1. Photo-dynamics of photoactivated adenylyl cyclase TpPAC from the spirochete bacterium Turneriella parva strain H(T).
- Author
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Penzkofer A, Tanwar M, Veetil SK, and Kateriya S
- Subjects
- Adenylyl Cyclases chemistry, Adenylyl Cyclases genetics, Amino Acid Sequence, Bacterial Proteins chemistry, Bacterial Proteins genetics, Flavins chemistry, Leptospiraceae radiation effects, Light, Molecular Sequence Data, Protein Stability radiation effects, Protein Structure, Tertiary, Quantum Theory, Recombinant Proteins biosynthesis, Recombinant Proteins chemistry, Recombinant Proteins isolation & purification, Sequence Alignment, Spectrometry, Fluorescence, Transition Temperature, Adenylyl Cyclases metabolism, Bacterial Proteins metabolism, Leptospiraceae enzymology
- 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)., (Copyright © 2015 Elsevier B.V. All rights reserved.)
- Published
- 2015
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