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Water structuring on the outer surface of protein molecules called the hydration shell is essential as well as the internal water structures for higher-order structuring of protein molecules and their biological activities in vivo. We now show the molecular-scale hydration structure measurements of native purple membrane patches composed of proton pump proteins by a noninvasive three-dimensional force mapping technique based on frequency modulation atomic force microscopy. We successfully resolved the ordered water molecules localized near the proton uptake channels on the cytoplasmic side of the individual bacteriorhodopsin proteins in the purple membrane. We demonstrate that the three-dimensional force mapping can be widely applicable for molecular-scale investigations of the solid–liquid interfaces of various soft nanomaterials.

The average conformation of the methyl-branched chains of archaeal lipid phosphatidyl glycerophosphate methyl ester (PGP-Me) was examined in a hydrated bilayer membrane based on the 2H nuclear magnetic resonance (NMR) of enantioselectively 2H-labeled compounds that were totally synthesized for the first time in this study. The NMR results in combination with molecular dynamics simulations revealed that the PGP-Me chain appeared to exhibit behavior different from that of typical membrane lipids such as dimyristoylphosphatidylcholine (DMPC). The C-C bonds of the PGP-Me chain adopt alternative parallel and tilted orientations to the membrane normal as opposed to a DMPC chain where all of the C-C bonds tilt in the same way on average. This characteristic orientation causes the intertwining of PGP-Me chains, which plays an important role in the excellent thermal and high-salinity stabilities of archaeal lipid bilayers and membrane proteins.

To investigate the effects of temperature and hydration on the dynamics of purple membrane (PM), we measured the broadband complex dielectric spectra from 0.5 GHz to 2.3 THz using a vector network analyzer and terahertz time-domain spectroscopy from 233 to 293 K. In the lower temperature region down to 83 K, the complex dielectric spectra in the THz region were also obtained. The complex dielectric spectra were analyzed through curve fitting using several model functions. We found that the hydrated states of one relaxational mode, which was assigned as the coupled motion of water molecules with the PM surface, began to overlap with the THz region at approximately 230 K. On the other hand, the relaxational mode was not observed for the dehydrated state. On the basis of this result, we conclude that the protein-dynamical-transition-like behavior in the THz region is due to the onset of the overlap of the relaxational mode with the THz region. Temperature hysteresis was observed in the dielectric spectrum at 263 K when the hydration level was high. It is suggested that the hydration water behaves similarly to supercooled liquid at that temperature. The third hydration layer may be partly formed to observe such a phenomenon. We also found that the relaxation time is slower than that of a globular protein, lysozyme, and the microscopic environment in the vicinity of the PM surface is suggested to be more heterogeneous than lysozyme. It is proposed that the spectral overlap of the relaxational mode and the low-frequency vibrational mode is necessary for the large conformational change of protein.

Over the past two decades orthogonal acceleration time-of-flight has been the de facto analyzer of choice for solution and membrane soluble protein native mass spectrometry (MS) studies; this however is gradually changing. Here we compare three MS instruments, the Q-ToF, the Orbitrap and the FT-ICR to analyze, under native instrument and buffer conditions, the 7-transmembrane helical protein bacteriorhodopsin-octylglucoside micelle complex and the empty nanodisc (MSP1D1-Nd) using both MS and tandem-MS modes of operation. Bacteriorhodopsin can be released from the octylglucoside-micelle efficiently on all three instruments (MS-mode of operation) producing a narrow charge state distribution (z = 8+ to 10+) by either increasing the source lens or collision cell (or HCD) voltages. A lower center-of-mass collision energy (0.20–0.41 eV) is required for optimal bacteriorhodopsin liberation on the FT-ICR, in comparison to the Q-ToF and Orbitrap instruments (0.29–2.47 eV). The empty MSP1D1-Nd can be measured with relative ease on a three instruments, resulting in a highly complex spectrum of overlapping, polydisperse charge state; a consequence of varying levels of phospholipid incorporation. There is a measurable difference in MSP1D1-Nd charge state distribution (z = 15+ to 26+), average molecular weight (141.7 to 169.6 kDa) and phospholipid incorporation number (143 to 184) under low activation conditions. Utilizing tandem-MS, bacteriorhodopsin can be effectively liberated from the octylglucoside-micelle by collisional (Q-ToF and FT-ICR) or continuous IRMPD activation (FT-ICR). MSP1D1-Nd spectral complexity can also be significantly reduced by tandem-MS (Q-ToF and FT-ICR) followed by mild collisional or continuous IRMPD activation, resulting in a spectrum in which the charge state and phospholipid incorporation levels can easily be determined.

