The incorporation of unnatural amino acids (UAAs) into proteins has become a common tool used to probe protein structure, function, and kinetics in a variety of systems. While it is generally accepted that the incorporation of UAAs does not result in significant perturbations to protein structure, relatively few structures with UAAs have been published showing this. We aim to obtain X-ray crystal structures of the green fluorescent protein (GFP) with the spectroscopic reporter UAAs 4-cyano-L-phenylalanine (pCNPhe), 4-azidomethyl-L-phenylalanine (pN3CH2Phe), or 4-ethynyl-L-phenylalanine (pCCPhe) incorporated at one or more unique sites in the protein � a solvent-accessible (134), semi-buried (150), and well-buried (75) site. These structures will allow us to determine how the environment around an incorporated UAA relates to the overall perturbation of the protein's structure. Crystal diffraction data was collected at beam line NE-CAT 24ID-E at Argonne National Laboratory for seven different GFP constructs. Initial structures of the 134/150 pCCPhe and 134 pCNPhe GFP constructs have been solved to 2.5 � and 2.2 � resolution, respectively, will be presented.

Assessing structural effects of unnatural amino acid incorporation into model system GFP

248th National Meeting (2014)

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