The effect of charged lipids on bacteriorhodopsin membrane reconstitution and its photochemical activities

Bacteriorhodopsin (BR) was reconstituted into artificial lipid membrane containing various charged lipid compositions. The proton pumping activity of BR under flash and continuous illumination, proton permeability across membrane, as well as the decay kinetics of the photocycle intermediate M₄₁₂ were studied. The results showed that lipid charges would significantly affect the orientation of BR inserted into lipid membranes. In liposomes containing anionic lipids, BRs were more likely to take natural orientation as in living cells. In neutral or positively charged liposomes, most BRs were reversely assembled, assuming an inside out orientation. Moreover, the lipids charges also affect BR’s M intermediate kinetics, especially the slow component in M intermediate decay. The half-life M_412s increased significantly in BRs in liposomes containing cationic lipids, while decreased in those in anionic liposomes.     

Reversible sheet-turn conformational change of a cell-penetrating peptide in lipid bilayers studied by solid-state NMR

The membrane-bound conformation of a cell-penetrating peptide, penetratin, is investigated using solid-state NMR spectroscopy. The ¹³C chemical shifts of ¹³C, ¹⁵N-labeled residues in the peptide indicate a reversible conformational change from β-sheet at low temperature to coil-like at high temperature. This conformational change occurs for all residues examined between positions 3 and 13, at peptide/lipid molar ratios of 1:15 and 1:30, in membranes with 25-50% anionic lipids, and in both saturated DMPC/DMPG (1,2-dimyristoyl-sn-glycero-3-phosphatidylchloline/1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol) membranes and unsaturated POPC/POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol) membranes. Thus, it is an intrinsic property of penetratin. The coil state of the peptide has C-H order parameters of 0.23-0.52 for C^α and C^β sites, indicating that the peptide backbone is unstructured. Moreover, chemical shift anisotropy lineshapes are uniaxially averaged, suggesting that the peptide backbone undergoes uniaxial rotation around the bilayer normal. These observations suggest that the dynamic state of penetratin at high temperature is a structured turn instead of an isotropic random coil. The thermodynamic parameters of this sheet-turn transition are extracted and compared to other membrane peptides reported to exhibit conformational changes. We suggest that the function of this turn conformation may be to reduce hydrophobic interactions with the lipid chains and facilitate penetratin translocation across the bilayer without causing permanent membrane damage.     

Mutual structural effect of bilirubin and model membranes by vibrational circular dichroism

In this study, vibrational circular dichroism (VCD) spectroscopy was employed for the first time to study the bilirubin (BR) interaction with model membranes and models for membrane proteins. An enantioselective interaction of BR with zwitterionic 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and sphingomyelin (SPM) liposomes was observed by VCD and electronic circular dichroism (ECD) complemented by absorption and fluorescence spectroscopy. The M-form of BR was preferentially recognized in the BR/DMPC system at concentration above 1 × 10^− 4 M, for lower concentrations the P-form of BR was recognized by the DMPC liposomes. The VCD spectra also showed that the SPM liposomes, which represent the main component of nerve cell membrane, were significantly more disturbed by the presence of BR than the DMPC liposomes—a stable association with a strong VCD signal was observed providing the explanations for the supposed BR neurotoxicity. The effect of time and pH on the BR/DMPC or SPM liposome systems was shown to be essential while the effect of temperature in the range of 15–70 °C was negligible demonstrating the surprisingly high temperature stability of BR when interacting with the studied membranes. The influence of a membrane protein was tested on a model consisting of poly-l-arginine (PLAG) bound in the α-helical form to the surface of 1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol) liposomes and sodium dodecyl sulfate micelles. VCD and also ECD spectra showed that a variety of BR diastereoisomers interacted with PLAG in such systems. In a system of PLAG with micelles composed of sodium dodecyl sulfate, the M-form of bound BR was observed.     

