Infoscience

Thesis

A new method for protein labeling with small molecules based on acyl carrier protein

A new method for the covalent and specific labeling of fusion proteins of carrier proteins (CPs) with small organic molecules has been developed in this work. This technology combines the convenience of expressing genetically tagged reporter proteins with the versatility of synthetic organic molecules. Moreover it promises to overcome some of the limitations of the currently used approaches. The method is based on the posttranslational modification of CPs by phosphopantetheine transferase (PPTase). In this reaction, the 4'-phosphopantetheine group of coenzyme A (CoA) is transferred to a serine residue of CP by PPTase. The PPTase can also use as substrates CoA derivatives that are modified in the thiol moiety by fluorophores or affinity reporter groups that are transferred to CP by PPTase in a covalent and irreversible manner. In this work, several CoA derivatives were synthesized by coupling of CoA with reporter groups functionalized by maleimide. The labeling method using the acyl carrier protein (ACP) and the PPTase (AcpS) from E. coli was applied to the in vitro labeling of purified proteins or in E. coli and yeast lysates, but also to the labeling of proteins expressed on cell surfaces of yeast and mammalian cells. The labeling reaction is fast, specific and quantitative. Pulse-chase labeling experiments with different fluorophores allowed the visualization of different protein generations on yeast cell surfaces. Thus, the method was demonstrated to be attractive for fluorescence microscopy. The second objective was to create a system for the selective labeling of different CPs with different CoA derivatives in the same sample, which requires PPTases with different specificities. The labeling must be performed sequentially, in order that each CP is labeled with only one CoA derivative. The pair peptidyl carrier protein (PCP) from B. brevis and the PPTase from B. subtilis (Sfp) was chosen as counterpart of the pair ACP / AcpS from E. coli. AcpS that is specific towards ACP is used for the first labeling reaction, and after a washing step to remove excess of substrate, the second labeling is performed with Sfp which is promiscuous. The system was successfully tested in vitro in solution and with proteins immobilized on microarrays, and on the surface of yeast and mammalian cells. Finally, the last objective was to reduce the size of the carrier protein (∼ 80 amino acids) to a minimal motif that is efficiently recognized by the PPTase. ACP and PCP were truncated before and after helix II whose residues are involved in the recognition by AcpS and Sfp. The fragments of ACP (aa 27-50) and PCP (aa 37-59) were labeled by AcpS and Sfp respectively, but the kinetics of labeling was slow. Two libraries were created with randomization of the six amino acids around the modified serine. Selections were performed using a phage display system based on the phagemid technology. Mt1 (32 aa) was modified by AcpS at the same rate as wild type ACP. Additional truncations of mt1 sequence yielded mt1.4 (16 aa) that was efficiently recognized by AcpS and weakly by Sfp. In conclusion, this labeling method should become an important tool for studies of cell surface proteins as well as for in vitro applications.

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