Tzvetana Lazarova - Academia.edu (original) (raw)
Papers by Tzvetana Lazarova
New heterocyclic and polycyclic aromatic retinal analogues (Fig. 1) were synthesised and their re... more New heterocyclic and polycyclic aromatic retinal analogues (Fig. 1) were synthesised and their recombination with bacterioopsin was studied.
Protein Science, 2005
The human adenosine A 2A receptor (A 2A R) belongs to one of the largest family of membrane prote... more The human adenosine A 2A receptor (A 2A R) belongs to one of the largest family of membrane proteins, the G-protein coupled receptors (GPCRs), characterized by seven transmembrane (TM) helices. Little is known about the determinants of their structures, folding, assembly, activation mechanisms, and oligomeric states. Previous studies in our group showed that peptides corresponding to all seven TM domains form stable helical structures in detergent micelles and lipid vesicles. However, the peptides behave differently; TM5 is the only peptide to have a ratio [u] 222 /[u] 208 obtained by circular dichroism (CD) spectroscopy >1. This finding suggested to us that TM5 might self-associate. In the present study, we investigate the unique properties of the TM5 domain. We performed detailed analyses of TM5 peptide behavior in membrane-mimetic environments using CD spectroscopy, fluorescence spectroscopy and Fo¨rster resonance energy transfer, and gel electrophoresis. We find that TM5 peptide has the ability to self-associate to form oligomeric structures in various hydrophobic milieus and that these oligomers are highly resistant to temperature and chemical denaturation. We also find that mutation of the full-length A 2A R at position M193, which is located in the fifth TM domain, noticeably alters A 2A R monomer:dimer ratio as observed on SDS-PAGE. Our results suggest that parallel association of TM5 dimers may play a role in the known adenosine A 2A receptor dimerization. This study represents the first evidence of an individual GPCR transmembrane domain self-association.
ChemBioChem, 2005
N-Heteroaryl retinals derived from indole, 1-indolizine and 3-indolizine (10 a-c) have been synth... more N-Heteroaryl retinals derived from indole, 1-indolizine and 3-indolizine (10 a-c) have been synthesized after their UV/Vis red-shifted absorption properties had been predicted by time-dependent density functional theory (TD-DFT) computations. The three new analogues form artificial pigments upon recombination with bacterioopsin: indolyl retinal 10 a undergoes fast and efficient reconstitution to form a species with a UV/Vis absorbance maximum similar to that of wild-type bacteriorhodopsin, whilst the indolizinyl retinals 10 b and 10 c also reconstitute in significant proportion to give noticeably red-shifted, although unstable, pigments. Significant changes in the pK(a) values of these artificial bacteriorhodopsins are interpreted as arising from nonoptimal binding-site occupancy by the chromophore due to steric constraints.
Bioelectrochemistry and Bioenergetics, 1995
The influence of “mild” trypsin digestion of pea thylakoid membranes on fluorescence emission and... more The influence of “mild” trypsin digestion of pea thylakoid membranes on fluorescence emission and excitation spectra was investigated. With increasing trypsin concentration the intensity of fluorescence emission maximum at 735 nm (attributed to PSI) was enhanced and the ratio F735/F685 increased from 1.47 ± 0.12 for control thylakoids up to 3.15 + 0.23 for thylakoids, treated with 8 μg tryp/mg
Biochemistry, 2002
Low-temperature Fourier transform infrared spectroscopy has been used to study mutants of Glu194 ... more Low-temperature Fourier transform infrared spectroscopy has been used to study mutants of Glu194 and Glu204, two amino acids that are involved in proton release to the extracellular side of bacteriorhodopsin. Difference spectra of films of E194Q, E204Q, E194Q/E204Q, E9Q/E194Q/E204Q, and E9Q/E74Q/E194Q/E204Q at 243, 277, and 293 K and several pH values were obtained by continuous illumination. A specific effect of Cl(-) ions was found for the mutants, promoting a N-like intermediate at alkaline pH and an O' intermediate at neutral or acid pH. The apparent pK(a) of Asp85 in the M intermediate was found to be decreased for E194Q in the presence of Cl(-) (pK(a) of 7.6), but it was unchanged for E204Q, as compared to wild-type. In the absence of Cl(-) (i.e., in the presence of SO(4)(2)(-)), mutation of Glu194 or of Glu204 produces M- (or M(N), M(G))-like intermediates under all of the conditions examined. The absence of N, O, and O' intermediates suggests a long-range effect of the mutation. Furthermore, it is suggested that Cl(-) acts by reaching the interior of the protein, rather than producing surface effects. The effect of low water content was also examined, in the presence of Cl(-). Similar spectra corresponding to the M(1) intermediate were found for dry samples of both mutants, indicating that the effects of the mutations or of Cl(-) ions are confined to the second part of the photocycle. The water O-H stretching data further confirms altered photocycles and the effect of Cl(-) on the accumulation of the N intermediate.
