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M. B. Dolinska, M. P. Chan, Y. V. Sergeev, P. T. Wingfield, F. J. Hejtmancik; The N-Terminal Extensions in Homo- and Hetero-Associations of β-Crystallins. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2112.
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β-crystallins are structural proteins existing at extremely high concentrations in mammalian lens, and are known to associate into oligomers under physiological conditions. Crystallins have N- and C-terminal extensions which are susceptible to age related in-vivo proteolytic processing which may enhance their associative properties. This study compares the oligomeric states of the wild-type recombinant human βA3- and mouse βB1-crystallins and their N-terminal truncated counterparts (βA3ΔN30 and βB1ΔN56).
All crystallins were expressed in E.coli and purified from the soluble fractions using ion-exchange and size-exclusion chromatography. Protein homo- and hetero-associations were characterized using analytical ultracentrifugation (AUC), size-exclusion chromatography (SEC), native gel electrophoresis, and tryptophan fluorescence.
AUC shows a homo-dimer with a dissociation constant (Kd) of 0.1 µM for βA3ΔN30, and a monomer-dimer equilibrium with a dissociation constant of 14.7 µM for full-length βA3. SEC and AUC analysis of βB1 also indicates a reversible monomer-dimer system with a Kd of 1.55 µM, whereas, βB1ΔN56 exhibited monomer-tetramer association with a Kd of 5.39 x 10-11 µM.
Deletion of N-terminal domain of βA3 significantly increases the binding affinity for dimer formation while the N-terminal deletion of βB1 has a more profound effect, resulting in homotetramer association. Interaction between βB1 and either βA3 or βA3ΔN30 resulted in heterotetramer formation. In contrast, the N-terminal truncated βB1 did not associate with either βA3 crystallin, presumably due to masking of interaction surfaces during homotetramer formation.
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