Tip-Enhanced Infrared Difference-Nanospectroscopy of the Proton Pump Activity of Bacteriorhodopsin in Single Purple Membrane Patches.

Photosensitive proteins embedded in the cell membrane (about 5 nm thickness) act as photoactivated proton pumps, ion gates, enzymes, or more generally, as initiators of stimuli for the cell activity. They are composed of a protein backbone and a covalently bound cofactor (e.g. the retinal chromophore in bacteriorhodopsin (BR), channelrhodopsin, and other opsins). The light-induced conformational changes of both the cofactor and the protein are at the basis of the physiological functions of photosensitive proteins. Despite the dramatic development of microscopy techniques, investigating conformational changes of proteins at the membrane monolayer level is still a big challenge. Techniques based on atomic force microscopy (AFM) can detect electric currents through protein monolayers and even molecular binding forces in single-protein molecules but not the conformational changes. For the latter, Fourier-transform infrared spectroscopy (FTIR) using difference-spectroscopy mode is typically employed, but it is performed on macroscopic liquid suspensions or thick films containing large amounts of purified photosensitive proteins. In this work, we develop AFM-assisted, tip-enhanced infrared difference-nanospectroscopy to investigate light-induced conformational changes of the bacteriorhodopsin mutant D96N in single submicrometric native purple membrane patches. We obtain a significant improvement compared with the signal-to-noise ratio of standard IR nanospectroscopy techniques by exploiting the field enhancement in the plasmonic nanogap that forms between a gold-coated AFM probe tip and an ultraflat gold surface, as further supported by electromagnetic and thermal simulations. IR difference-spectra in the 1450-1800 cm-1 range are recorded from individual patches as thin as 10 nm, with a diameter of less than 500 nm, well beyond the diffraction limit for FTIR microspectroscopy. We find clear spectroscopic evidence of a branching of the photocycle for BR molecules in direct contact with the gold surfaces, with equal amounts of proteins either following the standard proton-pump photocycle or being trapped in an intermediate state not directly contributing to light-induced proton transport. Our results are particularly relevant for BR-based optoelectronic and energy-harvesting devices, where BR molecular monolayers are put in contact with metal surfaces, and, more generally, for AFM-based IR spectroscopy studies of conformational changes of proteins embedded in intrinsically heterogeneous native cell membranes.

Fast and high-resolution mapping of elastic properties of biomolecules and polymers with bimodal AFM.

Fast, high-resolution mapping of heterogeneous interfaces with a wide elastic modulus range is a major goal of atomic force microscopy (AFM). This goal becomes more challenging when the nanomechanical mapping involves biomolecules in their native environment. Over the years, several AFM-based methods have been developed to address this goal. However, none of these methods combine sub-nanometer spatial resolution, quantitative accuracy, fast data acquisition speed, wide elastic modulus range and operation in physiological solutions. Here, we present detailed procedures for generating high-resolution maps of the elastic properties of biomolecules and polymers using bimodal AFM. This requires the simultaneous excitation of the first two eigenmodes of the cantilever. An amplitude modulation (AM) feedback acting on the first mode controls the tip-sample distance, and a frequency modulation (FM) feedback acts on the second mode. The method is fast because the elastic modulus, deformation and topography images are obtained simultaneously. The method is efficient because only a single data point per pixel is needed to generate the aforementioned images. The main stages of the bimodal imaging are sample preparation, calibration of the instrument, tuning of the microscope and generation of the nanomechanical maps. In addition, with knowledge of the deformation, bimodal AFM enables reconstruction of the true topography of the surface. It takes ~9 h to complete the whole procedure.

Imaging biological samples with atomic force microscopy.

Atomic force microscopy (AFM) is an invaluable tool both for obtaining high-resolution topographical images and for determining the values of mechanical and structural properties of specimens adsorbed onto a surface. AFM is useful in an array of fields and applications, from materials science to biology. It is an extremely versatile technique that can be applied to almost any surface-mounted sample and can be operated in ambient air, ultrahigh vacuum, and, most importantly for biology, liquids. AFM can be used to explore samples ranging in size from atoms to molecules, molecular aggregates, and cells. Individual biomolecules can be viewed and manipulated at the nanoscale, providing fundamental biological information. In particular, the study of the mechanical properties of biomolecular aggregates at the nanoscale constitutes an important source of data to elaborate mechanochemical structure/function models of single-particle biomachines, expanding and complementing the information obtained from bulk experiments.

Structural changes in bacteriorhodopsin caused by two-photon-induced photobleaching.

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.

In-focus electron microscopy of frozen-hydrated biological samples with a Boersch phase plate.

