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Srabani Karmakar, Tommi White, K Krishna Sharma; Structural and Functional Studies of Substrate Recognition by Alpha Crystallin and N terminal deletion mutant αBΔ54-61. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5588.
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Our objective is to study the substrate recognition mechanism of alpha crystallin and to find out the reason for enhancement in chaperone function in N-terminal deletion mutant of αB-crystallin (αBΔ54-61).
Chaperone-substrate complex was prepared by partial denaturation of a model substrate alcohol dehydrogenase (ADH) by incubating at 450 C in presence of α-crystallin. Size exclusion column chromatography (Superose 6) was used to isolate ADH-α-crystallin complex. The complex was negatively stained with uranyl acetate and Transmission electron microscopy (TEM) was used to study the three dimensional structure of the oligomeric α-crystallin as well as complex of α-crystallin and ADH. Single particle analysis of the negative staining images for α-crystallin and the complex of α-crystallin and ADH were carried out using EMAN2.1. We also performed single particle analysis of αBΔ54-61, a mutant that has enhanced chaperone activity and partially characterized earlier in lab and αB-crystallin wild-type (αB-wt).
The results showed that α-crystallin forms high molecular weight chaperone substrate complex at 1:2 ratios (how did you get this?) with partially denatured ADH. The complex elutes in the void volume of Superose 6 size exclusion column. The complex particles are greater than 25 nm in size and heterogeneous in structure. Two or three oligomers are found joined in different angles in the complex particles. The particles of αBΔ54-61 are smaller than the αB-wt reflecting their lesser oligomeric mass. Preliminary negative staining and single particle analysis showed structural heterogeneity.
The results suggest that α-crystallin interacts with substrates in multiple ways and forms heterogeneous complex. Enhancement of chaperone function in the deletion mutant might be due to the exposure of “new” substrate binding sites.
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