Purple membrane (PM), which is a membrane patch formed by the self-assembly of the membrane protein bacteriorhodopsin (bR) with archaeal lipids, is a good subject for studying the mechanism for the supramolecular structural formation of membrane proteins. Several studies have suggested that PM is not simply planar but that it has a curvature. Atomic force microscopy (AFM) studies also indicate the presence of dome-like structures (bumps) on the cytoplasmic surface of PM. PM must have a curvature to form the bump structures; therefore, bump formations will be related to a mechanism for supramolecular structural formation via self-assembly. To elucidate the effect of an asymmetric distribution of charged residues between two aqueous domains on the bump curvature, AFM topography of identical PM sheets were examined with variation of the solvent ionic strength and pH using a newly constructed solvent circulation system. The radius and height distributions of the bumps on the identical PM sheets indicated a linear correlation. The bump curvature, which was simply estimated by the slope of the distribution, became smaller with increasing KCl concentration, which suggests that tension at the cytoplasmic surface caused by electrostatic repulsive force between negatively charged amino acid residues becomes weaker by the electrostatic shielding effect. AFM observations revealed that the bump curvature remained even at high KCl concentration where the Debye length is within a few Angstroms; therefore, the contribution of the intrinsic difference between the domain sizes of bR between two sides was confirmed. Interestingly, the bump curvature was significantly increased by the addition of CaCl2 and then decreased with a similar dependency to KCl at higher CaCl2 concentration. The effect of pH on the bump curvature was also examined, where the curvature increased and reached a maximum at pH 9, while it decreased above pH 10, at which point the two-dimensional crystalline lattice of bR began to disassemble. These experimental results indicate that the bump curvature is strongly influenced by electrostatic interactions. A plausible model for bump structure formation by electrostatic repulsive force is presented based on these results.

Using steady-state spectroscopic and zeta potential methods, we have unraveled the interaction of the purple membrane (PM) and bacteriorhodopsin (bR) with various surfactants below their critical micelle concentrations. We found that the charged hydrophilic heads of ionic surfactants play a role in perturbing the structure and conformation of PM and bR and that ionic surfactants of opposite charges cause opposing effects. Specifically, the addition of a low concentration (0.2 mM) of the cationic surfactant cetyl trimethylammonium bromide (CTAB) is capable of neutralizing the negatively charged lipids on the PM surface via electrostatic forces. This results in increased hydrophobicity of PM that leads to the aggregation of PM. In contrast, denaturation of PM and bR was observed when the anionic surfactant sodium dodecyl sulfate (SDS) was added to the PM suspensions. The attachment of SDS to the PM surface increases the solubility of PM and causes a loose crystalline structure. As the SDS concentration is increased to more than 3 mM, the secondary structure of the constituents of bR is significantly distorted, and the protonated Schiff base is hydrolyzed to form free retinal. The addition of the neutral surfactant diethylene glycol mono-n-hexyl ether (C6E2) does not significantly influence the PM and bR, meaning most of their original properties are preserved. We conclude that the addition of surfactants might cause the aggregation or solubilization of the membrane protein, depending on the signs of the charged hydrophilic heads of the surfactants and the charges of the membrane protein surface. Aggregation results when the surfactant and protein have opposite charges, whereas solubilization results when the surfactant and protein have the same charge.

Protection of biological compounds, for example, enzymes, viruses, or even whole cells, against degradation is very important for many applications. Embedding of such compounds into polymer matrices is a straightforward common method. However, in biotechnology and medicine there is a great interest to prepare micro- and nanosized shells around the biocomponents in order to protect them and having only a minor increase in size. The PEGylation of biological macromolecules has gained attention because degradation by proteolytic enzymes is significantly retarded and, in turn, their bioavailability is enhanced. We found that PEGylation is also a powerful tool to protect biomaterials from degradation by small organic solvent molecules, in particular, ethanol. Methoxy-polyethylene glycol (MPEG) modified BR survives exposure to significant concentrations of ethanol, up to 30%, and preserves its photochromism, whereas unmodified PM is instantaneously denatured at such concentrations. This is useful for potential technical applications of BR but is of relevance for many other applications where biomaterials and, in particular, biomembranes may be exposed to solvents.

Inkjet printing is a versatile technique widely applied in biological microarray technology. Because of its photochemical and photophysical properties, bacteriorhodopsin (BR) from Halobacterium salinarum holds promise for applications in nanotechnology, and inkjet printing would simplify the transfer of BR to suitable substrates. Surfactants are essential parts of inkjet formulations tuning viscosity, rheology, and spreading behavior of the solution. However, many surfactants destabilize the structure of proteins and often cause denaturation accompanied by a complete loss of function. Inkjet printing of membrane proteins is particularly challenging and special care must be taken in the choice of the surfactant. For BR, the situation is complicated by the fact that the structural integrity of BR depends on its native membrane environment, the so-called purple membrane (PM). PM contains 10 lipid molecules per BR monomer and is very sensitive toward surfactants. In this work, we identified surfactants suitable for inkjet formulations containing PM. Initially, we screened a variety of technically relevant surfactants for compatibility with PM using the UV-vis absorption of the retinal chromophore as a natural probe. Promising candidates were selected, and their impact on the structure of PM and BR was analyzed using UV-vis spectroscopy, CD spectroscopy, and small-angle X-ray scattering (SAXS). We identified two surfactants compatible with PM and suitable for inkjet formulations. An inkjet formulation containing PM as dye component was developed. We demonstrate that the photochromic properties of BR are maintained upon inkjet printing.

