Detection of bacteriorhodopsin trimeric rotation at thermal phase transitions of purple membrane in suspension.

Bacteriorhodopsin (bR) of purple membrane (PM) is a retinal protein that forms aggregates in the form of trimers constituting, together with archaeal lipids, the crystalline structure of PM. The rotary motion of bR inside PM may be pertinent in understanding the essence of the crystalline lattice. An attempt has been made to determine the rotation of bR trimers which has been found to be detected solely at thermal phase transitions of PM, namely lipid, crystalline lattice and protein melting phase transitions. The temperature dependences of dielectric versus electronic absorption spectra of bR have been determined. The results suggest that the rotation of bR trimers, together with concomitant bending of PM, are most likely brought by structural changes in bR which might be driven by retinal isomerization and mediated by lipid. The rupturing of the lipid-protein contact might consequently lead to rotation of trimers associated with bending, curling or vesicle formation of PM. So the retinal reorientation may underlie the concomitant rotation of trimers. Most importantly, rotation of trimers might play a role, in terms of the essence of the crystalline lattice, in the functional activity of bR and may serve physiological relevance.

Imaging DNA Equilibrated onto Mica in Liquid Using Biochemically Relevant Deposition Conditions.

For over 25 years, imaging of DNA by atomic force microscopy has been intensely pursued. Ideally, such images are then used to probe the physical properties of DNA and characterize protein-DNA interactions. The atomic flatness of mica makes it the preferred substrate for high signal-to-noise ratio (SNR) imaging, but the negative charge of mica and DNA hinders deposition. Traditional methods for imaging DNA and protein-DNA complexes in liquid have drawbacks: DNA conformations with an anomalous persistence length ( p), low SNR, and/or ionic deposition conditions detrimental to preserving protein-DNA interactions. Here, we developed a process to bind DNA to mica in a buffer containing both MgCl2 and KCl that resulted in high SNR images of equilibrated DNA in liquid. Achieving an equilibrated 2D configuration ( i. e., p = 50 nm) not only implied a minimally perturbative binding process but also improved data quality and quantity because the DNA's configuration was more extended. In comparison to a purely NiCl2-based protocol, we showed that an 8-fold larger fraction (90%) of 680-nm-long DNA molecules could be quantified. High-resolution images of select equilibrated molecules revealed the right-handed structure of DNA with a helical pitch of 3.5 nm. Deposition and imaging of DNA was achieved over a wide range of monovalent and divalent ionic conditions, including a buffer containing 50 mM KCl and 3 mM MgCl2. Finally, we imaged two protein-DNA complexes using this protocol: a restriction enzyme bound to DNA and a small three-nucleosome array. We expect such deposition of protein-DNA complexes at biochemically relevant ionic conditions will facilitate biophysical insights derived from imaging diverse protein-DNA complexes.

Stationary Phase EPR Spectroscopy for Monitoring Membrane Protein Refolding by Conformational Response.

Protein production remains a major bottleneck in membrane protein structural biology. In many cases, large-scale recombinant protein expression is either unfeasible or impossible, driving structural biologists to explore new production avenues. Several membrane proteins have been successfully refolded from solubilized E. coli inclusion bodies. In recent years, a structure of the G-protein-coupled receptor CXCR1 was obtained using refolded material from E. coli inclusion bodies. However, aggregation during the refolding process is a common difficulty, which is often addressed by immobilization of the protein onto a solid support. Most spectroscopic methods are incompatible with these light-scattering matrices, which renders automated buffer exchange to screen refolding conditions impossible. This work explores a potential approach to overcome this problem by utilizing site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy of protein bound to standard, commercially available Ni-NTA agarose resin. With this approach, the correct protein fold is determined by activity, which is inferred from a protein conformational response to a known stimulant. EPR spectra at each state of the refolding workflow of spin-labeled Haloarcula marismortui bacteriorhodopsin-I (HmbRI) are obtained, and refolded fractions of HmbRI with this platform are quantitated using both protein from inclusion bodies and denatured recombinant protein from E. coli membranes. The stimulant used for HmbRI is visible light. The solid support allows for multiple refolding trials through buffer exchanges, and the EPR spectra are collected on the order of seconds under ambient conditions.

