Bio‐incorporation of Amino Acid Analogs into Target Proteins

Abstract

The 3D structure of a protein is vital for understanding how it functions in both normal conditions as well as in disease. X‐ray crystallography has been the traditional method of determining protein structure, however, the small atoms that compose amino acids are problematic in this approach. This phase problem can be solved through multi‐wavelength anomalous diffraction (MAD) if a heavy atom exists in the protein. Incorporating telluromethionine (TeMet), which includes a tellurium atom in place of the lighter sulfur atom, into proteins will assist in MAD measurements. This is done by providing only TeMet to an auxotrophic Escherichia coli strain that is unable to produce methionine, causing the synthesized proteins to include TeMet. However, it has been shown that TeMet is toxic to cells, and if the cells die, an insufficient amount of the target protein will be synthesized for crystallization. To reduce the toxic effects of TeMet, the E. coli cells have been cultured in chemically defined media with components necessary for cell growth that seem to be lost with incorporation of TeMet. By measuring the absorbance of the cultures over time, the growth curve of the cells can be constructed. Once the optimum growing conditions have been determined, the target protein, dihydrofolate reductase, will be isolated and characterized. Successful incorporation of TeMet will make solving the 3D structure of proteins more accessible, allowing for enhanced disease treatment and drug design.