Bacteriorhodopsin (BR) is the key protein of the halobacterial photosynthetic system. BR assembles into two-dimensional crystalline patches, the so-called purple membranes (PM), and acts as a light-driven proton pump converting light energy into the chemical energy of a proton gradient over the cell membrane. The two-photon absorption (TPA) of BR is so far not fully understood. Astonishingly high TPA cross sections have been reported, but the molecular mechanisms have not been elucidated. In this work, we address structural changes in BR and PM upon TPA, investigating its TPA photochemistry by spectroscopy, small-angle X-ray scattering, as well as electron and atomic force microscopy. We observe that TPA of BR leads to formation of an UV-absorbing N-retinyl-bacterioopsin state, which is accompanied by the loss of crystalline order in PM. FTIR and CD spectroscopy confirm that BR trimers as well as the secondary structure of the BR molecules are preserved. We demonstrate that excitation by TPA results in the photochemical reduction of the retinal Schiff base, which in turn causes a permanent asymmetric shape change of BR, similar to the one transiently observed during the photocycle-related opening and closing of the cytoplasmic proton half channel. This shape change causes PM sheets to merely roll up toward the extracellular side and causes the loss of crystallinity of PM. We present a model for the TPA photoresponse of BR, which also explains the irreversibility of the process in terms of a photochemical reduction of the Schiff base.

Purple membranes (PM) from Halobacterium salinarum have been discussed for several technical applications. These ideas started just several years after its discovery. The biological function of bacteriorhodopsin (BR), the only protein in PM, is the light-driven proton translocation across the membrane thereby converting light energy into chemical energy. The astonishing physicochemical robustness of this molecular assembly and the ease of its isolation triggered ideas for technical uses. All basic molecular functions of BR, that is, photochromism, photoelectrism, and proton pumping, are key elements for technical applications like optical data processing and data storage, ultrafast light detection and processing, and direct utilization of sunlight in adenosine 5'-triphospate (ATP) generation or seawater desalination. In spite of the efforts of several research groups worldwide, which confirmed the proof-of-principle for all these potential applications, only the photochromism-based applications have reached a technical level. The physical reason for this is that no fixation or orientation of the PMs is required. The situation is quite different for photoelectrism and proton pumping where the macroscopic orientation of PMs is a prerequisite. For proton pumping, in addition, the formation of artificial membranes which prevent passive proton leakage is necessary. In this manuscript, we describe a new class of PM variants with oppositely charged membrane sides which enable an almost 100% orientation on a surface, which is the key element for photoelectric applications of BR. As an example, the mutated BR, BR-E234R7, was prepared and analyzed. A nearly 100% self-orientation on mica was obtained.

A light-driven proton pump bacteriorhodopsin (bR) forms a two-dimensional hexagonal lattice with about 10 archaeal lipids per monomer bR on purple membrane (PM) of Halobacterium salinarum. In this study, we found that the weakening of the bR-lipid interaction on PM by addition of alcohol can be detected as the significant increase of protein solubility in a nonionic detergent, dodecyl β-D-maltoside (DDM). The protein solubility in DDM was also increased by bR-lipid interaction change accompanied by structural change of the apoprotein after retinal removal and was about 7 times higher in the case of completely bleached membrane than that of intact PM. Interestingly, the cyclic and milliseconds order of structural change of bR under light irradiation also led to increasing the protein solubility and had a characteristic light intensity dependence with a phase transition. These results indicate that there is a photointermediate in which bR-lipid interaction has been changed by its dynamic structural change. Because partial delipidation of PM by CHAPS gave minor influence for the change of the protein solubility compared to intact PM in both dark and light conditions, it is suggested that specific interactions of bR with some lipids which remain on PM even after delipidation treatment have a key role for the change of solubility in DDM induced by alcohol binding, ligand release, and photon absorption on bR.

Purple membrane (PM) from Halobacterium salinarum, which comprises bacteriorhodopsin (BR) and lipids only, has been employed by many groups as a model system to study the structure and dynamics of membrane proteins. Although the conformational dynamics of BR within PM has been extensively analyzed with subnanometer resolution by means of diffraction experiments and spectroscopic methods, as well, structural studies of dynamical transitions within single PMs are rare. In this work, we show that tapping-mode atomic force microscopy (TM-AFM) is ideally suited to study dynamical transitions within solid-supported PMs at the nanoscale. Time-dependent AFM analysis of solid-supported PMs shows that redistribution processes take place between a crystalline core region, featuring a height of approximately 5 nm, and a highly mobile rim region (approximately 4 nm in height). Furthermore, we discuss the influence of temperature and substrate on the equilibrium. The experiments are complemented by electrostatic force microscopy (EFM) of PM on mica. Beyond their importance for many physiological processes, dynamical transitions in biological membranes, as observed in this work, are of critical importance for all methods that make use of solid-supported membrane assemblies, either analytical tools or applications.