Characterization of the primary photochemistry of proteorhodopsin with femtosecond spectroscopy

Proteorhodopsin is an ion-translocating member of the microbial rhodopsin family. Light absorption by its retinal chromophore initiates a photocycle, driven by trans/cis isomerization, leading to transmembrane translocation of a proton toward the extracellular side of the cytoplasmic membrane. Here we report a study on the photoisomerization dynamics of the retinal chromophore of proteorhodopsin, using femtosecond time-resolved spectroscopy, by probing in the visible- and in the midinfrared spectral regions. Experiments were performed both at pH 9.5 (a physiologically relevant pH value in which the primary proton acceptor of the protonated Schiff base, Asp⁹⁷, is deprotonated) and at pH 6.5 (with Asp⁹⁷ protonated). Simultaneous analysis of the data sets recorded in the two spectral regions and at both pH values reveals a multiexponential excited state decay, with time constants of ∼0.2 ps, ∼2 ps, and ∼20 ps. From the difference spectra associated with these dynamics, we conclude that there are two chromophore-isomerizaton pathways that lead to the K-state: one with an effective rate of ∼(2 ps)⁻¹ and the other with a rate of ∼(20 ps)⁻¹. At high pH, both pathways are equally effective, with an estimated quantum yield for K-formation of ∼0.7. At pH 6.5, the slower pathway is less productive, which results in an isomerization quantum yield of 0.5. We further observe an ultrafast response of residue Asp²²⁷, which forms part of the counterion complex, corresponding to a strengthening of its hydrogen bond with the Schiff base on K-state formation; and a feature that develops on the 0.2 ps and 2 ps timescale and probably reflects a response of an amide II band in reaction to the isomerization process.     

Characterization of Membrane Protein Interactions by Isothermal Titration Calorimetry

Understanding the structure, folding, and interaction of membrane proteins requires experimental tools to quantify the association of transmembrane (TM) helices. Here, we introduce isothermal titration calorimetry (ITC) to measure integrin αIIbβ3 TM complex affinity, to study the consequences of helix–helix preorientation in lipid bilayers, and to examine protein-induced lipid reorganization. Phospholipid bicelles served as membrane mimics. The association of αIIbβ3 proceeded with a free energy change of − 4.61 ± 0.04 kcal/mol at bicelle conditions where the sampling of random helix–helix orientations leads to complex formation. At bicelle conditions that approach a true bilayer structure in effect, an entropy saving of > 1 kcal/mol was obtained from helix–helix preorientation. The magnitudes of enthalpy and entropy changes increased distinctly with bicelle dimensions, indicating long-range changes in bicelle lipid properties upon αIIbβ3 TM association. NMR spectroscopy confirmed ITC affinity measurements and revealed αIIbβ3 association and dissociation rates of 4500 ± 100 s^− 1 and 2.1 ± 0.1 s^− 1, respectively. Thus, ITC is able to provide comprehensive insight into the interaction of membrane proteins.     

Structural basis of diversification of fungal retinal proteins probed by site-directed mutagenesis of Leptosphaeria rhodopsin

Numerous fungal genomes encode homologs of bacteriorhodopsin (BR), but only two fungal rhodopsins were overexpressed and characterized spectroscopically. Neurospora rhodopsin (NR) is a slow-cycling sensory rhodopsin-like protein, while Leptosphaeria rhodopsin (LR) is a BR-like proton pump. Recently, we found that a conservative replacement of the cytoplasmic proton donor Asp150 by Glu converts LR into an NR-like protein. In this work, we search for structural reasons for the dramatic differences in their photochemistry by mutating the hydrogen-bonding partner of Asp150 (Thr87) and three additional residues (Thr233, Asp248, and Gly271) selected by comparison of the primary structures of NR and LR. We conclude that while these residues may contribute to the differences between LR and NR, they are not crucial for the optimization of the Schiff base reprotonation by Asp150, and that the dramatic effect of the D150E mutation is not a simple result of the introduction of a bulkier glutamate sidechain.     

Weakened coupling of conserved arginine to the proteorhodopsin chromophore and its counterion implies structural differences from bacteriorhodopsin

In wild-type proteorhodopsin (pR), titration of the chromophore's counterion Asp⁹⁷ occurs with a pK_a of 8.2±0.1. R94C mutation reduces this slightly to 7.0±0.2, irrespective of treatment with ethylguanidinium. This contrasts with the homologous archaeal protein bacteriorhodopsin (bR), where R82C mutation was previously shown to elevate the pK_a of Asp⁸⁵ by ∼5 units, while reconstitution with ethylguanidinium restores it nearly to the wild-type value of 2.5. We conclude there is much weaker electrostatic coupling between Arg⁹⁴ and Asp⁹⁷ in the unphotolyzed state of pR, in comparison to Arg⁸² and Asp⁸⁵ in bR. Therefore, while fast light-driven H⁺ release may depend on these two residues in pR as in bR, no tightly conserved pre-photolysis configuration of them is required.     

