Showing 1 - 3 of 3 results
1.
Photo-dynamics of BLUF domain containing adenylyl cyclase NgPAC3 from the amoeboflagellate Naegleria gruberi NEG-M strain.
Abstract:
The absorption and emission spectroscopic behavior of the photo-activated adenylyl cyclase NgPAC3 from the amoeboflagellate Naegleria gruberi NEG-M strain was studied. The flavin cofactor was found to be partly fully oxidized and partly fully reduced. The typical BLUF domain (BLUF = Blue Light sensor Using Flavin) oxidized flavin absorption photo-cycle dynamics with about 14 nm flavin absorption red-shift in the signaling state was observed. The quantum efficiency of signaling state formation was determined to be s = 0.66 ± 0.03. A bi-exponential signaling state recovery to the dark-adapted receptor state was observed with the time constants rec,f = 275 s and rec,sl = 45 min. The thermal irreversible protein unfolding was studied and an apparent protein melting temperature of ϑm ≈ 50 ◦C was found. The photodynamic behavior of NgPAC3 is compared with the behavior of the previously investigated photo-activated cyclases NgPAC1 (nPAC) and NgPAC2 from the same N. gruberi NEG-M strain. Purified recombinant NgPAC3 showed light-gated adenylate cyclase activity upon illumination with blue light. Its cyclase activity is compared with those of NgPAC1 and NgPAC2.
2.
Photo-dynamics and thermal behavior of the BLUF domain containing adenylate cyclase NgPAC2 from the amoeboflagellate Naegleria gruberi NEG-M strain.
Abstract:
The absorption and emission spectroscopic behavior of the photo-activated adenylate cyclase NgPAC2 from the amoeboflagellate Naegleria gruberi NEG-M strain was studied in the dark, during blue-light exposure and after blue-light exposure. The typical BLUF domain (BLUF = Blue Light sensor Using Flavin) flavin cofactor absorption and fluorescence photo-cycle dynamics was observed. For fresh samples a reversible concentration dependent protein oligomerization occurred showing up in free flavin binding and protein color center formation with increasing protein concentration. Thermal and temporal irreversible protein unfolding with loss of BLUF domain activity was investigated. Temperature dependent protein melting times and the apparent protein melting temperature were determined. The photodynamic behavior of the NgPAC2 is compared with the behavior of the previously investigated photo-activated cyclase NgPAC1 (nPAC) from the same N. gruberi NEG-M strain.
3.
Light modulation of cellular cAMP by a small bacterial photoactivated adenylyl cyclase, bPAC, of the soil bacterium Beggiatoa.
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Stierl, M
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Stumpf, P
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Udwari, D
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Gueta, R
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Hagedorn, R
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Losi, A
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Gärtner, W
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Petereit, L
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Efetova, M
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Schwarzel, M
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Oertner, TG
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Nagel, G
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Hegemann, P
Abstract:
The recent success of channelrhodopsin in optogenetics has also caused increasing interest in enzymes that are directly activated by light. We have identified in the genome of the bacterium Beggiatoa a DNA sequence encoding an adenylyl cyclase directly linked to a BLUF (blue light receptor using FAD) type light sensor domain. In Escherichia coli and Xenopus oocytes, this photoactivated adenylyl cyclase (bPAC) showed cyclase activity that is low in darkness but increased 300-fold in the light. This enzymatic activity decays thermally within 20 s in parallel with the red-shifted BLUF photointermediate. bPAC is well expressed in pyramidal neurons and, in combination with cyclic nucleotide gated channels, causes efficient light-induced depolarization. In the Drosophila central nervous system, bPAC mediates light-dependent cAMP increase and behavioral changes in freely moving animals. bPAC seems a perfect optogenetic tool for light modulation of cAMP in neuronal cells and tissues and for studying cAMP-dependent processes in live animals.
