Semi-synthetic proteins for catalytic and analytical applications

Synthetic cofactor;Protein labeling;Semi-synthetic protein;Myoglobin

Huettinger, Karl ; Chemistry and Biochemistry

University:Georgia Institute of Technology

Department:Chemistry and Biochemistry

关键词: Synthetic cofactor;    Protein labeling;    Semi-synthetic protein;    Myoglobin;   

Others  :  https://smartech.gatech.edu/bitstream/1853/33823/1/huettinger_karl_j_200905_phd.pdf

美国|英语

来源: SMARTech Repository

【 摘 要 】

Proteins have evolved over millions of years to serve a plethora of highly specialized functions in biological systems. Given the enormous diversity in structure and function, it is truly surprising that only 20 different amino acids are utilized as the building blocks of proteins. Furthermore, only a small set of metal cations that are biologically available are used as structural or catalytically active cofactors in proteins, whereas rare metal cations such as platinum, ruthenium or rhodium remain absent. In the 20th century myriad catalysts, based on non-biological transition metals, emerged that can facilitate numerous organic transformations. The goal of the thesis was to introduce new functions into proteins by attaching platinum metals and fluorescent metal sensors. Thus, semi-synthetic proteins for catalytic and analytical applications were generated.The replacement of organic solvents by environmentally benign solvents such as water is an imperative step towards achieving "green chemistry". The combination of small molecule catalysts with proteins may introduce new functions and take advantage of the benefits of "both worlds" while avoiding their potential drawbacks. Therefore semi-synthetic catalysts were developed for enantioselective organic reactions in aqueous medium.A suitable reaction, reaction conditions and catalytic system for later utilization in a semi-synthetic protein were designed, developed and characterized. Ruthenium porphyrins catalyzed cyclopropanation reactions with fair yields and high stereoselectivity in aqueous medium. The successful reaction in water was a crucial requirement for a catalytically active semi-synthetic protein. Mechanistic studies did not elucidate the actual catalytic species for the formation of the cyclopropanation product and the side-product diethyl maleate; however, new insights were gained from the analysis of potential reaction pathways. Moreover, studies of the influence of axial ligands, resembling likely residues coordinating to the ruthenium metal center in the active site of a semi-synthetic protein, on the carbene formation of ruthenium porphyrins illustrated that coordination of axial ligands may inhibit the catalytic activity.The generation of ruthenium porphyrin based semi-synthetic proteins and their subsequent catalysis of cyclopropanation reactions was carried out. Myoglobin and myoglobin mutants were successfully reconstituted with a heme-like ruthenium carbonyl porphyrin; however, none of the formed semi-synthetic proteins catalyzed the enantioselective cyclopropanation of styrene. Efforts to determine the reconstitution efficiency of the generated semi-synthetic were hampered by problems to purify the generated semi-synthetic proteins that are probably due to non-specific binding of the ruthenium porphyrin to the protein surface.The exploration of labile metal pools of the biologically relevant transition metals copper, iron and zinc in cells was the goal of developing semi-synthetic proteins for analytical applications. Combining fluorescent proteins with colored or fluorescent metal chelators by forming semi-synthetic proteins allows taking advantage of their beneficial properties while avoiding their downsides. This design offers an attractive platform for in vivo metal sensing.Plasmids encoding fluorescent proteins, targeting sequences and AGT or intein fusion domains (necessary for labeling) for eukaryotic and prokaryotic expression were generated. The targeting of intracellular compartments (mitochondria, nucleus and TGN) was successful (confirmed by light microscopy experiments with transfected mammalian cells). In vitro labeling experiments of expressed and purified fusion proteins with rhodamine derivatives succeeded with AGT based fusion proteins; however, labeling of fusion proteins by trans-splicing with split-inteins failed. A new Zinc(II)-chelator was attached to an AGT based protein and the resulting semi-synthetic protein exhibited strong changes of fluorescence in the presence of zinc(II). This represents an important step towards the goal of in vivo cell imaging of labile zinc(II) pools. Despite extensive efforts, all attempts failed to generate a chelator that forms Cu(I)-complexes with the 1:1 stochiometry (ligand:metal) that is necessary for metal sensing with semi-synthetic proteins.