Crystal structure of the eukaryotic light-driven proton-pumping rhodopsin, Acetabularia rhodopsin II, from marine alga

Acetabularia rhodopsin (AR) is a rhodopsin from the marine plant Acetabularia acetabulum. The opsin-encoding gene from A. acetabulum, ARII, was cloned and found to be novel but homologous to that reported previously. ARII is a light-driven proton pump, as demonstrated by the existence of a photo-induced current through Xenopus oocytes expressing ARII. The photochemical reaction of ARII prepared by cell-free protein synthesis was similar to that of bacteriorhodopsin (BR), except for the lack of light-dark adaptation and the different proton release and uptake sequence. The crystal structure determined at 3.2 Å resolution is the first structure of a eukaryotic member of the microbial rhodopsin family. The structure of ARII is similar to that of BR. From the cytoplasmic side to the extracellular side of the proton transfer pathway in ARII, Asp92, a Schiff base, Asp207, Asp81, Arg78, Glu199, and Ser189 are arranged in positions similar to those of the corresponding residues directly involved in proton transfer by BR. The side-chain carboxyl group of Asp92 appears to interact with the sulfhydryl group of Cys218, which is unique to ARII and corresponds to Leu223 of BR and to Asp217 of Anabaena sensory rhodopsin. The orientation of the Arg78 side chain is opposite to the corresponding Arg82 of BR. The putative absence of water molecules around Glu199 and Arg78 may disrupt the formation of the low-barrier hydrogen bond at Glu199, resulting in the “late proton release”.     

The effect of binding of spider-derived antimicrobial peptides, oxyopinins, on lipid membranes

Oxyopinins (Oxki1 and Oxki2) are antimicrobial peptides isolated from the crude venom of the wolf spider Oxyopes kitabensis. The effect of oxyopinins on lipid bilayers was investigated using high-sensitivity titration calorimetry and ³¹P solid-state NMR spectroscopy. High-sensitivity titration calorimetry experiments showed that the binding of oxyopinins was exothermic, and the binding enthalpies (ΔH) to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) small unilamellar vesicles (SUVs) were − 18.1 kcal/mol and − 15.0 kcal/mol for Oxki1 and Oxki2, respectively, and peptide partition coefficient (K_p) was found to be 3.9 × 10³ M^− 1. ³¹P NMR spectra of 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE) membranes in the presence of oxyopinins indicated that they induced a positive curvature in lipid bilayers. The induced positive curvature was stronger in the presence of Oxki2 than in the presence of Oxki1. ³¹P NMR spectra of phosphaditylcholine (PC) membranes in the presence of Oxki2 showed that Oxki2 produced micellization of membranes at low peptide concentrations, but unsaturated PC membranes or acidic phospholipids prevented micellization from occurring. Furthermore, ³¹P NMR spectra using membrane lipids from E. coli suggested that Oxki1 was more disruptive to bacterial membranes than Oxki2. These results strongly correlate to the known biological activity of the oxyopinins.     

Asp and Thr are critical for cation exchange mediated by NhaD, Na/H antiporter of Vibrio cholerae

The Vc-NhaD is an Na⁺/H⁺ antiporter from Vibrio cholerae belonging to a new family of bacterial Na⁺/H⁺ antiporters, the NhaD family. In the present work we mutagenized five conserved Asp and Glu residues and one conserved Thr residue to Ala in order to identify amino acids that are critical for the antiport activity. All mutations fall into two distinct groups: (i) four variants, Glu¹⁰⁰Ala, Glu²⁵¹Ala, Glu³⁴²Ala, and Asp³⁹³Ala, did not abolish antiport activity but shifted the pH optimum to more alkaline pH, and (ii) variants Asp³⁴⁴Ala, Asp³⁴⁴Asn, and Thr³⁴⁵Ala caused a complete loss of both Na⁺/H⁺ and Li⁺/H⁺ antiport activity whereas the Asp³⁴⁴Glu variant exhibited reduced Na⁺/H⁺ and Li⁺/H⁺ antiport activity. This is the first mutational analysis of the antiporter of NhaD type and the first demonstration of Thr residue being indispensable for Na⁺/H⁺ antiport. We discuss the possible role of Asp³⁴⁴ and Thr³⁴⁵ in the functioning of Vc-NhaD.     

