Abstract
Purpose: :
To determine the effects of truncations of various regions of human βB1-crystallin on its structural properties.
Methods: :
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.
Results: :
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.
Conclusions: :
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.
Keywords: crystallins • protein purification and characterization • protein structure/function