Agnieszka Polit

Agnieszka Polit
Jagiellonian University | UJ · Department of Physical Biochemistry

PhD

About

51
Publications
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1,196
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Publications

Publications (51)
Article
Full-text available
Numerous studies highlight the therapeutic potential of G protein-coupled receptor (GPCR) heterodimers, emphasizing their significance in various pathological contexts. Despite extensive basic research and promising outcomes in animal models, the translation of GPCR heterodimer-targeting drugs into clinical use remains limited. The complexities of...
Article
Heterotrimeric G proteins are responsible for signal transduction from G-protein-coupled receptors (GPCRs) to intracellular effectors. This process is only possible when G proteins are located on the inner side of the cell membrane due to the specific localization of GPCR receptors. The Gα subunit is directed to the cell membrane through several si...
Article
Full-text available
Background Specific interactions between G protein-coupled receptors (GPCRs) and G proteins play a key role in mediating signaling events. While there is little doubt regarding receptor preference for Gα subunits, the preferences for specific Gβ and Gγ subunits and the effects of different Gβγ dimer compositions on GPCR signaling are poorly underst...
Article
The potential-sensitive di-4-ANEPPDHQ dye is presently gaining popularity in structural studies of the lipid bilayer. Within the bilayer, dye environmental sensitivity originates from the excitation induced charge redistribution and is usually attributed to solvent relaxation. Here, di-4-ANEPPDHQ is utilized to compare the structure of neutral and...
Article
Full-text available
In highly organized multicellular organisms such as humans, the functions of an individual cell are dependent on signal transduction through G protein-coupled receptors (GPCRs) and subsequently heterotrimeric G proteins. As most of the elements belonging to the signal transduction system are bound to lipid membranes, researchers are showing increas...
Article
Full-text available
Background G protein-coupled receptor (GPCR) signaling via heterotrimeric G proteins plays an important role in the cellular regulation of responses to external stimuli. Despite intensive structural research, the mechanism underlying the receptor–G protein coupling of closely related subtypes of Gαi remains unclear. In addition to the structural ch...
Article
Lipid nanodiscs are macromolecular assemblies, where a scaffold protein is wrapped around a nanosized disc of a lipid bilayer, thus protecting the hydrocarbon chains at the disc edges from unfavorable interactions with water. These nanostructures have numerous applications in, e.g., nanotechnology and pharmaceutics, and in investigations of membran...
Article
Nanodiscs are suitable tool for studies of membrane proteins (MPs) due to their ability to mimic native biological membranes and several MP structures are solved in nanodiscs. Among the various cell membrane components, cholesterol (CHL) is known to regulate protein function and their concentration can reach up to 50 mol %. However, studies compris...
Article
Full-text available
Heterotrimeric G-proteins along with G-protein-coupled receptors (GPCRs) regulate many biochemical functions by relaying the information from the plasma membrane to the inside of the cell. The lipid modifications of Gα and Gγ subunits, together with the charged regions on the membrane interaction surface, provide a peculiar pattern for various hete...
Article
In proteins, proton exchange is caused due to the changes in the proton affinity (pKa) of ionizable groups that are engaged in conformational changes induced by the binding of a ligand. In addition, knowledge regarding the type and number of such ionizable groups is very important to understand the pH-dependent changes of the thermodynamic paramete...
Article
In recent years a wide range of studies have shown that G protein-coupled receptors modulate a variety of cell functions through the formation of dimers. For instance, there is growing evidence for the dimerization of bradykinin or dopamine receptors, both as homodimers and heterodimers. A discovery of direct interactions of angiotensin II receptor...
Article
Neuroleptic drugs are widely applied in effective treatment of schizophrenia and related disorders. The lipophilic character of neuroleptics, means that they tend to accumulate in the lipid membranes, impacting their functioning and processing. In this paper, the effect of four drugs, namely thioridazine, olanzapine, sulpiride and amisulpride on ne...
Article
Full-text available
Functional recombinant bovine β-lactoglobulin has been produced by expression in E. coli using an engineered protein gene and purified to homogeneity by applying a new protocol. Mutations L1A/I2S introduced into the protein sequence greatly facilitate in vivo cleavage of the N-terminal methionine, allowing correctly folded and soluble protein suita...
Article
Full-text available
