title: A general and efficient method for the site-specific dual-labeling of proteins for single molecule fluorescence resonance energy transfer.
abstract: A general strategy for the site-specific dual-labeling of proteins for single-molecule fluorescence resonance energy transfer is presented. A genetically encoded unnatural ketone amino acid was labeled with a hydroxylamine-containing fluorophore with high yield (>95%) and specificity. This methodology was used to construct dual-labeled T4 lysozyme variants, allowing the study of T4 lysozyme folding at single-molecule resolution. The presented strategy is anticipated to expand the scope of single-molecule protein structure and function studies.
authors.affiliation: Department of Molecular Biology, Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
authors.forename: Eric M
authors.initials: EM
authors.lastname: Brustad
authors.forename: Edward A
authors.initials: EA
authors.lastname: Lemke
authors.forename: Peter G
authors.initials: PG
authors.lastname: Schultz
authors.forename: Ashok A
authors.initials: AA
authors.lastname: Deniz
mesh_terms: D017122:Bacteriophage T4 / Q000201:enzymology; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006834:Hydrazines / Q000737:chemistry; D009113:Muramidase / Q000737:chemistry* / Q000378:metabolism; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: C417561:Alexa 488 hydrazide; D005456:Fluorescent Dyes; D006834:Hydrazines; D011506:Proteins; D009113:Muramidase