Biochemistry, 2005
The human adenosine A(2A) receptor (A(2A)R) is an integral membrane protein and a member of the G... more The human adenosine A(2A) receptor (A(2A)R) is an integral membrane protein and a member of the G-protein-coupled receptor (GPCR) superfamily, characterized by seven transmembrane (TM) helices. Although helix-helix association in the lipid bilayer is known to be an essential step in the folding of GPCRs, the determinants of their structures, folding, and assembly in the cell membrane are poorly understood. Previous studies in our group showed that while peptides corresponding to all seven TM domains of A(2A)R form stable helical structures in detergent micelles and lipid vesicles, they display significant variability in their helical propensity. This finding suggested to us that some TM domains might need to interact with other domains to properly insert and fold in hydrophobic environments. In this study, we assessed the ability of TM peptides to interact in pairwise combinations. We analyzed peptide interactions in hydrophobic milieus using circular dichroism spectroscopy and Förster resonance energy transfer. We find that specific interactions between TM helices occur, leading to additional helical content, especially in weakly helical TM domains, suggesting that some TM domains need a partner for proper folding in the membrane. The approach developed in this study will enable complete analysis of the TM domain interactions and the modeling of a folding pathway for A(2A)R.
Journal of Nanoscience and Nanotechnology, 2009
Bacteriorhodopsin (bR) is a membrane protein found in the archae Halobacterium salinarum. Here, w... more Bacteriorhodopsin (bR) is a membrane protein found in the archae Halobacterium salinarum. Here, we studied wild type bR and especially the triple mutant bR, 3Glu [E9Q/E194Q/E204Q], in combination with wide gap semiconductor TiO 2 for their suitability as efficient light harvester in solar cell. Our differential scanning calorimetry data show thermal robustness of bR wild type and 3Glu mutant, which make them good candidates as photosensitizer in solar cells. Molecular modeling indicates that binding of bR to the exposed oxygen atoms of anatase TiO 2 is favorable for electron transfer and directed by local, small distance interactions. A solar cell, based on bR wild type and bR triple mutant immobilized on nanocrystalline TiO 2 film was successfully constructed. The photocurrent density-photo voltage (J-V) characteristics of bio-sensitized solar cell (BSSC), based on the wild type bR and 3Glu mutant adsorbed on nanocrystalline TiO 2 film electrode were measured. The results show that the 3Glu mutant displays better photoelectric performance compared to the wild type bR, giving a short-circuit photocurrent density (J SC) of 0.09 mA/cm 2 and the open-circuit photovoltage (V OC) 0.35 V, under an illumination intensity of 40 mW/cm 2 .
The role of the extracellular Glu side chains of bacteriorhodopsin in the proton transport mechan... more The role of the extracellular Glu side chains of bacteriorhodopsin in the proton transport mechanism has been studied using the single mutants E9Q, E74Q, E194Q, and E204Q; the triple mutant E9Q/E194Q/E204Q; and the quadruple mutant E9Q/E74Q/E194Q/E204Q. Steady-state difference and deconvoluted Fourier transform infrared spectroscopy has been applied to analyze the Mand N-like intermediates in membrane films maintained at a controlled humidity, at 243 and 277 K at alkaline pH. The mutants E9Q and E74Q gave spectra similar to that of wild type, whereas E194Q, E9Q/E194Q/ E204Q, and E9Q/E74Q/E194Q/E204Q showed at 277 K a N-like intermediate with a single negative peak at 1742 cm, indicating that Asp and Asp are deprotonated. Under the same conditions E204Q showed a positive peak at 1762 cm and a negative peak at 1742 cm, revealing the presence of protonated Asp (in an M intermediate environment) and deprotonated Asp. These results indicate that in E194Q-containing mutants, the second in...
