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Site-directed spin labeling SDSL is a powerful tool for the characterization of protein structure and dynamics however its application in many systems is hampered by the reliance on unique and benign cysteine substitutions for the site-specific attachment of the spin label An elegant solution to this problem involves the use of genetically encoded unnatural amino acids UAAs containing reactive functional groups that are chemically orthogonal to those of the 20 amino acids found naturally in proteins These unique functional groups can then be selectively reacted with an appropriately functionalized spin probe In this chapter we detail the genetic incorporation of the ketone-bearing amino acid p-acetyl phenylalanine pAcPhe into recombinant proteins expressed in E coli Incorporation of pAcPhe is followed by chemoselective reaction of the ketone side chain with a hydroxylamine-functionalized nitroxide to afford the spin-labeled side chain K1 and we present two protocols for successful K1 labeling of proteins bearing site-specific pAcPhe We outline the basic requirements for pAcPhe incorporation and labeling with an emphasis on practical aspects that must be considered by the researcher if high yields of UAA incorporation and efficient labeling reactions are to be achieved To this end we highlight recent advances that have led to increased yields of pAcPhe incorporation and discuss the use of aniline-based catalysts allowing for facile conjugation of the hydroxylamine spin label under mild reaction conditions To illustrate the utility of K1 labeling in proteins where traditional cysteine-based SDSL methods are problematic we site-specifically K1 label the cellular prion protein at two positions in the C-terminal domain and determine the interspin distance using double electron-electron resonance EPR Recent advances in UAA incorporation and ketone-based bioconjugation in combination with the commercial availability of all requisite reagents should make K1 labeling an increasingly viable alternative to cysteine-based methods for SDSL in proteins 2015 Elsevier Inc All rights reserved

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Genetic Incorporation of the Unnatural Amino Acid p-Acetyl Phenylalanine into Proteins for Site-Directed Spin Labeling.