title: Site-directed spin labeling of a genetically encoded unnatural amino acid.
abstract: The traditional site-directed spin labeling (SDSL) method, which utilizes cysteine residues and sulfhydryl-reactive nitroxide reagents, can be challenging for proteins that contain functionally important native cysteine residues or disulfide bonds. To make SDSL amenable to any protein, we introduce an orthogonal labeling strategy, i.e., one that does not rely on any of the functional groups found in the common 20 amino acids. In this method, the genetically encoded unnatural amino acid p-acetyl-L-phenylalanine (p-AcPhe) is reacted with a hydroxylamine reagent to generate a nitroxide side chain (K1). The utility of this scheme was demonstrated with seven mutants of T4 lysozyme, each containing a single p-AcPhe at a solvent-exposed helix site; the mutants were expressed in amounts qualitatively similar to the wild-type protein. In general, the EPR spectra of the resulting K1 mutants reflect higher nitroxide mobilities than the spectra of analogous mutants containing the more constrained disulfide-linked side chain (R1) commonly used in SDSL. Despite this increased flexibility, site dependence of the EPR spectra suggests that K1 will be a useful sensor of local structure and of conformational changes in solution. Distance measurements between pairs of K1 residues using double electron electron resonance (DEER) spectroscopy indicate that K1 will also be useful for distance mapping.
authors.affiliation: Jules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095, USA.
authors.forename: Mark R
authors.initials: MR
authors.lastname: Fleissner
authors.forename: Eric M
authors.initials: EM
authors.lastname: Brustad
authors.forename: Tamás
authors.initials: T
authors.lastname: Kálai
authors.forename: Christian
authors.initials: C
authors.lastname: Altenbach
authors.forename: Duilio
authors.initials: D
authors.lastname: Cascio
authors.forename: Francis B
authors.initials: FB
authors.lastname: Peters
authors.forename: Kálmán
authors.initials: K
authors.lastname: Hideg
authors.forename: Sebastian
authors.initials: S
authors.lastname: Peuker
authors.forename: Peter G
authors.initials: PG
authors.lastname: Schultz
authors.forename: Wayne L
authors.initials: WL
authors.lastname: Hubbell
mesh_terms: D008958:Models, Molecular; D009154:Mutation; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics; D013113:Spin Labels
chemical_list: C476995:3-(4-acetylphenyl)-2-aminopropanoic acid; D013113:Spin Labels; D010649:Phenylalanine