The pathways through which amino acid sequences direct the intracellular folding of polypeptide chains into beta-sheets and beta-helices remain unclear. This limits the ability to extract information from human and other genome sequences. An additional problem in biomedical research and the biotechnology industry is the failure of many protein chains expressed from cloned genes to fold into their native state, instead associating into inclusion bodies. Related protein misfolding and aggregation processes underly a number of human amyloid and protein deposition diseases.A subclass of beta-sheets is the processive parallel beta-helix fold. For the parallel beta-helix P22 tailspike trimer, partially folded intermediates in both in vitro and in vivo folding and inclusion body pathways have been characterized. In the past period of GM17,980, we have resolved additional subunit assembly intermediates, and isolated and characterized three new classes of folding mutants (in addition to temperature sensitive folding mutants, and their global suppressors); buried hydrophobic core mutants, triple beta-helix assembly mutants, and cysteine folding mutants. These identity sets of residues directing difterent stages of chain folding and assembly. Using monoclonal antibodies, we identified a role for the ribosome itself in tailspike nascent chain folding within cells. A triple stranded beta-helical region has been shown to act as a molecular clamp in subunit assembly. An algorithm has been developed for efficiently predicting beta-helices, which identifies surface proteins of many human pathogens as beta-helices. Human gamma-D crystallin mutants associated with juvenile onset cataract have been expressed and characterized, giving insight into their molecular pathology. In the next period, we propose to: a) Identify additional hydrophobic stack residues controlling parallel beta-helix folding in both the talispike and monomeric chondroitinase B; b) Identify early in vitro intermediates in the folding of beta-helices; c) Identity sequences which control the formation of the interdigitated triple beta-helix that acts as a molecular clamp; d) Test whether predicted beta-helices of Helicobacter pylori have the beta-helix structure; e) Pursue the unfolding, refolding and aggregation of the all beta-sheet human aamma-D crvstallin, which forms lens cataracts.
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