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.

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.

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.

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.

Membrane proteomic analysis of Arabidopsis thaliana using alternative solubilization techniques.

This study presents a comparative proteomic analysis of the membrane subproteome of whole Arabidopsis seedlings using 2% Brij-58 or 60% methanol to enrich and solubilize membrane proteins for strong cation exchange fractionation and reversed-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 441 proteins were identified by our Brij-58 method, and 300 proteins were detected by our methanol-based solubilization approach. Although the total number of proteins obtained using the nonionic detergent was higher than the total obtained by organic solvent, the ratio of predicted membrane proteins to total proteins identified indicates up to an 18.6% greater enrichment efficiency using methanol. Using two different bioinformatics approaches, between 31.0 and 40.0% of the total proteins identified by the methanol-based method were classified as containing at least one putative transmembrane domain as compared to 22.0-23.4% for Brij-58. In terms of protein hydrophobicity as determined by the GRAVY index, it was revealed that methanol was more effective than Brij-58 for solubilizing membrane proteins ranging from -0.4 (hydrophilic) to +0.4 (hydrophobic). Methanol was also approximately 3-fold more effective than Brij-58 in identifying leucine-rich repeat receptor-like kinases. The ability of methanol to effectively solubilize and denature both hydrophobic and hydrophilic proteins was demonstrated using bacteriorhodopsin and cytochrome c, respectively, where both proteins were identified with at least 82% sequence coverage from a single reversed-phase LC-MS/MS analysis. Overall, our data show that methanol is a better alternative for identifying a wider range of membrane proteins than the nonionic detergent Brij-58.

Simulation of fluorescence anisotropy experiments: probing protein dynamics.

Time-resolved fluorescence anisotropy decay experiments on a protein-attached dye can probe local protein dynamics and steric restrictions, but are difficult to interpret at the structural level. Aiming at an atomistic description, we have carried out molecular dynamics simulations of such experiments. Our simulations describe an Alexa488 fluorescent dye maleimide derivative covalently attached via a single cysteine to the AB-loop of bacteriorhodopsin. Fluorescence anisotropy decay curves obtained from the simulations agree well with the measured ones. Three anisotropy decay components were resolved and assigned to: 1), the fast dynamics of the attached dye on the picosecond timescale; 2), the slower dynamics of the loop at the one nanosecond timescale; and 3), the overall tumbling of the molecule. For the biologically relevant 1-ns component we identified two processes from simulations, the motion of the flexible loop as well as slow conformational dynamics of the dye. These two processes are not separable by experiment alone. Furthermore, analysis of the correlation between the dye and the protein motion revealed which part and which motion of the protein is actually probed by the experiment. Finally, our simulations allowed us to test the usual and inevitable assumption underlying these types of spectroscopic measurements that the attached dye probe does not severely perturb the protein dynamics. For the case at hand, by comparison with a simulation of the dye-free protein, the perturbation was quantified and found to be small.

Microwave-assisted acid hydrolysis of proteins combined with liquid chromatography MALDI MS/MS for protein identification.

Simple and efficient digestion of proteins, particularly hydrophobic membrane proteins, is of significance for comprehensive proteome analysis using the bottom-up approach. We report a microwave-assisted acid hydrolysis (MAAH) method for rapid protein degradation for peptide mass mapping and tandem mass spectrometric analysis of peptides for protein identification. It uses 25% trifluoroacetic acid (TFA) aqueous solution to dissolve or suspend proteins, followed by microwave irradiation for 10 min. This detergent-free method generates peptide mixtures that can be directly analyzed by liquid chromatography (LC) matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) without the need of extensive sample cleanup. LC-MALDI MS/MS analysis of the hydrolysate from 5 microg of a model transmembrane protein, bacteriorhodopsin, resulted in almost complete sequence coverage by the peptides detected, including the identification of two posttranslational modification sites. Cleavage of peptide bonds inside all seven transmembrane domains took place, generating peptides of sizes amenable to MS/MS to determine possible sequence errors or modifications within these domains. Cleavage specificity, such as glycine residue cleavage, was observed. Terminal peptides were found to be present in relatively high abundance in the hydrolysate, particularly when low concentrations of proteins were used for MAAH. It was shown that these peptides could still be detected from MAAH of bacteriorhodopsin at a protein concentration of 1 ng/microl or 37 fmol/microl. To evaluate the general applicability of this method, it was applied to identify proteins from a membrane protein enriched fraction of cell lysates of human breast cancer cell line MCF7. With one-dimensional LC-MALDI MS/MS, a total of 119 proteins, including 41 membrane-associated or membrane proteins containing one to 12 transmembrane domains, were identified by MS/MS database searching based on matches of at least two peptides to a protein.