Potential Second-Harmonic Ghost Bands in Fourier Transform Infrared (FT-IR) Difference Spectroscopy of Proteins.

Fourier transform infrared (FT-IR) difference absorption spectroscopy is a common method for studying the structural and dynamical aspects behind protein function. In particular, the 2800-1800 cm-1 spectral range has been used to obtain information about internal (deuterated) water molecules, as well as site-specific details about cysteine residues and chemically modified and artificial amino acids. Here, we report on the presence of ghost bands in cryogenic light-induced FT-IR difference spectra of the protein bacteriorhodopsin. The presence of these ghost bands can be particularly problematic in the 2800-1900 cm-1 region, showing intensities similar to O-D vibrations from water molecules. We demonstrate that they arise from second harmonics from genuine chromophore bands located in the 1400-850 cm-1 region, generated by double-modulation artifacts caused from reflections of the IR beam at the sample and at the cryostat windows back to the interferometer (inter-reflections). The second-harmonic ghost bands can be physically removed by placing an optical filter of suitable cutoff in the beam path, but at the cost of losing part of the multiplexing advantage of FT-IR spectroscopy. We explored alternatives to the use of optical filters. Tilting the cryostat windows was effective in reducing the intensity of the second harmonic artifacts but tilting the sample windows was not, presumably by their close proximity to the focal point of the IR beam. We also introduce a simple numerical post-processing approach that can partially, but not fully, correct for second-harmonic ghost bands in FT-IR difference spectra.

A high sensitive and contaminant tolerant matrix for facile detection of membrane proteins by matrix-assisted laser desorption/ionization mass spectrometry.

Despite the significance of membrane proteins (MPs) in biological system is indisputable, their specific natures make them notoriously difficult to be analyzed. Particularly, the widely used Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) prefers analyses of hydrophilic cytosolic proteins and has a limited ionization efficiency towards hydrophobic MPs. Herein, a hydrophobic compound (E)-propyl α-Cyano-4-Hydroxyl Cinnamylate (CHCA-C3), a propyl-esterified derivative of α-cyano-4-hydroxycinnamic acid (CHCA), was applied as a contaminant tolerant matrix for high sensitivity MALDI-MS analyses of MPs. With CHCA-C3, the detection limits of hydrophobic peptides were 10- to 100-fold better than those using CHCA. Furthermore, high quality of spectra could be achieved in the presence of high concentration of chaotropes, salts and detergents, as well as human urinary and serum environment. Also, CHCA-C3 could generate uniform sample distribution even in the presence of contaminants. This high contaminant-resistance was revealed to be ascribed to the enhanced hydrophobicity of CHCA-C3 with a lower affinity towards hydrophilic contaminants. The application of CHCA-C3 is further demonstrated by the analysis of trypsin/CNBr digests of bacteriorhodopsin containing seven transmembrane domains (TMDs), which dramatically increased numbers of identified hydrophobic peptides in TMDs and sequence coverage (∼100%). Besides, a combined method by using CHCA-C3 with fluoride solvent and a patterned paraffin plate was established for analysis of integral MPs. We achieved a low detection limit of 10 fmol for integral bacteriorhodopsin, which could not be detected using traditional matrices such as 3,5-dimethoxy-4-hydroxycinamic acid, 2,5-dihydroxyacetophenone even at sample concentration of 10 pmol.

Glass is a Viable Substrate for Precision Force Microscopy of Membrane Proteins.

