Effects of controlled mutations on the N- and C-terminal extensions of chick lens βB1 crystallin

Abstract •

Background: While γ-crystallin exists as a monomer, β-crystallin, which unlike γ-crystallin contains N- and C-terminal arms, associates to form homodimers. • Methods: In order to answer the question of whether the extensions are involved in dimerisation of chick lens βB1 crystallin, we have developed a heterologous expression system for chicken βB1 crystallin in Escherichia coli, and produced three mutations by site-directed mutagenesis. We have substituted residues in the PAPA segment of the N-terminal extension, curtailed the N-terminal extension by five residues, and deleted 16 residues from the C-terminal extension. • Results: High-resolution gel filtration chromatography and non-denaturing gel electrophoresis show that the mutations did not influence dimerisation of the βB1 crystallin, while circular dichroism and tryptophan fluorescence indicated that the mutations did not have a major influence on βB1 crystallin structure or its heat stability. • Conclusions: Our experiments show that as with rat lens βB2 crystallin, dimerisation of βB1 crystallin is not affected by alterations to the conserved PAPA region and that the peptide linker region rather than the N- and C-terminal extensions must be important in dimerisation.