Efficient in-gel digestion procedure using 5-cyclohexyl-1-pentyl-beta-D-maltoside as an additive for gel-based membrane proteomics.

A cycloalkyl aliphatic saccharide, 5-cyclohexyl-1-pentyl-beta-D-maltoside (CYMAL-5), was evaluated as a novel additive in a high-throughput in-gel protein digestion system using 96-well plates. Addition of 0.1% CYMAL-5 (final concentration) during trypsin treatment was compatible with both matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis, and gave a better digestion efficiency than n-octylglucoside, which we previously reported. In-gel reduction and alkylation of Cys residues under denaturing conditions also improved the sequence coverage of peptides. In-gel tryptic digestion with the optimum combination of 0.5 mm thick gels, negative staining, alkylation under denaturing conditions (6 M guanidine hydrochloride), and digestion in the presence of CYMAL-5, gave excellent performance especially for membrane protein analysis, where recovery of hydrophobic peptides was markedly enhanced. The new protocol is simple and convenient, and should be widely applicable to gel-based proteomics.

On-column sample enrichment for capillary electrophoresis sheathless electrospray ionization mass spectrometry: evaluation for peptide analysis and protein identification.

Although several designs have been advanced for coupling sample enrichment devices to a sheathless electrospray ionization-mass spectrometry (MS) interface on a capillary electrophoresis (CE) column, most of these approaches suffer from difficulties in fabrication, and the CE separation efficiency is degraded as a result of the presence of coupling sleeves. We have developed a design that offers significant improvements in terms of ease of fabrication, durability, and maintenance of the integrity of the CE-separated analyte zones. Capillaries with different inside and outside diameters were evaluated to optimize the performance of the CE-MS system, resulting in a mass limit of detection of 500 amol for tandem MS analysis of a standard peptide using a 20-microm-i.d. capillary. The improved design incorporates an efficient method to preconcentrate a sample directly within the CE capillary followed by its electrophoretic separation and detection using a true zero dead-volume sheathless CE-MS interface. Testing of this novel CE-MS system showed its ability to characterize proteomic samples such as protein digests, in-gel-digested proteins, and hydrophobic peptides as well as to quantitate ICAT-labeled peptides.

Extraction method for analysis of detergent-solubilized bacteriorhodopsin and hydrophobic peptides by electrospray ionization mass spectrometry.

The analysis of integral membrane proteins or transmembrane peptides by electrospray ionization mass spectrometry (ESI-MS) is difficult since detergents, used to solubilize these hydrophobic proteins and peptides, severely suppress analyte ion formation. This problem has been addressed previously by precipitating the protein, removing the detergent, and resolubilizing the protein in a nonpolar solvent. Here, we demonstrate a method that avoids protein precipitation and resolubilization. Detergent-solubilized bacteriorhodopsin is extracted into a nonpolar solvent phase by adding a chloroform/methanol/water solvent mixture to the aqueous detergent solution. ESI mass spectra of the nonpolar, chloroform-rich phase were dominated by peaks due to bacterioopsin. Bacterioopsin precursors with partially cleaved leader sequences were seen in all mass spectra. Additional peaks were likely due to intact bacteriorhodopsin, i.e., bacterioopsin with the retinal prosthetic group attached, and to bacterioopsin associated with lipid molecules. A separation process that occurred in the fused-silica capillary leading to the electrospray tip was essential for obtaining ESI mass spectra of bacterioopsin. The extraction-into-chloroform procedure also worked well with hydrophobic, transmembrane-type peptides that were insoluble in other electrospray solvents, including 100% formic acid, and the method has application to transmembrane peptides formed from digests of integral membrane proteins.

