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R. Gupta, K. Srivastava, O.P. Srivastava; Folding/Refolding Properties of N–Terminal Extension and Domains of Human ßA3/A1 Crystallin . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2002.
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© ARVO (1962-2015); The Authors (2016-present)
To characterize the unfolding/refolding properties of wild–type (WT) ßA3/A1 and its truncated mutant proteins and to determine their interaction properties with human ßB1crystallin.
PCR–based truncations were performed to delete: (i) N–terminal 21 amino acids , named (ßA3/A1  mutant), (ii) N–terminal 22 amino acids (ßA3/A1  mutant), (iii) N–terminal extension (ßA3/A1 [NTE] mutant), (iv) N–terminal extension plus Motif I (ßA3/A1 [N+1] mutant), (v) N–terminal extension plus Motif I and II (ßA3/A1 [N+I+II] mutant), (vi) N–terminal extension plus Motif I, II and connecting peptide (ßA3/A1 [N+I+II+CP] mutant), (vii) Motif III and IV (ßA3/A1 [III+IV] mutant), (viii) Motif IV (ßA3/A1 [IV] mutant). Individual proteins were purified using a Ni2+–affinity column. Total intrinsic Tryptophan (Trp) fluorescence was recorded under native and denaturing conditions. Equilibrium unfolding/refolding was performed at varying urea concentrations at 370C using intrinsic Trp as a probe. The ratios of emission intensities of 360 nm over 350 nm against urea concentrations were plotted to analyze the results.
The purified WT–ßA3/A1 and the truncated mutant proteins showed a single band on SDS–PAGE. Under native and denatured conditions, WT–ßA3/A1 showed fluorescence emission maximum of 332 nm and 350 nm, respectively whereas the mutant proteins showed between 339–354 nm and between 332–362 nm, respectively. Equilibrium unfolding/refolding analyses of WT ßA3/A1 showed a single major unfolding transition–midpoint [(C1/2)urea] and the data were fitted to a two–state model. The refolding curve was super imposable on the unfolding curve indicating reversible kinetics. Deletion up to CP in ßA3/A1 ( i.e. N+I+II+CP mutant) generated the C–terminal domain and the deletion of motifs III and IV in ßA3/A1 (i.e. III+IV mutant) generated the N–terminal domain . Based on (C1/2)urea data and assuming unfolding as a qualitative measure of stability, the N–terminal domain was found to be relatively more stable than C–terminal domain. Hetero–oligomer containing WT–ßA3/A1 , truncated mutants (NTE and N+I) with WT–ßB1 resulted in oligomers of same size whereas truncation of motifs II to IV resulted in lower size oligomers.
WT–ßA3/A1 and the mutant proteins exhibited monophasic transition i.e. direct transition between native and unfolded states. WT–ßA3/A1 exhibited reversible refolding however, the mutants showed difference between unfolding/refolding transition curves. The motifs II to IV of ßA3/A1 interact with ßB1–crystallin during hetero–oligomer formation.
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