Though ubiquitous in optical microscopy, glass has long been overlooked as a specimen supporting surface for high resolution atomic force microscopy (AFM) investigations due to its roughness. Using bacteriorhodopsin from Halobacterium salinarum and the translocon SecYEG from Escherichia coli, we demonstrate that faithful images of 2D crystalline and non-crystalline membrane proteins in lipid bilayers can be obtained on microscope cover glass following a straight-forward cleaning procedure. Direct comparison between AFM data obtained on glass and on mica substrates show no major differences in image fidelity. Repeated association of the ATPase SecA with the cytoplasmic protrusion of SecYEG demonstrates that the translocon remains competent for binding after tens of minutes of continuous AFM imaging. This opens the door for precision long-timescale investigations of the active translocase in near-native conditions and, more generally, for integration of high resolution biological AFM with many powerful optical techniques that require non-birefringent substrates.

Improved protease digestion conditions for membrane protein detection.

This work presents improved protease digestion conditions for membrane protein detection. The enzymatic digest of bacteriorhodopsin (BR), a model membrane protein with seven transmembrane domains (TMDs) was investigated. An initial in-gel digestion identified 17% BR sequence coverage, including part of the seventh TMD. To improve sequence coverage, BR digest was tested with different concentrations of RapiGest, methanol (MeOH) and SDS using either trypsin or chymotrypsin. Two improved conditions, 0.01% SDS or the combination of 10% MeOH and 0.01% RapiGest, were chosen. Trypsin digestions in both conditions achieved more than 40% BR sequence coverage compared to 0% using standard digestion conditions. Peptides detected from trypsin and chymotrypsin digestions in the same condition were combined to maximize sequence coverage. The same conditions were applied to a different membrane protein with one TMD, Selenoprotein S, and proteins from Escherichia coli. For Selenoprotein S, a higher sequence coverage, including a peptide from the TMD, was detected from the improved condition compared to the typical condition. The application of both improved conditions to a membrane protein fraction of Escherichia coli resulted in the identification of 309 (SDS) and 329 (MeOH/RapiGest) unique proteins of which 140/309 and 148/329 were membrane proteins.

A new manual dispensing system for in meso membrane protein crystallization with using a stepping motor-based dispenser.

A reliable and easy to use manual dispensing system has been developed for the in meso membrane protein crystallization method. The system consists of a stepping motor-based dispenser with a new microsyringe system for dispensing, which allows us to deliver any desired volume of highly viscous lipidic mesophase in the range from ~50 to at least ~200 nl. The average, standard deviation, and coefficient of variation of 20 repeated deliveries of 50 nl cubic phase were comparable to those of a current robotic dispensing. Moreover, the bottom faces of boluses delivered to the glass crystallization plate were reproducibly circular in shape, and their centers were within about 100 µm from the center of the crystallization well. The system was useful for crystallizing membrane and soluble proteins in meso.

Isolation of a new Pseudomonas halophila strain possess bacteriorhodopsin-like protein by a novel method for screening of photoactive protein producing bacteria.

Bacteriorhodopsin (bR) is a transmembrane protein deposited in the purple membrane of Halobacterium salinarum which absorbs energy from photons to create a photo-induced proton gradient across the membrane. A bR molecule can be considered as a natural solar device transforming light into other types of energy and therefore is of interest for a wide range of applications including two and three-dimensional memory storage, optical data processing, artificial cells, holographic media, the artificial retina and photo sensor devices. H. salinarum is a slow-growing, halophilic Archaea present in red salt waters. The present study introduces a novel bR-like pigment from a new strain of Pseudomonas halophila (with registered accession number KC959570 in the NCBI databank) which has a very significant degree of light-dependent activity. This is the first report on the presence of functional bR-like protein in the Pseudomonas family. The isolate is a fast-growing, halophilic bacterium and is comparable with other photoactive protein producer microorganisms. Also, in the present study a novel isolation method for screen light-stimulating protein producing microorganisms is introduced. For this purpose 2,3,5-triphenyltetrazolium chloride (TTC) was employed for the first time as an artificial hydrogen acceptor in the proton-transfer processes. The TTC test is an easy and susceptible method for estimating hydrogen production during the proton transport process. This is the first report of the use of TTC for photo activity measurement and selection of bacteria containing light dependent proteins.