Role of extracellular glutamic acids in the stability and energy landscape of bacteriorhodopsin

Bacteriorhodopsin (BR), a specialized nanomachine, converts light energy into a proton gradient to power Halobacterium salinarum. In this work, we analyze the mechanical stability of a BR triple mutant in which three key extracellular residues, Glu⁹, Glu¹⁹⁴, and Glu²⁰⁴, were mutated simultaneously to Gln. These three Glu residues are involved in a network of hydrogen bonds, in cation binding, and form part of the proton release pathway of BR. Changes in these features and the robust photocycle dynamics of wild-type (WT) BR are apparent when the three extracellular Glu residues are mutated to Gln. It is speculated that such functional changes of proteins go hand in hand with changes in their mechanical properties. Here, we apply single-molecule dynamic force spectroscopy to investigate how the Glu to Gln mutations change interactions, reaction pathways, and the energy barriers of the structural regions of WT BR. The altered heights and positions of individual energy barriers unravel the changes in the mechanical and the unfolding kinetic properties of the secondary structures of WT BR. These changes in the mechanical unfolding energy landscape cause the proton pump to choose unfolding pathways differently. We suggest that, in a similar manner, the changed mechanical properties of mutated BR alter the functional energy landscape favoring different reaction pathways in the light-induced proton pumping mechanism.     

Proteorhodopsin is a light-driven proton pump with variable vectoriality

Proteorhodopsin, a homologue of archaeal bacteriorhodopsin (BR), belongs to a newly identified family of retinal proteins from marine bacteria, which could play an important role in the energy balance of the biosphere. We cloned the cDNA sequence of proteorhodopsin by chemical gene synthesis, expressed the protein in Escherichia coli cells, purified and reconstituted the protein in its functional active state. The photocycle characteristics were determined by time-resolved absorption and Fourier transform infrared (FT-IR) spectroscopy. The pH-dependence of the absorption spectrum indicates that the pK(a) of the primary acceptor of the Schiff base proton (Asp97) is 7.68. Generally, the photocycle of proteorhodopsin is similar to that of BR, although an L-like photocycle intermediate was not detectable. Whereas at pH>7 an M-like intermediate is formed upon illumination, at pH 5 no M-like intermediate could be detected. As the photocycle kinetics do not change between the acidic and alkaline state of proteorhodopsin, the only difference between these two forms is the protonation status of Asp97. This is corroborated by time-resolved FT-IR spectroscopy, which demonstrates that proton transfer from the retinal Schiff base to Asp97 is observed at alkaline pH, but the other vibrational changes are essentially pH-independent.After reconstitution into proteoliposomes, light-induced proton currents of proteorhodopsin were measured in a compound membrane system where proteoliposomes were adsorbed to planar lipid bilayers. Our results show that proteorhodopsin is a light-driven proton pump with characteristics similar to those of BR at alkaline pH. However, at acidic pH, the direction of proton pumping is inverted. Complementary experiments were carried out on proteorhodopsin expressed heterologously in Xenopus laevis oocytes under voltage clamp conditions.The following results were obtained. (1) At alkaline pH, proteorhodopsin mediates outwardly directed proton pumping like BR. (2) The direction of proton pumping can be inverted, when Asp97 is protonated. (3) The current can be inverted by changes of the polarity of the applied voltage. (4) The light intensity-dependence of the photocurrents leads to the conclusion that the alkaline form of proteorhodopsin shows efficient proton pumping after sequential excitation by two photons.     

The membrane-induced structure of melittin is correlated with the fluidity of the lipids

The effect of the bee toxin melittin on DMPC dynamics in fast-tumbling bicelles has been investigated. The ¹³C R₁ and ¹³C-¹H NOE relaxation parameters for DMPC were used to monitor the effect of melittin and cholesterol on lipid dynamics. It was found that melittin has the largest effect on the DMPC mobility in DMPC/DHPC bicelles, while less effect was observed in cholesterol-doped bicelles, or in bicelles made with CHAPS, indicating that the rigidity of the membrane affects the melittin-membrane interaction. CD spectra were analysed in terms of cooperativity of the α-helix to random coil transition in melittin, and these results also indicated similar differences between the bicelles. The study shows that bicelles can be used to investigate lipid dynamics by spin relaxation, and in particular of peptide-induced changes in membrane fluidity.     

