Physics (Internal)

Vibrational studies of biological chromophores: Raman spectroscopy of the Yellow Fluorescent Protein and ultrafast infrared spectroscopy of the bacterial blue light sensing protein, AppA.

This event is part of the Biophysics/Condensed Matter Seminar Series.

Abstract: Light driven reactions are fundamental to life on earth and include biological processes such as photosynthesis as well as the photophobic and phototaxic responses of organisms to sunlight. Biological systems interact with photons via chromophores that are bound within a protein matrix, and this work focuses on two light-activated systems that differ fundamentally in their response to light. In fluorescent proteins, protein-chromophore interactions have evolved to maximize the fluorescent quantum yield from the chromophore, while in the bacterial anti-repressor AppA photoexcitation causes structural changes in the protein that result in dissociation of a transcription factor and an inhibition of photosystem biosynthesis. Fluorescent proteins, such as the well-known green fluorescent protein (GFP), are used extensively in biology as genetically encoded imaging agents in live cells. Many mutants of GFP have been created to tailor absorption and emission maxima, including yellow fluorescent protein (YFP) a GFP variant that emits at 527 nm. In addition to fluorescence emission, irradiation of YFP leads to the formation of non-fluorescent states which can potentially complicate imaging applications. Steady-state vibrational spectroscopy has been used to determine that formation of the non-fluorescent state in YFP involves cis to trans isomerization of the 4-hydroxybenzylidene imidazolinone chromophore, a structural change that is normally inhibited by the protein matrix. In the blue-light-utilizing FAD (BLUF) protein AppA, the signaling state is formed within a nanosecond of excitation, and picosecond infrared absorption spectroscopy has been used to elucidate early structural events in the AppA photocycle. The flavin chromophore in AppA is rigid, raising questions concerning how excitation is coupled to changes in protein structure. In the time resolved infrared data, a mode is observed at 1666 cm-1 in the dark and light states of AppA whose origin is under debate. Using site-directed mutagenesis and isotope-labeling of the flavin cofactor, the 1666 cm-1 mode is assigned to the side chain of Q63, a residue that is hydrogen-bonded to the flavin. Photoexcitation of AppA is proposed to result in tautomerism followed by rotation of the Q63 amide side chain, resulting in alterations in hydrogen-bonding that lead to formation of the signaling state of the protein.