3HWL: Crystal Structure Of T4 Lysozyme With The Unnatural Amino Acid P- Acetyl-L-Phenylalanine Incorporated At Position 131

Citation:
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.
PDB ID: 3HWLDownload
MMDB ID: 78584
PDB Deposition Date: 2009/6/17
Updated in MMDB: 2009/12
Experimental Method:
x-ray diffraction
Resolution: 1.8  Å
Source Organism:
Similar Structures:
Biological Unit for 3HWL: dimeric; determined by software (PISA)
Molecular Components in 3HWL
Label Count Molecule
Proteins (2 molecules)
2
Lysozyme(Gene symbol: e)
Molecule annotation
Chemicals (10 molecules)
1
4
2
6
* Click molecule labels to explore molecular sequence information.

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