High Protein Density Research Articles

On the role of membrane anisotropy and BAR proteins in the stability of tubular membrane structures

Recent studies have demonstrated that actin filaments are not crucial for the short-term stability of tubular membrane protrusions originating from the cell surface. It has also been demonstrated that prominin nanodomains and curvature inducing I-BAR proteins could account for the stability of the membrane protrusion. Here we constructed an axisymmetric model of a membrane protrusion that excludes actin filaments in order to investigate the contributions of prominin nanodomains (rafts) and I-BAR proteins to the membrane protrusion stability. It was demonstrated that prominin nanodomains and I-BAR proteins can stabilize the membrane protrusion only over a specific range of spontaneous curvature. On the other hand, high spontaneous curvature and/or high density of I-BAR proteins could lead to system instability and to non-uniform contraction in the radial direction of the membrane protrusion. In agreement with previous studies, it was also shown that the isotropic bending energy of lipids is not sufficient to explain the stability of the observed tubular membrane protrusion without actin filaments.

Molecular Convergence of Bacterial and Eukaryotic Surface Order

The conservation of fluidity is a theme common to all cell membranes. In this study, an analysis of lipid packing was conducted via C-laurdan spectroscopy of cell surface membranes prepared from representative species of Bacteria and Eukarya. We found that despite their radical differences in composition (namely the presence and absence of membrane-rigidifying sterol) the membrane order of all taxa converges on a remarkably similar level. To understand how this similarity is constructed, we reconstituted membranes with either bacterial or eukaryotic components. We found that transmembrane segments of proteins have an important role in buffering lipid-mediated packing. This buffering ensures that sterol-free and sterol-containing membranes exhibit similar barrier properties.

Optimized Purification of a Heterodimeric ABC Transporter in a Highly Stable Form Amenable to 2-D Crystallization

Optimized protocols for achieving high-yield expression, purification and reconstitution of membrane proteins are required to study their structure and function. We previously reported high-level expression in Escherichia coli of active BmrC and BmrD proteins from Bacillus subtilis, previously named YheI and YheH. These proteins are half-transporters which belong to the ABC (ATP-Binding Cassette) superfamily and associate in vivo to form a functional transporter able to efflux drugs. In this report, high-yield purification and functional reconstitution were achieved for the heterodimer BmrC/BmrD. In contrast to other detergents more efficient for solubilizing the transporter, dodecyl-ß-D-maltoside (DDM) maintained it in a drug-sensitive and vanadate-sensitive ATPase-competent state after purification by affinity chromatography. High amounts of pure proteins were obtained which were shown either by analytical ultracentrifugation or gel filtration to form a monodisperse heterodimer in solution, which was notably stable for more than one month at 4°C. Functional reconstitution using different lipid compositions induced an 8-fold increase of the ATPase activity (k cat∼5 s−1). We further validated that the quality of the purified BmrC/BmrD heterodimer is suitable for structural analyses, as its reconstitution at high protein densities led to the formation of 2-D crystals. Electron microscopy of negatively stained crystals allowed the calculation of a projection map at 20 Å resolution revealing that BmrC/BmrD might assemble into oligomers in a lipidic environment.

Multilamellar spherical particles as potential sources of excessive light scattering in human age-related nuclear cataracts

