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Sipos Áron
Machine learning analysis of ultrafast fluorescence kinetics of NADH conformations in solutions

Aug 31 - csütörtök

10:30 – 10:45

Bioenergetika és fotobiofizika

E40

Machine learning analysis of ultrafast fluorescence kinetics of NADH conformations in solutions

Áron Sipos, Ferenc Sarlós, Rita Nagypál, László Zimányi and Géza I. Groma

Institute of Biophysics, Biological Research Centre Szeged, Eötvös Loránd Research Network, Szeged, Hungary

The structure of many different enzyme-bound forms of the essential coenzyme nicotinamide adenine dinucleotide is well characterized by X-ray diffraction data. Due to the limitations of this technique in solution, the unbound forms of the molecule need to be characterized by alternative methods, such as time-resolved fluorescence spectroscopy. In NADH the relative position of the nicotinamide and adenine groups has primary impact on the fluorescence kinetics of the excited nicotinamide group. In aqueous solution the molecule exists in an equilibrium of closed and open conformations, while the presence of methanol favors the latter.

The fluorescence kinetics of NADH were measured in water and methanol environments using fluorescence upconversion and time-correlated single photon counting in a large, 50 fs – 10 ns time window at different wavelengths for both environments.

To avoid the uncertainties of exponential fitting, the experimental data were fitted by a quasi-continuous set of time constants, applying regularization terms for favoring sparse solutions, i.e., a minimum number of nonzero amplitudes. For fine tuning the level of sparsity we developed a machine-learning method based on cross-validation and Bayesian optimization. This approach was found to be a powerful method for fluorescence kinetics analysis, avoiding any arbitrary or random parameters.

According to the above analysis the fast (<100 ps) part of the kinetics can be characterized by an unusually complex, three-step vibrational relaxation process. The slow part is well modelled either by the conventional distinct exponential terms or by distributed kinetics, corresponding to an equilibrium of a very high number of conformational states [1], as shown by the improved version of the analysis method.

Acknowledgment

This work has been supported by the National Research, Development and Innovation Office of Hungary; 2018-1.2.1-NKP-2018-00009; ÁS is grateful for the support of National Research, Development and Innovation Office of Hungary PD-121170.

References

[1] Zimányi L, Sipos Á, Sarlós F, Nagypál R, Groma GI (2021) PLoS ONE 16(8): e0255675.

Steinbach Gábor
Anisotropy imaging using RCM

Aug 29 - kedd

15:30 – 17:00

I. Poszterszekció

P26

Anisotropy imaging using RCM

Gábor Steinbach^1,2, Dávid Nagy³, Gábor Sipka^2,4 , Ábel Garab² , Győző Garab^2,4 and László Zimányi³

¹ ELKH, Biological Research Centre, Szeged, C ellular Imaging Laboratory

² Biofotonika R&D Ltd.

³ ELKH, Biological Research Centre, Szeged, Institute of Biophysics

⁴ ELKH, Biological Research Centre, Szeged, Institute of Plant Biology

Both the differential polarization attachments for LSMs and Re-scan Confocal Microscopy (RCM) facilitate revealing structures at the molecular level [1]. For subcellular structures, increasing the available resolution can be fundamental. The re-scan confocal microscopy (RCM) provides 4 times better signal to noise ratio (compared to conventional PMTs) using an sCMOS camera, moreover, due to the second scanner, the image resolution increases by the factor of 1.4, while the axial resolution is identical with the LSMs [2].

With additional polarization elements, the RCM enables the 2D and 3D microscopic mapping of the anisotropy of samples via measuring fluorescence-detected linear dichroism (FDLD). The usage of the liquid crystal modulator for the RCM and the high frequency modulation (photoelastic modulator – PEM) are synchronised with the imaging. The DP-LSMs and the pRCM (RCM equipped with polarization attachment) are suitable for obtaining unique structural information on the anisotropic molecular organization of biological samples and intelligent materials [3, 4] in 2D and 3D.

References

[1] Steinbach et al. (2019) European Biophysics Journal 48: 457-463., doi: 10.1007/s00249-019-01365-4

[2] De Luca GMR et al. (2017) Methods Appl Fluoresc 5:015002., doi: 10.1088/2050-6120/5/1/015002

[3] Simonović Radosavljević J et al. (2021) Int. J. Mol. Sci. 22: 7661, doi: 10.3390/ijms22147661

[4] Pleckaitis M et al. (2022) Nano Research, 15: 5527-5537., doi: 10.1007/s12274-021-4048-x

Zimányi László
Spectral and redox properties of a mouse cytochrome b561 protein suggest transmembrane electron transfer function

Aug 31 - csütörtök

09:50 – 10:10

Bioenergetika és fotobiofizika

E38

Spectral and redox properties of a mouse cytochrome b561 protein suggest transmembrane electron transfer function

Alajos Bérczi¹, Zsuzsanna Márton¹, Krisztina Laskay¹, András Tóth^1,2, Gábor Rákhely^1,2, Ágnes Duzs^1,2, Krisztina Sebők-Nagy¹, Tibor Páli¹ and László Zimányi¹

¹ Biological Research Centre, Szeged, Institute of Biophysics

² University of Szeged, Faculty of Science and Informatics, Department of Biotechnology

Cytochrome b561 proteins (CYB561s) are integral membrane proteins with 6 trans membrane domains, two heme b redox centers, one on each side of the host membrane. The major characteristics of these proteins are their ascorbate reducibility and transmembrane electron transferring capability. More than one CYB561 can be found in a wide range of animal and plant phyla and they are localized in membranes different from the membranes participating in bioenergization. Two homologous proteins, both in humans and rodents, are thought to participate via yet unidentified way in cancer pathology. The recombinant forms of the human tumor suppressor 101F6 protein (Hs_CYB561D2 and its mouse ortholog (Mm_CYB561D2 have already been studied in some details. However, nothing has yet been published about the physical- chemical properties of their homologues (Hs_CYB561D1 in humans and Mm_CYB561D1 in mice). The tissue specificity, location and function of this protein is unknown. Here we present optical, redox and structural properties of the recombinant Mm_CYB561D1 obtained based on various spectroscopic methods and homology modeling. In a new model calculation we show that traditional evaluation of the redox titration of these proteins cannot unequivocally provide the midpoint redox potentials of the individual heme centers without further assumptions. The interaction with ascorbate, the redox properties, the (limited) sequence homology and the modelled 3D structure all substantiate the function of Mm_CYB561D1 as a transmembrane electron transporter with ascorbate as the primary electron donor [1].

References

[1] Bérczi A, Márton Z, Laskay K, Tóth A, Rákhely G, Duzs Á, Sebők-Nagy K, Páli T, Zimányi L (2023)

Molecules 28: 2261.