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. 2008 Dec 31;130(52):17664-5.
doi: 10.1021/ja807430h.

A general and efficient method for the site-specific dual-labeling of proteins for single molecule fluorescence resonance energy transfer

Eric M Brustad  1 , Edward A Lemke, Peter G Schultz, Ashok A Deniz
Affiliations

A general and efficient method for the site-specific dual-labeling of proteins for single molecule fluorescence resonance energy transfer

Eric M Brustad et al. J Am Chem Soc. .

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.

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Figures

Figure 1
Figure 1
a) Oxime ligation with an unnatural ketone amino acid. b) Fluorescence image of site-specific labeled T4 lysozyme after SDS-PAGE separation. c) p-acetylphenylalanine (1)
Figure 2
Figure 2
a) smFRET histograms of double-labeled T4L*K83(1)/T157C. b) contour plot of double-labeled T4L*K83(1)/T157C smFRET unfolding histograms. c) T4L*K83(1)/T157C single molecule unfolding curve d) Peak shift of folded T4L*S38(1)/T157C at low GdmCl concentrations.
See this image and copyright information in PMC

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