Gloeobacter Rhodopsin,Limitation of Proton Pumping at High Electrochemical Load

We studied the photocurrents of a cyanobacterial rhodopsin Gloeobacter violaceus (GR) in Xenopus laevis oocytes and HEK-293 cells. This protein is a light-driven proton pump with striking similarities to marine proteorhodopsins, including the D121-H87 cluster of the retinal Schiff base counterion and a glutamate at position 132 that acts as a proton donor for chromophore reprotonation during the photocycle. Interestingly, at low extracellular pH_o and negative voltage, the proton flux inverted and directed inward. Using electrophysiological measurements of wild-type and mutant GR, we demonstrate that the electrochemical gradient limits outward-directed proton pumping and converts it into a purely passive proton influx. This conclusion contradicts the contemporary paradigm that at low pH, proteorhodopsins actively transport H⁺ into cells. We identified E132 and S77 as key residues that allow inward directed diffusion. Substitution of E132 with aspartate or S77 with either alanine or cysteine abolished the inward-directed current almost completely. The proton influx is likely caused by the pK_a of E132 in GR, which is lower than that of other microbial ion pumping rhodopsins. The advantage of such a low pK_a is an acceleration of the photocycle and high pump turnover at high light intensities.     

High-Resolution Orientation and Depth of Insertion of the Voltage-Sensing S4 Helix of a Potassium Channel in Lipid Bilayers

The opening and closing of voltage-gated potassium (Kv) channels are controlled by several conserved Arg residues in the S4 helix of the voltage-sensing domain. The interaction of these positively charged Arg residues with the lipid membrane has been of intense interest for understanding how membrane proteins fold to allow charged residues to insert into lipid bilayers against free-energy barriers. Using solid-state NMR, we have now determined the orientation and insertion depth of the S4 peptide of the KvAP channel in lipid bilayers. Two-dimensional ¹⁵N correlation experiments of macroscopically oriented S4 peptide in phospholipid bilayers revealed a tilt angle of 40° and two possible rotation angles differing by 180° around the helix axis. Remarkably, the tilt angle and one of the two rotation angles are identical to those of the S4 helix in the intact voltage-sensing domain, suggesting that interactions between the S4 segment and other helices of the voltage-sensing domain are not essential for the membrane topology of the S4 helix. ¹³C-³¹P distances between the S4 backbone and the lipid ³¹P indicate a ∼ 9 Å local thinning and 2 Å average thinning of the DMPC (1,2-dimyristoyl-sn-glycero-3-phosphochloline)/DMPG (1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol) bilayer, consistent with neutron diffraction data. Moreover, a short distance of 4.6 Å from the guanidinium C^ζ of the second Arg to ³¹P indicates the existence of guanidinium phosphate hydrogen bonding and salt bridges. These data suggest that the structure of the Kv gating helix is mainly determined by protein-lipid interactions instead of interhelical protein-protein interactions, and the S4 amino acid sequence encodes sufficient information for the membrane topology of this crucial gating helix.     

Gloeobacter Rhodopsin,Limitation of Proton Pumping at High Electrochemical Load

We studied the photocurrents of a cyanobacterial rhodopsin Gloeobacter violaceus (GR) in Xenopus laevis oocytes and HEK-293 cells. This protein is a light-driven proton pump with striking similarities to marine proteorhodopsins, including the D121-H87 cluster of the retinal Schiff base counterion and a glutamate at position 132 that acts as a proton donor for chromophore reprotonation during the photocycle. Interestingly, at low extracellular pH_o and negative voltage, the proton flux inverted and directed inward. Using electrophysiological measurements of wild-type and mutant GR, we demonstrate that the electrochemical gradient limits outward-directed proton pumping and converts it into a purely passive proton influx. This conclusion contradicts the contemporary paradigm that at low pH, proteorhodopsins actively transport H⁺ into cells. We identified E132 and S77 as key residues that allow inward directed diffusion. Substitution of E132 with aspartate or S77 with either alanine or cysteine abolished the inward-directed current almost completely. The proton influx is likely caused by the pK_a of E132 in GR, which is lower than that of other microbial ion pumping rhodopsins. The advantage of such a low pK_a is an acceleration of the photocycle and high pump turnover at high light intensities.     

