Document Detail

Site-directed spin labeling of a genetically encoded unnatural amino acid.
MedLine Citation:
PMID:  19995976     Owner:  NLM     Status:  MEDLINE    
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.
Mark R Fleissner; Eric M Brustad; Tamás Kálai; Christian Altenbach; Duilio Cascio; Francis B Peters; Kálmán Hideg; Sebastian Peuker; Peter G Schultz; Wayne L Hubbell
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.     Date:  2009-12-07
Journal Detail:
Title:  Proceedings of the National Academy of Sciences of the United States of America     Volume:  106     ISSN:  1091-6490     ISO Abbreviation:  Proc. Natl. Acad. Sci. U.S.A.     Publication Date:  2009 Dec 
Date Detail:
Created Date:  2010-01-18     Completed Date:  2010-02-18     Revised Date:  2013-05-31    
Medline Journal Info:
Nlm Unique ID:  7505876     Medline TA:  Proc Natl Acad Sci U S A     Country:  United States    
Other Details:
Languages:  eng     Pagination:  21637-42     Citation Subset:  IM    
Jules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095, USA.
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MeSH Terms
Models, Molecular
Phenylalanine / analogs & derivatives*,  chemistry,  genetics
Spin Labels*
Grant Support
R01 EY005216-29/EY/NEI NIH HHS; R01 GM062159-10/GM/NIGMS NIH HHS; R01EY05216/EY/NEI NIH HHS; R01GM062159/GM/NIGMS NIH HHS; RT32EY007026/EY/NEI NIH HHS
Reg. No./Substance:
0/3-(4-acetylphenyl)-2-aminopropanoic acid; 0/Spin Labels; 63-91-2/Phenylalanine
Erratum In:
Proc Natl Acad Sci U S A. 2010 Mar 23;107(12):5693
Note: Peuker, Sebastian [added]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

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