OPTIMALLY STABLE PROTEINS FROM PEPTIDE LIBRARIES

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The long-term objective of this application is to design proteins that can be selected ex-vivo for their high stability. Such proteins will be resistant to proteolysis, and will tolerate surface modifications that will reduce their immunogenicity. As such, they will be ideal candidates for therapeutic applications requiring the disruption of endogenous protein-protein interactions. Successful completion of the proposed research will reveal amino-acid sequences that confer optimal folding stability on small globular proteins. An in vitro chemical assembly and selection strategy provides candidate sequences for subsequent in vivo expression. Specifically, assembly of peptide secondary structural elements into a pre-determined fold is accomplished using exchange-labile metal-ligand chemistry. The subunits exchange partners under thermodynamic control, and the most stable associations are isolated and their amino acid sequences determined. DNA cassettes are constructed based on the sequence information returned from the in vitro selection using synthetic trinucleotides. Overexpressed proteins are isolated and characterized. Of particular interest are the folding stabilities of these de novo proteins, and their folding kinetics as compared with natural proteins of similar structure. The creation of proteins with extremely stable folded structures will open the door to protein-based drugs for applications in therapeutic strategies where small molecules are ineffective.