Összes szerző

---

Nagy Péter

az alábbi absztraktok szerzői között szerepel:

Bihariné Batta Ágnes
Improved estimation of the ratio of detection efficiencies of excited acceptors and donors for FRET measurements

Aug 29 - kedd

15:30 – 17:00

I. Poszterszekció

P05

Improved estimation of the ratio of detection efficiencies of excited acceptors and donors for FRET measurements

Ágnes Batta^1,2, Tímea Hajdu¹ and Péter Nagy¹

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

² Doctoral School of Molecular Medicine, University of Debrecen

Förster resonance energy transfer (FRET) is a radiationless interaction between a donor and an acceptor whose distance dependence makes it a sensitive tool for studying the oligomerization and the structure of proteins. When FRET is determined by measuring the sensitized emission of the acceptor, a parameter characterizing the ratio of detection efficiencies of an excited acceptor versus an excited donor is invariably involved in the formalism. For FRET measurements involving fluorescent antibodies or other external labels, this parameter, designated by α, is usually determined by comparing the intensity of a known number of donors and acceptors in two independent samples leading to a large statistical variability if the sample size is small. Here, we present a method that improves precision by applying microbeads with a calibrated number of antibody binding sites and a donor-acceptor mixture in which donors and acceptors are present in a certain, experimentally determined ratio. A formalism is developed for determining α and the superior reproducibility of the proposed method compared to the conventional approach is demonstrated. Since the novel methodology does not require sophisticated calibration samples or special instrumentation, it can be widely applied for the quantification of FRET experiments in biological research. [1]

References

[1] Batta Á, Hajdu T, Nagy P, Cytometry Part A, 2023. DOI: 10.1002/cyto.a.24728

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.

Szatmári Tímea
Effect of rational modification of disordered domains of the epidermal growth factor receptor on its biophysical characteristics

Aug 30 - szerda

15:30 – 17:00

II. Poszterszekció

P54

Effect of rational modification of disordered domains of the epidermal growth factor receptor on its biophysical characteristics

Tímea Szatmári¹, Péter Nagy¹

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

The epidermal growth factor receptor (EGFR) belongs to the ErbB receptor tyrosine kinase family. The extracellular domain of EGFR consists of four subdomains (I-IV). Upon ligand binding, the extracellular (EC) domain of the receptor is rearranged, resulting in exposition of the dimerization arm of subunit II providing an opportunity to form homodimers or heterodimers. The 3D structure determined by the amino acid sequence affects protein function. Proteins in which the formation of an ordered structure is not complete are called intrinsically disordered proteins. Recently, an algorithm (FuzPred) was developed that predicts the tendency of proteins to form disordered regions and their structural changes upon interacting with other proteins. The amino acid sequence of the EGFR was analyzed by FuzPred, which determines the tendency of distinct sequence regions to constitute globular (assuming ordered conformations), disordered (intrinsically disordered adopting ordered conformation upon binding), or fuzzy regions (remaining disordered even in their bound state). The following mutations were designed based on the software prediction that alter the fuzziness and the molecular interactions of EGFR domain II. 1. Mutation T274G is predicted to result in increased dynamics and destabilization of the dimerization site. It still forms a dimer, but the interaction is weak. 2. Mutation Q276F is expected to decrease dynamics of the dimerization arm and create a rigid binding site. As a result, it is unable to form a dimer due to lack of flexibility. 3. Mutation K284Q is expected to decrease dynamics while strengthening the binding site. This mutation promotes dimerization. We successfully generated all three mutants (dark and EGFP tagged on the C terminal of the proteins) by site-directed mutagenesis and stably transfected CHO cells expressing the EGFP-tagged mutant EGFRs. We investigated the ligand binding and the cooperativity of the wild-type EGFR and its mutant variants. CHO cells transfected with the wild-type or the mutant EGFRs were labeled with a concentration series of fluorescently labeled EGF (TAMRA-EGF). Our results suggest that those mutants that were predicted to be less prone to dimerization bind EGF less cooperatively and with a slightly lower affinity. In our further experiment we would like to examine the dimerization process (FRET, N&B) itself and consequent transmembrane signaling.

Tóth Gabriella
Examinations of cellular uptake of cell penetrating peptides in vitro and in vivo

Aug 30 - szerda

15:30 – 17:00

II. Poszterszekció

P59

Examinations of cellular uptake of cell penetrating peptides in vitro and in vivo

Gabriella Tóth¹, Gyula Batta^1,2, Levente Kárpáti³, Árpád Szöőr¹, István Mándity³, Péter Nagy¹

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

²University of Debrecen, Faculty of Science and Technology, Institute of Biotechnology, Department of Genetics and Applied Microbiology

³Semmelweis University, Faculty of Pharmacy, Institute of Organic Chemistry

Cell-penetrating peptides (CPPs) are peptides that enter cells by endocytosis and/or directly through the cell membrane. CPPs in general have been considered potential carriers of molecules that have difficulties entering cells. This is the feature that we would like to exploit and thereby establishing the opportunity for CPPs to have therapeutic applications in the long term. Our previously published results have shown that we can increase the cellular uptake and endosomal release of CPPs with statins. Our goal was to modify them and test if it is possible to make them enter the cells more efficiently. We also aimed to test the biodistribution of CPPs in mice after intravenous administration. We examined the cellular uptake and endosomal release by flow cytometry and confocal microscopy in SKBR-3 and MDA-MB-231 cell lines, while for the in vivo experiments a mouse model was applied. Fluorescently-labeled CPPs were used both in the in vivo and in vitro experiments. We compared the differences in the biophysical properties of the original and the modified CPPs, and we found that the cellular uptake of the modified version is more effective. There is a difference between the enhancement in the uptake of CPPs labeled by the pH-sensitive naphthofluorescein or Alexa Fluor 532. In the case of in vivo experiments, we found that peptides enter the mouse organs, including the liver, for which we have shown that CPPs is present in the intracellular space of hepatocytes. CPPs hold promise for increasing the efficiency and specificity of drug delivery to cells.