His-75 in Proteorhodopsin, a Novel Component in Light-driven Proton Translocation by Primary Pumps

Proteorhodopsins (PRs), photoactive retinylidene membrane proteins ubiquitous in marine eubacteria, exhibit light-driven proton transport activity similar to that of the well studied bacteriorhodopsin from halophilic archaea. However, unlike bacteriorhodopsin, PRs have a single highly conserved histidine located near the photoactive site of the protein. Time-resolved Fourier transform IR difference spectroscopy combined with visible absorption spectroscopy, isotope labeling, and electrical measurements of light-induced charge movements reveal participation of His-75 in the proton translocation mechanism of PR. Substitution of His-75 with Ala or Glu perturbed the structure of the photoactive site and resulted in significantly shifted visible absorption spectra. In contrast, His-75 substitution with a positively charged Arg did not shift the visible absorption spectrum of PR. The mutation to Arg also blocks the light-induced proton transfer from the Schiff base to its counterion Asp-97 during the photocycle and the acid-induced protonation of Asp-97 in the dark state of the protein. Isotope labeling of histidine revealed that His-75 undergoes deprotonation during the photocycle in the proton-pumping (high pH) form of PR, a reaction further supported by results from H75E. Finally, all His-75 mutations greatly affect charge movements within the PR and shift its pH dependence to acidic values. A model of the proteorhodopsin proton transport process is proposed as follows: (i) in the dark state His-75 is positively charged (protonated) over a wide pH range and interacts directly with the Schiff base counterion Asp-97; and (ii) photoisomerization-induced transfer of the Schiff base proton to the Asp-97 counterion disrupts its interaction with His-75 and triggers a histidine deprotonation.A variety of unicellular microorganisms contain primary proton pumps that convert solar energy into a transmembrane electrochemical proton gradient, which is subsequently used by membrane ATP synthases to generate chemical energy. Well known examples of such pumps are the haloarchaeal rhodopsins, photoactive, seven-helix membrane proteins, which include the well studied proton pump bacteriorhodopsin (BR)⁴ from Halobacterium salinarum and BR homologs in other haloarchaea. Recently, a much larger new family of light-driven proton pumps, the proteorhodopsins (PRs), was identified in marine proteobacteria throughout the oceans (1–3). Despite the diverse properties of PRs, including different visible absorption maxima and photocycle rates (4–6), they all share with BR several key conserved residues as well as an all-trans-retinylidene chromophore in their unphotolyzed state, which is covalently bound to transmembrane helix G via a protonated Schiff base linkage.Many of the molecular events that occur in PRs following light activation are similar to those of BR, including an initial ultrafast all-trans→13-cis-retinal isomerization, which triggers a sequence of protein conformational changes, including several intramolecular proton transfer reactions. The two key carboxylate groups involved in proton pumping in helix C of BR are conserved in PRs, and in the first found and most commonly studied PR, the Monterey Bay variant eBAC31A08, also known as green-absorbing proteorhodopsin (GPR), the helix C residues Asp-97 and Glu-108 undergo protonation changes during the photocycle similar to those of the homologous carboxylate residues in BR. Initial FTIR studies on GPR identified the role of Asp-97 as the Schiff base counterion and proton acceptor during Schiff base deprotonation and concomitant M formation and Glu-108 as the proton donor that reprotonates the Schiff base during N formation (7, 8). Studies of other variants indicate these roles of the two carboxylic acid residues are general in the proteorhodopsin family.⁵One major difference between BR and the PRs is the presence of a highly conserved histidine residue at position 75, near the middle of transmembrane helix B in the latter pigments. The His-75 homolog is not present in BR nor thus far found in other microbial rhodopsins (9). The proximity of His-75 to the protein active site and specifically to the Schiff base counterion Asp-97 inferred from the x-ray crystal structure of BR suggests its involvement in spectral tuning of the visible absorption (10) and potentially PR photochemical reactions. Because the pK_a of histidine in solution is close to neutral pH (11), its imidazole group often plays a major role in intramolecular proton transfers in enzymes, including NADPH oxidase (12), alcohol dehydrogenase (13), carbonic anhydrase II (14), and serine proteases (15).In this study we have used a combination of time-resolved FTIR difference spectroscopy, visible absorption spectroscopy, isotope labeling, kinetic charge displacement measurements, and site-directed mutagenesis to study the role of His-75 in GPR. We report evidence that protonated His-75 interacts directly with Asp-97 in the unphotolyzed protein and during the photocycle undergoes a deprotonation in response to the protonation of Asp-97.     