The membrane protein bacteriorhodopsin in its native membrane bound form (purple membrane) was adsorbed and incorporated into polyelectrolyte multilayered films, and adsorption was in situ monitored by optical waveguide light-mode spectroscopy. The formation of a single layer or a double layer of purple membranes was observed when adsorbed on negatively or positively charged surfaces, respectively. The purple membrane patches adsorbed on the polyelectrolyte multilayers were also evidenced by atomic force microscopy images. The driving forces of the adsorption process were evaluated by varying the ionic strength of the solution as well as the purple membrane concentration. At high purple membrane concentration, interpenetrating polyelectrolyte loops might provide new binding sites for the adsorption of a second layer of purple membranes, whereas at lower concentrations only a single layer is formed. Negative surfaces do not promote a second protein layer adsorption. Driving forces other than just electrostatic ones, such as hydrophobic forces, should play a role in the polyelectrolyte/purple membrane layering. The subtle interplay of all these factors determines the formation of the polyelectrolyte/purple membrane matrix with a presumably high degree of orientation for the incorporated purple membranes, with their cytoplasmic, or extracellular side toward the bulk on negatively or positively charged polyelectrolyte, respectively. The structural stability of bacteriorhodopsin during adsorption onto the surface and incorporation into the polyelectrolyte multilayers was investigated by Fourier transform infrared spectroscopy in attenuated total reflection mode. Adsorption and incorporation of purple membranes within polyelectrolyte multilayers does not disturb the conformational majority of membrane-embedded alpha-helix structures of the protein, but may slightly alter the structure of the extramembraneous segments or their interaction with the environment. This high stability is different from the lower stability of the predominantly beta-sheet structures of numerous globular proteins when adsorbed onto surfaces.

Below 195 K, the bacteriorhodopsin photocycle could not be adequately described with exponential kinetics [Dioumaev, A. K., and Lanyi, J. K. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 9621-9626] but required distributed kinetics, previously found in hemoglobin and myoglobin at temperatures below the vitrification point of the surrounding solvent. The aim of this study is to determine which factors cause the switch from this low-temperature regime to the conventional kinetics observed at ambient temperature. The photocycle was monitored by time-resolved FTIR between 180 and 280 K, using the D96N mutant. Depending on the temperature, decay and temporal redistribution of two or three intermediates (L, M, and N) were observed. Above approximately 245 K, an abrupt change in the kinetic behavior of the photocycle takes place. It does not affect the intermediates present but greatly accelerates their decay. Below approximately 240 K, a kinetic pattern with partial decay that cannot be explained by conventional kinetics, but suggesting distributed kinetics, was dominant, while above approximately 250 K, there were no significant deviations from exponential behavior. The approximately 245 K critical point is/=10 K below the freezing point of interbilayer water, and we were unable to correlate it with any FTIR-detectable transition of the lipids. Therefore, we attribute the change from distributed to conventional kinetics to a thermodynamic phase transition in the protein. Most probably, it is related to the freezing and thawing of internal fluctuations of the protein, known as the dynamic phase transition, although in bacteriorhodopsin the latter is usually believed to take place at least 15 K below the observed critical temperature of approximately 245 K.

Bacteriorhodopsin (BR) undergoes a conformational change during the photocycle and the proton transport through the membrane. For the first time, we could demonstrate by direct imaging of freely suspended native purple membranes (PMs) that the flat disk-like shape of PMs changes dramatically as soon as most of the BRs are in a state characterized by a deprotonated Schiff base. Light-induced shape changes are easily observed with mutated BRs of the BR-D96N type, i.e., all variants which show an increased M 2 lifetime. On the other hand, large-scale shape changes are induced by pH changes with PM containing mutated BRs of the BR-D85T type, where Asp85 is replaced for a neutral amino acid. In such PMs, all BRs are titrated simultaneously and the resulting shape of the membranes depends on the initial shape only. As the majority of PMs in the "flat" state are more or less round disks, the bent membranes often comprise bowl-like and tube-like bent structures. The method presented here enables one to derive size changes of membrane-embedded BRs on the single molecule level from "macroscopic", easily accessible data like the curvature radii observed in cryo-SEM. The potential of BR as a pH-controlled and/or light-controlled microscaled biological actuator needs further consideration.

Molecular electronics is very much about contacts, and thus understanding of any generic contact effect is essential to its advance. For example, it is still not obvious in how far variations in electrode roughness of macroscopic contacts can lead to rectification. Here we report an investigation of this contact effect on electronic transport properties using metal-insulator-metal planar junctions with a 5 nm thick bacteriorhodopsin-based insulator as model system. We demonstrate that the experimentally observed rectifying behavior is not an intrinsic property of the molecules used, but rather of the local contact quality. Even a slight increase in surface roughness of the bottom electrode gives rise to distinct rectifying behavior in these and, by extrapolation, possibly other molecular junctions.

The bulky and amphiphilic nature of tryptophan residues makes them particularly interesting components of proteins. In bacteriorhodopsin, four of the eight tryptophan residues are in the active site, forming parts of the retinal binding pocket. In this work, we use solid-state NMR to study the interactions of the tryptophan residues in wild-type bacteriorhodopsin, in the resting state, and in critical intermediates of the proton-motive photocycle. The range of the chemical shifts of the indole nitrogens suggests that all eight of them are hydrogen bonded. Using difference spectroscopy, we isolate several changes in these hydrogen bonds in the early and late M states. As found earlier for the peptide backbone, some perturbations found in the early M state relax in the transition to the late M state while new perturbations arise. Interestingly, Rotational Echo DOuble Resonance (REDOR) difference spectroscopy of (20- 13 C)retinal,- (indole- 15 N)Trp-bR shows that indole of Trp182 is not involved in the significant hydrogen bond perturbations. We also use REDOR to measure dipolar interactions in (20- 13 C)retinal,(indole- 15 N)Trp- bR, and thereby determine the distance between the C20 of retinal and the indole nitrogen of Trp182. The internuclear distance changes only slightly from the light-adapted state (3.36 ( 0.2 A) to the early M state (3.16 ( 0.4 A).

