Magdalena Makarska-Bialokoz

Application of BRET for Studying G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) constitute one of the largest classes of cell surface receptors. GPCR biology has been a subject of widespread interest owing to the functional relevance of these receptors and their potential importance in the development of new drugs. At present, over 30% of all launched drugs target these receptors. GPCRs have been considered for a long time to function as monomeric entities and the idea of GPCR dimerization and oligomerization was initially accepted with disbelief. However, a significant amount of experimental and molecular modeling evidence accumulated during the last several years suggests that the process of GPCRs dimer or oligomer formation is a general phenomenon, in some cases even essential for receptor function. Among the many methods to study GPCR dimerization and oligomerization, modern biophysical techniques such as those based on resonance energy transfer (RET) and particularly bioluminescence resonance energy transfer (BRET) have played a leading role. RET methods are commonly applied as non-destructive indicators of specific protein-protein interactions (PPIs) in living cells. Data from numerous BRET experiments support the idea that the process of GPCR oligomerization may be relevant in many physiological and pathological conditions. The application of BRET to the study of GPCRs is not only limited to the assessment of receptor oligomerization but also expands to the investigation of the interactions of GPCRs with other proteins, including G proteins, G protein-coupled receptor kinases, β-arrestins or receptor tyrosine kinases, as well as to the characterization of GPCR activation and signaling. In this review, we briefly summarize the fundaments of BRET, discuss new trends in this technology and describe the wide range of applications of BRET to study GPCRs.

Spectroscopic study of associated systems formed between water-soluble cationic porphyrins or their copper (II) complexes and nucleic building blocks

AbstractThe association process between two water soluble cationic porphyrins, 5,10,15,20-tetrakis[4-(trimethyl-ammonio)phenyl]-21H,23H-porphine tetra-p-tosylate (H2TTMePP) and 5,10,15,20-tetrakis(1-methyl-4-pyridyl)-21H,23H-porphine tetra-p-tosylate (H2TMePyP), as well as their Cu (II) complexes, with five series of nucleic agents has been studied using UV-VIS spectroscopy in aqueous solutions. During the titration with nucleic compounds the bathochromic effect of porphyrins absorption spectra can be observed as well as the hypochromicity of the Soret maximum. The association constants were calculated using a curve-fitting procedure (KAC of the order of magnitude of 103–105 mol−1). It has been shown that the interactions of H2TTMePP with nucleic agents are much stronger than interactions of H2TMePyP, which is most likely related to the kind and the size of the porphyrin substituent groups partaking in the process of stacking. The strength of the observed associated systems increases generally in a series: nucleic base < nucleoside < nucleotide.

Analysis of the binding interaction in uric acid - Human hemoglobin system by spectroscopic techniques

The binding interaction between human hemoglobin and uric acid has been studied for the first time, by UV-vis absorption and steady-state, synchronous and three-dimensional fluorescence techniques. Characteristic effects observed for human hemoglobin intrinsic fluorescence during interaction with uric acid at neutral pH point at the formation of stacking non-covalent and non-fluorescent complexes. All the calculated parameters, the binding, fluorescence quenching and bimolecular quenching rate constants, as well as Förster resonance energy transfer parameters confirm the existence of static quenching. The results of synchronous fluorescence measurements indicate that the fluorescence quenching of human hemoglobin originates both from Trp and Tyr residues and that the addition of uric acid could significantly hinder the physiological functions of human hemoglobin.

