Cysteine residue is not essential for CPM protein thermal-stability assay

Zhaoshuai Wang, Cui Ye, Xinyi Zhang, Yinan Wei

Research output: Contribution to journalArticlepeer-review

11 Scopus citations


A popular thermal-stability assay developed especially for the study of membrane proteins uses a thiol-specific probe, 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM). The fluorescence emission of CPM surges when it forms a covalent bond with the side chain of a free Cys, which becomes more readily accessible upon protein thermal denaturation. Interestingly, the melting temperatures of membrane proteins determined using the CPM assay in literature are closely clustered in the temperature range 45–55 °C. A thorough understanding of the mechanism behind the observed signal change is critical for the accurate interpretation of the protein unfolding. Here we used two α-helical membrane proteins, AqpZ and AcrB, as model systems to investigate the nature of the fluorescence surge in the CPM assay. We found that the transition temperatures measured using circular-dichroism (CD) spectroscopy and the CPM assay were significantly different. To eliminate potential artifact that might arise from the presence of detergent, we monitored the unfolding of two soluble proteins. We found that, contrary to current understanding, the presence of a sulfhydryl group was not a prerequisite for the CPM thermal-stability assay. The observed fluorescence increase is probably caused by binding of the fluorophore to hydrophobic patches exposed upon protein unfolding.

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Original languageEnglish
JournalAnalytical and Bioanalytical Chemistry
Issue number13
StateAccepted/In press - Mar 14 2015

Bibliographical note

Funding Information:
We thank Dr Trevor Creamer (University of Kentucky) for helpful discussions. This work was supported by the National Science Foundation (MCB 1158036, YW), National Institute of Health (1R21AI103717, YW), and Kentucky Science and Engineering Foundation (KSEF-148-502-14-332, YW).

Publisher Copyright:
© 2015 Springer-Verlag Berlin Heidelberg


  • Maleimide
  • Membrane-protein folding
  • Protein denaturation
  • Thiol-specific labeling

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry


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