Zeitschrift für Naturforschung C
Three spin-labelled fatty acids were used to detect the dynamics of lipid bilayer of apomem brane... more Three spin-labelled fatty acids were used to detect the dynamics of lipid bilayer of apomem branes and purple membranes. It was found that ESR spectra of spin labels bound to apo membranes showed a temperature-induced changes rather similar to those seen with purple membranes. At the same time, however, the values of hyperfine splitting parameter 2Tm were lower as compared to purple membranes. The results pointed out that the removal of the retinal from purple membranes affects the dynamics of lipid bilayer and apomembranes were more rigid structure than those of purple membranes.
Proceedings of the National Academy of Sciences
Infrared spectroscopy has been used in the past to probe the dynamics of internal proton transfer... more Infrared spectroscopy has been used in the past to probe the dynamics of internal proton transfer reactions taking place during the functional mechanism of proteins but has remained mostly silent to protonation changes in the aqueous medium. Here, by selectively monitoring vibrational changes of buffer molecules with a temporal resolution of 6 µs, we have traced proton release and uptake events in the light-driven proton-pump bacteriorhodopsin and correlate these to other molecular processes within the protein. We demonstrate that two distinct chemical entities contribute to the temporal evolution and spectral shape of the continuum band, an unusually broad band extending from 2,300 to well below 1,700 cm−1. The first contribution corresponds to deprotonation of the proton release complex (PRC), a complex in the extracellular domain of bacteriorhodopsin where an excess proton is shared by a cluster of internal water molecules and/or ionic E194/E204 carboxylic groups. We assign the s...
![Research paper thumbnail of {"__content__"=>"Mapping the Interface of a GPCR Dimer: A Structural Model of the A Adenosine and D Dopamine Receptor Heteromer.", "sub"=>[{"__content__"=>"2A"}, {"__content__"=>"2"}]}](https://attachments.academia-assets.com/69408918/thumbnails/1.jpg)
](Frontiers in pharmacology, 2018
The A adenosine (AR) and D dopamine (DR) receptors form oligomers in the cell membrane and allost... more The A adenosine (AR) and D dopamine (DR) receptors form oligomers in the cell membrane and allosteric interactions across the AR-DR heteromer represent a target for development of drugs against central nervous system disorders. However, understanding of the molecular determinants of AR-DR heteromerization and the allosteric antagonistic interactions between the receptor protomers is still limited. In this work, a structural model of the AR-DR heterodimer was generated using a combined experimental and computational approach. Regions involved in the heteromer interface were modeled based on the effects of peptides derived from the transmembrane (TM) helices on AR-DR receptor-receptor interactions in bioluminescence resonance energy transfer (BRET) and proximity ligation assays. Peptides corresponding to TM-IV and TM-V of the AR blocked heterodimer interactions and disrupted the allosteric effect of AR activation on DR agonist binding. Protein-protein docking was used to construct a m...
Biochemistry Usa, 2004
Human adenosine A(2)a receptor is a member of the G-protein-coupled receptor (GPCR) superfamily o... more Human adenosine A(2)a receptor is a member of the G-protein-coupled receptor (GPCR) superfamily of seven-helix transmembrane (TM) proteins. To test general models for membrane-protein folding and to identify specific features of folding and assembly for this representative member of an important and poorly understood class of proteins, we synthesized peptides corresponding to its seven TM domains. We assessed the ability of the peptides to insert into micelles and vesicles and measured secondary structure for each peptide in aqueous and membrane-mimetic environments. CD spectra indicate that each of the seven TM peptides form thermally stable, independent alpha-helical structures in both micelles and vesicles. The helical content of the peptides depends on the nature of the membrane-mimetic environment. Four of the peptides (TM3, TM4, TM5, and TM7) exhibit very high-helical structure, near the predicted maximum for their TM segments. The TM1 peptide also adopts relatively high alpha-helical structures. In contrast, two of peptides, TM2 and TM6, display low alpha helicity. Similarly, the ability of the peptides to insert into membrane-mimetic environments, assayed by intrinsic tryptophan fluorescence and fluorescence quenching, varied markedly. Most peptides exhibit higher alpha helicity in anionic sodium dodecyl sulfate than in neutral dodecyl-beta-D-maltoside micelles, and TM2 was disordered in zwiterionic DMPC but was alpha-helical in negatively charged DMPC/DMPG vesicles. These findings strongly suggest that electrostatic interactions between lipids and peptides control the insertion of the peptides and may be involved in membrane-protein-folding events. The measured helical content of these TM domains does not correlate with the predicted helicity based on amino acid sequence, pointing out that, while hydrophobic interactions can be a major determinant for folding of TM peptides, other factors, such as electrostatic interactions or helix-helix interactions, may play significant roles for specific TM domains. Our results represent a comprehensive analysis of helical propensities for a human GPCR and support models for membrane-protein folding in which interactions between TM domains are required for proper insertion and folding of some TM helix domains. The tendency of some peptides to self-associate, especially in aqueous environments, underscores the need to prevent improper interactions during folding and refolding of membrane proteins in vivo and in vitro.