Investigation of the binding affinity in vitamin B12—Bovine serum albumin system using various spectroscopic methods

The binding affinity between vitamin B12 (VitB12) and bovine serum albumin (BSA) has been investigated in aqueous solution at pH=7.4, employing UV-vis absorption and steady-state, synchronous and three-dimensional fluorescence spectra techniques. Representative effects noted for BSA intrinsic fluorescence resulting from the interactions with VitB12 confirm the formation of π-π stacked non-covalent and non-fluorescent complexes in the system VitB12-BSA. All the determined parameters, the binding, fluorescence quenching and bimolecular quenching rate constants (of the order of 104Lmol-1, 103Lmol-1 and 1011Lmol-1s-1, respectively), as well as Förster resonance energy transfer parameters validate the mechanism of static quenching. The interaction with VitB12 induces folding of the polypeptide chains around Trp residues of BSA, resulting in a more hydrophobic surrounding. Presented outcomes suggest that the addition of VitB12 can lead to the more organized BSA conformation and its more folded tertiary structure, what could influence the physiological functions of bovine serum albumin, notably in case of its overuse or abnormal metabolism.

Interactions of hemin with bovine serum albumin and human hemoglobin: A fluorescence quenching study

The binding interactions between hemin (Hmi) and bovine serum albumin (BSA) or human hemoglobin (HHb), respectively, have been examined in aqueous solution at pH=7.4, applying UV-vis absorption, as well as steady-state, synchronous and three-dimensional fluorescence spectra techniques. Representative results received for both BSA and HHb intrinsic fluorescence proceeding from the interactions with hemin suggest the formation of stacking non-covalent and non-fluorescent complexes in both the Hmi-BSA and Hmi-HHb systems, with highly possible concurrent formation of a coordinate bond between a group on the protein surface and the metal in Hmi molecule. All the values of calculated parameters, the binding, fluorescence quenching and bimolecular quenching rate constants point to the involvement of static quenching in both the systems studied. The blue shift in the synchronous fluorescence spectra imply the participation of both tryptophan and tyrosine residues in quenching of BSA and HHb intrinsic fluorescence. Depicted outcomes suggest that hemin is supposedly able to influence the physiological functions of BSA and HHb, the most important blood proteins, particularly in case of its overuse.

Computational Analysis of Chlorophyll Structure and UV-Vis Spectra: A Student Research Project on the Spectroscopy of Natural Complexes

ABSTRACT Chlorophyll was extracted from fresh spinach leaves (Spinacia L.) and its absorption spectrum was recorded. Computational methods were used to optimize the geometry and energy of chlorophyll as well as to compute UV-Vis spectra of chlorophylls a and b and to compare them with the spectrum of chlorophyll extract. Chlorophyll bands are well predicted, especially with B3LYP/CIS and TD-B3LYP methods. This research project forms a modern approach to the process of spectroscopy teaching—students can apply quantum-mechanics calculations for understanding of reaction mechanisms with the involvement of biologically active compounds and learn to interpret the absorption spectra of chlorophyll extract obtained by themselves.

Novel Antibacterial Compounds and their Drug Targets - Successes and Challenges

Infectious diseases are one of the most important and urgent health problems in the world. According to the World Health Organization (WHO) statistics, infectious and parasitic diseases are a cause of about 16% of all deaths worldwide and over 40% of deaths in Africa. A considerable progress that has been made during last hundred years in the fight against infectious diseases, in particular bacterial infections, can be attributed mainly to three factors: (1) the general improvement of living conditions, in particular sanitation; (2) development of vaccines and (3) development of efficient antibacterial drugs. Although considerable progress in reduction of the number of cases of bacterial infections, especially in lethal cases, has been made, continued cases and outbreaks of these diseases persist, which is caused by different contributing factors. Indeed, during last sixty years antibacterial drugs were used against various infectious diseases caused by bacterial pathogens with an undoubtable success. The most fruitful period for antibiotic development lasted from 40s to 60s of the last century and resulted in the majority of antibiotics currently on the market, which were obtained by screening actinomycetes derived from soil. Although the market for antibacterial drugs is nowadays greater than 25 billion US dollars per year, novel antibacterial drugs are still demanded due to developed resistance of many pathogenic bacteria against current antibiotics. In the last five years, one can observe a dramatic increase in cases of resistant bacteria strains (e.g. Klebsiella pneumoniae and E. coli) which are responsible for difficult to treat pneumonia and infections of urinary tract. The development of resistant bacteria strains is a side effect of antibiotic application for treatment: the infections become untreatable as a result of the existence of antibiotic-tolerant persisters. In this review, we discuss the challenges in antibacterial drug discovery, including the molecular basis of drug resistance, drug targets for novel antibacterial drugs, and new compounds (since year 2010) from different chemical classes with antibacterial activity, focusing on structure-activity relationships.