In-gel total protein quantification using a ninhydrin-based method.

Precise in-gel quantification of total protein amount of bands or spots in gels is the basis of subsequent biochemical, molecular biological and immunological analyses. Though several methods have been designed to evaluate relative amounts of proteins, these methods are of limited reliability because (semi-) quantifications depend on the amount of protein migrating into the gel and different proteins may lead to different absorptions/intensities of stained bands or spots. In the present study, we described a method to quantify both, hydrophilic and hydrophobic proteins using in-gel digestion with proteinase K, subsequent extraction and acid hydrolysis followed by the use of the ninhydrin reaction. The protocol is accurate and compatible with mass spectrometric characterization of proteins. Reproducible in-gel protein quantification was performed from SDS-PAGE and IEF/SDS-PAGE gels using bovine serum albumin as a standard protein. Bacteriorhodopsin separated on SDS-PAGE gel was quantified in addition in order to show that the method is also suitable for quantification of hydrophobic protein. This protocol for reliable in-gel protein quantification, which not only provides "arbitrary units of optical density", can also be completed in a minimum of 4 days or maximum 1 week depending on the type of electrophoresis with the disadvantage of being time consuming.

Insight into a single halobacterium using a dual-bacteriorhodopsin system with different functionally optimized pH ranges to cope with periplasmic pH changes associated with continuous light illumination.

The light-driven outward proton transporter assists energy production via an ATP synthase system best exemplified by the bacteriorhodopsin (BR) from Halobacterium salinarum, HsBR. As the only archaea able to survive in the resource-limited ecosystem of the Dead Sea, Haloarcula marismortui has been reported to have a unique dual-BR system, consisting of HmBRI and HmBRII, instead of only a single BR in a cell (solo-BR). The contribution of this dual-BR system to survival was investigated. First, native H. marismortui and H. salinarum cells were tested in water that had been adjusted to mimic the conditions of Dead Sea water. These archaea were shown to accumulate protons and reduce pH in their periplasmic regions, which disabled further proton transportation functionality in H. salinarum but not in H. marismortui. Then, pH-dependent photocurrent measurements using purified BR proteins demonstrated that HsBR and HmBRI were functional at pH > 5.0 and that HmBRII was functional at pH > 4.0. Our results indicate that the dual-HmBR system is composed of two BRs with different optimal functional pH ranges and together they maintain light-driven proton transport activity under pH > 4.0, which might contribute the survival of H. marismortui under the acidic pH of the Dead Sea.

Evaluation of 2,6-dihydroxyacetophenone as matrix-assisted laser desorption/ionization matrix for analysis of hydrophobic proteins and peptides.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is widely used for analysis of macromolecules like peptides and proteins. The analysis procedure is generally simple but must be adapted to the characteristics of the analytes. Therefore, specific matrices suitable for, e.g., hydrophobic proteins and peptides that are difficult to analyze would be preferable in order to optimize the outcome. In the present work, 2,6-dihydroxyacetophenone (DHAP) was shown to be beneficial in comparison to DHB for intact bacteriorhodopsin (BR) as well as for chemically digested BR.

Benzophenone-containing fatty acids and their related photosensitive fluorescent new probes: design, physico-chemical properties and preliminary functional investigations.

