Identification of Specific Effect of Chloride on the Spectral Properties and Structural Stability of Multiple Extracellular Glutamic Acid Mutants of Bacteriorhodopsin.

In the present work we combine spectroscopic, DSC and computational approaches to examine the multiple extracellular Glu mutants E204Q/E194Q, E204Q/E194Q/E9Q and E204Q/E194Q/E9Q/E74Q of bacteriorhodopsin by varying solvent ionic strength and composition. Absorption spectroscopy data reveal that the absorption maxima of multiple EC Glu mutants can be tuned by the chloride concentration in the solution. Visible Circular dichroism spectra imply that the specific binding of Cl- can modulate weakened exciton chromophore coupling and reestablish wild type-like bilobe spectral features of the mutants. The DSC data display reappearance of the reversible thermal transition, higher Tm of denaturation and an increase in the enthalpy of unfolding of the mutants in 1 M KCl solutions. Molecular dynamics simulations indicate high affinity binding of Cl- to Arg82 and to Gln204 and Gln194 residues in the mutants. Analysis of the experimental data suggests that simultaneous elimination of the negatively charged side chain of Glu194 and Glu204 is the major cause for mutants' alterations. Specific Cl- binding efficiently coordinates distorted hydrogen bonding interactions of the EC region and reconstitutes the conformation and structure stability of mutated bR in WT-like fashion.

Helical unwinding and side-chain unlocking unravel the outward open conformation of the melibiose transporter.

Molecular dynamics simulations have been used to study the alternate access mechanism of the melibiose transporter from Escherichia coli. Starting from the outward-facing partially occluded form, 2 out of 12 simulations produced an outward full open form and one partially open, whereas the rest yielded fully or partially occluded forms. The shape of the outward-open form resembles other outward-open conformations of secondary transporters. During the transporter opening, conformational changes in some loops are followed by changes in the periplasm region of transmembrane helix 7. Helical curvature relaxation and unlocking of hydrophobic and ionic locks promote the outward opening of the transporter making accessible the substrate binding site. In particular, FRET studies on mutants of conserved aromatic residues of extracellular loop 4 showed lack of substrate binding, emphasizing the importance of this loop for making crucial interactions that control the opening of the periplasmic side. This study indicates that the alternate access mechanism for the melibiose transporter fits better into a flexible gating mechanism rather than the archetypical helical rigid-body rocker-switch mechanism.

The Melibiose Transporter of Escherichia coli: CRITICAL CONTRIBUTION OF LYS-377 TO THE STRUCTURAL ORGANIZATION OF THE INTERACTING SUBSTRATE BINDING SITES.

We examine the role of Lys-377, the only charged residue in helix XI, on the functional mechanism of the Na(+)-sugar melibiose symporter from Escherichia coli. Intrinsic fluorescence, FRET, and Fourier transform infrared difference spectroscopy reveal that replacement of Lys-377 with either Cys, Val, Arg, or Asp disables both Na(+) and melibiose binding. On the other hand, molecular dynamics simulations extending up to 200-330 ns reveal that Lys-377 (helix XI) interacts with the anionic side chains of two of the three putative ligands for cation binding (Asp-55 and Asp-59 in helix II). When Asp-59 is protonated during the simulations, Lys-377 preferentially interacts with Asp-55. Interestingly, when a Na(+) ion is positioned in the Asp-55-Asp-59 environment, Asp-124 in helix IV (a residue essential for melibiose binding) reorients and approximates the Asp-55-Asp-59 pair, and all three acidic side chains act as Na(+) ligands. Under these conditions, the side chain of Lys-377 interacts with the carboxylic moiety of these three Asp residues. These data highlight the crucial role of the Lys-377 residue in the spatial organization of the Na(+) binding site. Finally, the analysis of the second-site revertants of K377C reveals that mutation of Ile-22 (in helix I) preserves Na(+) binding, whereas that of melibiose is largely abolished according to spectroscopic measurements. This amino acid is located in the border of the sugar-binding site and might participate in sugar binding through apolar interactions.

Engineering a Robust Photovoltaic Device with Quantum Dots and Bacteriorhodopsin.