Photochemistry and Photobiology, Mar 1, 2009
Glu194 is a residue located at the end of F helix on the extracellular side of the light-induced ... more Glu194 is a residue located at the end of F helix on the extracellular side of the light-induced proton pump bacteriorhodopsin (BR). Currently, it is well recognized that Glu194 and Glu204 residues, along with water clusters, constitute the proton release group of BR. Here we report that the replacement of Glu194 for Gln affects not only the photocycle of the protein but also has tremendous effect on the all-trans to 13-cis thermal isomerization. We studied the pH dependence of the dark adaptation of the E194Q mutant and performed HPLC analysis of the isomer compositions of the light-and partially darkadapted states of the mutant at several pH values. Our data confirmed that E194Q exhibits extremely slow dark adaptation over a wide range of pH. HPLC data showed that a significantly larger concentration of all-trans isomer was present in the samples of the E194Q mutant even after prolonged dark adaptation. After 14 days in the dark the 13-cis to all-trans ratio was 1:3 in the mutant, compared to 2:1 in the wild type. These data clearly indicate the involvement of Glu194 in control of the rate of all-trans to 13-cis thermal isomerization.
The chromophore fluorescence of bacteriorhodopsin (BR) has been studied by using visible lines of... more The chromophore fluorescence of bacteriorhodopsin (BR) has been studied by using visible lines of an argon and He-Ne laser at room temperature. This fluorescence is extremely weak and appears as an unstructured band in the 650-880 nm range. The same line shape of ...
Biochemistry, 2010
G protein-coupled receptors (GPCRs)1 constitute the largest family of integral membrane proteins ... more G protein-coupled receptors (GPCRs)1 constitute the largest family of integral membrane proteins present in all eukaryotic cells, yet relatively little information is known pertaining to their structure, folding, and stability. In this work, we describe several approaches to characterize conformational stability of the human adenosine A 2 a receptor (hA 2 aR). Thermal and chemical denaturation were not reversible, yet clear differences in the unfolding behavior were observed upon ligand binding via circular dichroism and fluorescence spectrometry. We found that the stability of hA 2 aR was increased upon incubation with the agonist N 6-cyclohexyladenosine or the antagonist theophylline. When extracellular disulfide bonds were reduced with a chemical reducing agent, the ligand-binding activity decreased by ~40%, but reduction of these bonds did not compromise the unfolding transition observed via urea denaturation. Overall, these approaches offer a general strategy for characterizing the effect of surfactant and ligand effects on the stability of GPCRs.
The following multiple Glu extracellular mutants of bacteriorhodopsin have been studied, with res... more The following multiple Glu extracellular mutants of bacteriorhodopsin have been studied, with respect to their possible use in bioelectronic applications: the quadruple E9Q+E74Q+E194Q+E204Q, the triple E9Q+E194Q+E204Q and the double E194Q+E204Q. The wavelength of the absorption maximum of the quadruple mutant is very sensitive to pH and salts, covering a wide wavelength range, from about 500 to 600 nm. It shows a high accessibility for small molecules like hydroxylamine. The triple mutant shows properties very similar to the quadruple mutant. The mutant E194Q+E204Q possesses intermediate properties in between wild type and the quadruple mutant. It has a wavelength maximum at 530-540 nm in water and at neutral pH, and shows increased accessibility for hydroxylamine. All mutants are stable at extreme pH values. Thermal stability was monitored using DSC. In water at neutral pH, small differences were detected with respect to the main transition, the E9Q+E74Q+E194Q+E204Q and E9Q+E194Q+E204Q mutants showing a T m about 6 ºC lower than wild type. None of the multiple mutants possess the pre-transition that is found at about 82 ºC for the wild type. Functional studies using flash photolysis showed that the E9Q+E74Q+E194Q+E204Q mutant, like E9Q or E204Q, presents increased yield of the O 640 intermediate. Light-dark adaptation was also found to be strongly altered in all multiple mutants. As an example of a new application of the purple membrane, formation of a wild type film over a microchip makes its electrical response independent on pH in the range 4-8, thus having the characteristics of a reference electrode for pH measurements. Formation of a film using the E9Q+E74Q+E194Q+E204Q mutant nearly eliminates the sharp decrease of the electric signal obtained below pH 4. Thus, the quadruple mutant shows a promising behaviour for extending the useful range down to acid pH values.
