The work proposed in Project 1 is based on the first observation that one can design short Catalytic AMyloid-forming Peptides (CAMPs) to catalyze chemical reactions with high efficiency in addition to their own self-assembly. The goals of the proposed work are: 1. to understand the mechanism of peptide self-assembly and how the resulting amyloid fibril tunes the properties of the metal ion for catalysis; 2. using 2D-IR, to establish structural models for the CAMPs to set important structural and functional reference points for the broad community of scientists interested in the role of amyloids in protein folding, catalysis and health. 3. to examine the ability of CAMPs to effectively utilize different metals to catalyze redox reactions. 4. to develop complex multidomain CAMPs with tunable structural features and degree of assembly. Development and characterization of catalytic amyloids will advance several fields of biomedical importance. The structure- activity relationships and structural insights generated in the proposed work will help us better understand the mechanisms of amyloid toxicity and will improve our knowledge of the structures adopted by more complex amyloid-forming proteins. In addition to its practical value this research program will have a profound impact on our understanding of the fundamental aspects of catalysis. Project 2. The ability of pathogens to neutralize drugs via a newly developed catalytic activity is one of the mechanisms of drug resistance. Therefore, deeper understanding of the factors that determine the ability of proteins to catalyze new chemical transformations is of paramount importance.
We aim to determine the factors that guide evolution of protein function at a molecular level and use these principles to create catalysts for chemical transformations not found in nature. Specifically, we will: 1. combine minimalist computational approach with sophisticated protein engineering tools to test the limits of protein evolvability; 2. create new protein catalysts for a number of different chemical transformations; 3. establish a new method to efficiently guide directed evolution. Project 3 aims to develop a new environmentally insensitive fluorescent probe to study the mechanism of pore formation by antimicrobial peptides and to elucidate the mechanism of proton conductance by influenza A M2 and hepatitis C p7 ion channels. Specifically we will: 1. gain a thorough understanding of the fluorescence characteristics of AzAla, an unnatural amino acid that contains azulene; 2. develop efficient protocols for in vivo incorporation of AzAla into proteins; 3. use AzAla to determine the pKa values of the key proton-conducting histidine residues in M2 and p7.
The proposed work will advance our understanding of the mechanisms of amyloid toxicity, paving the way to new approaches in treatment of neurodegenerative diseases. Through deeper understanding of the factors that determine the ability of proteins to catalyze new chemical transformations the proposed work will help combat drug resistance. The proposed work will also aid in development of new drugs for treatment of influenza and hepatitis C.
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