Structural analysis of membrane-bound retrovirus capsid proteins.

We have developed a system for analysis of histidine-tagged (His-tagged) retrovirus core (Gag) proteins, assembled in vitro on lipid monolayers consisting of egg phosphatidylcholine (PC) plus the novel lipid DHGN. DHGN was shown to chelate nickel by atomic absorption spectrometry, and DHGN-containing monolayers specifically bound gold conjugates of His-tagged proteins. Using PC + DHGN monolayers, we examined membrane-bound arrays of an N-terminal His-tagged Moloney murine leukemia virus (M-MuLV) capsid (CA) protein, His-MoCA, and in vivo studies suggest that in vitro-derived His-MoCA arrays reflect some of the Gag protein interactions which occur in assembling virus particles. The His-MoCA proteins formed extensive two-dimensional (2D) protein crystals, with reflections out to 9.5 A resolution. The image-analyzed 2D projection of His-MoCA arrays revealed a distinct cage-like network. The asymmetry of the individual building blocks of the network led to the formation of two types of hexamer rings, surrounding protein-free cage holes. These results predict that Gag hexamers constitute a retrovirus core substructure, and that cage hole sizes define an exclusion limit for entry of retrovirus envelope proteins, or other plasma membrane proteins, into virus particles. We believe that the 2D crystallization method will permit the detailed analysis of retroviral Gag proteins and other His-tagged proteins.     

Two-dimensional crystallization of streptavidin by nonspecific binding to a surface film: study with a scanning electron microscope.

A two-dimensional (2D) crystal of streptavidin has been obtained by a nonspecific binding method. The protein molecules were bound and formed a dense packing on the film of poly(1-benzyl-L-histidine) spread at the surface of protein solution. The surface film was moderately heated to stimulate crystallization of bound streptavidin. A potential of this method for obtaining 2D crystals of soluble proteins is demonstrated. The present 2D crystal structure of streptavidin resembles that previously obtained by specific binding to biotinylated lipid. We show in addition that the 2D array of protein with usual size approximately 50 A can be imaged using a high resolution scanning electron microscope (HR-SEM) and subject to structural analysis at low resolution. Various limitations in HR-SEM degrade considerably the image quality. However, the usability of a bulk plate as specimen support would make HR-SEM a convenient tool, when such a substrate must be considered in application of protein arrays, and if an intrinsic low resolution is acceptable.     

A maximum likelihood approach to two-dimensional crystals

Maximum likelihood (ML) processing of transmission electron microscopy images of protein particles can produce reconstructions of superior resolution due to a reduced reference bias. We have investigated a ML processing approach to images centered on the unit cells of two-dimensional (2D) crystal images. The implemented software makes use of the predictive lattice node tracking in the MRC software, which is used to window particle stacks. These are then noise-whitened and subjected to ML processing. Resulting ML maps are translated into amplitudes and phases for further processing within the 2dx software package. Compared with ML processing for randomly oriented single particles, the required computational costs are greatly reduced as the 2D crystals restrict the parameter search space. The software was applied to images of negatively stained or frozen hydrated 2D crystals of different crystal order. We find that the ML algorithm is not free from reference bias, even though its sensitivity to noise correlation is lower than for pure cross-correlation alignment. Compared with crystallographic processing, the newly developed software yields better resolution for 2D crystal images of lower crystal quality, and it performs equally well for well-ordered crystal images.     

Application of multiplexed cysteine‐labeled complex protein sample for 2D electrophoretic gel alignment

The analysis of cellular subproteomes by 2DE is hampered by the difficulty of aligning gel images from samples that have very different protein composition. Here, we present a sensitive and cost‐effective fluorescent labeling method for analyzing protein samples that is not dependent on their composition. The alignment is guided by inclusion of a complex mixture of proteins that is co‐run with the sample. Maleimide‐conjugated fluorescent dyes Dy‐560 and Dy‐635 are used to label the cysteine residues of the sample of interest and the alignment standard, respectively. The two differently labeled mixtures are then combined and separated on a 2D gel and, after selective fluorescence detection, an unsupervised image registration process is used to align the protein patters. In a pilot study, this protocol significantly improved the accuracy of alignment of nuclear proteins with total cellular proteins.     