Hydrophobic photoaffinity labeling is a powerful strategy to identify hydrophobic segments within molecules, in particular membrane proteins. Here we report the design and synthesis of a novel family of fluorescent and photosensitive lipid tools, which have a common amino acid scaffold functionalized by three groups: (i) a first fatty acid chain grafted with a photoactivatable benzophenone moiety (Fatty Acid BenzoPhenone, FABP), (ii) a second fatty acid chain to ensure anchoring into a half-bilayer or hydrophobic environment, and (iii) a fluorescent carboxytetramethylrhodamine headgroup (CTMR) to detect the photolabeled compound. We present data of the synthesis and characterization of three lipid tools whose benzophenone ring is situated at various distances from the central scaffold. We could therefore establish structure/properties relationships dependent upon the depth of insertion of benzophenone into the membrane. Our lipid tools were extensively characterized both physico- and bio-chemically, and we assessed their functionality in vitro using bacterioRhodopsin (bR). We thus provide the scientific community with novel and reliable tools for the identification and study of hydrophobic regions in proteins.

A gene-fusion strategy for stoichiometric and co-localized expression of light-gated membrane proteins.

The precise co-localization and stoichiometric expression of two different light-gated membrane proteins can vastly improve the physiological usefulness of optogenetics for the modulation of cell excitability with light. Here we present a gene-fusion strategy for the stable 1:1 expression of any two microbial rhodopsins in a single polypeptide chain. By joining the excitatory channelrhodopsin-2 with the inhibitory ion pumps halorhodopsin or bacteriorhodopsin, we demonstrate light-regulated quantitative bi-directional control of the membrane potential in HEK293 cells and neurons in vitro. We also present synergistic rhodopsin combinations of channelrhodopsin-2 with Volvox carteri channelrhodopsin-1 or slow channelrhodopsin-2 mutants, to achieve enhanced spectral or kinetic properties, respectively. Finally, we demonstrate the utility of our fusion strategy to determine ion-turnovers of as yet uncharacterized rhodopsins, exemplified for archaerhodopsin and CatCh, or to correct pump cycles, exemplified for halorhodopsin.

Quantum dot enhancement of bacteriorhodopsin-based electrodes.

Nanoscale sensing arrays utilizing the unique properties of the optical protein bacteriorhodopsin and colloidal semiconductor quantum dots are being developed for toxin detection applications. This paper describes an innovative method to activate bacteriorhodopsin-based electrodes with the optical output of quantum dots, producing an enhanced electrical response from the protein. Results show that the photonic emission of CdSe/ZnS quantum dots is absorbed by the bacteriorhodopsin retinal and initiates the proton pumping sequence, resulting in an electrical output from a bacteriorhodopsin-based electrode. It is also shown that activated quantum dots in sub-10nm proximity to bacteriorhodopsin further amplify the photovoltaic response of the protein by approximately 23%, compared to without attached quantum dots, suggesting direct energy transfer mechanisms beyond photonic emission alone. The ability of quantum dots to activate nanoscale regions on bacteriorhodopsin-based electrodes could allow sub-micron sensing arrays to be created due to the ability to activate site-specific regions on the array.

Membrane protein analysis using an improved peptic in-solution digestion protocol.

In the proteomic analysis of membrane proteins, less-specific proteases have become a promising tool to overcome fundamental limitations of trypsin with its unique specificity for basic residues. Pepsin is well-known to be utilized for specific applications that require acidic conditions, but in terms of membrane protein identification and characterization, it has been disregarded for the most part. This work presents an optimization of an existing peptic digest protocol for the analysis of membrane proteins using bacteriorhodopsin from purple membranes as reference.

Site-specific difference 2D-IR spectroscopy of bacteriorhodopsin.

We demonstrate the extension of the principle of difference Fourier transform infrared (FTIR) spectroscopy to difference 2D-IR spectroscopy. To this end, we measure difference 2D-IR spectra of the protein bacteriorhodopsin in its early J- and K-intermediates. By comparing with the static 2D-IR spectrum of the protonated Schiff base of all-trans retinal, we demonstrate that the 2D-IR spectrum of the all-trans retinal chromophore in bacteriorhodopsin can be measured with the background from the remainder of the protein completely suppressed. We discuss several models to interpret the detailed line shape of the difference 2D-IR spectrum.

A tin oxide transparent electrode provides the means for rapid time-resolved pH measurements: application to photoinduced proton transfer of bacteriorhodopsin and proteorhodopsin.

