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R. Gupta, K. Srivastava, O.P. Srivastava; Potential Role of N–Terminal Extension and Individual Motifs (I to IV) on Structural Stability of Human ßA3/A1 Crystallin . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3877.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine the effects of deletion of either N–terminal extension, connecting peptide or individual motifs I to IV on structural stability of ßA3/A1 crystallin. Methods: Seven human ßA3/A1 crystallin deletion mutants were generated by sequentially deleting: (i) N–terminal 22 amino acids (ßA3/A1  mutant), (ii) N–terminal extension (ßA3/A1 [N] mutant), (iii) N–terminal extension plus motif I (ßA3/A1 [N+1] mutant), (iv) N–terminal extension plus motif I and II (ßA3/A1 [N+I+II] mutant), (v) N–terminal extension plus motif I, II and connecting peptide (ßA3/A1 [N+I+II+CP] mutant), (vi) motif III and IV (ßA3/A1 [III+IV] mutant), (vii) motif IV (ßA3/A1 [IV] mutant). WT–ßA3/A1 and each of the mutants were expressed in E.coli. WT–ßA3/A1 was purified using ion–exchange chromatography whereas the mutants were purified using ProBond nickel affinity column under denaturating conditions. The secondary and tertiary structural changes in the crystallin preparations were determined by far–UV circular dichroism (CD) spectroscopic and intrinsic Tryptophan (Trp) fluorescence, analyses respectively. Results: DNA sequencing and MALDI–TOF analyses confirmed deletions of the desired regions in the mutants. SDS–PAGE gel analyses showed that the WT–ßA3/A1 crystallin was expressed in soluble fraction, ßA3/A1  and ßA3/A1 [N] mutants in both soluble and inclusion bodies whereas the remaining mutants were found only in the inclusion bodies. Following purification, each mutant protein showed a single band on SDS–PAGE. Far–UV CD spectra showed changes in ß–sheet structures in all the mutants compared to WT–ßA3/A1 crystallin. Intrinsic Trp fluorescence showed the emission maxima between 341–352 nm in the mutant proteins suggesting that their paired Trp residues in each domain were less quenched compared to WT–ßA3/A1 crystallin. Conclusions: WT–ßA3/A1 crystallin upon deletion of N–terminal 22 amino acids or N–terminal extension remained soluble whereas mutants with deletion of motifs I to IV became water insoluble. These mutants exhibited changes in their secondary and tertiary structures compared to WT–ßA3/A1 crystallin.
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