3D multicolor super-resolution imaging offers improved accuracy in neuron tracing

The connectivity among neurons holds the key to understanding brain function. Mapping neural connectivity in brain circuits requires imaging techniques with high spatial resolution to facilitate neuron tracing and high molecular specificity to mark different cellular and molecular populations. Here, we tested a three-dimensional (3D), multicolor super-resolution imaging method, stochastic optical reconstruction microscopy (STORM), for tracing neural connectivity using cultured hippocampal neurons obtained from wild-type neonatal rat embryos as a model system. Using a membrane specific labeling approach that improves labeling density compared to cytoplasmic labeling, we imaged neural processes at 44 nm 2D and 116 nm 3D resolution as determined by considering both the localization precision of the fluorescent probes and the Nyquist criterion based on label density. Comparison with confocal images showed that, with the currently achieved resolution, we could distinguish and trace substantially more neuronal processes in the super-resolution images. The accuracy of tracing was further improved by using multicolor super-resolution imaging. The resolution obtained here was largely limited by the label density and not by the localization precision of the fluorescent probes. Therefore, higher image resolution, and thus higher tracing accuracy, can in principle be achieved by further improving the label density.     

Localization of c-di-GMP-Binding Protein with the Linear Terminal Complexes of Acetobacter xylinum

Specific labeling of a single row of cellulose-synthesizing complexes (terminal complexes, TC subunits, TCs, or TC arrays) in Acetobacter xylinum by antibodies raised against a 93-kDa protein (the cyclic dignanylic acid-binding protein) has been demonstrated by using the sodium dodecyl sulfate (SDS)-freeze-fracture labeling (FRL) technique. The antibodies to the 93-kDa protein specifically recognized the TC subunits on the protoplasmic fracture (PF) face of the outer membrane in A. xylinum; however, nonlabeled TCs were also observed. Two types of TC subunits (particles or pits) are observed on the PF face of the outer membrane: (i) immunogold-labeled TCs showing a line of depressions (pits) with an indistinct particle array and (ii) nonlabeled TC subunits with a distinct single row of particle arrays. The evidence indicates that the labeling patterns differ with respect to the presence or absence of certain TC subunits remaining attached to the replica after SDS treatment. This suggests the presence of at least two TC components, one in the outer membrane and the other in the cytoplasmic membrane. If the TC component in the outer membrane is preferentially fractured and remains attached to the ectoplasmic fracture face (or outer leaflet) of the outer membrane, subsequent replica formation reveals a pit or depression with positive antibody labeling on the PF face of the outer membrane. If the TC component in the outer membrane remains with the PF face (or inner leaflet) of the outer membrane, the innermost TC component is removed during SDS treatment and labeling does not occur. SDS-FRL of TCs in A. xylinum has enabled us to provide the first topological molecular analysis of component proteins in a cellulose-synthesizing TC structure in a prokaryotic organism.     

Characterization by electron microscopy of isolated particles and two-dimensional crystals of the CP47-D1-D2-cytochrome b-559 complex of photosystem II

A photosystem II complex containing the reaction center proteins D1 and D2, a 47-kDa chlorophyll-binding protein (CP47), and cytochrome b-559 was isolated with high yield, purity, and homogeneity; small but well-ordered two-dimensional crystals were prepared from the particles. The crystals and the isolated particles were analyzed by electron microscopy using negatively stained specimens. The information of 20 different digitized crystals was combined by alignment programs based on correlation methods to obtain a final average. The calculated diffraction pattern, with spots up to a resolution of 2.5 nm, and the optical diffraction pattern of a single crystal indicate that the plane group is p22121 (also called p2gg) and that the unit cell is rectangular with parameters of 23.5 x 16.0 nm, containing four stain-excluding monomers (two face-up and two face-down). In projection, the monomers have an asymmetrical shape with a length of 10 nm, a maximal width of 7.5 nm, and a height of 6 nm; their molecular mass is 175 +/- 40 kDa.     

Two-dimensional crystallization of the ryanodine receptor Ca2+ release channel on lipid membranes

The ryanodine receptor (RyR) is the largest known membrane protein with a total molecular mass of 2.3 x 10(3) kDa. Well ordered, two-dimensional (2D) crystals are an essential prerequisite to enable RyR structure determination by electron crystallography. Conventionally, the 2D crystallization of membrane proteins is based on a 'trial-and-error' strategy, which is both time-consuming and chance-directed. By adopting a new strategy that utilizes protein sequence information and predicted transmembrane topology, we successfully crystallized the RyR on positively charged lipid membranes. Image processing of negatively stained crystals reveals that they are well ordered, with diffraction spots of IQ < or = 4 extending to approximately 20 angstroms, the resolution attainable in negative stain. The RyR crystals obtained on the charged lipid membrane have characteristics consistent with 2D arrays that have been observed in native sarcoplasmic reticulum of muscle tissues. These crystals provide ideal materials to enable structural analysis of RyR by high-resolution electron crystallography. Moreover, the reconstituted native-like 2D array provides an ideal model system to gain structural insights into the mechanism of RyR-mediated Ca2+ signaling processes, in which the intrinsic ability of RyR oligomers to organize into a 2D array plays a crucial role.     