The goal of this project was to determine the relative refractive index (RI) of the interior of multilamellar bodies (MLBs) compared to the adjacent cytoplasm within human nuclear fiber cells. MLBs have been characterized previously as 1-4 μm diameter spherical particles covered by multiple lipid bilayers surrounding a cytoplasmic core of variable density. Age-related nuclear cataracts have more MLBs than transparent donor lenses and were predicted to have high forward scattering according to Mie scattering theory, assuming different RIs for the MLB and cytoplasm. In this study quantitative values of relative RI were determined from specific MLBs in electron micrographs of thin sections and used to calculate new Mie scattering plots. Fresh lenses were Vibratome sectioned, immersion fixed and en bloc stained with osmium tetroxide and uranyl acetate, or uranyl acetate alone, prior to dehydration and embedding in epoxy or acrylic resins. Thin sections 70 nm thick were cut on a diamond knife and imaged without grid stains at 60 kV using a CCD camera on a transmission electron microscope (TEM). Integrated intensities in digital electron micrographs were related directly to protein density, which is linearly related to RI for a given substance. The RI of the MLB interior was calculated assuming an RI value of 1.42 for the cytoplasm from the literature. Calculated RI values for MLBs ranged from 1.35 to 1.53. Thus, some MLBs appeared to have interior protein densities similar to or less than the adjacent cytoplasm whereas others had significantly higher densities. The higher density MLBs occurred preferentially in older and more advanced cataracts suggesting a maturation process. The bilayer coats were more often observed in MLBs from transparent donors and early stage cataracts indicating that bilayer loss was part of the MLB maturation, producing large low-density spaces around dense MLB cores. These spaces were frequently observed in advanced cataracts from India as large low-density crescents and annular rings. Predicted scattering from Mie plots using particles with dense cores and low-density rims was higher than reported previously, although the most important factor was the relative RI, not the MLB coat or lack thereof. In conclusion, the measurements confirm the high protein density and RI of some MLB interiors compared to adjacent cytoplasm. This high RI ratio used in the Mie calculations suggests that for 2000 MLBs/mm³, about half that reported for early stage nuclear cataracts from the US, the forward scattering could be more than 30% of the incident light. Therefore, the extent of forward scattering and its influence on macular visual acuity could be important components of ophthalmological evaluations of cataract patients.

Efficient Light Harvesting by Photosystem II Requires an Optimized Protein Packing Density in Grana Thylakoids

A recently developed technique for dilution of the naturally high protein packing density in isolated grana membranes was applied to study the dependence of the light harvesting efficiency of photosystem (PS) II on macromolecular crowding. Slight dilution of the protein packing from 80% area fraction to the value found in intact grana thylakoids (70%) leads to an improved functionality of PSII (increased antenna size, enhanced connectivity between reaction centers). Further dilution induces a functional disconnection of light-harvesting complex (LHC) II from PSII. It is concluded that efficient light harvesting by PSII requires an optimal protein packing density in grana membranes that is close to 70%. We hypothesize that the decreased efficiency in overcrowded isolated grana thylakoids is caused by excited state quenching in LHCII, which has previously been correlated with neoxanthin distortion. Resonance Raman spectroscopy confirms this increase in neoxanthin distortion in overcrowded grana as compared with intact thylakoids. Furthermore, analysis of the changes in the antenna size in highly diluted membranes indicates a lipid-induced dissociation of up to two trimeric LHCII from PSII, leaving one trimer connected. This observation supports a hierarchy of LHCII-binding sites on PSII.

Abstract 3999: Human proteome arrays for auto-antibody identification in clinical cancer studies

Abstract Early detection, identification and classification of cancers still present considerable challenges. Innovations in several areas of research have provided novel and powerful approaches with which to tackle these challenges. Autoantibodies to tumor antigens have been found in cancer patients. Such autoantibodies are potentially ideal diagnostic biomarkers, as they are present in serum, are stable, and can be measured using established serological techniques. In order to discover new autoantibody responses in cancer patients, we have developed an approach based on the generation of protein arrays for systematic identification of auto-reactive antibodies. Our approach makes use of several key technologies: sequence-curated clone libraries, in-vitro protein expression, and a sensitive detection technology based on a proprietary combination of electrochemiluminescence detection and patterned arrays (Multi-Array® Technology, Meso Scale Diagnostics). Using this combination of technologies we plan to generate immobilized arrays of >1,000 proteins, with a high density of proteins per well in a 96-well microtiter plate format. The array of human proteins is selected from our collection of 60,000 mammalian clones (characterized by sequencing). The protein arrays will be used to screen cancer patient sera and controls. During the initial development of this platform we optimized the protocols for in-vitro protein expression and labeling, specifically for immobilization on our Multi-Array plates. With this approach we have demonstrated detection of antigen-specific antibodies down to a concentration of 0.1 ng/mL. We have also validated this approach in a model system, with the detection of auto-reactive antibodies in the sera from 29 patients with a range of auto-immune diseases (Sjogren's syndrome, mixed connective tissue disease, scleroderma, systemic lupus erythematosus, and primary bilary cirrhosis), using 9 cloned autoimmune antigens. In parallel, we also optimized approaches for spotting in-vitro expressed proteins in high density spot/well arrays. In these studies we evaluated a selection of electrode surfaces and protein coupling methods, in combination with various spotting methods, and demonstrated serological measurements in a high density spot/well array. This effort was supported with funding from the NCI #1R44CA130391-1A1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3999.

