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.

Conformational selection and adaptation to ligand binding in T4 lysozyme cavity mutants

“…Introduction of the R1 side chain at surface sites in a protein with a single stable conformation generally leads to decreases, or more rarely, small increases in stability as measured by T m (16,34). For R1 at contact sites between helices, such as 130R1 in the WT′ protein, destabilization is observed due to unfavorable steric interactions (16,28).…”

Optimized clinical performance of growth hormone with an expanded genetic code

“…Building on previous structural work characterizing the hGH receptor-binding interface (7-9), 19 surfaceexposed amino acids distal from the site 1 and 2 receptor-binding domains were selected for substitution with p-acetylphenylalanine (pAcF) and subsequent PEG attachment. pAcF is a nonnative phenylalanine derivative with a p-acetyl group that has chemical reactivity orthogonal to the common 20 amino acids and efficiently forms a covalent oxime bond with an amino-oxy terminated PEG polymer (10,11). Its structural similarity to native amino acids Phe and Tyr reduces the likelihood of conformational perturbations that could lead to loss of function, instability, or immunogenicity.…”

Structural insights into conformational changes of a cyclic nucleotide-binding domain in solution from Mesorhizobium loti K1 channel

“…One way to provide such structural information is the use of double electron-electron resonance (DEER) technique (36,37). If combined with rapid-mixing techniques, DEER could provide a distance map of selected residues along the reaction pathway and, thereby, reveal the pattern of conformational movements.…”