7A projection map of the S-layer protein sbpA obtained with trehalose-embedded monolayer crystals

Two-dimensional crystallization on lipid monolayers is a versatile tool to obtain structural information of proteins by electron microscopy. An inherent problem with this approach is to prepare samples in a way that preserves the crystalline order of the protein array and produces specimens that are sufficiently flat for high-resolution data collection at high tilt angles. As a test specimen to optimize the preparation of lipid monolayer crystals for electron microscopy imaging, we used the S-layer protein sbpA, a protein with potential for designing arrays of both biological and inorganic materials with engineered properties for a variety of nanotechnology applications. Sugar embedding is currently considered the best method to prepare two-dimensional crystals of membrane proteins reconstituted into lipid bilayers. We found that using a loop to transfer lipid monolayer crystals to an electron microscopy grid followed by embedding in trehalose and quick-freezing in liquid ethane also yielded the highest resolution images for sbpA lipid monolayer crystals. Using images of specimens prepared in this way we could calculate a projection map of sbpA at 7A resolution, one of the highest resolution projection structures obtained with lipid monolayer crystals to date.     

Two-dimensional crystals of apoferritin

A simple 2D crystallization method using unfolded protein film as a supporting film of crystals was described, which allows modification of protein surfaces by injecting chemical reagents into the subphase after the crystal formation. As an example, glutaraldehyde was used to cross-link adjacent proteins and then stabilize protein crystals. The second layer of other proteins can also be formed on the apoferritin array using cross-linkers.The array of apoferritin is not only beneficial for electron crystallography but also for practical applications. For example, apoferritin produces a mineral core with a size which can be adjusted by the size to the cavity (i.e. 6 nm). Fabrication of such a small size of well defined fine particles is currently not easy using physical or chemical procedures. Using apoferritin, however, it is easy to produce uniform fine particles. If the core is designed to add interesting properties such as magnetism it is possible to make the highest class of magnetic film with ferritin 2D crystals.Basic researches toward practical applications of 2D protein crystal is now under way in various fields. The well defined size and function of protein molecules will benefit to many applications. The function and crystalline order can be designed by site-directed mutagenesis with the development of protein engineering.     

Metabolic labeling of human primary retinal pigment epithelial cells for accurate comparative proteomics

Metabolic labeling was evaluated, using both 13C6-Arg and 13C6, 15N2-Lys amino acids, for a primary human retinal pigment epithelial cell (hRPE) culture prepared from an autopsy eye of an 81 year old donor. Satisfactory incorporation (>90%) was achieved with both stable isotope labeled amino acids after four passages (roughly 7 population doublings). The degree of incorporation was found to be efficient with both amino acids as well as in different proteins. The presence of 10% whole serum in the culture medium did not interfere with the incorporation of the exogenous stable isotope labeled amino acids. Metabolic labeling of these human primary retinal pigment epithelial cells was further tested to quantify protein ratios between proliferating and resting cells using a combination of 2-DG and MALDI-TOF-TOF/MS analysis. Using computational data processing and analysis, we obtained accurate protein ratio measurement for every single identified protein (156 proteins) in the 2-Dg array. Of these 156 proteins, 12 proteins were found significantly increased in dividing versus resting cells by at least a factor of 1.5 while 13 other proteins were found increased in resting versus dividing cells by at least the same fold. Most of these differentially expressed proteins are directly involved in cell proliferation, protein synthesis, and actin-remodeling and differentiation.     

Quantitative analysis of the yeast proteome by incorporation of isotopically labeled leucine

Quantitative comparison of protein expression levels in 2D gels is complicated by the variables associated with protein separation and mass spectrometric responses. Metabolic labeling allows cells from different experiments to be mixed prior to analysis. This approach has been reported for prokaryotic cells. Here, we demonstrate that metabolic labeling can also be successfully applied to the eukaryote Saccharormyces cerevisiae. Yeast leucine auxotrophs grown on synthetic complete media containing natural abundance Leu or D10-Leu were mixed prior to 2D gel separation and MALDI analysis of the digested proteins. D10-Leu labeling provided an effective internal calibrant for peptide MS analysis, and the number of Leu residues yielded an additional parameter for peptide identification at low mass resolution (1000). Metabolic incorporation of D10-Leu into yeast proteins was found to be quantitative since the intensities of the peptide peaks corresponded to those expected on the basis of the percent label in the media. Thus, D10-Leu labeling should provide reliable data for comparing proteomes both quantitatively and qualitatively from wild-type and nonessential-gene-null-mutant strains of S. cerevisiae. Given the central role played by yeast in our understanding of eukaryotic gene and protein expression, it is anticipated that the quantitative expressional proteomic method outlined here will have widespread applications.     

