Spin-Labeling of Proteins via Genetically Encoded Unnatural Amino Acids



Some of the major challenges of protein structure elucidation are proteins that do not crystallize well for x-ray studies and one that give overly convoluted NMR spectra. One way of resolving this issue is through the addition of radical spin-labels for study via electron paramagnetic resonance (EPR). This is usually done through disulfide linkages between a nitroxide spin-label and a cysteine residue of a protein, which allows for continuous wave and pulse EPR measurements. A wealth of information about the system is gained to provide structural insights. However, this requires one to remove native cysteines, which can significantly alter the overall structure and function of the protein. In this investigation, we use the unnatural amino acid, p-azido-phenylalanine, and an alkyne spin probe to “click” our probe to the model protein, T4 lysozyme. This ensures minimal structural perturbation, and allows this method to be used in proteins with native cysteines and disulfide bonds. Through our studies, we observed that this method works, but requires further refinement, and that it is also superior to the cysteine-based method due to the rigidity of the “click” bond, which gives more accurate measurements. Through further study, we hope to expand this method to more complex systems, and streamline a process for quick and easy unnatural protein labeling which will allow researchers to more easily study protein structure and dynamics.



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