Összes szerző

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Mocsár Gábor

az alábbi absztraktok szerzői között szerepel:

Goda Katalin
Crosstalk between nucleotide and substrate binding in ABCG2

Aug 30 - szerda

09:00 – 09:20

Membránok és membránfehérjék biofizikája

E21

Crosstalk between nucleotide and substrate binding in ABCG2

Zsuzsanna Gyöngy^1,2, Gábor Mocsár¹, Zsuzsanna Ritter^1,2, Thomas Stockner³, Gergely Szakács^4,5, Katalin Goda¹

¹ Department of Biophysics, Faculty of Medicine, University of Debrecen, Hungary

² Doctoral School of Molecular Cell and Immune Biology, University of Debrecen

³ Institute of Pharmacology, Center for Physiology and Pharmacology, Medical University of Vienna, Austria

⁴ Institute of Enzymology, Research Centre for Natural Sciences, Budapest, Hungary

⁵ Institute of Cancer Research, Medical University of Vienna, Austria

ABCG2 is an exporter type ABC protein that can expel numerous chemically unrelated xeno- and endobiotics from cells. When expressed in tumor cells and tumor stem cells, it may cause multidrug resistance contributing to the failure of chemotherapy. A better understanding of the molecular mechanism of ABCG2 may provide new therapeutic targets to improve the treatment of drug resistant tumors. In the present work, we designed fluorescence-based assays to investigate the affinity of ABCG2 to transported drugs and nucleotides in live or semi-permeabilized cells. Using the conformation-sensitive antibody 5D3, we show that the switch from the 5D3-reactive inward-facing (IF) to a 5D3-dim outward-facing (OF) conformation is induced by nucleotide binding, and this conformational transition is accelerated by substrates and hindered by the known inhibitor Ko143. The drop of 5D3 binding occurred simultaneously with the decrease of substrate binding, suggesting that the high-to-low switch in drug binding affinity might coincide with the transition from the IF to the OF conformation. Low substrate binding persists in the vanadate-trapped post-hydrolysis state, indicating that dissociation of the ATP hydrolysis products is required to reset the high substrate affinity IF conformation of ABCG2.

Acknowledgment

We are grateful for the financial supports by the Hungarian National Research, Development and Innovations Office (NKFIH; https://nkfih.gov.hu/english), grant number K124815 and GINOP-2.2.1-15-2017-00079. GS was supported by a Momentum Grant of the Hungarian Academy of Sciences.

Nagy Péter
The effect of fluorescence labeling on the function and dynamical properties of antibodies

Aug 30 - szerda

14:18 – 14:36

Sejtanalitika biofizikai megközelítéssel

E33

The effect of fluorescence labeling on the function and dynamical properties of antibodies

Tímea Hajdu¹, Gábor Mocsár¹, István Rebenku¹, Ágnes Batta¹, Bálint Bécsi², Ferenc Erdődi² and Peter Nagy¹

¹ Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen

² Department of Medical Chemistry, Faculty of Medicine, University of Debrecen

Fluorescent antibodies have been the cornerstone of cell biological investigations in the last couple of decades due to their relatively straightforward application. While fluorescence labeling has been shown to deteriorate the affinity of antibodies and the fluorescence properties of the dyes, an effect that may significantly affect the reliability of quantitative biophysical measurements [1], the background and further implications of these findings have not been explored. Here we show that fluorescence labeling of antibodies not only deteriorates their epitope binding capability, but functions linked to other IgG domains, and the extent of these effects reveals remarkably similar dependence on the degree of labeling. The melting temperature of the Fab and Fc domains of unlabeled and fluorescently-tagged antibodies were identical according to differential scanning fluorometry implying that the overall stability of antibody domains is not affected by fluorescence labeling. According to time-dependent measurements, the decay rate of fluorescence anisotropy increased by the degree of labeling suggesting that the wagging motion of antibody domains in accelerated by the presence of the fluorophores. This conclusion is corroborated by FRET measurements between the Fc domain and the IgG-bound epitope in which the steady-state energy transfer efficiency was higher in antibodies with a high degree of labeling implying that the Fc and epitope-binding domains approach each other more closely in highly labeled antibodies on average over time. The investigations suggest that the effect of fluorescence labeling on all antibody functions may be due to altered antibody dynamics.

Acknowledgment

The project was supported by research grants from the National Research, Development and Innovation Office (K138075, ANN133421).

References

[1] Szabó Á, Szatmári T, Ujlaky-Nagy L, Rádi I, Vereb G, Szöllősi J, Nagy P (2018) Biophys J, 114: 668-700.