title: Contributions of hydrophobic domain interface interactions to the folding and stability of human gammaD-crystallin.
abstract: Human gammaD-crystallin (HgammaD-Crys) is a monomeric eye lens protein composed of two highly homologous beta-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 HgammaD-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.
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Building 68, Room 330, 31 Ames Street, Cambridge, MA 02139, USA.
authors.forename: Shannon L
authors.initials: SL
authors.lastname: Flaugh
authors.forename: Melissa S
authors.initials: MS
authors.lastname: Kosinski-Collins
authors.forename: Jonathan
authors.initials: J
authors.lastname: King
mesh_terms: D002942:Circular Dichroism; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D009154:Mutation; D011489:Protein Denaturation; D017510:Protein Folding; D020673:Protein Renaturation; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D038222:gamma-Crystallins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D038222:gamma-Crystallins
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