Quantification of molecules in 1H-NMR metabolomics with formate as a concentration standard

Quantitative analysis of metabolites is important in (1)H-nuclear magnetic resonance (NMR)-based metabolomics of plasma. Human plasma contains a high density of proteins which heavily adsorb the commonly-used standard compound of sodium 3-(trimethylsilyl) propionate 2, 2, 3, 3-d(4) (TSP). We have evaluated calcium formate as an alternative standard in 1D single-pulse (1)H-NMR spectra to quantify plasma metabolites. Formate did not interact with either plasma metabolites or proteins under adequate conditions. Linear relations between the signal intensities and the added formate have been demonstrated in (1)H spectra. The quantifications of glucose and creatinine by this method have shown good accordance with biochemical analysis. Calcium formate is applicable as a concentration standard to NMR metabolomics of plasma.

Functional Consequences of Lipid-Mediated Interaction Between Rhodopsin Molecules

In the retina, rhodopsin is densely packed in a rod outer segment disk membrane rich in phospholipids with PC and PE headgroups. Increasing rhodopsin packing density in model PC membranes has been shown to alter metarhodopsin-II (MII) formation. This observation is deemed to be the consequence of rhodopsin association promoted by non-specific properties of the membrane. Here, we studied the effect of rhodopsin packing density on MII formation in membranes characterized by different intrinsic curvature and different interfacial hydrogen bonding propensity. Rhodopsin was reconstituted into a series of POPC bilayers doped with DOPC, di-and mono-methylated DOPE or DOPE at rhodopsin/lipid ratios ranging from 1:250 to 1:70. The level of rhodopsin activation and rate of MII formation were determined by steady-state and time-resolved UV/vis spectroscopy. In PC membranes, lower rhodopsin concentrations shift the MI/MII equilibrium towards MII and result in a faster rate of MII formation, in agreement with previous findings. On the contrary, in membranes rich in lipids with PE headgroups, the MII concentration is independent of rhodopsin packing density and rates of MII formation, while reduced, show only a slight dependence on rhodopsin crowding. In addition, at low or high protein density, the amount of MII formed depends to a larger extent on the ability of the annular PE headgroups to establish hydrogen bonds with the MII state than on changes in membrane curvature elasticity. These results show clearly that MII formation and interaction between rhodopsin molecules depend strongly on interactions between annular lipids and rhodopsin, highlighting the fundamental role of the first layer of lipids surrounding the protein.

Functional Reconstitution of Membrane Proteins Monitored by Isothermal Titration Calorimetry

Membrane protein reconstitution denotes the transfer of a purified (but usually inactive) membrane protein from detergent micelles into lipid bilayers. The aim is to restore the native protein fold and function in a well-defined membrane environment. The reconstitution yield critically depends on a wide range of parameters, including temperature, pH, ionic strength, as well as the type and concentration of detergent, lipid, protein, and additives. Moreover, it is of paramount importance to initiate the reconstitution process from a suitable lipid-to-detergent ratio. Unfortunately, however, assessing the success of a reconstitution experiment has thus far been limited to a trial-and-error approach, which has substantially slowed progress in the field.To address this problem, we have established high-sensitivity isothermal titration calorimetry (ITC) as a powerful tool for monitoring the reconstitution of membrane proteins into lipid vesicles. Using ITC, the complex changes in the physical state of a protein/lipid/detergent mixture during reconstitution can be followed in a non-invasive and fully automated manner. Here we exemplify this approach for the prokaryotic potassium channel KcsA, which we first purified in detergent micelles and then reconstituted into stable proteoliposomes at very high protein densities. Electrophysiological experiments performed in planar lipid membranes confirmed that KcsA regained its functional activity upon reconstitution.

