Tzvetana Lazarova

Opening the Schiff base moiety of bacteriorhodopsin by mutation of the four extracellular Glu side chains

The quadruple bacteriorhodopsin (BR) mutant E9Q+E74Q+E194Q+E204Q shows a λ max of about 500 nm in water at neutral pH and a great influence of pH and salts on the visible absorption spectrum. Accessibility to the Schiff base is strongly increased, as detected by the rapid bleaching effect of hydroxylamine in the dark as well as in light. Both the proton release kinetics and the photocycle are altered, as indicated by a delayed proton release after proton uptake and changed M kinetics. Moreover, affinity of the color‐controlling cation(s) is found to be decreased. We suggest that the four Glu side chains are essential elements of the extracellular structure of BR.

Fourier Transform Infrared Evidence for Early Deprotonation of Asp85 at Alkaline pH in the Photocycle of Bacteriorhodopsin Mutants Containing E194Q

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 M- and 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(-1), indicating that Asp(85) and Asp(96) are deprotonated. Under the same conditions E204Q showed a positive peak at 1762 cm(-1) and a negative peak at 1742 cm(-1), revealing the presence of protonated Asp(85) (in an M intermediate environment) and deprotonated Asp(96). These results indicate that in E194Q-containing mutants, the second increase in the Asp(85) pK(a) is inhibited because of lack of deprotonation of the proton release group. Our data suggest that Glu(194) is the group that controls the pK(a) of Asp(85).

Analysis of Adenosine A2a Receptor Stability: Effects of Ligands and Disulfide Bonds †

G protein-coupled receptors (GPCRs) constitute the largest family of integral membrane proteins present in all eukaryotic cells, yet relatively little information about their structure, folding, and stability has been published. In this work, we describe several approaches to characterizing the conformational stability of the human adenosine A(2)a receptor (hA(2)aR). Thermal denaturation 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.

Stable interactions between the transmembrane domains of the adenosine A2A receptor

G‐protein‐coupled receptors (GPCRs) must properly insert and fold in the membrane to adopt a stable native structure and become biologically active. The interactions between transmembrane (TM) helices are believed to play a major role in these processes. Previous studies in our group showed that specific interactions between TM helices occur, leading to an increase in helical content, especially in weakly helical TM domains, suggesting that helix–helix interactions in addition to helix–lipid interactions facilitate helix formation. They also demonstrated that TM peptides interact in a similar fashion in micelles and lipid vesicles, as they exhibit relatively similar thermal stability and α‐helicity inserted in SDS micelles to that observed in liposomes. In this study, we perform an analysis of pairwise interactions between peptides corresponding to the seven TM domains of the human A2A receptor (A2AR). We used a combination of Förster resonance energy transfer (FRET) measurement and circular dichroism (CD) spectroscopy to detect and analyze these interactions in detergent micelles. We found that strong and specific interactions occur in only seven of the 28 possible peptide pairs. Furthermore, not all interactions, identified by FRET, lead to a change in helicity. Our results identify stabilizing contacts that are likely related to the stability of the receptor and that are consistent with what is known about the three‐dimensional structure and stability of rhodopsin and the β2 adrenergic receptor.

Synthesis of N-Heteroaryl Retinals and their Artificial Bacteriorhodopsins

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 pKa values of these artificial bacteriorhodopsins are interpreted as arising from nonoptimal binding‐site occupancy by the chromophore due to steric constraints.

Study of membrane-induced conformations of substance P: detection of extended polyproline II helix conformation.

We study the conformation of substance P (SP), a ligand of neurokinin 1 receptor, and its analogue [Trp8]SP in membrane-mimetic media to provide further insights into membrane-ligand interactions and the factors determining and modulating the peptide structure. CD data revealed that the neuropeptide attains α-helical fold in negatively charged SDS micelles and DMPG liposomes but not in zwitterionic DMPC. The fluorescence experiments reported that the Trp side chain of [Trp8]SP inserts into the hydrophobic core of the SDS micelles and DMPG liposomes but faces the DMPC hydrophilic region, indicating that electrostatic interactions between membrane and SP are essential for the α-helical fold. Formation of extended polyproline II (PPII) helical structure in aqueous solutions and in submicellar concentrations of SDS and DMPC liposomes was confirmed by comparing CD spectra at increasing temperatures. Moreover, in all conditions where PPII conformation was detected, the Trp was totally exposed to the bulk. The PPII structure may be vital for recognition processes of SP by neurokinin receptors.