It was found that bacterial transglutaminase (TGase) facilitates selective cross-linking of bacteriorhodopsin (BR) in purple membrane (PM) form under mild conditions. Fluorescent probes were used to detect that the membrane protein BR may act as a glutamine donor as well as a lysine donor for TGase. The binding sites were determined to be Gln-3 as the reactive glutamine, and Lys-129 is the corresponding lysine residue. Upon incubation of PM with TGase, cross-linking of PM patches can be achieved without an additional spacer molecule. To our knowledge, this is the first time that an intermembrane cross-linking of membrane-bound proteins is reported. Furthermore, this finding may provide the ability to achieve covalent linkage of complete purple membrane patches to synthetic polymers.

A novel glycolipid of mass 1935 and a phospholipid of mass 1522 are the main residual lipids (along with traces of PGP-Me, S-TGD-1, and PG) specifically associated with "delipidated" bacteriorhodopsin fractions BR I and BR II, prepared by Triton X-100 treatment of purple membrane (PM), from a genetically engineered strain (L33) of Halobacterium salinarum, and chromatography on phenyl-Sepharose CL-4B. The novel glycolipid and phospholipid are components of the PM matrix not previously described. The TLC isolated and purified novel glycolipid and phospholipid were shown, by chemical degradation, mass spectrometry, and NMR analyses, to have the structure, respectively, of a phosphosulfoglycolipid, 3-HSO(3)-Galp-beta1,6Manp-alpha1,2Glcp-alpha1,1-[sn-2, 3-di-O-phytanylglycerol]-6-[phospho-sn-2,3-di-O-phytanylglycero l], and of a glycerol diether analogue of bisphosphatidylglycerol (cardiolipin), sn-2,3-di-O-phytanyl-1-phosphoglycerol-3-phospho-sn-2, 3-di-O-phytanylglycerol.

Light absorbed by bacteriorhodopsin (bR) leads to a proton being released at the extracellular surface of the purple membrane. Structural studies as well as studies of mutants of bR indicate that several groups form a pathway for proton transfer from the Schiff base to the extracellular surface. These groups include D85, R82, E204, E194, and water molecules. Other residues may be important in tuning the initial state pK(a) values of these groups and in mediating light-induced changes of the pK(a) values. A potentially important residue is R134: it is located close to E194 and might interact electrostatically to affect the pK(a) of E194 and light-induced proton release. In this study we investigated effects of the substitution of R134 with a histidine on light-induced proton release and on the photocycle transitions associated with proton transfer. By measuring the light-induced absorption changes versus pH, we found that the R134H mutation results in an increase in the pK(a) of the proton release group in both the M (0.6 pK unit) and O (0.7 pK unit) intermediate states. This indicates the importance of R134 in tuning the pK(a) of the group that, at neutral and high pH, releases the proton upon M formation (fast proton release) and that, at low pH, releases the proton simultaneously with O decay (slow proton release). The higher pK(a) of the proton release group found in R134H correlates with the slowing of the rate of the O --> bR transition at low pH and probably is the cause of this slowing. The pH dependence of the fraction of the O intermediate is altered in R134H compared to the WT but is similar to that in the E194D mutant: a very small amount of O is present at neutral pH, but the fraction of O increases greatly upon decreasing the pH. These results provide further support for the hypothesis that the O --> bR transition is controlled by the rate of deprotonation of the proton release group. These data also provide further evidence for the importance of the R134-E194 interaction in modulating proton release from D85 after light has led to its being protonated.

A spin-label at site 101 in the C-D loop of bacteriorhodopsin was previously found to detect a conformational change during the M --> N transition [Steinhoff, H. -J., Mollaaghababa, R., Altenbach, C., Hideg, K., Krebs, M. P., Khorana, H. G., and Hubbell, W. L. (1994) Science 266, 105-107]. We have extended these time-resolved electron paramagnetic resonance studies in purple membranes by analyzing conformational changes detected by a spin-label at another site in the C-D loop (103), and at sites in the A-B loop (35), the D-E loop (130), and the E-F loop (160). In addition, we have investigated the motion detected by a spin-label at site 101 in a D96A mutant background that has a prolonged M intermediate. We find that among the examined sites, only spin-labels in the C-D loop detect a significant change in the local environment after the rise of M. Although the D96A mutation dramatically prolongs the lifetime of the M intermediate, it does not perturb either the structure of bacteriorhodopsin or the nature of the light-activated conformational change detected by a spin-label at site 101. In this mutant, a conformational change is detected during the lifetime of M, when no change in the 410 nm absorbance is observed. These results provide direct structural evidence for the heterogeneity of the M population in real time, and demonstrate that the motion detected at site 101 occurs in M, prior to Schiff base reprotonation.

