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 qualitativ

SUBMITTER: Fleissner MR 

PROVIDER: S-EPMC2799802 | biostudies-literature | 2009 Dec

REPOSITORIES: biostudies-literature