NIH Award #GM17980, "Folding annd Misfolding of Parallel Beta-Helix Proteins"
Summary: 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.
A protein is a long polymer of amino acids, the sequence of which is determined by the genetic code of DNA (another long polymer). But unlike DNA, most proteins adopt a highly complex final structure, driven entirely by the interactions of amino acid side chains with other side chains and the surrounding solvent. Proteins fold very quickly, despite the enormous (try 10E90!) number of potential starting conformations. Hence it is expected that protein folding follows a directed pathway, reducing the effective number of conformations. The trick is to figure out what determines the pathway: what interactions of the side chains are necessary and sufficient for directing the polypeptide chain to the final native state?
Our laboratory has described the in vitro and in vivo folding pathways for the tailspike protein of bacteriophage P22. The native structure of tailspike, comprised of three polypeptide chains of 666 amino acids each, is dominated by its extended, repeating b-coil motif, and the overall resemblance of the C-terminal 5/6ths of the protein to a fish (see image above).
Want to learn more about protein folding/misfolding? Read this general-audience article .
Under favorable conditions, most proteins have no problem quickly folding to their native structures. However, there are some proteins which appear unable to fold without the presence of other helper proteins, called chaperones. In the absence of chaperones, these proteins will fail to achieve their native state and instead may associate with other unfolded polypeptide chains to form large aggregate structures (or in vivo, this may result in inclusion body formation). A similar scenario can occur when a protein acquires a mutation, a genetic change resulting in the alteration of one of the amino acid sites on the chain. Misfolding can also occur when a protein is subjected to unfavorable conditions, such as extremes of heat or pH. Clearly, understanding the physical basis for why folding has failed in these cases can provide clues as to the interactions vital for successful protein folding.
The King lab has characterized over 70 genetically-isolated point mutations in the tailspike sequence, and demonstrated that, for the most part, these mutations have no effects on native protein stability. The mutations do, however, have wide-ranging effects on the ability of the protein to fold to its native structure, and hence are assumed to affect the stability or structure of a folding intermediate.
Want to learn more about protein folding/misfolding? Read this general-audience article .
Viruses have long been known as sources of human disease, but have now also become important tools of biological research used in DNA cloning, targeted gene therapy, and phage display of novel proteins. Although viruses are extremely diverse in their life cycles and infectious mechanisms, most share a common structure, consisting of an inner core of condensed nucleic acid enclosed within a spherical protein capsid. The structures of these capsids are based upon icosahedral symmetry.
In the case of herpesviruses and adenoviruses, as well as the double-stranded DNA bacteriophages such as P22, the initial product of the viral assembly pathway is not an infectious virion but a closed shell that does not contain DNA. These precursor shells, or procapsids, include proteins not found in the mature virion, but essential for their production. These proteins are termed "scaffolding proteins". Our laboratory has described the assembly pathway for P22, focussing on the role of the scaffolding protein in virus assembly.