Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 105, Issue 31, Pages 10779-10784Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0802737105
Keywords
FRET; protein folding; heme
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Funding
- NIGMS NIH HHS [R01-GM63170, R01 GM063170, R01 GM063170-07, R01 GM063170-06] Funding Source: Medline
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FRET between the zinc porphyrin (ZnP) chromophore in zinc-substituted cytochrome c(Zn-cyt c) and an Alexa Fluor dye attached to specific surface sites was used to characterize Zn-cyt c unfolding. The use of ZnP as a fluorescent acceptor eliminates the need to doubly label the protein with exogenous dyes to perform FRET experiments in which both donor and acceptor fluorescence is monitored. The requirement for attachment of only one dye also minimizes perturbation to the protein. This sensitive technique allowed for the determination of distances between the label placed at six different sites and ZnP through a range of denaturant concentrations. Fitting of the data to a three-state model provides distances in the unfolding intermediate. The use of ZnP as a fluorescent acceptor of energy in FRET has a significant potential for application to a range of other systems including heme-binding proteins and proteins to which a covalently attached heme tag may be added.
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