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Quantification of free cysteines in membrane and soluble proteins using a fluorescent dye and thermal unfolding

Nature Protocols volume 8, pages 2090–2097 (2013)Cite this article

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Abstract

Cysteine is an extremely useful site for selective attachment of labels to proteins for many applications, including the study of protein structure in solution by electron paramagnetic resonance (EPR), fluorescence spectroscopy and medical imaging. The demand for quantitative data for these applications means that it is important to determine the extent of the cysteine labeling. The efficiency of labeling is sensitive to the 3D context of cysteine within the protein. Where the label or modification is not directly measurable by optical or magnetic spectroscopy, for example, in cysteine modification to dehydroalanine, assessing labeling efficiency is difficult. We describe a simple assay for determining the efficiency of modification of cysteine residues, which is based on an approach previously used to determine membrane protein stability. The assay involves a reaction between the thermally unfolded protein and a thiol-specific coumarin fluorophore that is only fluorescent upon conjugation with thiols. Monitoring fluorescence during thermal denaturation of the protein in the presence of the dye identifies the temperature at which the maximum fluorescence occurs; this temperature differs among proteins. Comparison of the fluorescence intensity at the identified temperature between modified, unmodified (positive control) and cysteine-less protein (negative control) allows for the quantification of free cysteine. We have quantified both site-directed spin labeling and dehydroalanine formation. The method relies on a commonly available fluorescence 96-well plate reader, which rapidly screens numerous samples within 1.5 h and uses <100 μg of material. The approach is robust for both soluble and detergent-solubilized membrane proteins.

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Figure 1: Schematic diagram of the thermofluor assay.
Figure 2: Thermofluor profile.
Figure 3: Comparison of thermal fluorescence with CW-EPR-derived efficiencies of cysteine modification and MS.

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Acknowledgements

We acknowledge the Biomedical Sciences Research Complex Mass Spectrometry and Proteomics Facility for mass spectroscopy analysis and assistance with writing the manuscript; B. Bode and R. Ward for discussing the manuscript; A. Plechanovová at the University of Dundee for providing the plasmid for UbcH5a; and J. Chalker at the University of Oxford for providing the dibromide reagent. The UK Biotechnology and Biological Sciences Research Council (BB/H017917/1), the Wellcome Trust (WT081862) and EaStCHEM (E.B. studentship) funded this work.

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Author notes

  1. Gregor Hagelueken

    Present address: Present address: Institute of Physical and Theoretical Chemistry, University of Bonn, Bonn, Germany.,

  2. Emma Branigan and Christos Pliotas: These authors contributed equally to this work.

Authors and Affiliations

  1. Biomedical Sciences Research Complex, University of St. Andrews, St. Andrews, UK

    Emma Branigan, Christos Pliotas, Gregor Hagelueken & James H Naismith

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  1. Emma Branigan
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Contributions

E.B., C.P. and G.H. performed experiments. E.B., C.P., G.H. and J.H.N. analyzed the data. E.B., C.P. and J.H.N. wrote the paper.

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Correspondence to James H Naismith.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 cwEPR spectra for spin-labeled MscS mutants.

cwEPR spectra for MscS (a) V32R1 (b) S58R1, (c) D67R1, (d) L124R1 and (e) S147R1 and the positive control (f) 4-amino TEMPO with magnetic field (x axis) plotted against intensity (y axis) for each mutant.

Supplementary Figure 2 Thermofluor profile at low concentration.

Thermofluor profile for MscS S147R1 showing the negative control in blue, the positive control in red and the spin-labeled sample in green. Repeat measurements are denoted with (1) a smooth line and (2) a dashed line. The temperature at which the fluorescence intensity was used is highlighted with a black line.

Source data

Supplementary information

Supplementary Methods

Double integration of cwEPR spectra and mass spectroscopy. (PDF 53 kb)

Supplementary Table 1

Efficiency of conversion of cysteine. Table of data presented in Figure 3a showing the percentage of spin-labeling efficiency and the conversion to dehydroalanine calculated using the fluorescence assay and cwEPR (where appropriate), including associated error estimations. (PDF 74 kb)

Supplementary Figure 1

cwEPR Spectra for Spin Labeled MscS Mutants. (PDF 178 kb)

Supplementary Figure 2

Thermofluor Profile at Low Concentration. (PDF 2108 kb)

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Branigan, E., Pliotas, C., Hagelueken, G. et al. Quantification of free cysteines in membrane and soluble proteins using a fluorescent dye and thermal unfolding. Nat Protoc 8, 2090–2097 (2013). https://doi.org/10.1038/nprot.2013.128

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