We present a route toward a radical improvement in solar cell efficiency using resonant energy transfer and sensitization of semiconductor metal oxides with a light-harvesting quantum dot (QD)/bacteriorhodopsin (bR) layer designed by protein engineering. The specific aims of our approach are (1) controlled engineering of highly ordered bR/QD complexes; (2) replacement of the liquid electrolyte by a thin layer of gold; (3) highly oriented deposition of bR/QD complexes on a gold layer; and (4) use of the Forster resonance energy transfer coupling between bR and QDs to achieve an efficient absorbing layer for dye-sensitized solar cells. This proposed approach is based on the unique optical characteristics of QDs, on the photovoltaic properties of bR, and on state-of-the-art nanobioengineering technologies. It permits spatial and optical coupling together with control of hybrid material components on the bionanoscale. This method paves the way to the development of the solid-state photovoltaic device with the efficiency increased to practical levels.

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.

The substitution of Arg149 with Cys fixes the melibiose transporter in an inward-open conformation.

The melibiose transporter from Escherichia coli (MelB) can use the electrochemical energy of either H(+), Na(+) or Li(+) to transport the disaccharide melibiose to the cell interior. By using spectroscopic and biochemical methods, we have analyzed the role of Arg149 by mutagenesis. According to Fourier transform infrared difference and fluorescence spectroscopy studies, R149C, R149Q and R149K all bind substrates in proteoliposomes, where the protein is disposed inside-out. Analysis of right-side-out (RSO) and inside-out (ISO) membrane vesicles showed that the functionally active R149Q and R149K mutants could bind externally added fluorescent sugar analog in both types of vesicles. In contrast, the non-transporting R149C mutant does bind the fluorescent sugar analog as well as melibiose and Na(+) in ISO, but not in RSO vesicles. Therefore, the mutation of Arg149 into cysteine restrains the orientation of transporter to an inward-open conformation, with the inherent consequences of a) reducing the frequency of access of outer substrates to the binding sites, and b) impairing active transport. It is concluded that Arg149, most likely located in the inner (cytoplasmic) half of transmembrane helix 5, is critically involved in the reorientation mechanism of the substrate-binding site accessibility in MelB.

Studying substrate binding to reconstituted secondary transporters by attenuated total reflection infrared difference spectroscopy.

The determination of protein conformational changes induced by the interaction of substrates with secondary transporters is an important step toward the elucidation of their transport mechanism. Since conformational changes in a protein alter its vibrational patterns, they can be detected with high sensitivity by infrared difference (IR(diff)) spectroscopy without the need for external probes. We describe a general procedure to obtain substrate-induced IR(diff) spectra by alternating perfusion of buffers over an attenuated total reflection (ATR) crystal containing an adhered film of a membrane protein reconstituted in lipids. As an example, we provide specific protocols to obtain melibiose and Na(+)-induced ATR-IR(diff) spectra of reconstituted melibiose permease, a sodium/melibiose co-transporter from E. coli. The presented methodology is applicable in principle to any membrane protein, provided that it can be purified and reconstituted in functional form, and appropriate substrates are available.

Probing a polar cluster in the retinal binding pocket of bacteriorhodopsin by a chemical design approach.

Bacteriorhodopsin has a polar cluster of amino acids surrounding the retinal molecule, which is responsible for light harvesting to fuel proton pumping. From our previous studies, we have shown that threonine 90 is the pivotal amino acid in this polar cluster, both functionally and structurally. In an attempt to perform a phenotype rescue, we have chemically designed a retinal analogue molecule to compensate the drastic effects of the T90A mutation in bacteriorhodopsin. This analogue substitutes the methyl group at position C(13) of the retinal hydrocarbon chain by and ethyl group (20-methyl retinal). We have analyzed the effect of reconstituting the wild-type and the T90A mutant apoproteins with all-trans-retinal and its 20-methyl derivative (hereafter, 13-ethyl retinal). Biophysical characterization indicates that recovering the steric interaction between the residue 90 and retinal, eases the accommodation of the chromophore, however it is not enough for a complete phenotype rescue. The characterization of these chemically engineered chromoproteins provides further insight into the role of the hydrogen bond network and the steric interactions involving the retinal binding pocket in bacteriorhodopsin and other microbial sensory rhodopsins.

Structural features of the C-terminus from the human neurokinin-1 receptor.