The G protein-coupled receptors (GPCRs), one of the largest protein families, are essential components of the most commonly used signal-transduction systems in cells. These receptors, often using common pathways, may cooperate in the regulation of signal transmission to the cell nucleus. Recent scientific interests increasingly focus on the coopera...
Poster
A review poster describing FLIM-FRET microscopy and its use with the previously presented results as an example.
Poster
The aim of this study was to investigate interactions between G-proteins αs and β1 subunits in living HEK293 cells using fluorescence lifetime microscopy (FLIM) combined with Förster Resonance Energy Transfer (FRET) measurements.
Article
HtrA2(Omi) protease is involved in the maintenance of mitochondrial homeostasis and stimulation of apoptosis as well as in development of cancer and neurodegenerative disorders. The protein is a homotrimer whose subunits comprise serine protease domain (PD) and PDZ regulatory domain. In the basal, inactive state, a tight interdomain interface limit...
Article
Interactions between bovine and goat β-lactoglobulin and tetracaine and pramocaine were investigated with isothermal titration calorimetry, X-ray crystallography and molecular modelling. Tetracaine and pramocaine binding to lactoglobulin is an entropy driven endothermic reaction. In this work, we found that determined association constants and ther...
Article
Full-text available
Bacterial HtrAs are proteases engaged in extracytoplasmic activities during stressful conditions and pathogenesis. A model prokaryotic HtrA (HtrA/DegP from Escherichia coli) requires activation to cleave its substrates efficiently. In the inactive state of the enzyme, one of the regulatory loops, termed LA, forms inhibitory contacts in the area of...
Article
The details of the interaction between G-proteins and the GPCRSs have been subjected to extensive investigation with structural and functional assays, but still many fundamental questions regarding this macromolecular assembly and its mechanism remain unanswered. In the context of current structural data we investigated interactions of dopamine D1...
Article
Goat β-lactoglobulin (GLG), lipocalin protein sharing high sequence similarity to bovine β-lactoglobulin (BLG), has been structurally and thermodynamically characterized. Two crystal forms of GLG have been obtained, trigonal (P3121) and orthorhombic (P21212), with unique molecular packing, not observed previously for BLG. In the trigonal structure,...
Article
Studies of the membrane proteins suggest their close interaction with the lipid surroundings. Membrane proteins and their activities are affected by the composition and structure of lipid bilayer, therefore adequate surroundings for studied protein are crucial for model membrane to ensure its biological relevance. In recent years nanodiscs which ar...
Article
Full-text available
Bacterial HtrAs are serine proteases engaged in extracytoplasmic protein quality control, and are required for the virulence of several pathogenic species. The proteolytic activity of HtrA (DegP) from Escherichia coli, a model prokaryotic HtrA, is stimulated by stressful conditions; the regulation of this process is mediated by the LA, LD, L1, L2,...
Article
Isoforms A (LGB-A) and B (LGB-B) of bovine lactoglobulin, the milk protein, differ in positions 64 (D↔G) and 118 (V↔A). Interactions of LGB-A and LGB-B with sodium dodecyl sulfate (SDS), dodecyltrimethylammonium chloride (DTAC) and lauric acid (LA), 12-carbon ligands possessing differently charged polar groups, were investigated using isothermal ti...
Article
Binding of 18-carbon unsaturated oleic and linoleic acid to lactoglobulin, the milk protein, has been studied for the first time by isothermal titration calorimetry (ITC) and X-ray crystallography. Crystal structures determined to resolution 2.10Å have revealed presence of single fatty acid molecule bound in β-barrel, the primary binding site, with...
Article
Full-text available
HtrA2(Omi), belonging to the high-temperature requirement A (HtrA) family of stress proteins, is involved in the maintenance of mitochondrial homeostasis and in the stimulation of apoptosis, as well as in cancer and neurodegenerative disorders. The protein comprises a serine protease domain and a postsynaptic density of 95 kDa, disk large, and zonu...
Article
Lactoglobulin is a globular milk protein for which physiological function has not been clarified. Due to its binding properties lactoglobulin might serve as a carrier for bioactive molecules. Binding of 12-, 14-, 16- and 18-carbon saturated fatty acids to bovine β-lactoglobulin has been characterised by isothermal titration calorimetry and X-ray cr...
Article
Full-text available
The impact of the illumination with white linearly polarized light (WLPL) of two commercially available cellulases from Trichoderma reesei on their activity in hydrolysis of microcrystalline cellulose was studied. Enzymes were illuminated with WLPL for 60 min and 120 min and for each native and illuminated enzyme sample specific activity and kineti...
Article
Lactoglobulin is a natural protein present in bovine milk and common component of human diet, known for binding with high affinity wide range of hydrophobic compounds, among them fatty acids 12-20 carbon atoms long. Shorter fatty acids were reported as not binding to β-lactoglobulin. We used X-ray crystallography and fluorescence spectroscopy to sh...
