Protein engineering through in vivo incorporation of phenylalanine analogs

unnatural amino acids

Carrico, Isaac Sheridan ; Tirrell, David A. (advisor)

University:California Institute of Technology

Department:Chemistry and Chemical Engineering

关键词: unnatural amino acids;   

Others  :  https://thesis.library.caltech.edu/3375/1/Chapter1.pdf

美国|英语

来源: Caltech THESIS

【 摘 要 】

Proteins mediate the bulk of biochemical functions within the cell.These biopolymers control processes utilizing specific arrangements of the natural twenty amino acids.Expanding the set of amino acids available could both aid in the study of these macromolecules as well as significantly increase their functional capabilities.A set of enzymes known as aminoacyl tRNA-synthetases lies at the heart of the fidelity of translation, the process by which genetic information gets decoded into proteins.These synthetases accurately charge a specific tRNA with its cognate amino acid in the presence of the other nineteen natural amino acids.Interestingly these enzymes demonstrate a much higher level of promiscuity with unnatural amino acids.However, acceptable amino acids are limited to those that bear steric and electronic resemblance to the natural analog.Our efforts to expand the substrate set of phenylalanyl-tRNA (PheRS) synthetase are described in Chapters 2-4.We redesigned the catalytic site of PheRS computationally.These results combined with an already known mutant allowed us to rationally create a third mutant.All three mutants were characterized for their ability to activate a large panel of unnatural amino acids in vitro.Further, we were able to confirm the in vivo incorporation of a number of these analogs.In vitro and in vivo results were consistent and defined an expanded substrate set for the described mutants.This substrate set includes a number of analogs that are dramatically different from phenylalanine both sterically and electronically, as well as a number which contain chemical moieties valuable to protein engineering efforts.One example is para-azidophenylalanine (pN3Phe), which provides access to photochemistry as well as modified Staudinger ligations and copper mediated electrocyclizations.In Chapter 5 we describe utilization pN3Phe as a photocrosslinking reagent.Our aim was to create photochemically crosslinkable artificial extracellular matrix proteins for the production of synthetic vascular grafts.These proteins, produced in E. coli, were diblocks of endothelial cell binding domains and structural domains including the pN3Phe site.Photochemical crosslinking of these constructs provided moduli well within the range presented by the natural vascular wall.Chapter 6 describes our ability to photopattern films composed of the above protein.Photopatterning provided a means to spatially array endothelial cells, based upon a number of controllable processing parameters of such films.The final chapter details the utilization of incorporated unnatural amino acids, particularly para-iodophenylalanine, para-acetylphenylalanine and homopropargylglycine, to access Pd(0) catalyzed cross-coupling chemistry.We demonstrated this chemistry exhibits the characteristics necessary for chemoselective ligations.Futher, we demonstrated the selective modification of proteins incorporating all of the above analogs.