The efficient folding of polypeptide sequences into stable structures is one of the most fascinating and fundamentally important biorecognition phenomena. Studies of the thermodynamics of this process provide insights into the factors that are required for avid binding of peptide drugs and hormones to their receptors. As a result, there is an increasing interest in the de novo design of protein-like scaffolds and the redesign of protein domains to be of the minimum size required for efficient self-assembly. The present proposal addresses peptide structuring requisites as a fold optimization and de novo design problem and also at a more fundamental level (the thermodynamics and rates of secondary structure formation and how these influence the rates and efficiency of protein folding, the quantitation of the deltaG increments due to specific hydrophobic and H-bonding interactions in miniprotein constructs). Some of the key experiments proposed should provide: a) the thermodynamic parameters for the formation of isolated alpha helices and beta hairpins and b) the relative importance of the hydrophobic effect for the stabilization of secondary structures. Some of the initial studies will be conducted in aqueous fluoroalcohol media that appear to accentuate the hydrophobic effect. However, the designed peptides proposed should allow the extension of these studies to strictly aqueous medium. The measurements of the rates of both alpha helix and beta hairpin formation will employ isotope-edited T-jump FT-IR. Stable alpha helices are promising units of scaffolding for designed folds; the helix/coil model developed at U.W. will be extended and reparameterized as a helix design tool and several key questions concerning intrinsic helical propensities and the stabilization increments associated with sidechain interactions will be addressed experimentally. Several novel miniprotein folds are proposed for study. One series consists of non-crosslinked eicosamers that fold cooperatively to produce a hydrophobic cage about a tryptophan ring. Mutants of this structure will be employed for the deltadeltaG measurements, as a model for developing strategies for the optimization of hydrophobic clusters, and as a test case for computer simulations of protein folding. A smaller effort will be directed at the construction and optimization of a betaalphaalpha miniprotein (a parallel beta sheet resulting from the association of beta strands at each end of an alpha helix). These constructs will mimic some features of the B1 domain of protein L but will have a left-handed crossover which has never been observed in nature. The studies described should provide insights and algorithms that will be useful in other peptide structure and ligand/receptor interface optimization efforts. The tryptophan cage fold, which will be examined extensively, is a unique intramolecular example of a binding motif that is common in intermolecular biorecognition phenomena. As a result, this project will provide strategies for designing more stable scaffolds of predictable structure for artificial enzymes and principles for the design of more potent biomolecules.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Bio-Organic and Natural Products Chemistry Study Section (BNP)
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Wehrle, Janna P
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University of Washington
Schools of Arts and Sciences
United States
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Sivanesam, Kalkena; Andersen, Niels H (2016) Modulating the Amyloidogenesis of ?-Synuclein. Curr Neuropharmacol 14:226-37
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