Applications of Polymer Brushes in Protein Analysis and Purification

This review examines the application of polymer brush-modified flat surfaces, membranes, and beads for protein immobilization and isolation. Modification of porous substrates with brushes yields membranes that selectively bind tagged proteins to give 99% pure protein at capacities as high as 100 mg of protein per cubic centimeter of membrane. Moreover, enrichment of phosphopeptides on brush-modified matrix-assisted laser desorption/ionization (MALDI) plates allows detection and characterization of femtomole levels of phosphopeptides by MALDI mass spectrometry. Because swollen hydrophilic brushes can resist nonspecific protein adsorption while immobilizing a high density of proteins, they are attractive as substrates for protein microarrays. This review highlights the advantages of polymer brush-modified surfaces over self-assembled monolayers and identifies some research needs in this area.

Investigation Of Electrogenic Partial Reactions In Detergent-solubilized Na,K-ATPase

Electrochromic styryl dyes are in use now for almost two decades to detect ion movements in various P-type ATPases. The extremely hydrophobic dye molecules have a high partition coefficient in favor of the hydrophobic core of lipid phase of membrane preparations. Fluorescence changes are obtained by modification of local electric fields in the membrane dielectric produced by ions bound to or released from binding sites of the ion pumps. To obtain significant signals a prerequisite is a high density of active proteins in the membranes. This limitation could be overcome by solubilization of the Na,K-ATPase in mixed micelles of protein/lipid/detergent obtained by incubation of microsomal membranes from rabbit kidney with dodecyl maltoside. In this preparation the specific enzyme activity of the Na,K-ATPase was reduced compared to that in native membranes. This effect was assigned to the highly affected lipid environment of the singularized proteins which may be depleted of specific lipid components and the content of dodecyl maltoside. The fluorescence changes which were detected with the styryl dye RH421 showed smaller amplitudes than in the case of purified membrane preparations, however, the responses on Na+ binding, Na+ release upon enzyme phosphorylation and conformation transition, and subsequent K+ binding in the E2P conformation were clearly detectable. Na+ binding affinity and its dependence on Mg2+ concentration and buffer pH, as well as K+ affinity were comparable to the results obtained with native preparations. The transfer of the method to solubilized ion pumps will allow investigations of mutants isolated from cell in which they were expressed in low density. In addition, this approach will possibly extend method also to the investigation of ion channel molecules by electrochromic styryl dyes.

A new role of the rDNA and nucleolus in the nucleus—rDNA instability maintains genome integrity

The nucleolus is a region of the nucleus with high protein density and it acts as a ribosome factory. The nucleolus contains a distinct region of the genome, the ribosomal RNA gene repeats (rDNA) that supply ribosomal RNA (rRNA) molecules. The rDNA is the most-abundant gene and occupies a large part of the genome, for example, there are thousands of rDNA copies in the genomes of plant cells. Therefore, it is natural to suppose that the condition of the rDNA, such as its stability, might affect cellular functions. Here I would like to propose a new model regarding the roles of the rDNA and nucleolus. The key point of this model is that they act to preserve genome stability and trigger aging.

Significance of molecular crowding in grana membranes of higher plants for light harvesting by photosystem II

Significance of molecular crowding in grana thylakoids of higher plants on photosystem II function was studied by 'titrating' the naturally high protein density by fusing unilamellar liposomes of the native lipid mixture with isolated grana membranes (BBY). The incorporation of lipids was monitored by equilibrium density gradient centrifugation and two-dimensional thin layer chromatography. The excitonic coupling between light-harvesting (LHC) II and photosystem (PS) II was analysed by chlorophyll a fluorescence spectroscopy. The fluorescence parameters Fv/Fm and Fo clearly depend on the protein density indicating the importance of molecular crowding for establishing an efficient excitonic protein network. In addition the strong dependency of Fo on the protein density reveals weak interactions between LHCII complexes which could be important for dynamic adjustment of the photosynthetic apparatus in higher plants.

