Applications of Genetically Encoded Unnatural Amino Acids for Protein Structure Mapping by Site-Directed Spin Labeling

Abstract

Site-directed spin labeling (SDSL) is an important method for obtaining structural information on proteins that are refractory to other techniques. In this method, interspin distances are used as structural constraints for protein structure mapping, and here we present two applications of genetically encoded unnatural amino acids for such studies. One application is orthogonal labeling, in which an unnatural amino acid bearing a unique functional group is used to specifically introduce spin labels into proteins that are not amenable to the traditional, thiol-based SDSL method. Recently, a strategy based on p-acetyl-L-phenylalanine was reported [PNAS 2009 106(51):21637], in which the ketone was used to generate a ketoxime-linked spin label (K1). Here, we present an analogous strategy based on p-azido-L-phenylalanine, which can be specifically modified using “click” chemistry by a cyclooctyne-nitroxide reagent to generate a triazole-linked nitroxide side chain (C1). In contrast to the previous orthogonal labeling strategy, the azide-cyclooctyne reaction proceeds uncatalyzed at pH 7, and spectra of C1 mutants of T4 lysozyme (T4L) reflect a more ordered nitroxide motion than the analogous K1 mutant. A second application involves the Cu(II) binding unnatural amino acid (2,2’-bipyridin-5-yl)alanine (BpyAla), which enhances the longitudinal relaxation rate (1/T1) of a nearby nitroxide when copper is bound, thereby allowing average interspin distances to be measured at ambient temperature using saturation recovery EPR. In contrast to other methods for paramagnetic metal introduction, BpyAla can be introduced via a single mutation into any protein topology. Studies of T4L mutants containing BpyAla and a disulfide-linked nitroxide indicate that interspin distances of up to ∼30Å are accessible to T1 based EPR methods compared to ∼20Å for traditional T2 based methods. Both applications are expected to enhance distance mapping capability by EPR spectroscopy.