Effects of controlled mutations on the N- and C-terminal extensions of chick lens beta B1 crystallin.
BACKGROUND
While gamma-crystallin exists as a monomer, beta-crystallin, which unlike gamma-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 beta B1 crystallin, we have developed a heterologous expression system for chicken beta B1 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 beta B1 crystallin, while circular dichroism and tryptophan fluorescence indicated that the mutations did not have a major influence on beta B1 crystallin structure or its heat stability.


CONCLUSIONS
Our experiments show that as with rat lens beta B2 crystallin, dimerisation of beta B1 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.
Coop
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Division of Cell and Molecular Biology, School of Animal and Microbial Sciences, University of Reading, Berkshire, UK. m.j.c.crabbe@rdg.ac.uk
Goode
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Sumner
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Crabbe
M J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002645:Chickens; D002942:Circular Dichroism; D003459:Crystallins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D015870:Gene Expression; D007908:Lens, Crystalline / Q000737:chemistry*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D013050:Spectrometry, Fluorescence
D003459:Crystallins; D004247:DNA