We report the implementation of an electrostatic Einzel lens (Boersch) phase plate in a prototype transmission electron microscope dedicated to aberration-corrected cryo-EM. The combination of phase plate, C(s) corrector and Diffraction Magnification Unit (DMU) as a new electron-optical element ensures minimal information loss due to obstruction by the phase plate and enables in-focus phase contrast imaging of large macromolecular assemblies. As no defocussing is necessary and the spherical aberration is corrected, maximal, non-oscillating phase contrast transfer can be achieved up to the information limit of the instrument. A microchip produced by a scalable micro-fabrication process has 10 phase plates, which are positioned in a conjugate, magnified diffraction plane generated by the DMU. Phase plates remained fully functional for weeks or months. The large distance between phase plate and the cryo sample permits the use of an effective anti-contaminator, resulting in ice contamination rates of <0.6 nm/h at the specimen. Maximal in-focus phase contrast was obtained by applying voltages between 80 and 700 mV to the phase plate electrode. The phase plate allows for in-focus imaging of biological objects with a signal-to-noise of 5-10 at a resolution of 2-3 nm, as demonstrated for frozen-hydrated virus particles and purple membrane at liquid-nitrogen temperature.

Effect of antimicrobial peptide-amide: indolicidin on biological membranes.

Indolicidin, a cationic antimicrobial tridecapeptide amide, is rich in proline and tryptophan residues. Its biological activity is intensively studied, but the details how indolicidin interacts with membranes are not fully understood yet. We report here an in situ atomic force microscopic study describing the effect of indolicidin on an artificial supported planar bilayer membrane of dipalmitoyl phosphatidylcholine (DPPC) and on purple membrane of Halobacterium salinarum. Concentration dependent interaction of the peptide and membranes was found in case of DPPC resulting the destruction of the membrane. Purple membrane was much more resistant against indolicidin, probably due to its high protein content. Indolicidin preferred the border of membrane disks, where the lipids are more accessible. These data suggest that the atomic force microscope is a powerful tool in the study of indolicidin-membrane interaction.

Temperature-dependent phase transitions in zeptoliter volumes of a complex biological membrane.

Phase transitions in purple membrane have been a topic of debate for the past two decades. In this work we present studies of a reversible transition of purple membrane in the 50-60 °C range in zeptoliter volumes under different heating regimes (global heating and local heating). The temperature of the reversible phase transition is 52 ± 5 °C for both local and global heating, supporting the hypothesis that this transition is mainly due to a structural rearrangement of bR molecules and trimers. To achieve high resolution measurements of temperature-dependent phase transitions, a new scanning probe microscopy-based method was developed. We believe that our new technique can be extended to other biological systems and can contribute to the understanding of inhomogeneous phase transitions in complex systems.

Experimental evidence for membrane-mediated protein-protein interaction.

Membrane proteins diffuse within the membrane, form oligomers and supramolecular assemblies. Using high-speed atomic force microscopy, we present direct experimental measure of an in-membrane-plane interaction potential between membrane proteins. In purple membranes, ATP-synthase c-rings formed dimers that temporarily dissociated. C-ring dimers revealed subdiffusive motion, while dissociated monomers diffused freely. C-rings center-to-center distance probability distribution allowed the calculation and modeling of an in-membrane-plane energy landscape that presented repulsion at 80 Å, most stable dimer association at 103 Å (-3.5 k(B)T strength), and dissociation at 125 Å (-1 k(B)T strength). This first experimental data of nonlabeled membrane protein diffusion and the corresponding in-membrane-plane interaction energy landscape characterized membrane protein interaction with an attractive range of several k(B)T that reaches to a radius of ∼50 Å within the membrane plane.

Ultrathin conductive carbon nanomembranes as support films for structural analysis of biological specimens.

Ultrathin carbon nanomembranes (CNM) have been tested as supports for both cryogenic high-resolution transmission electron microscopy (cryo-EM) as well as atomic force microscopy (AFM) of biological specimens. Purple membrane (PM) from Halobacterium salinarum, a 2-D crystalline monolayer of bacteriorhodopsin (BR) and lipids, was used for this study. Due to their low thickness of just 1.6 nm CNM add virtually no phase contrast to the transmission pattern. This is an important advantage over commonly used amorphous carbon support films which become instable below a thickness of approximately 20 nm. Moreover, the electrical conductivity of CNM can be tuned leading to conductive carbon nanomembranes (cCNM). cCNM support films were analyzed for the first time and were found to ideally meet all requirements of cryo-EM of insulating biological samples. A projection map of PM on cCNM at 4 A resolution has been calculated which proves that the structural integrity of biological samples is preserved up to the high-resolution range. CNM have also proven to be suitable supports for AFM analysis of biological samples. PM on CNM was imaged at molecular resolution and single molecule force spectra were recorded which show no differences compared to force spectra of PM obtained with other substrates. This is the first demonstration of a support film material which meets the requirements of both, cryo-EM and AFM, thus enabling comparative structural studies of biomolecular samples with unchanged sample-substrate interactions. Beyond high-resolution cryo-EM of biological samples, cCNM are attractive new substrates for other biophysical techniques which require conductive supports, i.e. scanning tunneling microscopy (STM) and electrostatic force microscopy (EFM).