New heterocyclic and polycyclic aromatic retinal analogues (Fig. 1) were synthesised and their re... more New heterocyclic and polycyclic aromatic retinal analogues (Fig. 1) were synthesised and their recombination with bacterioopsin was studied.
Protein Science, 2005
The human adenosine A 2A receptor (A 2A R) belongs to one of the largest family of membrane prote... more The human adenosine A 2A receptor (A 2A R) belongs to one of the largest family of membrane proteins, the G-protein coupled receptors (GPCRs), characterized by seven transmembrane (TM) helices. Little is known about the determinants of their structures, folding, assembly, activation mechanisms, and oligomeric states. Previous studies in our group showed that peptides corresponding to all seven TM domains form stable helical structures in detergent micelles and lipid vesicles. However, the peptides behave differently; TM5 is the only peptide to have a ratio [u] 222 /[u] 208 obtained by circular dichroism (CD) spectroscopy >1. This finding suggested to us that TM5 might self-associate. In the present study, we investigate the unique properties of the TM5 domain. We performed detailed analyses of TM5 peptide behavior in membrane-mimetic environments using CD spectroscopy, fluorescence spectroscopy and Fo¨rster resonance energy transfer, and gel electrophoresis. We find that TM5 peptide has the ability to self-associate to form oligomeric structures in various hydrophobic milieus and that these oligomers are highly resistant to temperature and chemical denaturation. We also find that mutation of the full-length A 2A R at position M193, which is located in the fifth TM domain, noticeably alters A 2A R monomer:dimer ratio as observed on SDS-PAGE. Our results suggest that parallel association of TM5 dimers may play a role in the known adenosine A 2A receptor dimerization. This study represents the first evidence of an individual GPCR transmembrane domain self-association.
ChemBioChem, 2005
N-Heteroaryl retinals derived from indole, 1-indolizine and 3-indolizine (10 a-c) have been synth... more N-Heteroaryl retinals derived from indole, 1-indolizine and 3-indolizine (10 a-c) have been synthesized after their UV/Vis red-shifted absorption properties had been predicted by time-dependent density functional theory (TD-DFT) computations. The three new analogues form artificial pigments upon recombination with bacterioopsin: indolyl retinal 10 a undergoes fast and efficient reconstitution to form a species with a UV/Vis absorbance maximum similar to that of wild-type bacteriorhodopsin, whilst the indolizinyl retinals 10 b and 10 c also reconstitute in significant proportion to give noticeably red-shifted, although unstable, pigments. Significant changes in the pK(a) values of these artificial bacteriorhodopsins are interpreted as arising from nonoptimal binding-site occupancy by the chromophore due to steric constraints.
Bioelectrochemistry and Bioenergetics, 1995
The influence of “mild” trypsin digestion of pea thylakoid membranes on fluorescence emission and... more The influence of “mild” trypsin digestion of pea thylakoid membranes on fluorescence emission and excitation spectra was investigated. With increasing trypsin concentration the intensity of fluorescence emission maximum at 735 nm (attributed to PSI) was enhanced and the ratio F735/F685 increased from 1.47 ± 0.12 for control thylakoids up to 3.15 + 0.23 for thylakoids, treated with 8 μg tryp/mg
Biochemistry, 2002
Low-temperature Fourier transform infrared spectroscopy has been used to study mutants of Glu194 ... more Low-temperature Fourier transform infrared spectroscopy has been used to study mutants of Glu194 and Glu204, two amino acids that are involved in proton release to the extracellular side of bacteriorhodopsin. Difference spectra of films of E194Q, E204Q, E194Q/E204Q, E9Q/E194Q/E204Q, and E9Q/E74Q/E194Q/E204Q at 243, 277, and 293 K and several pH values were obtained by continuous illumination. A specific effect of Cl(-) ions was found for the mutants, promoting a N-like intermediate at alkaline pH and an O' intermediate at neutral or acid pH. The apparent pK(a) of Asp85 in the M intermediate was found to be decreased for E194Q in the presence of Cl(-) (pK(a) of 7.6), but it was unchanged for E204Q, as compared to wild-type. In the absence of Cl(-) (i.e., in the presence of SO(4)(2)(-)), mutation of Glu194 or of Glu204 produces M- (or M(N), M(G))-like intermediates under all of the conditions examined. The absence of N, O, and O' intermediates suggests a long-range effect of the mutation. Furthermore, it is suggested that Cl(-) acts by reaching the interior of the protein, rather than producing surface effects. The effect of low water content was also examined, in the presence of Cl(-). Similar spectra corresponding to the M(1) intermediate were found for dry samples of both mutants, indicating that the effects of the mutations or of Cl(-) ions are confined to the second part of the photocycle. The water O-H stretching data further confirms altered photocycles and the effect of Cl(-) on the accumulation of the N intermediate.
