title: Photo-dynamics of photoactivated adenylyl cyclase TpPAC from the spirochete bacterium Turneriella parva strain H(T).
abstract: The photoactivated adenylyl cyclase TpPAC from the spirochete bacterium Turneriella parva was synthesized and the purified recombinant protein was characterized by biochemical and optical spectroscopic methods. TpPAC consists of a BLUF domain (BLUF = Blue Light sensor Using Flavin) and an adenylyl cyclase homology domain (CHD). A light induced cAMP cyclase activity of ≈ 53.3 nmolmg(-1)min(-1) was measured while in the dark the cyclase activity was approximately a factor of 240 lower. The photo-cycling dynamics of the BLUF domain of TpPAC was studied by absorption spectra, fluorescence quantum distribution, and fluorescence lifetime measurements. The quantum efficiency of BLUF domain signaling state formation was found to be ϕs ≈ 0.59. A three-component exponential recovery of the signaling state to the receptor state was observed with the time constants τrec,1 = 4.8s, τrec,2 = 34.2s, and τrec,3 = 293s at 21.3 °C. The protein thermal stability was studied by stepwise sample heating and cooling. An apparent TpPAC melting temperature of ϑm ≈ 46 °C was determined. The photo-degradation of TpPAC in the signaling state was studied by prolonged intense light exposure at 455 nm. An irreversible flavin photo-degradation was observed with quantum yield ϕD ≈ 8.7 × 10(-6).
authors.affiliation: Fakultät für Physik, Universität Regensburg, Universitätsstraße 31, D-93053 Regensburg, Germany. Electronic address: alfons.penzkofer@physik.uni-regensburg.de.
authors.forename: Alfons
authors.initials: A
authors.lastname: Penzkofer
authors.affiliation: Department of Biochemistry, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021, India.
authors.forename: Meenakshi
authors.initials: M
authors.lastname: Tanwar
authors.affiliation: Department of Biochemistry, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021, India.
authors.forename: Sindhu Kandoth
authors.initials: SK
authors.lastname: Veetil
authors.affiliation: School of Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India.
authors.forename: Suneel
authors.initials: S
authors.lastname: Kateriya
mesh_terms: D000262:Adenylyl Cyclases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005415:Flavins / Q000737:chemistry; D016953:Leptospiraceae / Q000201:enzymology* / Q000528:radiation effects; D008027:Light; D008969:Molecular Sequence Data; D055550:Protein Stability / Q000528:radiation effects; D017434:Protein Structure, Tertiary; D011789:Quantum Theory; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence; D044366:Transition Temperature
chemical_list: D001426:Bacterial Proteins; D005415:Flavins; D011994:Recombinant Proteins; D000262:Adenylyl Cyclases
keywords: Apparent protein melting temperature; BLUF domain photocycle; Optogenetics; Photo-degradation; Photoactivated adenylyl cyclase TpPAC from Turneriella parva; cAMP cyclase activity