The Folding Mechanism of the Intracellular Lipocalins

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9405282 Ropson The purpose of this study is to examine the kinetic folding mechanism of several members of the intracellular lipocalin family of proteins. These proteins share remarkable structural similarity by x-ray crystallography despite as little as 20% sequence identity. The experiments proposed here will determine if proteins that share the same structure also share the same folding mechanism, even when there are low levels of homology in their sequence. A multifaceted approach will be used to address this problem on all levels of structural organization. Stopped- flow circular dichroism will be used to examine global changes in secondary structure during folding and unfolding. In addition, for proteins in this family that have proton NMR assignments, backbone amide-hydrogen exchange labeling will be used to determine when the specific hydrogen bonds that form the secondary structure and stabilized during the folding process. Formation of tertiary structure during folding will be examined by monitoring tryptophan fluorescence and by site-specific labeling of the protein with fluorine and fluorine NMR. This unique method allows the changes in tertiary contacts at specific sites during folding to be monitored, both at equilibrium and kinetically by stopped-flow NMR. Fluorine NMR has already identified an unusual intermediate that has little if any secondary structure for one member of the family, intestinal fatty-acid binding protein. This intermediate is highly populated at equilibrium at levels of denaturant sufficient to completely unfold the protein by other spectroscopic methods. These studies will be extended to other members of the lipocalin family to determine if the same types of intermediates are present on their folding pathways as well. %%% The mechanism of protein folding continues to be a elusive problem in biochemistry. It is not currently known how the amino acid sequence of a protein determines the final structure of the protein and the structures of the intermediates that might be present during the formation of that structure. This proposal uses a variety of physical methods to determine the mechanism of folding for the intracellular lipocalin family of proteins. The study of this family of proteins will improve our understanding of protein folding in general, and of -sheet proteins in particular, which will have benefits for the production of improved protein products by the biotechnology industry. ***