Biophysical studies of the membrane location of the voltage-gated sensors in the HsapBK and KvAP K channels

The membrane location of two fragments in two different K⁺-channels, the KvAP (from Aeropyrum pernix) and the HsapBK (human) corresponding to the putative “paddle” domains, has been investigated by CD, fluorescence and NMR spectroscopy. Both domains interact with q = 0.5 phospholipid bicelles, DHPC micelles and with POPC vesicles. CD spectra demonstrate that both peptides become largely helical in the presence of phospholipid bicelles. Fluorescence quenching studies using soluble acrylamide or lipid-attached doxyl-groups show that the arginine-rich domains are located within the bilayered region in phospholipid bicelles. Nuclear magnetic relaxation parameters, T₁ and ¹³C-¹H NOE, for DMPC in DMPC/DHPC bicelles and for DHPC in micelles showed that the lipid acyl chains in the bicelles become less flexible in the presence of either of the fragments. An even more pronounced effect is seen on the glycerol carbons. ²H NMR spectra of magnetically aligned bicelles showed that the peptide derived from KvAP had no or little effect on bilayer order, while the peptide derived from HsapBK had the effect of lowering the order of the bilayer. The present study demonstrates that the fragments derived from the full-length proteins interact with the bilayered interior of model membranes, and that they affect both the local mobility and lipid order of model membrane systems.     

Predicted bacteriorhodopsin from Exiguobacterium sibiricum is a functional proton pump

The predicted Exigobacterium sibiricum bacterirhodopsin gene was amplified from an ancient Siberian permafrost sample. The protein bacteriorhodopsin from Exiguobacterium sibiricum (ESR) encoded by this gene was expressed in Escherichia coli membrane. ESR bound all-trans-retinal and displayed an absorbance maximum at 534 nm without dark adaptation. The ESR photocycle is characterized by fast formation of an M intermediate and the presence of a significant amount of an O intermediate. Proteoliposomes with ESR incorporated transport protons in an outward direction leading to medium acidification. Proton uptake at the cytoplasmic surface of these organelles precedes proton release and coincides with M decay/O rise of the ESR.     

Multiple Tight Phospholipid-Binding Modes of α-Synuclein Revealed by Solution NMR Spectroscopy

'In dopaminergic neurons, α-synuclein (αS) partitions between a disordered cytosolic state and a lipid-bound state. Binding of αS to membrane phospholipids is implicated in its functional role in synaptic regulation, but also impacts fibril formation associated with Parkinson's disease. We describe here a solution NMR study in which αS is added to small unilamellar vesicles of a composition mimicking synaptic vesicles; the results provide evidence for multiple distinct phospholipid-binding modes of αS. Exchange between the free state and the lipid-bound αS state, and between different bound states is slow on the NMR timescale, being in the range of 1-10 s^− 1. Partitioning of the binding modes is dependent on lipid/αS stoichiometry, and tight binding with slow-exchange kinetics is observed at stoichiometries as low as 2:1. In all lipid-bound states, a segment of residues starting at the N-terminus of αS adopts an α-helical conformation, while succeeding residues retain the characteristics of a random coil. The 40 C-terminal residues remain dynamically disordered, even at high-lipid concentrations, but can also bind to lipids to an extent that appears to be determined by the fraction of cis X-Pro peptide bonds in this region. While lipid-bound αS exhibits dynamic properties that preclude its direct observation by NMR, its exchange with the NMR-visible free form allows for its indirect characterization. Rapid amide-amide nuclear Overhauser enhancement buildup points to a large α-helical conformation, and a distinct increase in fluorescence anisotropy attributed to Tyr39 indicates an ordered environment for this “dark state.” Titration of αS with increasing amounts of lipids suggests that the binding mode under high-lipid conditions remains qualitatively similar to that in the low-lipid case. The NMR data appear incompatible with the commonly assumed model where αS lies in an α-helical conformation on the membrane surface and instead suggest that considerable remodeling of the vesicles is induced by αS.