Biochemistry, 2005
The human adenosine A(2A) receptor (A(2A)R) is an integral membrane protein and a member of the G... more The human adenosine A(2A) receptor (A(2A)R) is an integral membrane protein and a member of the G-protein-coupled receptor (GPCR) superfamily, characterized by seven transmembrane (TM) helices. Although helix-helix association in the lipid bilayer is known to be an essential step in the folding of GPCRs, the determinants of their structures, folding, and assembly in the cell membrane are poorly understood. Previous studies in our group showed that while peptides corresponding to all seven TM domains of A(2A)R form stable helical structures in detergent micelles and lipid vesicles, they display significant variability in their helical propensity. This finding suggested to us that some TM domains might need to interact with other domains to properly insert and fold in hydrophobic environments. In this study, we assessed the ability of TM peptides to interact in pairwise combinations. We analyzed peptide interactions in hydrophobic milieus using circular dichroism spectroscopy and Förster resonance energy transfer. We find that specific interactions between TM helices occur, leading to additional helical content, especially in weakly helical TM domains, suggesting that some TM domains need a partner for proper folding in the membrane. The approach developed in this study will enable complete analysis of the TM domain interactions and the modeling of a folding pathway for A(2A)R.
Journal of Nanoscience and Nanotechnology, 2009
Bacteriorhodopsin (bR) is a membrane protein found in the archae Halobacterium salinarum. Here, w... more Bacteriorhodopsin (bR) is a membrane protein found in the archae Halobacterium salinarum. Here, we studied wild type bR and especially the triple mutant bR, 3Glu [E9Q/E194Q/E204Q], in combination with wide gap semiconductor TiO 2 for their suitability as efficient light harvester in solar cell. Our differential scanning calorimetry data show thermal robustness of bR wild type and 3Glu mutant, which make them good candidates as photosensitizer in solar cells. Molecular modeling indicates that binding of bR to the exposed oxygen atoms of anatase TiO 2 is favorable for electron transfer and directed by local, small distance interactions. A solar cell, based on bR wild type and bR triple mutant immobilized on nanocrystalline TiO 2 film was successfully constructed. The photocurrent density-photo voltage (J-V) characteristics of bio-sensitized solar cell (BSSC), based on the wild type bR and 3Glu mutant adsorbed on nanocrystalline TiO 2 film electrode were measured. The results show that the 3Glu mutant displays better photoelectric performance compared to the wild type bR, giving a short-circuit photocurrent density (J SC) of 0.09 mA/cm 2 and the open-circuit photovoltage (V OC) 0.35 V, under an illumination intensity of 40 mW/cm 2 .
The role of the extracellular Glu side chains of bacteriorhodopsin in the proton transport mechan... more The role of the extracellular Glu side chains of bacteriorhodopsin in the proton transport mechanism has been studied using the single mutants E9Q, E74Q, E194Q, and E204Q; the triple mutant E9Q/E194Q/E204Q; and the quadruple mutant E9Q/E74Q/E194Q/E204Q. Steady-state difference and deconvoluted Fourier transform infrared spectroscopy has been applied to analyze the Mand N-like intermediates in membrane films maintained at a controlled humidity, at 243 and 277 K at alkaline pH. The mutants E9Q and E74Q gave spectra similar to that of wild type, whereas E194Q, E9Q/E194Q/ E204Q, and E9Q/E74Q/E194Q/E204Q showed at 277 K a N-like intermediate with a single negative peak at 1742 cm, indicating that Asp and Asp are deprotonated. Under the same conditions E204Q showed a positive peak at 1762 cm and a negative peak at 1742 cm, revealing the presence of protonated Asp (in an M intermediate environment) and deprotonated Asp. These results indicate that in E194Q-containing mutants, the second in...
