Beta-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 Beta-sheet and Beta-helix folding 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 beta-structures. The Greek key Beta-sandwich of the eye lens crystallins is needed for lens transparency and avoiding cataract. The parallel Beta-helix is found in viral adhesins, microbial virulence factors and auto transporters of significant bacterial pathogens. The Beta-sandwich proteins under study are the monomeric Human gammaD-and gammaS-crystallins. The Beta-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 Beta-strand packing. Major goals include elucidating the sequence control of Beta-sheet folding; determining the difference between the arrangement of Beta-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 Beta-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 Beta-helix folding represent a novel class of potential antimicrobial agents. ? ? ?

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Macromolecular Structure and Function B Study Section (MSFB)
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Basavappa, Ravi
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Massachusetts Institute of Technology
Schools of Arts and Sciences
United States
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Raytcheva, Desislava A; Haase-Pettingell, Cameron; Piret, Jacqueline et al. (2014) Two novel proteins of cyanophage Syn5 compose its unusual horn structure. J Virol 88:2047-55
Zhu, Bin; Tabor, Stanley; Raytcheva, Desislava A et al. (2013) The RNA polymerase of marine cyanophage Syn5. J Biol Chem 288:3545-52
Moreau, Kate L; King, Jonathan A (2012) Cataract-causing defect of a mutant ýý-crystallin proceeds through an aggregation pathway which bypasses recognition by the ýý-crystallin chaperone. PLoS One 7:e37256
Takata, Takumi; Haase-Pettingell, Cameron; King, Jonathan (2012) The C-terminal cysteine annulus participates in auto-chaperone function for Salmonella phage P22 tailspike folding and assembly. Bacteriophage 2:36-49
Moreau, Kate L; King, Jonathan A (2012) Protein misfolding and aggregation in cataract disease and prospects for prevention. Trends Mol Med 18:273-82
Raytcheva, Desislava A; Haase-Pettingell, Cameron; Piret, Jacqueline M et al. (2011) Intracellular assembly of cyanophage Syn5 proceeds through a scaffold-containing procapsid. J Virol 85:2406-15
Kong, Fanrong; King, Jonathan (2011) Contributions of aromatic pairs to the folding and stability of long-lived human ýýD-crystallin. Protein Sci 20:513-28
Knee, Kelly M; Goulet, Daniel R; Zhang, Junjie et al. (2011) The group II chaperonin Mm-Cpn binds and refolds human ?D crystallin. Protein Sci 20:30-41
Acosta-Sampson, Ligia; King, Jonathan (2010) Partially folded aggregation intermediates of human gammaD-, gammaC-, and gammaS-crystallin are recognized and bound by human alphaB-crystallin chaperone. J Mol Biol 401:134-52
Das, Payel; King, Jonathan A; Zhou, Ruhong (2010) beta-Strand interactions at the domain interface critical for the stability of human lens gammaD-crystallin. Protein Sci 19:131-40

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