P-sheet structure is the major fold in thousands of proteins. It is also the structural theme in amyloid fibers, and non-amyloid polymeric aggregates implicated in a variety of human diseases. Deciphering the amino acid'sequence control of (3-sheet and P-helixfolding continues to be a pressing biomedical goal. This proposal focuses on elucidating the amino acid sequence control and processes in the folding and misfolding of two important classes of p-structures. The Greek key (3-sandwichof the eye lens crystallins is needed for lens transparency and avoiding cataract. The parallel p-helix is found in viral adhesins, microbial virulence factors and auto transporters of significantbacterial pathogens. The (3-sandwichproteins under study are the monomeric Human vD-and vS-crystallins. The p-helical proteins under study are the trimeric P22 tailspike and monomeric chondroitinase B.These proteins refold in the test tube without chaperones or other helpers, and also exhibit competing off pathway polymerization both to polymeric aggregates and to amyloid fibers. The experiments pay particular attention to the role of buried hydrophobic and aromatic residues, utilizing the fluorescence quenching of tryptophans as a reporter of p-strand packing. Major goals include elucidating the sequence control of P-sheet folding; determining the difference between the arrangement of p-strands and sheets in the native state, and their arrangement in the aggregated and amyloid states; characterizing the features of the perturbed or partially unfolded conformers which render them precursors of the aggregated and amyloid states; and screening small molecule libraries for their ability to inhibit or alter both the productive folding reactions and the off-pathway aggregation and amyloid reactions. Relevance: a) Elucidation of the amino acid sequence control of p-sandwich folding will improve extraction of information from genome sequences, enhance the ability to design new proteins, and improve prediction and prevention of protein misfolding that plagues biomedical research and biotechnology; b)Resonance Raman characterization of polymerized misfolded subunits offers a new route for developing early diagnosis and detection in protein deposition diseases; c)Small molecule inhibitors of crystallin aggregation and amyloid formation open up new therapeutic approaches for the prevention protein deposition disease such as cataract, and d) Inhibitors of processive P-helix folding represent a novel class of potential antimicrobial agents.
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