compared to wild type, these mutants also had reduced rates for productive refolding of the N-td but not the C-td. These data suggest a refolding pathway where the domain interface residues of the refolded C-td act as a nucleating center for refolding of the N-td. Specificity of domain interface interactions is likely important for preventing incorrect associations in the high protein concentrations of the lens nucleus

Folding and stability of the isolated Greek key domains of the long-lived human lens proteins γD-crystallin and γS-crystallin

The transparency of the eye lens depends on the high solubility and stability of the lens crystallin proteins. The monomeric γ-crystallins and oligomeric β-crystallins have paired homologous double Greek key domains, presumably evolved through gene duplication and fusion. Prior investigation of the refolding of human γD-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 γD-crystallin, as well as the isolated domains of human γS-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°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 γD-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

Interdomain side-chain interactions in human γD crystallin influencing folding and stability

Human γD crystallin (HγD-Crys) is a two domain, β-sheet eye lens protein that must remain soluble throughout life for lens transparency. Single amino acid substitutions of HγD-Crys are associated with juvenile-onset cataracts. Features of the interface between the two domains conserved among γ-crystallins are a central six-residue hydrophobic cluster, and two pairs of interacting residues flanking the cluster. In HγD-Crys these pairs are Gln54/Gln143 and Arg79/Met147. We previously reported contributions of the hydrophobic cluster residues to protein stability. In this study alanine substitutions of the flanking residue pairs were constructed and analyzed. Equilibrium unfolding/refolding experiments at 37°C revealed a plateau in the unfolding/refolding transitions, suggesting population of a partially folded intermediate with a folded C-terminal domain (C-td) and unfolded N-terminal domain (N-td). The N-td was destabilized by substituting residues from both domains. In contrast, the C-td was not significantly affected by substitutions of either domain. Refolding rates of the N-td were significantly decreased for mutants of either domain. In contrast, refolding rates of the C-td were similar to wild type for mutants of either domain. Therefore, domain interface residues of the folded C-td probably nucleate refolding of the N-td. We suggest that these residues stabilize the native state by shielding the central hydrophobic cluster from solvent. Glutamine and methionine side chains are among the residues covalently damaged in aged and cataractous lenses. Such damage may generate partially unfolded, aggregation- prone conformations of HγD-Crys that could be significant in cataract