Characterization of a transient unfolding intermediate in a core mutant of γS-crystallin.
In many age-related and neurological diseases, formerly native proteins aggregate via formation of a partially unfolded intermediate. γS-Crystallin is a highly stable structural protein of the eye lens. In the mouse Opj cataract, a non-conservative F9S mutation in the N-terminal domain core of γS allows the adoption of a native fold but renders the protein susceptible to temperature- and concentration-dependent aggregation, including fibril formation. Hydrogen/deuterium exchange and denaturant unfolding studies of this mutant protein (Opj) have suggested the existence of a partially unfolded intermediate in its aggregation pathway. Here, we used NMR and fluorescence spectroscopy to obtain evidence for this intermediate. In 3.5 M urea, Opj forms a stable and partially unfolded entity that is characterized by an unstructured N-terminal domain and a largely intact C-terminal domain. Under physiologically relevant conditions, Carr-Purcell-Meiboom-Gill T(2)-relaxation dispersion experiments showed that the N-terminal domain residues were in conformational exchange with a loosely structured intermediate with a population of 1-2%, which increased with temperature. This provides direct evidence for a model in which proteins of native fold can explore an intermediate state with an increased propensity for formation of aggregates, such as fibrils. For the crystallins, this shows how inherited sequence variants or environmentally induced modifications can destabilize a well-folded protein, allowing the formation of intermediates able to act as nucleation sites for aggregation and the accumulation of light-scattering centers in the cataractous lens.
Biochemistry Department, Ohio State University, Columbus, OH 43210, USA.
Bryon
B
Mahler
Kiran
K
Doddapaneni
Ian
I
Kleckner
Chunhua
C
Yuan
Graeme
G
Wistow
Zhengrong
Z
Wu
D000818:Animals; D002386:Cataract / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D011487:Protein Conformation; D011489:Protein Denaturation; D058767:Protein Unfolding; D038222:gamma-Crystallins / Q000737:chemistry* / Q000235:genetics
C504673:Crygs protein, mouse; D038222:gamma-Crystallins