Objective
In the lens, beta crystallins are found as oligomers, ranging from dimers to octamers. The first step in oligomeric assembly is the interaction of the N-terminal and C-terminal domains from 2 separate polypeptides which ensures dimerization. Protein engineering studies have been carried out to determine whether the N-terminal and C-terminal arms of the beta crystallins contribute to stability or higher assembly. The betaB2 crystallin was chosen as subject for these experiments since it is the only beta crystallin of which the structure has been solved crystallographically. In vivo betaB2 crystallin is found not only as a homodimer but also as a heterodimer with one of the acidic beta crystallins.
Deletion of the N-terminal arm of betaB2 crystallin did not appear to affect dimerization, as expected from novel crystallographic data, which showed that the N-terminal arm mediated interaction between tetramers. Unexpectedly, upon first analysis, the N-terminal domain (betaB2-86stop) appeared to form a tetramer. The structural interpretation of this finding was not clear.
Data pointed to a role of the N-terminal extension of the beta crystallins in tetramer interactions. It is noteworthy that the sequence of the N-terminal and C-terminal extensions was generally much less conserved than that of the domains. A prime example of this difference in evolutionary rates of change between arms and domains was betaA4 crystallin. The amino acid identity between calf and Xenopus sequences in the N-terminal arm was only 30%, whilst in the domains it was 78%. Expression of rat beta crystallins in the Xenopus lens thus placed these in a mutant environment with respect to the N-terminal arms and thus provided a test of the extent to which the interactions between the N-terminal arms were sequence specific.
A solid experimental basis for the expression of heterologous genetic information in the tadpole eye lens has been established and manipulation of the transparency of the lens has been shown to be possible.
To elucidate the molecular basis for the complex inter-molecular interactions shown by the lens-specific beta-crystallins, models of such complex aggregates will be constructed, based upon the crystallographically determined structure of the betaB2-crystallin dimer. The predictions of these studies will be tested by protein engineering of the beta-crystallins. The basic modules of these proteins will be mutated or interchanged with particular emphasis on the role played by the N-terminal extension in oligomer formation. The role of the beta-crystallin aggregates in the functioning of the lens will be determined in transgenic XENOPUS tadpoles expressing either mutant beta-crystallin genes or anti-sense RNA constructs (to silence the endogenous gene).
Fields of science (EuroSciVoc)
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
- natural sciences biological sciences biochemistry biomolecules proteins
- natural sciences biological sciences genetics RNA
- natural sciences chemical sciences organic chemistry amines
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Coordinator
6500 GL NIJMEGEN
Netherlands
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