Article
In the present study, detailed information is presented on the hetero-dimerization of the serotonin 5-HT(2A) receptor and the dopamine D(2) receptor. Biophysical approaches (fluorescence spectroscopy as well as fluorescence lifetime microscopy) were used to determine the degree of fluorescence resonance energy transfer (FRET) between cyan and yello...
Article
Within the coding region of the dopamine D(1) receptor (D(1)R), two synonymous polymorphisms, D(1)R(G198A) and D(1)R(G1263), have been identified and postulated to correlate with the schizophrenia phenotype. Binding studies revealed that the density of these genetic variants was much lower than the density of wild type D(1)R in the human embryonic...
Article
The present investigation was undertaken to characterize mechanism of thermal activation of serine protease HtrA (DegP) from Escherichia coli. We monitored the temperature-induced structural changes within the regulatory loops L1, L2 and LA using a set of single-Trp HtrA mutants. The accessibility of each Trp residue to aqueous medium at temperatur...
Article
We investigated the influence of an epitope from the third intracellular loop (ic3) of the dopamine D(2) receptor, which contains adjacent arginine residues (217RRRRKR222), and an acidic epitope from the C-terminus of the dopamine D(1) receptor (404EE405) on the receptors' localization and their interaction. We studied receptor dimer formation usin...
Article
Full-text available
In Escherichia coli, cyclic AMP receptor protein (CRP) is known to regulate the transcription of about 100 genes. The signal to activate CRP is the binding of cyclic AMP. It has been suggested that binding of cAMP to CRP leads to a long-distance signal transduction from the N-terminal cAMP-binding domain to the C-terminal domain of the protein, whi...
Article
Full-text available
The concept that G protein-coupled receptors (GPCRs) function as oligomers has been widely accepted, however, different methodologies often used to study the phenomenon of GPCR interactions do not allow, as yet, for any generalization as to whether di- or oligomers are formed constitutively or are ligand-promoted. Here, we report on the use of thre...
Article
Evidence for hetero-oligomerization has recently been provided for various G protein-coupled receptors. In this paper, we have studied the possibility that dopamine D(1) and D(2) receptors physically interact with each other. Human dopamine D(1) and D(2) receptors were fluorescently tagged with derivatives of green fluorescence protein and transien...
Article
The cAMP receptor protein, allosterically activated by cAMP, regulates the expression of more than 100 genes in Escherichia coli. CRP is a homodimer of two-domain subunits. It has been suggested that binding of cAMP to CRP leads to a long-distance signal transduction from the N-terminal cAMP binding domain to the C-terminal domain of the protein re...
Article
Cyclic AMP receptor protein (CRP) regulates the expression of more then 100 genes in Escherichia coli. It is known that the allosteric activation of CRP by cAMP involves a long-distance signal transmission from the N-terminal cAMP-binding domain to the C-terminal domain of CRP responsible for the interactions with specific sequences of DNA. In this...
Article
Two-component monomolecular layers were formed with DPPC and two stereoisomers of zeaxanthin 9-cis and 13-cis at the argon-water interface. Very distinct over-additivity which represents affection of a lipid arrangement in the membrane has been observed in the case of zeaxanthin 9-cis (maximum at 20 mol%) but not in the case of zeaxanthin 13-cis. T...
Article
Full-text available
The cAMP receptor protein (CRP) regulates the expression of several genes in Escherichia coli. The protein is a homodimer, and each monomer is folded into two distinct structural domains. After allosteric transitions resulting from the binding of cAMP, CRP specifically binds to DNA and activates transcription. We have used stopped-flow fluorometry...
Article
cAMP receptor protein (CRP), allosterically activated by cAMP, regulates the expression of several genes in Escherichia coli. As binding of cAMP leads to undefined conformational changes in CRP, we performed a steady-state and time-resolved fluorescence study to show how the binding of the ligand influences the structure and dynamics of the protein...
Article
Cyclic AMP receptor protein (CRP) regulates the expression of more than 100 genes in Escherichia coli when complexed with cyclic AMP. Dynamic light scattering (DLS) and fluorescence decay anisotropy measurements of CRP were performed in solution, in the absence and presence of cAMP. We have also measured the effect of DNA sequences, including lac a...
Article
Full-text available
Cyclic AMP receptor protein (CRP) regulates the expression of several genes in Escherichia coli. The ability of CRP to bind specific DNA sequences and stimulate transcription is achieved as result of binding of an allosteric ligand: cAMP. Stopped-flow fluorimetry was employed to study the kinetics of the conformational changes in CRP induced by cAM...

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