GLUTAMATE RECEPTOR PLASTICITY AT EXCITATORY SYNAPSES IN THE BRAIN

1. Synapse plasticity, defined as an activity dependent change in the strength of synapses, was first described in 1973 and, since those seminal experiments were reported, the field of synapse plasticity has expanded into one of the most widely studied areas in neuroscience. 2. Significant effort has been focused on determining the expression mechanisms of the changes in synapse strength. The present review will focus on the changes in the post-synaptic expression of glutamate receptors that have been shown to occur during the expression of synapse plasticity. 3. Biochemical studies of excitatory synapses in the central nervous system have revealed a high density of proteins concentrated at dendritic spines. These proteins appear to play critical roles in synaptic structure, plasticity and in trafficking receptors to synapses. 4. There is growing evidence that synapse plasticity could be the cellular basis of certain forms of learning and memory. Determining the behavioural correlates of this fundamental synaptic process will continue to be addressed in current and future research.

Macromolecular crowding and its influence on possible reaction mechanisms in photosynthetic electron flow

The diffusion of plastoquinol and its binding to the Qo site of the cyt bf complex in the course of photosynthetic electron transport was studied by following the sigmoidal flash-induced re-reduction kinetics of P700 after previous oxidation of the intersystem electron carriers. The data resulting from these experiments were matched with a simulation of electron transport using Monte Carlo techniques. The simulation was able to account for the experimental observations. Two different extreme cases of reaction mechanism at the Qo site were compared: a diffusion limited collisional mechanism and a non-diffusion limited tight binding mechanism. Assuming a tight binding mechanism led to best matches due to the high protein density in thylakoids. The varied parameters resulted in values well within the range of published data. The results emphasise the importance of structural characteristics of thylakoids in models of electron transport.

A Bacterial Arginine-Agmatine Exchange Transporter Involved in Extreme Acid Resistance

The arginine-dependent extreme acid resistance response of Escherichia coli operates by decarboxylating arginine. AdiC, a membrane antiporter, catalyzes arginine influx coupled to efflux of the decarboxylation product agmatine, effectively exporting a proton in each turnover. Using the adiC coding sequence under control of a tetracycline promoter in an E. coli vector, we expressed and purified the transport-protein with a yield of approximately 10 mg/liter bacterial culture. Glutaraldehyde cross-linking experiments indicate that the protein is a homodimer in detergent micelles and lipid membranes. Purified AdiC reconstituted into liposomes exchanges arginine and agmatine in a strictly coupled, electrogenic fashion. Kinetic analysis yields K(m) approximately 80 microm for Arg, in the same range as its dissociation constant determined by isothermal titration calorimetry.

High-Resolution AFM of Membrane Proteins Directly Incorporated at High Density in Planar Lipid Bilayer

The heterologous expression and purification of membrane proteins represent major limitations for their functional and structural analysis. Here we describe a new method of incorporation of transmembrane proteins in planar lipid bilayer starting from 1 pmol of solubilized proteins. The principle relies on the direct incorporation of solubilized proteins into a preformed planar lipid bilayer destabilized by dodecyl- β-maltoside or dodecyl- β-thiomaltoside, two detergents widely used in membrane biochemistry. Successful incorporations are reported at 20°C and at 4°C with three bacterial photosynthetic multi-subunit membrane proteins. Height measurements by atomic force microscopy (AFM) of the extramembraneous domains protruding from the bilayer demonstrate that proteins are unidirectionally incorporated within the lipid bilayer through their more hydrophobic domains. Proteins are incorporated at high density into the bilayer and on incubation diffuse and segregate into protein close-packing areas. The high protein density allows high-resolution AFM topographs to be recorded and protein subunits organization delineated. This approach provides an alternative experimental platform to the classical methods of two-dimensional crystallization of membrane proteins for the structural analysis by AFM. Furthermore, the versatility and simplicity of the method are important intrinsic properties for the conception of biosensors and nanobiomaterials involving membrane proteins.

