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• W2018635548 abstract "We apply the photon counting histogram (PCH) model, a fluorescence technique with single-molecule sensitivity, to study pH-induced conformational changes of cytochrome c. PCH is able to distinguish different protein conformations based on the brightness of a fluorophore sensitive to its local environment. We label cytochrome c through its single free cysteine with tetramethylrhodamine-5-maleimide (TMR), a fluorophore with specific brightnesses that we associate with specific protein conformations. Ensemble measurements demonstrate two different fluorescence responses with increasing pH: (i) a decrease in fluorescence intensity caused by the alkaline transition of cytochrome c (pH 7.0-9.5), and (ii) an increase in intensity when the protein unfolds (pH 9.5-10.8). The magnitudes of these two responses depend strongly on the molar ratio of TMR used to label cytochrome c. Using PCH we determine that this effect arises from the proportion of a nonfunctional conformation in the sample, which can be differentiated from the functional conformation. We further determine the causes of each ensemble fluorescence response: (i) during the alkaline transition, the fluorophore enters a dark state and discrete conformations are observed, and (ii) as cytochrome c unfolds, the fluorophore incrementally brightens, but discrete conformations are no longer resolved. Moreover, we also show that functional TMR-cytochrome c undergoes a response of identical magnitude regardless of the proportion of nonfunctional protein in the sample. As expected for a technique with single-molecule sensitivity, we demonstrate that PCH can directly observe the most relevant conformation, unlike ensemble fluorometry." @default.
• W2018635548 created "2016-06-24" @default.
• W2018635548 creator A5000788964 @default.
• W2018635548 creator A5012643108 @default.
• W2018635548 creator A5024513735 @default.
• W2018635548 date "2005-11-28" @default.
• W2018635548 modified "2023-10-16" @default.
• W2018635548 title "Cytochrome <i>c</i> conformations resolved by the photon counting histogram: Watching the alkaline transition with single-molecule sensitivity" @default.
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• W2018635548 doi "https://doi.org/10.1073/pnas.0508975102" @default.
• W2018635548 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/1308922" @default.
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