Összes szerző

Lukács András

az alábbi absztraktok szerzői között szerepel:

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.

Lukács András
Single amino acid mutation decouples photochemistry of the BLUF domain from the enzymatic function of OaPAC and drives the enzyme to a switched-on state

Aug 29 - kedd

15:30 – 17:00

I. Poszterszekció

P17

Single amino acid mutation decouples photochemistry of the BLUF domain from the enzymatic function of OaPAC and drives the enzyme to a switched-on state 

Emőke Bódis¹, Jinnette Tolentino Collado², Mihály Szűcs¹, Zsuzsanna Fekete¹, Elek Telek¹, Kinga Pozsonyi¹, Sofia M. Kapetanaki¹, Greg Greetham³, Peter J. Tonge², Stephen R. Meech⁴, and András Lukács¹

¹ Department of Biophysics, Medical School, University of Pécs, 7624 Pécs, Hungary

² Department of Chemistry, Stony Brook University, New York, 11794, United States.

³ Central Laser Facility, Research Complex at Harwell, Rutherford Appleton Laboratory, Didcot, U.K

 ⁴ School of Chemistry, University of East Anglia, Norwich, NR4 7TJ, U.K.

Photoactivated adenylate cyclases (PACs) are light-activated enzymes that combine a BLUF (blue-light using flavin) domain and an adenylate cyclase domain that are able to increase the levels of the important second messenger cAMP (cyclic adenosine monophosphate) upon blue-light excitation [1-3]. The light-induced changes in the BLUF domain are transduced via a mechanism that has not been established yet to the adenylate cyclase domain. One critical residue in the vicinity of the flavin in BLUF proteins is the glutamine amino acid close to the N5 of the flavin[4-6]. The role of this residue has been investigated extensively both experimentally and theoretically. However, its role in the activity of the photoactivated adenylate cyclase, OaPAC has never been addressed. In this work, we have applied ultrafast transient visible and infrared spectroscopies to study the photochemistry of the Q48E OaPAC mutant. This mutation decelerated the primary electron transfer process but switched the enzyme in to a permanent ‘on’ state, able to increase the cAMP levels under dark conditions compared to the wild-type OaPAC. Differential scanning calorimetry measurements pointed out to a less compact structure for the mutant. These findings provide insight into the important elements in PACs and how their fine tuning may result to the design of optogenetic devices.

Acknowledgment

A.L. acknowledges funding from the Hungarian National Research and Innovation Office (K-137557) and was supported by PTE ÁOK-KA-2021 E.T was supported by PTE ÁOK-KA-2022-09.

References

[1] Iseki M, Park SY. Photoactivated Adenylyl Cyclases: Fundamental Properties and Applications. Adv Exp Med Biol. 2021;1293:129-39.

[2] Ohki M, Sugiyama K, Kawai F, Tanaka H, Nihei Y, Unzai S, et al. Structural insight into photoactivation of an adenylate cyclase from a photosynthetic cyanobacterium. Proceedings of the National Academy of Sciences of the United States of America. 2016;113:6659-64.

[3] Collado J, Iuliano J, Pirisi K, Jewlikar S, Adamczyk K, Greetham G, et al. Unraveling the Photoactivation Mechanism of a Light-Activated Adenylyl Cyclase Using Ultrafast Spectroscopy Coupled with Unnatural Amino Acid Mutagenesis. Acs Chemical Biology. 2022;17:2643-54.

[4] Udvarhelyi A, Domratcheva T. Glutamine rotamers in BLUF photoreceptors: a mechanistic reappraisal. J Phys Chem B. 2013;117:2888-97.

[5] Domratcheva T, Hartmann E, Schlichting I, Kottke T. Evidence for Tautomerisation of Glutamine in BLUF Blue Light Receptors by Vibrational Spectroscopy and Computational Chemistry. Sci Rep. 2016;6:22669.

[6] Hontani Y, Mehlhorn J, Domratcheva T, Beck S, Kloz M, Hegemann P, et al. Spectroscopic and Computational Observation of Glutamine Tautomerization in the Blue Light Sensing Using Flavin Domain Photoreaction. J Am Chem Soc. 2023;145:1040-52.

Lukács András
Conformational flexibility in a photoactivated adenylate cyclase studied by fluorescence spectroscopy and solution X-ray scattering

Aug 29 - kedd

09:10 – 09:30

Molekuláris biofizika

E03

Conformational flexibility in a photoactivated adenylate cyclase studied by fluorescence spectroscopy and solution X-ray scattering

1,2, , , András Kengyel, Matteo Levantino, Caroline Maas, Dihia Moussaoui, Ildikó Pécsi, Petra Pernot, Kevin Pounot, Giorgio Schiro, Mark Tully, Kinga Ujfalusi-Pozsonyi, Jovana Vitas, Martin Weik, András Lukács

Sofia Maria Kapetanaki^1,2, Emőke Bódis¹, Miklós Nyitrai¹, András Kengyel¹, Matteo Levantino³, Caroline Maas², Dihia Moussaoui³, Ildikó Pécsi¹, Petra Pernot³, Kevin Pounot^2,3, Giorgio Schirò², Mark Tully³, Kinga Ujfalusi-Pozsonyi¹, Jovana Vitas², Martin Weik², András Lukács¹

¹ Department of Biophysics, Medical School, University of Pécs, 7624 Pécs, Hungary

² CEA–Institut de Biologie Structurale, Grenoble, 38044 France

³ European Synchrotron Radiation Facility, Grenoble, 38043 France

The photoactivated adenylate cyclase from the photosynthetic cyanobacterium Oscillatoria acuminata OaPAC is a homodimeric enzyme comprising of a N-terminal domain that senses blue light using flavin (BLUF) [1] and a C-terminal class III adenylate cyclase (AC) domain that catalyses the formation of cAMP from ATP (adenosine triphosphate) [2,3]. cAMP is a universal regulator of metabolism and gene expression in all life forms [4]. Modulating the cellular concentration of cAMP has emerged in the focus of modern optogenetic applications and therapeutic approaches. Recent crystallographic studies have indicated that the activation mechanism of OaPAC involves only small movements. In this study [5], we apply small-angle X-ray scattering (SAXS) [6] and time-resolved solution X-ray scattering [7] in combination with other biophysical techniques to investigate the substrate induced-conformational changes of OaPAC in solution. The implications of our work to the function of the enzyme are discussed.

Acknowledgment

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

References

[1] Fujisawa, T. and Masuda, S. (2018) Light-induced chromophore and protein responses and mechanical signal transduction of BLUF proteins Biophys. Rev. 10, 327-337.

[2] Ohki, M. et al. (2016) Structural insight into photoactivation of an adenylate cyclase from a photosynthetic cyanobacterium Proc. Natl. Acad. Sci. 113, 6659-6664.

[3] Ohki, M. et al. (2017) Molecular mechanism of photoactivation of a light-regulated adenylate cyclase Proc. Natl. Acad. Sci. 114, 8562-8567.

[4] Zaccolo, M., Zerio, A., and Lobo, M.J. (2021) Subcellular Organization of the cAMP Signaling Pathway Pharmacol.Rev. 73, 278–309.

[5] Kapetanaki, S.M. et al. (unpublished results)

[6] Da Vela Stefano and Svergun, D. (2020) Methods, development and applications of small-angle X-ray scattering to characterize biological macromolecules in solution Curr. Res. Struct. Biol. 2, 164-170.

[7] Cho, H.S., Schotte, F., Stadnytskyi, V., and Anfinrud, P. (2021) Time-resolved X-ray scattering studies of proteins Curr. Opin. Struct. Biol. 70, 99-107.