The neurokinin-1 receptor (NK1R) is a G-protein coupled receptor found in the central and peripheral nervous systems of vertebrates, and is responsible for many physiological processes. The C-terminus domain seems to be essential for coupling to the corresponding G-protein and ß-arrestin, and is important for receptor desensitization, internalization and recycling. We have focused our study on expression of the human NK1R (hNK1R) C-terminus in Escherichia coli, and its purification and characterization, in order to elucidate its structural properties. CD and Fourier transform infrared spectroscopy showed that the hNK1R C-terminus, rather than having a random structure, has well-defined secondary-structure patterns. The presence of three tyrosine residues in the primary sequence of the hNK1R C-terminus facilitated the use of UV and fluorescence spectroscopy techniques which revealed tyrosine fluorescence and UV absorption at anomalous wavelengths. In their entirety, the results show that the hNK1R C-terminus has clearly defined secondary (25% α-helix, 27% unordered structure and 48% ß-sheets and ß-turns) and tertiary structures which, it is believed, are tightly related to its multiple functions.

Combination of extended X-ray absorption fine structure spectroscopy with lipidic cubic phases for the study of cation binding in bacteriorhodopsin.

We have performed a quantitative X-ray absorption fine structure analysis of bacteriorhodopsin in purple membrane patches and in lipidic cubic phases regenerated with Mn(2+). Lipidic cubic phases and purple membrane results have been compared, demonstrating that the lipidic cubic phase process does not introduce relevant distortions in the local geometry of the cation binding sites. For both samples, we have observed similarities for Mn(2+) coordination in terms of type, number, and average distances of surrounding atoms, indicating a first coordination shell composed by 6 O atoms, and 3/4 C atoms located in the second coordination shell.

Probing specific molecular processes and intermediates by time-resolved Fourier transform infrared spectroscopy: application to the bacteriorhodopsin photocycle.

We present a general approach for probing the kinetics of specific molecular processes in proteins by time-resolved Fourier transform infrared (IR) spectroscopy. Using bacteriorhodopsin (bR) as a model we demonstrate that by appropriately monitoring some selected IR bands it is possible obtaining the kinetics of the most important events occurring in the photocycle, namely changes in the chromophore and the protein backbone conformation, and changes in the protonation state of the key residues implicated in the proton transfers. Besides confirming widely accepted views of the bR photocycle, our analysis also sheds light into some disputed issues: the degree of retinal torsion in the L intermediate to respect the ground state; the possibility of a proton transfer from Asp85 to Asp212; the relationship between the protonation/deprotonation of Asp85 and the proton release complex; and the timing of the protein backbone dynamics. By providing a direct way to estimate the kinetics of photocycle intermediates the present approach opens new prospects for a robust quantitative kinetic analysis of the bR photocycle, which could also benefit the study of other proteins involved in photosynthesis, in phototaxis, or in respiratory chains.

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.

Structural insights into the activation mechanism of melibiose permease by sodium binding.

The melibiose carrier from Escherichia coli (MelB) couples the accumulation of the disaccharide melibiose to the downhill entry of H(+), Na(+), or Li(+). In this work, substrate-induced FTIR difference spectroscopy was used in combination with fluorescence spectroscopy to quantitatively compare the conformational properties of MelB mutants, implicated previously in sodium binding, with those of a fully functional Cys-less MelB permease. The results first suggest that Asp55 and Asp59 are essential ligands for Na(+) binding. Secondly, though Asp124 is not essential for Na(+) binding, this acidic residue may play a critical role, possibly by its interaction with the bound cation, in the full Na(+)-induced conformational changes required for efficient coupling between the ion- and sugar-binding sites; this residue may also be a sugar ligand. Thirdly, Asp19 does not participate in Na(+) binding but it is a melibiose ligand. The location of these residues in two independent threading models of MelB is consistent with their proposed role.

In-plane and out-of-plane infrared difference spectroscopy unravels tilting of helices and structural changes in a membrane protein upon substrate binding.