Zeitschrift für Naturforschung C
Three spin-labelled fatty acids were used to detect the dynamics of lipid bilayer of apomem brane... more Three spin-labelled fatty acids were used to detect the dynamics of lipid bilayer of apomem branes and purple membranes. It was found that ESR spectra of spin labels bound to apo membranes showed a temperature-induced changes rather similar to those seen with purple membranes. At the same time, however, the values of hyperfine splitting parameter 2Tm were lower as compared to purple membranes. The results pointed out that the removal of the retinal from purple membranes affects the dynamics of lipid bilayer and apomembranes were more rigid structure than those of purple membranes.
Proceedings of the National Academy of Sciences
Infrared spectroscopy has been used in the past to probe the dynamics of internal proton transfer... more Infrared spectroscopy has been used in the past to probe the dynamics of internal proton transfer reactions taking place during the functional mechanism of proteins but has remained mostly silent to protonation changes in the aqueous medium. Here, by selectively monitoring vibrational changes of buffer molecules with a temporal resolution of 6 µs, we have traced proton release and uptake events in the light-driven proton-pump bacteriorhodopsin and correlate these to other molecular processes within the protein. We demonstrate that two distinct chemical entities contribute to the temporal evolution and spectral shape of the continuum band, an unusually broad band extending from 2,300 to well below 1,700 cm−1. The first contribution corresponds to deprotonation of the proton release complex (PRC), a complex in the extracellular domain of bacteriorhodopsin where an excess proton is shared by a cluster of internal water molecules and/or ionic E194/E204 carboxylic groups. We assign the s...
Frontiers in pharmacology, 2018
The A adenosine (AR) and D dopamine (DR) receptors form oligomers in the cell membrane and allost... more The A adenosine (AR) and D dopamine (DR) receptors form oligomers in the cell membrane and allosteric interactions across the AR-DR heteromer represent a target for development of drugs against central nervous system disorders. However, understanding of the molecular determinants of AR-DR heteromerization and the allosteric antagonistic interactions between the receptor protomers is still limited. In this work, a structural model of the AR-DR heterodimer was generated using a combined experimental and computational approach. Regions involved in the heteromer interface were modeled based on the effects of peptides derived from the transmembrane (TM) helices on AR-DR receptor-receptor interactions in bioluminescence resonance energy transfer (BRET) and proximity ligation assays. Peptides corresponding to TM-IV and TM-V of the AR blocked heterodimer interactions and disrupted the allosteric effect of AR activation on DR agonist binding. Protein-protein docking was used to construct a m...
Biochemistry Usa, 2004
Human adenosine A(2)a receptor is a member of the G-protein-coupled receptor (GPCR) superfamily o... more Human adenosine A(2)a receptor is a member of the G-protein-coupled receptor (GPCR) superfamily of seven-helix transmembrane (TM) proteins. To test general models for membrane-protein folding and to identify specific features of folding and assembly for this representative member of an important and poorly understood class of proteins, we synthesized peptides corresponding to its seven TM domains. We assessed the ability of the peptides to insert into micelles and vesicles and measured secondary structure for each peptide in aqueous and membrane-mimetic environments. CD spectra indicate that each of the seven TM peptides form thermally stable, independent alpha-helical structures in both micelles and vesicles. The helical content of the peptides depends on the nature of the membrane-mimetic environment. Four of the peptides (TM3, TM4, TM5, and TM7) exhibit very high-helical structure, near the predicted maximum for their TM segments. The TM1 peptide also adopts relatively high alpha-helical structures. In contrast, two of peptides, TM2 and TM6, display low alpha helicity. Similarly, the ability of the peptides to insert into membrane-mimetic environments, assayed by intrinsic tryptophan fluorescence and fluorescence quenching, varied markedly. Most peptides exhibit higher alpha helicity in anionic sodium dodecyl sulfate than in neutral dodecyl-beta-D-maltoside micelles, and TM2 was disordered in zwiterionic DMPC but was alpha-helical in negatively charged DMPC/DMPG vesicles. These findings strongly suggest that electrostatic interactions between lipids and peptides control the insertion of the peptides and may be involved in membrane-protein-folding events. The measured helical content of these TM domains does not correlate with the predicted helicity based on amino acid sequence, pointing out that, while hydrophobic interactions can be a major determinant for folding of TM peptides, other factors, such as electrostatic interactions or helix-helix interactions, may play significant roles for specific TM domains. Our results represent a comprehensive analysis of helical propensities for a human GPCR and support models for membrane-protein folding in which interactions between TM domains are required for proper insertion and folding of some TM helix domains. The tendency of some peptides to self-associate, especially in aqueous environments, underscores the need to prevent improper interactions during folding and refolding of membrane proteins in vivo and in vitro.