Mass spectrometric analysis of integral membrane proteins: application to complete mapping of bacteriorhodopsins and rhodopsin.

Integral membrane proteins have not been readily amenable to the general methods developed for mass spectrometric (or internal Edman degradation) analysis of soluble proteins. We present here a sample preparation method and high performance liquid chromatography (HPLC) separation system which permits online HPLC-electrospray ionization mass spectrometry (ESI-MS) and -tandem mass spectrometry (MS/MS) analysis of cyanogen bromide cleavage fragments of integral membrane proteins. This method has been applied to wild type (WT) bacteriorhodopsin (bR), cysteine containing mutants of bR, and the prototypical G-protein coupled receptor, rhodopsin (Rh). In the described method, the protein is reduced and the cysteine residues pyridylethylated prior to separating the protein from the membrane. Following delipidation, the pyridylethylated protein is cleaved with cyanogen bromide. The cleavage fragments are separated by reversed phase HPLC using an isopropanol/acetonitrile/aqueous TFA solvent system and the effluent peptides analyzed online with a Finnigan LCQ Ion Trap Mass Spectrometer. With the exception of single amino acid fragments and the glycosylated fragment of Rh, which is observable by matrix assisted laser desorption ionization (MALDI)-MS, this system permits analysis of the entire protein in a single HPLC run. This methodology will enable pursuit of chemical modification and crosslinking studies designed to probe the three dimensional structures and functional conformational changes in these proteins. The approach should also be generally applicable to analysis of other integral membrane proteins.

Analysis of hydrophobic proteins and peptides by electrospray ionization mass spectrometry.

During the last decade mass spectrometry has become an essential tool for the analysis of peptides and proteins. Electrospray ionization mass spectrometry (ESIMS) is one of several recently developed techniques for the determination of accurate molecular masses of proteins, peptides, and other biopolymers up to > 100 kDa. Up to the present, analyses have been performed mainly on biopolymers that are soluble in aqueous solutions. Mass spectrometric analyses of very hydrophobic species, such as membrane proteins, have seldom been reported in the literature. This is mainly due to the incompatibility between most mass spectrometric techniques and detergents and/or salts which are required to retain such proteins in solution. Hydrophobic proteins (for example, bacterioopsin) and peptides are in general not soluble in the solutions (methanol/water or acetonitrile/water) typically used for ESIMS, and most detergents and chaotropes interfere with the analysis. We have developed sample handling protocols and solvent systems that are compatible with instrumental requirements and also are capable of retaining very hydrophobic peptides and proteins in solution. Chloroform/methanol/water mixtures were found to work well with, e.g., bacterioopsin, and also to be compatible with samples dissolved in hexafluoroisopropanol and 70-95% formic acid.

Topography of surface-exposed amino acids in the membrane protein bacteriorhodopsin determined by proteolysis and micro-sequencing.

The topography of membrane-surface-exposed amino acids in the light-driven proton pump bacteriorhodopsin (BR) was studied. By limited proteolysis of purple membrane with papain or proteinase K, domains were cleaved, separated by SDS-PAGE, and electroblotted onto polyvinylidene difluoride (PVDF) membranes. Fragments transferred were sequenced in a gas-phase sequencer. Papain cleavage sites at Gly-65, Gly-72, and Gly-231, previously only deduced from the apparent molecular weight of the digestion fragments, could be confirmed by N-terminal micro-sequencing. By proteinase K, cleavage occurred at Gln-3, Phe-71, Gly-72, Tyr-131, Tyr-133, and Ser-226, i.e., in regions previously suggested to be surface-exposed. Additionally, proteinase-K cleavage sites at Thr-121 and Leu-127 were identified, which are sites predicted to be in the alpha-helical membrane-spanning segment D. Our results, especially that the amino acids Gly-122 to Tyr-133 are protruding into the aqueous environment, place new constraints on the amino-acid folding of BR across the purple membrane. The validity of theoretical prediction methods of the secondary structure and polypeptide folding for membrane proteins is challenged. The results on BR show that micro-sequencing of peptides separated by SDS-PAGE and blotted to PVDF can be successfully applied to the study of membrane proteins.

