Detection of Forster Cycle in Single Molecules of Green Fluorescent Protein
Abstract
Green Fluorescent Protein (GFP) from the jellyfish Aequorea victoria earned a Nobel Prize in 2008 for being an exceptionally exploitable biological marker in the living systems. The dominant absorption band at 395 nm of GFP consists of a protonated form (A) of the chromophore and involves a protolytic reaction that forms an excited anion of the intermediate form (I*) from the photo-excited protonated form (A*), which upon fast relaxation to the intermediate (I) ground state repopulates the protonated (A) form. The present work reports the observation of this proton movement cycle in the excited state of GFP (i.e. A&rarrA*&rarrI*&rarrI&rarrA, Frster cycle) by acquiring vibrational spectra of single GFP molecule using "nanometal-on-semiconductor" SERS substrates and applying a "pump and probe" technique. A new peak at 1510 cm-1 prominently exists with the protonated form (1560 cm-1). Statistical analysis of the GFP population reveals a higher probability (about 9 times) of observing both 1510 cm-1 and 1560 cm-1 peak over the probability of finding both 1510 cm-1 and 1530 cm-1 in one `spectral jump'. We recognize this unique Raman peak at 1510 cm-1 as a marker for the intermediate form of the chromophore. This peak is also observed both in `Cis' and `Trans' configuration of the chromophore, but considerably more with `Cis' configuration.
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- OSU Theses [15752]