High-speed atomic force microscopy shows dynamic molecular processes in photoactivated bacteriorhodopsin.

Dynamic changes in protein conformation in response to external stimuli are important in biological processes, but it has proved difficult to directly visualize such structural changes under physiological conditions. Here, we show that high-speed atomic force microscopy can be used to visualize dynamic changes in stimulated proteins. High-resolution movies of a light-driven proton pump, bacteriorhodopsin, reveal that, upon illumination, a cytoplasmic portion of each bacteriorhodopsin monomer is brought into contact with adjacent trimers. The bacteriorhodopsin-bacteriorhodopsin interaction in the transiently formed assembly engenders both positive and negative cooperative effects in the decay kinetics as the initial bacteriorhodopsin recovers and, as a consequence, the turnover rate of the photocycle is maintained constant, on average, irrespective of the light intensity. These results confirm that high-resolution visualization is a powerful approach for studying elaborate biomolecular processes under realistic conditions.

Automated setpoint adjustment for biological contact mode atomic force microscopy imaging.

Contact mode atomic force microscopy (AFM) is the most frequently used AFM imaging mode in biology. It is about 5-10 times faster than oscillating mode imaging (in conventional AFM setups), and provides topographs of biological samples with sub-molecular resolution and at a high signal-to-noise ratio. Unfortunately, contact mode imaging is sensitive to the applied force and intrinsic force drift: inappropriate force applied by the AFM tip damages the soft biological samples. We present a methodology that automatically searches for and maintains high resolution imaging forces. We found that the vertical and lateral vibrations of the probe during scanning are valuable signals for the characterization of the actual applied force by the tip. This allows automated adjustment and correction of the setpoint force during an experiment. A system that permanently performs this methodology steered the AFM towards high resolution imaging forces and imaged purple membrane at molecular resolution and live cells at high signal-to-noise ratio for hours without an operator.

Contact-mode high-resolution high-speed atomic force microscopy movies of the purple membrane.

High-speed atomic force microscopy (HS-AFM) is becoming a reference tool for the study of dynamic biological processes. The spatial and time resolutions of HS-AFM are on the order of nanometers and milliseconds, respectively, and allow structural and functional characterization of biological processes at the single-molecule level. In this work we present contact-mode HS-AFM movies of purple membranes containing two-dimensional arrays of bacteriorhodopsin (bR). In high-resolution movies acquired at a 100 ms frame acquisition time, the substructure on individual bR trimers was visualized. In regions in between different bR arrays, dynamic topographies were observed and interpreted as motion of the bR trimers. Similarly, motion of bR monomers in the vicinity of lattice defects in the purple membrane was observed. Our findings indicate that the bR arrays are in a mobile association-dissociation equilibrium. HS-AFM on membranes provides novel perspectives for analyzing the membrane diffusion processes of nonlabeled molecules.

Assembly of purple membranes on polyelectrolyte films.

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.

Light- and pH-dependent conformational changes in protein structure induce strong bending of purple membranes--active membranes studied by cryo-SEM.

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.

Asymmetric distribution of biotin labeling on the purple membrane.

This work examined the biotin modification of bacteriorhodopsin (BR) in the purple membrane (PM). The results of flash kinetic absorption measurements showed that photocycle was maintained in biotinylated BR. Biotinylated BR also maintained its photoelectric activity, as indicated by the photoelectric response of the bilayer lipid membrane (BLM). Atomic force microscopy (AFM) of stretavidiin-bound biotin revealed that biotin molecules covered both surfaces of the, but the amount of biotinylated BR on the extracellular (EC) surface was markedly higher than on the cytoplasmic (CP) surface. Further studies showed that, after reaction with fluorescamine (FL), biotin labeling occurred only on the CP surface. These results are informative for future work on bioconjugation of BR as well as work on oriented assembly and the design of BR-based photoelectric devices.

Hydration dependence of active core fluctuations in bacteriorhodopsin.