Attenuated total reflection infrared (ATR-IR) difference spectroscopy stands out because of its ability to provide information on the interaction of substrates with membrane proteins in their native lipid bilayer environment. We show how the study and interpretation of the structural changes in membrane proteins upon substrate binding is simplified by obtaining ATR-IR difference spectra with polarized light and then computing the difference spectra in the z and x,y directions, where structural and orientation changes give specific difference absorbance patterns. In combination with a maximum-entropy band-narrowing method and some simple spectroscopic rules, the present approach allows us to unambiguously identify changes in the tilt of some helices in the secondary transporter melibiose permease following melibiose binding in the presence of sodium, suggesting the formation of an occluded state during the transport mechanism of the substrates.

The role and selection of the filter function in Fourier self-deconvolution revisited.

Overlapped bands often appear in applications of infrared spectroscopy, for instance in the analysis of the amide I band of proteins. Fourier self-deconvolution (FSD) is a popular band-narrowing mathematical method, allowing for the resolution of overlapped bands. The filter function used in FSD plays a significant role in the factor by which the deconvolved bands are actually narrowed (the effective narrowing), as well as in the final signal-to-noise degradation induced by FSD. Moreover, the filter function determines, to a good extent, the band-shape of the deconvolved bands. For instance, the intensity of the harmful side-lobule oscillations that appear in over-deconvolution depends importantly on the filter function used. In the present paper we characterized the resulting band shape, effective narrowing, and signal-to-noise degradation in infra-, self-, and over-deconvolution conditions for several filter functions: Triangle, Bessel, Hanning, Gaussian, Sinc2, and Triangle2. We also introduced and characterized new filters based on the modification of the Blackmann filter. Our conclusion is that the Bessel filter (in infra-, self-, and mild over-deconvolution), the newly introduced BL3 filter (in self- and mild/moderate over-deconvolution), and the Gaussian filter (in moderate/strong over-deconvolution) are the most suitable filter functions to be used in FSD.

Alteration of sugar-induced conformational changes of the melibiose permease by mutating Arg141 in loop 4-5.

The melibiose permease (MelB) from Escherichia coli couples the uptake of melibiose to that of Na+, Li+, or H+. In this work, we applied attenuated total reflection Fourier transform infrared (ATR-FTIR) difference spectroscopy to obtain information about the structural changes involved in substrate interaction with the R141C mutant and with the wild-type MelB reacted with N-ethylmaleimide (NEM). These modified permeases have the ability to bind the substrates but fail to transport them. It is shown that the sugar-induced ATR-FTIR difference spectra of the R141C mutant are different from those corresponding to the Cys-less permease from which it is derived. There are alterations of peaks assigned to turns and beta-structures located most likely in loop 4-5. In addition, and quite notably, a peak at 1659 cm(-1), assigned to changes at the level of one alpha-helix subpopulation, disappears in the melibiose-induced difference spectrum in the presence of Na+, suggesting a reduction of the conformational change capacity of the mutated MelB. These helices may involve structural components that couple the cation- and sugar-binding sites. On the other hand, MelB-NEM difference spectra are proportionally less disrupted than the R141C ones. Hence, the transport cycle of these two permeases, modified at two different loops, is most likely impaired at a different stage. It is proposed that the R141C mutant leads to the generation of a partially defective ternary complex that is unable to catalyze the subsequent conformational change necessary for substrate translocation.

Study on the feasibility of bacteriorhodopsin as bio-photosensitizer in excitonic solar cell: a first report.

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 TiO2 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 TiO2 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 TiO2 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 TiO2 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/cm2 and the open-circuit photovoltage (V(oc)) 0.35 V, under an illumination intensity of 40 mW/cm2.

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.

Calcium binding to the purple membrane: A molecular dynamics study.

The purple membrane (PM) is a specialized membrane patch found in halophilic archaea, containing the photoreceptor bacteriorhodopsin (bR). It is long known that calcium ions bind to the PM, but their position and role remain elusive to date. Molecular dynamics simulations in conjunction with a highly detailed model of the PM have been used to investigate the stability of calcium ions placed at three proposed cation binding sites within bR, one near the Schiff base, one in the region of the proton release group, and one near Glu9. The simulations suggest that, of the sites investigated, the binding of calcium ions was most likely at the proton release group. Binding in the region of the Schiff base, while possible, was associated with significant changes in local geometry. Calcium ions placed near Glu9 in the interior of bR (simultaneously to a Ca(2+) near the Schiff base and another one near the Glu194-Glu204 site) were not stable. The results obtained are discussed in relation to recent experimental observations and theoretical considerations.