Transient proton inflows during illumination of anaerobic Halobacterium halobium cells.

In Halobacterium halobium strain R1 containing both bacteriorhodopsin (bR) and halorhodopsin (hR), the light-driven proton uptake has been experimentally resolved into three transient inflows which are superimposed on the larger proton outflow. Under anaerobic conditions the early proton uptake consists of two components: (i) an inflow which can be blocked using the ATPase inhibitor, Dio-9, and (ii) an inflow which can be abolished by low concentrations (less than 125 nM) of triphenyltin chloride (TPT) with no inhibition of ATP synthesis. At pH 6 these two inflows are approximately equal in magnitude and duration. Measurements of buffering capacity and internal pH indicate that Dio-9 does not alter the passive proton-hydroxyl permeability of the cell membrane and that TPT at these low concentrations slightly decreases it. At later times of illumination (iii) another transient light-driven proton inflow occurs. This inflow is most evident during the first illumination after cells have been stored for extended times in the dark. The internal potassium concentration is not changed by storage, but apparently sodium is taken up, and we attribute the third inflow to sodium extrusion in exchange for protons. These results demonstrate the existence of three distinct triggered secondary proton inflows through the cell membrane. The proton inflow, which can be inhibited by Dio-9, correlates with proton-dependent ATP synthesis. The second inflow, which disappears in the presence of low TPT concentrations, is a passive proton uptake through an otherwise unidentified channel in response to electrogenic chloride pumping by bacteriorhodopsin and/or halorhodopsin. The third system correlates with the Na+/H+ antiporter function that has been demonstrated in H. halobium cell envelope vesicles. In contrast to observations on hR-containing vesicles, which can develop substantial Cl- gradients, the electroneutral OH-/Cl- exchange function can be demonstrated in intact cells only at TPT concentrations greater than 500 nM.

Biochemical characterization of halorhodopsin in native membranes.

Procedures are described for selectively radiolabeling the protein moiety (haloopsin) or the chromophoric prosthestic group (retinal) of the light-driven chloride pump halorhodopsin in intact cells of Halobacterium halobium. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autofluorography, two retinal-binding polypeptides are observed to band near the known molecular weight of the halorhodopsin chromophoric polypeptide (25,000). Synthesis of one of these polypeptides is controlled by retinal and is sufficient for generation of complete halorhodopsin function. The other is constitutively produced by the cells and differs chemically from the haloopsin protein as indicated by differences in their V8 protease digestion patterns. V8 protease cleavage of haloopsin in its native membrane is compared with that of the protein in denaturing and nondenaturing detergents. Protease cleavage sites available in the denatured haloopsin molecule are hidden in its native membrane-integrated conformation and in nondenaturing detergent micelles. Treatment with a variety of proteases indicates susceptibility of a short terminal region of the haloopsin chain in its native conformation.

Box-shaped halophilic bacteria.

Three morphologically similar strains of halophilic, box-shaped procaryotes have been isolated from brines collected in the Sinai, Baja California (Mexico), and southern California (United States). Although the isolates in their morphology resemble Walsby's square bacteria, which are a dominant morphological type in the Red Sea and Baja California brines, they are probably not identical to them. The cells show the general characteristics of extreme halophiles and archaebacteria. They contain pigments similar to bacteriorhodopsin which apparently mediate light-driven ion translocation and photophosphorylation.