We used neutron scattering and specific hydrogen-deuterium labeling to investigate the thermal dynamics of isotope-labeled amino acids and retinal, predominantly in the active core and extracellular moiety of bacteriorhodopsin (BR) in the purple membrane and the dynamical response to hydration. Measurements on two neutron spectrometers allowed two populations of motions to be characterized. The lower amplitude motions were found to be the same for both the labeled amino acids and retinal of BR and the global membrane. The larger amplitude dynamics of the labeled part, however, were found to be more resilient than the average membrane, suggesting their functional importance. The response to hydration was characterized, showing that the labeled part of BR is not shielded from hydration effects. The results suggest that the inhibition of high-amplitude motions by lowering hydration may play a key role in the slowing down of the photocycle and the proton pumping activity of BR.

Electron cryo-microscopy of biological specimens on conductive titanium-silicon metal glass films.

Thin films of the metal glass Ti88Si12 were produced by evaporation and characterized by AFM and conductivity measurements. Thin Ti88Si12 support films for electron microscopy were prepared by coating standard EM grids with evaporated films floated off mica, and characterized by electron imaging and electron diffraction. At room temperature, the specific resistance of a thin TiSi film was 10(6) times lower than that of an amorphous carbon film. At 77K, the specific resistance of TiSi films decreased, whereas that of carbon became immeasurably high. The effective scattering cross-section of TiSi and amorphous carbon for 120 kV electrons is roughly equal, but TiSi films for routine use can be approximately 10 times thinner due to their high mechanical strength, so that they would contribute less background noise to the image. Electron diffraction of purple membrane on a TiSi substrate confirmed that the support film was amorphous, and indicated that the high-resolution order of the biological sample was preserved. Electron micrographs of TiSi films tilted by 45 degrees relative to the electron beam recorded at approximately 4 K indicated that the incidence of beam-induced movements was reduced by 50% compared to amorphous carbon film under the same conditions. The success rate of recording high-resolution images of purple membranes on TiSi films was close to 100%. We conclude that TiSi support films are ideal for high-resolution electron cryo-microscopy (cryo-EM) of biological specimens, as they reduce beam-induced movement significantly, due to their high electrical conductivity at low temperature and their favorable mechanical properties.

Bacteriorhodopsin (bR) as an electronic conduction medium: current transport through bR-containing monolayers.

Studying electron transport (ET) through proteins is hampered by achieving reproducible experimental configurations, particularly electronic contacts to the proteins. The transmembrane protein bacteriorhodopsin (bR), a natural light-activated proton pump in purple membranes of Halobacterium salinarum, is well studied for biomolecular electronics because of its sturdiness over a wide range of conditions. To date, related studies of dry bR systems focused on photovoltage generation and photoconduction with multilayers, rather than on the ET ability of bR, which is understandable because ET across 5-nm-thick, apparently insulating membranes is not obvious. Here we show that electronic current passes through bR-containing artificial lipid bilayers in solid "electrode-bilayer-electrode" structures and that the current through the protein is more than four orders of magnitude higher than would be estimated for direct tunneling through 5-nm, water-free peptides. We find that ET occurs only if retinal or a close analogue is present in the protein. As long as the retinal can isomerize after light absorption, there is a photo-ET effect. The contribution of light-driven proton pumping to the steady-state photocurrents is negligible. Possible implications in view of the suggested early evolutionary origin of halobacteria are noted.

Chemically induced enhancement of the opto-electronic response of Halobacterium purple membrane monolayer.

Acetylation of purple membranes (PM) significantly enhances the surface photovoltage that they exhibit, if adsorbed as a monolayer on a solid surface; we suggest that this increase is due to the improved orientation of the PM on the surface.

Circular dichroism of heterochromophoric and partially regenerated purple membrane: search for exciton coupling.

In order to determine the origin of the bisignate CD spectra of native purple membrane, heterochromophoric analogues containing bacteriorhodopsin regenerated with native all-trans-retinal and retinal analogues were investigated. The data collected for the purple membrane samples containing two different chromophores suggest the additive character of the CD spectra. This conclusion was supported by a series of spectra using 5,6-dihydroretinal and 3-dehydroretinal and by using 33% regenerated PM in buffer and in presence of osmolytes. Our results support the idea of conformational heterogeneity of the chromophores in the bR in the trimer, suggesting that the three bR subunits in the trimer are not conformationally equal, and therefore, the bisignate CD spectrum of bR in the purple membrane occurs rather due to a superposition of the CD spectra from variously distorted bR subunits in the trimer than interchromophoric exciton-coupling interactions.