Photochemistry and Photobiology, Mar 1, 2009
Glu194 is a residue located at the end of F helix on the extracellular side of the light-induced ... more Glu194 is a residue located at the end of F helix on the extracellular side of the light-induced proton pump bacteriorhodopsin (BR). Currently, it is well recognized that Glu194 and Glu204 residues, along with water clusters, constitute the proton release group of BR. Here we report that the replacement of Glu194 for Gln affects not only the photocycle of the protein but also has tremendous effect on the all-trans to 13-cis thermal isomerization. We studied the pH dependence of the dark adaptation of the E194Q mutant and performed HPLC analysis of the isomer compositions of the light-and partially darkadapted states of the mutant at several pH values. Our data confirmed that E194Q exhibits extremely slow dark adaptation over a wide range of pH. HPLC data showed that a significantly larger concentration of all-trans isomer was present in the samples of the E194Q mutant even after prolonged dark adaptation. After 14 days in the dark the 13-cis to all-trans ratio was 1:3 in the mutant, compared to 2:1 in the wild type. These data clearly indicate the involvement of Glu194 in control of the rate of all-trans to 13-cis thermal isomerization.
The chromophore fluorescence of bacteriorhodopsin (BR) has been studied by using visible lines of... more The chromophore fluorescence of bacteriorhodopsin (BR) has been studied by using visible lines of an argon and He-Ne laser at room temperature. This fluorescence is extremely weak and appears as an unstructured band in the 650-880 nm range. The same line shape of ...
Biochemistry, 2010
G protein-coupled receptors (GPCRs)1 constitute the largest family of integral membrane proteins ... more G protein-coupled receptors (GPCRs)1 constitute the largest family of integral membrane proteins present in all eukaryotic cells, yet relatively little information is known pertaining to their structure, folding, and stability. In this work, we describe several approaches to characterize conformational stability of the human adenosine A 2 a receptor (hA 2 aR). Thermal and chemical denaturation were not reversible, yet clear differences in the unfolding behavior were observed upon ligand binding via circular dichroism and fluorescence spectrometry. We found that the stability of hA 2 aR was increased upon incubation with the agonist N 6-cyclohexyladenosine or the antagonist theophylline. When extracellular disulfide bonds were reduced with a chemical reducing agent, the ligand-binding activity decreased by ~40%, but reduction of these bonds did not compromise the unfolding transition observed via urea denaturation. Overall, these approaches offer a general strategy for characterizing the effect of surfactant and ligand effects on the stability of GPCRs.
The following multiple Glu extracellular mutants of bacteriorhodopsin have been studied, with res... more The following multiple Glu extracellular mutants of bacteriorhodopsin have been studied, with respect to their possible use in bioelectronic applications: the quadruple E9Q+E74Q+E194Q+E204Q, the triple E9Q+E194Q+E204Q and the double E194Q+E204Q. The wavelength of the absorption maximum of the quadruple mutant is very sensitive to pH and salts, covering a wide wavelength range, from about 500 to 600 nm. It shows a high accessibility for small molecules like hydroxylamine. The triple mutant shows properties very similar to the quadruple mutant. The mutant E194Q+E204Q possesses intermediate properties in between wild type and the quadruple mutant. It has a wavelength maximum at 530-540 nm in water and at neutral pH, and shows increased accessibility for hydroxylamine. All mutants are stable at extreme pH values. Thermal stability was monitored using DSC. In water at neutral pH, small differences were detected with respect to the main transition, the E9Q+E74Q+E194Q+E204Q and E9Q+E194Q+E204Q mutants showing a T m about 6 ºC lower than wild type. None of the multiple mutants possess the pre-transition that is found at about 82 ºC for the wild type. Functional studies using flash photolysis showed that the E9Q+E74Q+E194Q+E204Q mutant, like E9Q or E204Q, presents increased yield of the O 640 intermediate. Light-dark adaptation was also found to be strongly altered in all multiple mutants. As an example of a new application of the purple membrane, formation of a wild type film over a microchip makes its electrical response independent on pH in the range 4-8, thus having the characteristics of a reference electrode for pH measurements. Formation of a film using the E9Q+E74Q+E194Q+E204Q mutant nearly eliminates the sharp decrease of the electric signal obtained below pH 4. Thus, the quadruple mutant shows a promising behaviour for extending the useful range down to acid pH values.