Fourier transform near-infrared resonance Raman spectroscopy can be used to obtain information on the bacteriochlorophyll a (BChl a) molecules responsible for the redmost absorption band in photosynthetic complexes from purple bacteria. This technique is able to distinguish distortions of the bacteriochlorin macrocycle as small as 0.02 A, and a systematic analysis of those vibrational modes sensitive to BChl a macrocycle conformational changes was recently published [Näveke et al. (1997) J. Raman Spectrosc. 28, 599-604]. The conformation of the two BChl a molecules constituting the primary electron donor in bacterial reaction centers, and of the 850 and 880 nm-absorbing BChl a molecules in the light-harvesting LH2 and LH1 proteins, has been investigated using this technique. From this study it can be concluded that both BChl a molecules of the primary electron donor in the photochemical reaction center are in a conformation close to the relaxed conformation observed for pentacoordinate BChl a in diethyl ether. In contrast, the BChl a molecules responsible for the long-wavelength absorption transition in both LH1 and LH2 antenna complexes are considerably distorted, and furthermore there are noticeable differences between the conformations of the BChl molecules bound to the alpha- and beta-apoproteins. The molecular conformations of the pigments are very similar in all the antenna complexes investigated.

The purple membrane of Halobacterium salinarium is a two-dimensional lattice of lipids and the integral membrane protein bacteriorhodopsin (BR). To determine whether helix-helix interactions within the membrane core stabilize this complex, we substituted amino acid residues at the helix-helix interface between BR monomers and examined the assembly of the protein into the lattice. Lattice assembly was demonstrated to fit a cooperative self-assembly model that exhibits a critical concentration in vivo. Using this model as the basis for a quantitative assay of lattice stability, bulky substitutions at the helix-helix interface between BR monomers within the membrane core were shown to be destabilizing, probably due to steric clash. Ala substitutions of two residues at the helix-helix interface also reduced stability, suggesting that the side chains of these residues participate in favorable van der Waals packing interactions. However, the stabilizing interactions were restricted to a small region of the interface, and most of the substitutions had little effect. Thus, the contribution of helix-helix interactions to lattice stability appears limited, and favorable interactions between other regions of neighboring BR monomers or between BR and lipid molecules must also contribute.

The capacitive photoelectric current responses of the halorhodopsins from Halobacterium salinarum (shR) and from Natronobacterium pharaonis (phR) were studied using membrane fragments adsorbed onto a thin polyester film. The electric current of shR was not much affected by ionic strength or cations present in the medium (Na+, K+, Li+, Mg2+, or Ca2+), but was greatly influenced by the Cl- concentration. It increased biphasically as the Cl- concentration increased from 0 to 5 M, then decreased and almost vanished at around 10 or 12 M. Apparent Kd's of about 0.1 and 6 M were deduced for the Kd of Cl- uptake sites. We had to assume a sigmoidal increase of Cl- binding with a Hill coefficient of about 8 at the cytoplasmic, Cl- release site(s). The half-maximum Cl- concentration for the sigmoidal binding was about 7.5 M. The electric current of phR had a maximum around 30 mM Cl- and biphasically decreased at higher Cl- concentrations. The apparent Kd for the Cl- uptake site was 5 mM. The biphasic decrease in the transport activity was explained by assuming a sum of simple hyperbolic type binding (Kd = 0.2 M) and sigmoidally increasing binding with a Hill coefficient of 10 on the cytoplasmic side. The half-maximum concentration of the latter cooperative binding was 5.6 M. This great difference between the apparent affinity of the release site of shR and that of phR can explain the previously reported difference between the Cl- dependency of their photocycles. These results also suggest that there may be multiple Cl- binding sites in the Cl- transport pathway. A simple sequence of Cl- transport steps based on a multiion channel model is proposed.

The photosynthetic reaction center of Rhodopseudomonas viridis, a purple bacterium, contains a tetraheme cytochrome subunit. After its photoinduced oxidation, the primary donor, P, is reduced by the nearby heme (c559) of the tetraheme subunit in about 200 ns. This heme, in turn, is reduced by another heme (c556) of the subunit in about 2 micro(s). The midpoint potentials of P, c559, and c556 are known to be +500, +380, and +320 mV, respectively. The reduction kinetics of P+ are strongly biphasic in living cells, membrane fragments, and isolated reaction centers. We show here that this biphasicity reflects a small equilibrium constant (lower than 10) for the electron-transfer reaction between P and c559, which arises from a significant difference between the operating redox potentials of the P+/P and c559+/c559 couples and their equilibrium midpoint potentials. This difference is partly due to the effect of the permanent transmembrane potential, which arises from the cell metabolism, and to significant electrostatic interactions which develop between the electron carriers of the reaction center. Interestingly, the kinetic parameters of P+ reduction in decoupled cells or membrane fragments are identical to those reported for isolated reaction centers. We estimate an interaction of about 20 mV between c556 and c559 and about 90 mV between c559 and P. Consequently, the operating redox potential of the P+/P couple is 410 mV.