An electrochemical cell was previously reported in which bacteriorhodopsin (BR, purple membrane) was adsorbed on the surface of a transparent SnO(2) electrode, and illumination resulted in potential or current changes (Koyama et al., Science 265:762-765, 1994; Robertson and Lukashev, Biophys. J. 68:1507-1517, 1995; Koyama et al., Photochem. Photobiol. 68:400-406, 1998). In this paper, we concluded that pH changes caused by proton transfer by the deposited BR or proteorhodopsin (PR) films lead to the flash-induced potential change in the SnO(2) electrode. Thus, the signals originate from BR and PR acting as light-driven proton pumps. This conclusion was drawn from the following observations. (1) The relation between the potential of a bare electrode and pH is linear for a wide pH range. (2) The flash-induced potential changes decrease with an increase in the buffer concentration. (3) The action spectrum of PR agrees well with the absorption spectrum. (4) The present electrode can monitor the pH change in the time range from 10 ms to several hundred milliseconds, as deduced by comparing the SnO(2) signal with the signals of pH-sensitive dyes. Using this electrode system, flash-induced proton transfer by BR was measured for a wide pH range from 2 to 10. From these data, we reconfirmed various pK(a) values reported previously, indicating that the present method can give the correct pK(a) values. This is the first report to estimate these pK(a) values directly from the proton transfer. We then applied this method to flash-induced proton transfer of PR. We observed proton uptake followed by release for the pH range from 4 to 9.5, and in other pH ranges, proton release followed by uptake was observed.

Structural characterization of an integral membrane protein in its natural lipid environment by oxidative methionine labeling and mass spectrometry.

Membrane proteins represent formidable challenges for many analytical techniques. Studies on these systems are often carried out after surfactant solubilization. Unfortunately, such a non-natural protein environment can affect conformation and stability, and it offers only partial protection against aggregation. This work employs bacteriorhodopsin (BR) as a model system for in situ structural studies on a membrane protein in its natural lipid bilayer. BR-containing purple membrane suspensions were exposed to hydroxyl radicals, generated by nanosecond laser photolysis of dilute aqueous H(2)O(2). The experiments rely on the premise that oxidative labeling occurs mainly at solvent-exposed side chains, whereas sites that are sterically protected will react to a much lesser extent. Following .OH exposure, the protein was analyzed by tryptic peptide mapping and electrospray tandem mass spectrometry. Oxidative labeling of BR was found to occur only at its nine Met residues. This is in contrast to the behavior of previously studied water-soluble proteins, which generally undergo modifications at many different types of residues. In those earlier experiments the high reactivity of Met has hampered its use as a structural probe. In contrast, the Met oxidation pattern observed here is in excellent agreement with the native BR structure. Extensive labeling is seen for Met32, 68, and 163, all of which are located in solvent-exposed loops. The remaining six Met residues are deeply buried and show severalfold less oxidation. Our results demonstrate the usefulness of Met oxidative labeling for structural studies on membrane proteins, especially when considering that many of these species are methionine-rich. The introduction of additional Met residues as conformational probes, as well as in vivo structural investigations, represents exciting future extensions of the methodology described here.

Membrane protein separation and analysis by supercritical fluid chromatography-mass spectrometry.

Membrane proteins comprise 25-30% of the human genome and play critical roles in a wide variety of important biological processes. However, their hydrophobic nature has compromised efforts at structural characterization by both X-ray crystallography and mass spectrometry. The detergents that are generally used to solubilize membrane proteins interfere with the crystallization process essential to X-ray studies and cause severe ion suppression effects that hinder mass spectrometric analysis. In this report, the use of supercritical fluid chromatography-mass spectrometry for the separation and analysis of integral membrane proteins and hydrophobic peptides is investigated. It is shown that detergents are rapidly and effectively separated from the proteins and peptides, yielding them in a state suitable for direct mass spectrometric analysis.