SNARE-Mediated Lipid Mixing Depends on the Physical State of the Vesicles

Reconstitution experiments have suggested that N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins constitute a minimal membrane fusion machinery but have yielded contradictory results, and it is unclear whether the mechanism of membrane merger is related to the stalk mechanism that underlies physiological membrane fusion. Here we show that reconstitution of solubilized neuronal SNAREs into preformed 100 nm liposomes (direct method) yields proteoliposomes with more homogeneous sizes and protein densities than the standard reconstitution method involving detergent cosolubilization of proteins and lipids. Standard reconstitutions yield slow but efficient lipid mixing at high protein densities and variable amounts of lipid mixing at moderate protein densities. However, the larger, more homogenous proteoliposomes prepared by the direct method yield almost no lipid mixing at moderate protein densities. These results suggest that the lipid mixing observed for standard reconstitutions is dominated by the physical state of the membrane, perhaps due to populations of small vesicles (or micelles) with high protein densities and curvature stress created upon reconstitution. Accordingly, changing membrane spontaneous curvature by adding lysophospholipids inhibits the lipid mixing observed for standard reconstitutions. Our data indicate that the lipid mixing caused by high SNARE densities and/or curvature stress occurs by a stalk mechanism resembling the mechanism of fusion between biological membranes, but the neuronal SNAREs are largely unable to induce lipid mixing at physiological protein densities and limited curvature stress.

Simulation of Diffusion Time of Small Molecules in Protein Crystals

A simple model for evaluation of diffusion times of small molecule into protein crystals has been developed, which takes into account the physical and chemical properties both of protein crystal and the diffusing molecules. The model also includes consideration of binding and the binding affinity of a ligand to the protein. The model has been validated by simulation of experimental set-ups of several examples found in the literature. These experiments cover a wide range of situations: from small to relatively large diffusing molecules, crystals having low, medium, or high protein density, and different size. The reproduced experiments include ligand exchange in protein crystals by soaking techniques. Despite the simplifying assumptions of the model, theoretical and experimental data are in agreement with available data, with experimental diffusion times ranging from a few seconds to several hours. The method has been used successfully for planning intermediate cryotrapping experiments in maltodextrin phosphorylase crystals.

Transversal and Lateral Exciton Energy Transfer in Grana Thylakoids of Spinach

The excitation energy transfer between photosystem (PS) II complexes was studied in isolated grana disks and thylakoids using chlorophyll a fluorescence induction measurements in the presence of DCMU under stacked and destacked conditions. Destacking of grana was achieved using a sonication protocol in a buffer without MgCl(2). The degree of stacking was controlled and quantified by atomic force microscopy and by the concomitant absorption changes. As expected from the literature, intact thylakoids showed a strong dependency of the connectivity of PSII centers, the F(m)/F(o) ratio as well as the fraction of PSIIbeta centers on the MgCl(2) concentration. In contrast, these parameters did not change in isolated grana disks. In particular, the connectivity remained constantly high irrespective of the degree of destacking. These differences were explained by the high protein density in grana disks, which hinders separation and mixing of proteins sufficiently to change energy transfer properties. Due to the occurrence of stroma lamella in intact thylakoids, intermixing of PSII and PSI is possible and allows for changes in F(m)/F(o) ratio as is the separation of LHCII from PSII, thus leading to an increase in the fraction of PSIIbeta. Even if mixing and separation of proteins are impaired in isolated grana disks, destacking should lead to a decrease in connectivity if transversal excitation energy transfer between two opposite membranes is significant. Because the connectivity is constant over all degrees of destacking employed, we conclude that the energy transfer in granas is mainly lateral.