We have engineered a photosynthetically competent mutant of the purple non-sulfur bacterium Rhodobacter capsulatus which seeks to mimic the behavior of the primary electron donor (P) of the plant photosystem II (PS II) reaction center (RC). To construct this mutant (denoted D1-ILMH), four residues in the bacterial L subunit were mutagenized, such that an 11-residue segment was made identical to the analogous segment from the D1 subunit of PS II. The electronic properties of the bacteriochlorophyll (Bchl) dimer which constitutes the primary donor are substantially altered by these modifications, to the degree that the dimer becomes functionally much more "monomeric". The changes include (1) an increase in the values of the zero-field splitting (ZFS) parameters, as measured by electron paramagnetic resonance (EPR), for the spin-polarized triplet state, 3P, from /D/ = 185 x 10(-4) cm(-1) and /E/ = 31 x 10(-4) cm(-1) in wild-type (WT) chromatophore membranes to /D/ = 200 x 10(-4) cm(-1) and /E/ = 44 x 10(-4) cm(-1) in the mutant and (2) an increase in the EPR line width of the oxidized state, P+, from 0.97 mT in WT to 1.09 mT in D1-ILMH RCs. However, unlike the PS II primary donor (P680), the orientation of 3P in the D1-ILMH mutant is the same as in WT bacteria and does not display the unusual orientation found for PS II. And whereas the redox couple P/P+ has a very high midpoint potential in PS II, P/P+ in the D1-ILMH mutant has a lower midpoint (90 mV more negative) than in WT Rb. capsulatus. In addition, Raman measurements indicate that the hydrogen bond between HisL168 and the C2 acetyl carbonyl oxygen of the Bchl on the active electron transfer pathway (P(A)) is absent in the mutant, due to the fact that HisL168 in the WT sequence has been replaced by a leucine in D1-ILMH. However, the Raman data also reveal the presence of a new hydrogen bond in the D1-ILMH RCs, between the C9 keto carbonyl oxygen of P(A) and an unknown hydrogen-bond donor. Thus, although the protein environment around one of the Bchls of the special pair is significantly changed in D1-ILMH, the chimeric RC does not, as a result of these changes, have a primary donor that is oriented like the one in PS II.

Brief exposure of purple membrane (PM) to dilute Triton X-100 eliminates the actinic light effect on the relative amounts of fast M (Mf) and slow M (Ms) intermediates and alters the character and kinetics of the photocycle, without destroying the native BR trimers (Mukhopadhyay et al., 1994). Particular membrane lipids are removed during the Triton treatment, and adding back an extract of membrane lipids can repair most of the affected photocycle behavior (Dracheva et al., 1996). This paper defines conditions which are important in the reconstitution procedure, using a group of quantitative parameters which measure the extents of damage and repair. Circular dichroism in both the UV and visible ranges shows that Triton can disturb both the secondary structure of BR and its ability to polymerize into trimers. Whereas the damage to protein conformation could be reversed by lipids alone, the formation of trimers and recovery of normal photocycle behavior required both lipids and a high salt concentration.

Excitation energy transfer steps in membranes of Heliobacillus mobilis were directly monitored by transient absorption spectroscopy with a time resolution of 30 fs under selective excitation within the inhomogeneously broadened bacteriochlorophyll g QY band. The initial anisotropy was found to be0.4, indicating that the pigments are excitonically coupled. After initial decay of this anisotropy in50 fs, major sub-picosecond components associated with spectral equilibration were identified, corresponding to uphill energy transfer with a 300 fs time constant (812 nm excitation) and downhill energy transfer with 100 and 500 fs components (770 nm excitation). These equilibrations are ascribed predominantly to single excitation transfer steps, as anisotropy measurements showed that equilibration within spectrally similar pigments occurs on the same time scale as spectral equilibration, a situation which contrasts with that in photosystem I. Downhill energy transfer occurs to a significant extent directly to an energetically heterogeneous population of excited states as well as in a sequential way via gradually lower-lying pools of bacteriochlorophyll g. This finding supports a description in which all pigments, including the bluemost absorbing, are spatially organized in a random way rather than in clusters of spectrally similar species. Spectral equilibration is not entirely completed prior to formation of the primary radical pair P798 + A0-, which was found to proceed in a multiexponential way (time constants of 5 and 30 ps). No indication for the formation of radical species other than P798 + A0- on the time scale up to 100 ps was found.