Non-covalent interactions between thio-caffeine derivatives and water-soluble porphyrin in ethanol-water environment

To determine the binding interactions and ability to form the non-covalent systems, the association process between 5,10,15,20-tetrakis[4-(trimethylammonio)phenyl]-21H,23H-porphine tetra-p-tosylate (H2TTMePP) and a series of five structurally diverse thio-caffeine analogues has been studied in ethanol and ethanol-water solutions, analyzing its absorption and steady-state fluorescence spectra. The porphyrin fluorescence lifetimes in the systems studied were established as well. During the titration with thio-caffeine compounds the slight bathochromic effect and considerable hypochromicity of the porphyrin Soret band maximum can be noted. The fluorescence quenching effect observed for interactions in H2TTMePP - thio-caffeine derivative systems, as well as the order of binding and fluorescence quenching constants (of 105-103mol-1) suggest the existence of the mechanism of static quenching due to the formation of non-covalent and non-fluorescent stacking complexes. In all the systems studied the phenomenon of the fractional accessibility of the fluorophore for the quencher was observed as well. Additionally, the specific binding interactions, due to the changes in reaction environment polarity, can be observed. It was found that thio-caffeine compounds can quench the porphyrin fluorescence according to the structure of thio-substituent in caffeine molecule. The obtained results can be potentially useful from scientific, therapeutic or environmental points of view.

Comparative study of binding interactions between porphyrin systems and aromatic compounds of biological importance by multiple spectroscopic techniques: A review

The specific spectroscopic and redox properties of porphyrins predestine them to fulfill the role of sensors during interacting with different biologically active substances. Monitoring of binding interactions in the systems porphyrin-biologically active compound is a key question not only in the field of physiological functions of living organisms, but also in environmental protection, notably in the light of the rapidly growing drug consumption and concurrently the production of drug effluents. Not always beneficial action of drugs on natural porphyrin systems induces to further studies, with commercially available porphyrins as the model systems. Therefore the binding process between several water-soluble porphyrins and a series of biologically active compounds (e.g. caffeine, guanine, theophylline, theobromine, xanthine, uric acid) has been studied in different aqueous solutions analyzing their absorption and steady-state fluorescence spectra, the porphyrin fluorescence lifetimes and their quantum yields. The magnitude of the binding and fluorescence quenching constants values for particular quenchers decreases in a series: uric acid > guanine > caffeine > theophylline > theobromine > xanthine. In all the systems studied there are characters of static quenching, as a consequence of the π-π-stacked non-covalent and non-fluorescent complexes formation between porphyrins and interacting compounds, accompanied simultaneously by the additional specific binding interactions. The porphyrin fluorescence quenching can be explain by the photoinduced intermolecular electron transfer from aromatic compound to the center of the porphyrin molecule, playing the role of the binding site. Presented results can be valuable for designing of new fluorescent porphyrin chemosensors or monitoring of drug traces in aqueous solutions. The obtained outcomes have also the toxicological and medical importance, providing insight into the interactions of the water-soluble porphyrins with biologically active substances.

Naphthalene sorption on red clay and halloysite modified by quaternary ammonium salts

Sorption of naphthalene on modified red clay and halloysite was carried out successfully. It was established that the process was of pseudo-second-order mechanism. The maximum sorption capacities were 9.23 mg/g and 112.79 mg/g for red clay and halloysite, respectively.