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Download or read book Development and Benchmarking of Methods for Computational Design, and Experimental Characterization, of Proteins that Bind Small-Molecule Ligands written by Amanda Lynne Loshbaugh and published by . This book was released on 2019 with total page 144 pages. Available in PDF, EPUB and Kindle. Book excerpt: I present computational and experimental methods relating to the design of binding interactions involving proteins, including interactions of protein/small molecule, dimeric protein/protein, and tertiary protein/small molecule/protein systems. In chapter 2, I describe a benchmark comparison of flexible backbone design methods in Rosetta. Three methods, (1) BackrubEnsemble, (2) CoupledMoves, and (3) FastDesign, were tested for their ability to recapitulate observed protein sequence profiles assumed to represent the fitness landscapes of protein/protein and protein/small molecule binding interactions. We found that CoupledMoves, which combines backbone flexibility and sequence exploration into a single acceptance step during the sampling trajectory, better recapitulates sequence profiles than BackrubEnsemble and FastDesign, which separate backbone flexibility and sequence design into separate acceptance steps during the sampling trajectory. In chapter 3, I describe the screening and characterization of a chemically induced dimer (CID) that detects and responds to the presence of ibuprofen. The protein tool is composed of a sensor module and a reporter module, which are modular and can be interchanged. The sensor module is a heterodimer whose interface contains an ibuprofen binding site transplanted by computational design from a monomeric protein, such that ibuprofen binding induces heterodimerization. The reporter module is a protein complementation system whose complementation is induced by dimerization of the sensor domain. I present two methods to individually screen hundreds of designed CIDs targeting various proteins, (1) using a growth-based reporter module in E coli, and (2) using a luminescent reporter in a cell-free protein expression system. The work presented here represents methodological advances for both the computational and experimental design of protein binding interactions.