Solid-state NMR (SSNMR) is established as a powerful tool for extracting structural parameters from biological and chemical systems.1-3 However, the inherent low sensitivity of NMR often limits the scope of structural studies. With dynamic nuclear polarization (DNP) it is possible to address this issue since signal intensities can be enhanced by 2 to 3 orders of magnitude. This gain in sensitivity is accomplished by transferring polarization from electrons in exogenous paramagnetic molecules to nuclear spins via microwave irradiation at or near the EPR Larmor frequency.4 The size of the DNP signal enhancement is dependent on a number of factors including the nuclear spin lattice relaxation time, T1, which is maximized by performing experiments at low temperatures. Systems with an abundance of methyl groups, such as virus particles and membrane proteins, often have short T1’s even at low temperatures and could be particularly challenging candidates for DNP experiments. However, these are also some of the most interesting cases for SSNMR structural studies since they often cannot be examined with either solution NMR or X-ray diffraction. In this communication we demonstrate that it is possible to efficiently polarize archetypal examples of each of the systems mentioned above-namely the viral particle fd and the purple membrane containing bacteriorhodopsin (bR) and its accompanying lipids. Furthermore, by comparing the DNP signal enhancements in the 15N and 31P spectra of fd bacteriophage, we show that 1H spin diffusion evenly distributes the enhanced polarization throughout a large macromolecular assembly. These results suggest that DNP may be a generally applicable approach for sensitivity enhancement in SSNMR experiments. We have been developing DNP techniques for studies at high magnetic fields [initially at 5 T and more recently at 9 T (140 and 250 GHz EPR and 211 and 380 MHz 1H NMR frequencies)]. To date, our most successful polarization transfer experiments are based on thermal mixing5,6 with the nitroxide radical 4-aminoTEMPO doped into water/glycerol. The experiment is implemented as illustrated in Figure 1 and consists of microwave irradiation and cross polarization from 1H to a low-γ nucleus. We have achieved 13C signal enhancements of ∼400 in H2O/ glycerol mixtures at 13 K using a microwave cavity7 and ∼50 in the 15N magic angle spinning (MAS) spectrum of T4 lysozyme at 50 K,8 both at 5 T. Filamentous fd bacteriophages are long rod-shaped viruses comprising a 6408 nucleotide single-stranded DNA genome surrounded by 2700 copies of the R-helical pVIII (major coat) protein. The dimensions of a single virion are 8000 × 65 A.9 Due to the shape anisotropy and the alignment of the coat proteins approximately parallel to the virion long axis, the phage particles orient in a magnetic field, enabling high-resolution static SSNMR experiments.10,11 The Y21M mutation of the coat protein decreases the conformational heterogeneity of the coat protein assembly and increases the NMR spectral resolution for oriented samples.12 Since the fd virus is ∼90 wt % protein and 10 wt % DNA, the 15N NMR spectrum is dominated by signals from the coat protein backbone. In contrast, the 31P spectrum is due exclusively to the encapsulated DNA.10 Powder patterns for uniformly 15N-labeled Y21M fd bacteriophage in TEMPO/water/glycerol are shown in Figure 2 b and c at 13 K and 5 T with and without DNP, respectively. Since the fd bacteriophage orients in a magnetic field, the sample was frozen prior to insertion into the precooled probe to obtain a powder pattern. The resulting signal is fit with Ω ) 168 ppm and κ ) -0.75, representative of an amide backbone (Figure 2 a). The narrow peak on the upfield edge of the experimental powder pattern arises from the amino groups of the five lysine residues and the N terminus. The signal enhancement due to DNP is 26, representing a factor of approximately 675 reduction in signal averaging time. An additional factor of 20 in signal intensity is gained from the increased Bolztmann polarization by performing NMR experiments at 15 K versus room temperature. The 1H T1 ) 15 s at 13 K. The high number of methylated amino side chains on the coat protein contribute to this short T1; specifically, the 50-residue major coat protein of Y21M fd contains 10 Ala, 4 Ile, 2 Leu, 2 Met, 3 Thr, and 4 Val residues.9 Despite the short 1H T1 at low temperatures, a substantial signal enhancement is observed with DNP. To use DNP as a sensitivity enhancement tool for structural studies it is desirable to have uniform signal enhancements throughout the sample. In a typical DNP experiment, the bulk protons are polarized followed by cross-polarization (CP) from 1H’s to a low-γ nucleus prior to detection (Figure 1). Since 1H’s are highly abundant, we have argued that rapid spin-diffusion distributes the enhanced polarization throughout the solvent and solute.8 Thus, with the fd bacteriophage 1H spin diffusion should mediate the diffusion of polarization from the solvent to the encapsulated DNA, through the 20 A layer of coat proteins. Figure 2 e and f show 31P powder patterns with and without DNP acquired immediately after the 15N experiment (with no change in temperature) and a similar signal enhancement is observed, ∼26. Detection of equal DNP enhancements on the coat protein and DNA core confirms that 1H spin diffusion is an efficient mechanism for uniformly distributing enhanced polarization * Address correspondence to this author. ‡ Department of Chemistry, MIT. † Current address: Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093. § Department of Chemistry, Brandeis University. (1) Opella, S. J. Nat. Struct. Biol. 1997, 4, 845-848. (2) Griffin, R. G. Nat. Struct. Biol. 1998, 5, 508-512. (3) van Beek, J. D.; Beaulieu, L.; Schafer, H.; Demura, M.; Asakura, T.; Meier, B. H. Nature 2000, 405, 1077-1079. (4) Abragam, A. The Principles of Nuclear Magnetism; Clarendon: Oxford, England, 1961. (5) Wenckebach, W. T.; Swanenburg, T. J. B.; Poulis, N. J. Phys. Rep. 1974, 14, 181-255. (6) Duijvestijn, M. J.; Wind, R. A.; Smidt, J. Physica B 1986, 138, 147170. (7) Weis, V.; Bennati, M.; Rosay, M.; Bryant, J. A.; Griffin, R. G. J. Magn. Reson. 1999, 140, 293-299. (8) Hall, D. A.; Maus, D. C.; Gerfen, G. J.; Inati, S. J.; Becerra, L. R.; Dahlquist, F. W.; Griffin, R. G. Science 1997, 276, 930-2. (9) Marvin, D. A.; Hale, R. D.; Nave, C. J. Mol. Biol. 1994, 235, 260286. (10) Cross, T. A.; Tsang, P.; Opella, S. J. Biochemistry 1983, 22, 721726. (11) Cross, T. A.; Frey, M. H.; Opella, S. J. J. Am. Chem. Soc. 1983, 105, 7471-7473. (12) Tan, W. M.; Jelinek, R.; Opella, S. J.; Malik, P.; Terry, T. D.; Perham, R. N. J. Mol. Biol. 1999, 286, 787-796. 1010 J. Am. Chem. Soc. 2001, 123, 1010-1011