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J. M. Chaves, K. Srivastava, C. Asomugha, R. Gupta, O. Srivastava; Effects of Truncations of N- and C-Terminal Domains on Human Lens Deamidated A-Crystallin. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2038. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To compare the effects of deamidation alone, truncation alone or both truncation plus deamidation on structural and functional properties of human lens αA-crystallin.
Human wild type (WT) αA, previously cloned in pDIRECT, and human deamidated αA (αA-N101D, αA-N123D, αA-N101/123D) generated using QUIK change XL system, were used as templates to generate the following eight N (residue number 1-63)-terminally or C (residue number 140-173)-terminally truncated αA mutants and deamidated plus N- or C-terminally truncated mutants: (i) αA-NT (N-terminally truncated), (ii) αA-N101D-NT, (iii) αA-N123D-NT, (iv) αA-N101/123D-NT, (v) αA-CT (C-terminally truncated), (vi) αA-N101D-CT, (vii) αA-N123D-CT and (viii) αA-N101/123D-CT. The mutant proteins containing six His-tags were recovered in either soluble fraction or in inclusion bodies. Each mutant protein was purified using a Ni2+-affinity column and their purity was determined by SDS-PAGE analysis. Next, biophysical properties of the proteins were determined and related to their chaperone function.
Restriction enzyme digestion, DNA sequencing and MALDI-TOF mass spectrometric analyses confirmed the desired deletions of the αA-crystallin mutants. Intrinsic Trp and total fluorescence spectral studies suggested changed microenvironments in the WT and deamidated mutant proteins following N- or C-terminal truncations. On ANS-binding, relatively increased fluorescence quenching and red shifts were observed on N- or C-terminal truncations in the deamidated mutant proteins, suggesting changed hydrophobic patches following truncations. Far-UV CD spectral analyses also showed changes in the secondary structures after N- or C-terminal truncations in the deamidated mutant proteins. Dynamic light scattering analyses using MALS showed that the truncations resulted in higher oligomer sizes compared to deamidation alone. The chaperone activity was decreased on deamidation, but was substantially restored after N- or C-terminal truncations in the mutant proteins.
N- or C-terminal truncation of deamidated αA-crystallin species resulted in major structural changes. The chaperone activity was lost due to deamidation, but was substantially restored after N- or C-terminal truncations in the deamidated αA mutant proteins.
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