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Photo-dynamics of photoactivated adenylyl cyclase TpPAC from the spirochete bacterium Turneriella parva strain HT

Paper ID Volume ID Publish Year Pages File Format Full-Text
30109 44460 2015 13 PDF Available
Title
Photo-dynamics of photoactivated adenylyl cyclase TpPAC from the spirochete bacterium Turneriella parva strain HT
Abstract

•Photoactivated adenylyl cyclase TpPAC from Turneriella parva was synthesized.•Adenylyl cyclase activity of TpPAC in dark and light adapted states was measured.•The primary photo-cycle dynamics of TpPAC BLUF domain was studied quantitatively.•Quantum efficiency of BLUF domain signaling state formation was found to be 0.59.•Photo-degradation of flavin in BLUF domain led to reduced flavin–protein adduct.

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 nmol mg− 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 = 4.8 s, τrec,2 = 34.2 s, and τrec,3 = 293 s 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.

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Keywords
Photoactivated adenylyl cyclase TpPAC from Turneriella parva; BLUF domain photocycle; cAMP cyclase activity; Apparent protein melting temperature; Photo-degradation; Optogenetics
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Photo-dynamics of photoactivated adenylyl cyclase TpPAC from the spirochete bacterium Turneriella parva strain HT
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Photochemistry and Photobiology B: Biology - Volume 153, December 2015, Pages 90–102
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us