3HWL

Crystal Structure of T4 lysozyme with the unnatural amino acid p-Acetyl-L-Phenylalanine incorporated at position 131

Summary for 3HWL

• Entry DOI: 10.2210/pdb3hwl/pdb
• Related: 139L 1T6H
• Descriptor: Lysozyme, AZIDE ION, CHLORIDE ION, ... (4 entities in total)
• Functional Keywords: unnatural amino acid, p-acetyl-phenylalanine, antimicrobial, bacteriolytic enzyme, glycosidase, hydrolase
• Biological source: Enterobacteria phage T4
• Total number of polymer chains: 1
• Total formula weight: 18929.95

Authors

Fleissner, M.R.,Cascio, D.,Schultz, P.G.,Hubbell, W.L. (deposition date: 2009-06-17, release date: 2009-12-08, Last modification date: 2023-12-27)

Primary citation

Fleissner, M.R.,Brustad, E.M.,Kalai, T.,Altenbach, C.,Cascio, D.,Peters, F.B.,Hideg, K.,Peuker, S.,Schultz, P.G.,Hubbell, W.L.
Site-directed spin labeling of a genetically encoded unnatural amino acid.
Proc.Natl.Acad.Sci.USA, 106:21637-21642, 2009

PubMed 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.

PubMed: 19995976
DOI: 10.1073/pnas.0912009106

Experimental method

X-RAY DIFFRACTION (1.8 Å)