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C. O. Asomugha, R. Gupta, J. M. Chaves, K. Srivastava, O. P. Srivastava; Effects of Truncation of N- and C- Terminal Domains on Deamidated Human Alpha B Crystallin. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2037.
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© ARVO (1962-2015); The Authors (2016-present)
To compare the effects of deamidation alone, truncation alone, or both deamidation plus truncation on structural and functional properties of human lens αB-crystallin.
Human wild type (WT) αB, previously cloned in pDIRECT, and human deamidated αB (αB N78D, αB N146D, and αB N78/146D) generated using QUIK change XL system, were used as templates to generate the following eight N (residue number 1-67)-terminally or C (residue number 151-175)-terminally truncated αB, or deamidated (N- or C-terminally truncated) mutants: αB-NT (N-terminally truncated), αB N78D-NT, αB N146D-NT, αB N78/146D-NT, αB-CT (C-terminally truncated), αB N78D-CT, αB N146D-CT, αB N78/146D-CT. Proteins with N-terminal truncation and/or deamidation were recovered in the soluble fraction, while similar C-terminally truncated and/or deamidated proteins were present mostly in the inclusion bodies. The expressed proteins containing six His tags were individually purified using Ni2+-affinity column under native or denaturing conditions. Each purified protein was characterized for its biophysical properties and chaperone function.
Restriction enzyme digestion, DNA sequencing, and MALDI-TOF mass spectrometric analyses confirmed desired deletions in WT and mutant proteins. On SDS-PAGE analysis, each protein showed a single major band with desired molecular weight. Total protein fluorescence and tryptophan fluorescence mostly showed red shifts and quenching, suggesting changes in the microenvironment following truncation in deamidated mutant proteins. On ANS-binding, greater red shifts and quenching were observed with N-terminal truncations than with C-terminal truncations compared to proteins with deamidations alone. Far UV spectra showed secondary structural changes upon both N- and C-terminal truncations. Dynamic light scattering data showed that N-terminally truncated/deamidated proteins produced bigger oligomers than C-terminally truncated/deamidated proteins. Chaperone activity was greatly reduced on N- or C-terminal truncations in the deamidated proteins.
In general, WT αB and deamidated αB mutant proteins with N- and C-terminal truncations showed greater structural and functional changes compared with deamidations alone. The N-terminal truncation caused greater structural changes than C-terminal truncation in deamidated αB-crystallin.
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