SYNTHESIS OF NEW HETEROCYCLIC AND POLYCYCLIC AROMATIC RETINALS AND THEIR BACTERIORHODOPSIN ANALOGUES

Abstract New heterocyclic and polycyclic aromatic retinal analogues (Fig.1) were synthesised and their recombination with bacterioopsin was studied.

Coupling between the retinal thermal isomerization and the Glu194 residue of bacteriorhodopsin.

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 dark‐adapted 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 effect of triple glutamic mutations E9Q/E194Q/E204Q on the structural stability of bacteriorhodopsin.

In the present study, we report on the structural features of the bacteriorhodopsin triple mutant E9Q/E194Q/E204Q (3Glu) of bacteriorhodopsin by combining experimental and molecular dynamics (MD) approaches. In 3Glu mutant, Glu9, Glu194 and Glu204 residues located at the extracellular side of the protein were mutated altogether to glutamines. UV‐visible and differential scanning calorimetry experiments served as diagnostic tools for monitoring the resistance against thermal stress of the active site and the tertiary structures of the 3Glu. The analyses of the UV‐visible thermal difference spectra demonstrate that the spectral forms at room temperature and the thermal unfolding path differ in the wild‐type bacteriorhodopsin and the 3Glu. Even with these spectral differences, the thermal unfolding of the active site occurs at rather similar melting temperatures in both proteins. A noteworthy consequence of the mutations is the altered two‐dimensional packing revealed by the lack of the pre‐transition peak in differential scanning calorimetry traces of 3Glu mutant, as previously detected in wild‐type and the corresponding single mutants. The infrared spectroscopy data agree with the loss of paracrystalinity, illustrating a substantial conversion of αII to αI helical conformation in the 3Glu mutant. Molecular dynamics simulations show higher dynamics flexibility of most of the extracellular regions of 3Glu, which may account for the somewhat lower tertiary structural stability of the mutated protein. Finally, hydrogen bond analysis reveals that the mutated Glu194 and Glu204 residues create ~ 50% less hydrogen bonds with water molecules compared to wild‐type bacteriorhodopsin. These results exemplify the role of the water hydrogen‐bonding network for structural integrity and conformational flexibility of bacteriorhodopsin.

Cross‐Linking of Transmembrane Helices Reveals a Rigid‐Body Mechanism in Bacteriorhodopsin Transport

In membrane proteins, the mechanisms driving transmembrane (TM) helices through the conformational changes needed to accomplish the translocation of substrates or for intraprotein signal transduction are still a matter of debate. Do the helices follow an alternating mechanical rocker-switch mechanism or do they act as soft structures, which would allow a Brownian ratchet mechanism? Are the interruptions or the hinges in the middle of transmembrane helices necessary to allow individual movements of parts of the helices? The activation of the G-protein-coupled receptor (GPCR) subfamily of 7TM helical receptors seems to imply that transmembrane helices VI and VII move apart (cytoplasmic side) or together (extracellular side) following a global toggle-switch mechanism. This mechanism also relies on see-saw movements around proline bends to allow the independent movement of different halves of the TM helices. Bacteriorhodopsin (bR), as a structural homologue of 7TM GPCRs, is a suitable model to further explore these questions. In bR, conformational changes triggered by the absorption of a photon by the retinal chromophore are responsible for the active transport of a proton, through a series of intermediate transport steps named K, L, M, N, and O and known as the photocycle. Although the first half of the photocycle (K–M) involves minor and subtle structural changes, the major conformational changes happen in the second half, in which the proton-transfer events swap from the extracellular to the cytoplasmic domain. It has been proposed that an outward rotation around the conserved Pro186 of helix F, accompanied by a tilt of helix G, is required for water entry during the M–N intermediate transition. It is believed that this would facilitate the reprotonation of both the Schiff base and Asp96. Conformational changes of the E–F loop during the photocycle have also been described. X-ray diffraction, spin labeling, and fluorescence experiments have provided experimental details of such movements, although the functional relevance of these changes has been questioned. An attractive approach to get insight into the nature of these movements is to restrict the conformational changes occurring between the cytoplasmic ends of helices F and G in the protein. With this purpose, we introduced two cysteines at the strategic positions 166 (end of helix F) and 228 (end of helix G) and induced a disulfide bond in the double mutant E166C/A228C (Figure 1 and the Materials and Methods