Összes szerző

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Raics Katalin

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Bódis Emőke
Accelerated electron transfer and increased enzymatic activity in genetically modified photoactivable adenylate cyclase OaPAC

Aug 31 - csütörtök

10:10 – 10:30

Bioenergetika és fotobiofizika

E39

Accelerated electron transfer and increased enzymatic activity in genetically modified photoactivable adenylate cyclase OaPAC

Emoke Bodis¹, Katalin Raics¹, Katalin Pirisi ¹, Zsuzsanna Fekete¹, Nikolett Kis-Bicskei¹, Ildiko Pecsi ¹, Kinga Pozsonyi Ujfalusi ¹, Elek Telek ¹, Marten H. Vos ², and Andras Lukacs ¹

¹ Department of Biophysics, Medical School, University of Pecs, Szigeti str. 12, 7624 Pecs, Hungary.

² Laboratoire d’Optique et Biosciences, Ecole Polytechnique, 91128 Palaiseau cedex, France

Photoactivable adenylate cyclases (PAC) are light activated enzymes that combine blue light sensing capacity with the ability to convert adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP) and pyrophosphate (PPi) in a light-dependent manner. In most of the known PACs blue light regulation is provided by a BLUF domain which undergoes a structural reorganization after blue-light absorption. This minor structural change then is translated towards the C-terminal of the protein, inducing a larger conformational change that results in the ATP conversion to cAMP. As cAMP is a key second messenger in numerous signal transduction pathways regulating various cellular functions, photoactivable adenylate cyclases are of great interest in optogenetic studies. The optimal optogenetic device must be “silent” in the dark and highly responsive upon light illumination. OaPAC is a very good candidate as its basal activity is very small in the dark and the conversion rates increase 20-fold upon light illumination. In this paper, we studied the effect of replacing D67 to N, in the BLUF domain. This mutation was found to accelerate the primary electron transfer process in the photosensing domain of the protein, as has been predicted. Furthermore, it resulted in a longer lived signalling state, which was formed with a lower quantum yield. We hypothesized that the more effective electron transfer correlates with a more efficient cAMP production. Our studies show that D67N OaPAC mutant has a slightly higher conversion of ATP to cAMP compared to the wild-type OaPAC which points in the direction that by fine tuning the electron transfer process more responsive PACs and optogenetic devices can be generated.

Acknowledgements

A.L. acknowledges funding from the Hungarian National Research and Innovation Office (K-137557) and was supported by PTE ÁOK-KA-2021.