EFFECTS OF CONTROLLED MUTATIONS ON THE N-TERMINAL AND C-TERMINAL EXTENSIONS OF CHICK LENS BETA-B1 CRYSTALLIN

Background: While gamma-crystallin exists as a monomer, beta-crystalli n, which unlike gamma-crystallin contains N- and C-terminal arms, asso ciates to form homodimers. Methods: In order to answer the question of whether the extensions are involved in dimerisation of chick lens bet a B1 crystallin, we have developed a heterologous expression system fo r chicken beta B1 crystallin in Escherichia coli, and produced three m utations by site-directed mutagenesis. We have substituted residues in the PAPA segment of the N-terminal extension, curtailed the N-termina l extension by five residues, and deleted 16 residues from the C-termi nal extension. Results: High-resolution gel filtration chromatography and non-denaturing gel electrophoresis show that the mutations did not influence dimerisation of the beta B1 crystallin, while circular dich roism and tryptophan fluorescence indicated that the mutations did not have a major influence of beta B1 crystallin structure or its heat st ability. Conclusions: Our experiments show that as with rat lens beta B2 crystallin, dimerisation of beta B1 crystallin is not affected by a lterations to the conserved PAPA region and that the peptide linker re gion rather than the N- and C-terminal extensions must be important in dimerisation.