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O. P. Srivastava, K. Srivastava, R. Gupta, J. M. Chaves; Truncations of Different Regions of Human βB1-Crystallin Alter its Structural Properties. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2110.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the effects of truncations of various regions of human βB1-crystallin on its structural properties.
The following seven truncated mutants of βB1-crystallin were generated and expressed in E. coli: (i) βB1-ΔNT (N-terminal arm truncated; contains residue no. 59-252), (ii) βB1-Δ[NT+I] (N-terminal arm + motif I truncated, contains residue no. 99-252), (iii) βB1-Δ[NT+I+II] (N-terminal arm + motif I + motif II truncated; contains residue no. 144-252), (iv) βB1-Δ[NT+I+II+CP] (N-terminal arm+ motif I + motif II + connecting peptide truncated; contains residue no. 149-252) (v) βB1-ΔCT (C-terminal extension truncated, contains residue no. 1-234), (vi) βB1-Δ[CT+IV] (C-terminal extension plus motif IV truncated, contains residue no. 1-190) and (vii) βB1-Δ[CT+III+IV] (C-terminal extension + motif III + motif IV truncated, contains residue no. 1-148). Following purification of wild type (WT)-βB1 crystallin and its mutant proteins by Ni2+-affinity column chromatography, their structural properties were compared by biophysical methods.
The specific deletions in the mutant proteins were confirmed by DNA sequencing and by the QTRAP mass spectrometric method. The solubility of βB1-crystallin was maximally affected on deletion of the C-terminal domain than the N-terminal domain. The Trp microenvironment of βB1 was maximally affected on truncation of the N-teminal arm plus motifs I+II+connecting peptide. The 8-anilino-1-naphthalenesulfate (ANS)-binding results showed relatively greater exposure of hydrophobic patches in the crystallin on deletion of the N-terminal domain. On far-UV circular dichroism spectral analyses, the deletion of regions of the N-terminal domain showed relatively greater loss of β-sheet structure compared to the deletion of regions of the C-terminal domain. This was especially true when the connecting peptide was also deleted with the N-terminal domain.
The results show that the C-terminal domain is important for the solubility of human βB1-crystallin. However, compared to the C-terminal domain, the N-terminal domain seems to play a relatively greater role in maintaining the native structure of human βB1-crystallin.
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