Multiphase method for automatic alignment of transmission electron microscope images using markers

In order to successfully perform the 3D reconstruction in electron tomography, transmission electron microscope images must be accurately aligned or registered. So far, the problem is solved by either manually showing the corresponding fiducial markers from the set of images or automatically using simple correlation between the images on several rotations and scales. The present solutions, however, share the problem of being inefficient and/or inaccurate. We therefore propose a method in which the registration is automated using conventional colloidal gold particles as reference markers between images. We approach the problem from the computer vision viewpoint; hence, the alignment problem is divided into several subproblems: (1) finding initial matches from successive images, (2) estimating the epipolar geometry between consecutive images, (3) finding and localizing the gold particles with subpixel accuracy in each image, (4) predicting the probable matching gold particles using the epipolar constraint and its uncertainty, (5) matching and tracking the gold beads through the tilt series, and (6) optimizing the transformation parameters for the whole image set. The results show not only the reliability of the suggested method but also a high level of accuracy in alignment, since practically all the visible gold markers can be used.     

7 Å projection map of the S-layer protein sbpA obtained with trehalose-embedded monolayer crystals

Two-dimensional crystallization on lipid monolayers is a versatile tool to obtain structural information of proteins by electron microscopy. An inherent problem with this approach is to prepare samples in a way that preserves the crystalline order of the protein array and produces specimens that are sufficiently flat for high-resolution data collection at high tilt angles. As a test specimen to optimize the preparation of lipid monolayer crystals for electron microscopy imaging, we used the S-layer protein sbpA, a protein with potential for designing arrays of both biological and inorganic materials with engineered properties for a variety of nanotechnology applications. Sugar embedding is currently considered the best method to prepare two-dimensional crystals of membrane proteins reconstituted into lipid bilayers. We found that using a loop to transfer lipid monolayer crystals to an electron microscopy grid followed by embedding in trehalose and quick-freezing in liquid ethane also yielded the highest resolution images for sbpA lipid monolayer crystals. Using images of specimens prepared in this way we could calculate a projection map of sbpA at 7 Å resolution, one of the highest resolution projection structures obtained with lipid monolayer crystals to date.     

Classification and Reconstruction of a Heterogeneous Set of Electron Microscopic Images: A Case Study of GroEL–Substrate Complexes

Image analysis methods were used to separate images of a large macromolecular complex, the chaperonin GroEL, in a preparation in which it is partially liganded to a nonnative protein substrate, glutamine synthetase. The relatively small difference (6%) in size between the chaperonin in its free and complexed forms, and the absence of gross changes in overall conformation, made separation of the two types of particles challenging. Different approaches were evaluated and used for alignment and classification of images, both in two common projections and in three dimensions, yielding 2D averages and a 3D reconstruction. The results of 3D analysis describe the conformational changes effected by binding of this particular protein substrate and demonstrate the utility of 2D analysis as an indicator of structural change in this system.     

Classification and reconstruction of a heterogeneous set of electron microscopic images: a case study of GroEL-substrate complexes

Image analysis methods were used to separate images of a large macromolecular complex, the chaperonin GroEL, in a preparation in which it is partially liganded to a nonnative protein substrate, glutamine synthetase. The relatively small difference ( approximately 6%) in size between the chaperonin in its free and complexed forms, and the absence of gross changes in overall conformation, made separation of the two types of particles challenging. Different approaches were evaluated and used for alignment and classification of images, both in two common projections and in three dimensions, yielding 2D averages and a 3D reconstruction. The results of 3D analysis describe the conformational changes effected by binding of this particular protein substrate and demonstrate the utility of 2D analysis as an indicator of structural change in this system.     

Printing protein arrays from DNA arrays

We describe a method, DNA array to protein array (DAPA), which allows the 'printing' of replicate protein arrays directly from a DNA array template using cell-free protein synthesis. At least 20 copies of a protein array can be obtained from a single DNA array. DAPA eliminates the need for separate protein expression, purification and spotting, and also overcomes the problem of long-term functional storage of surface-bound proteins.     

Light sharing in multi-flat-panel-PMT PEM detectors

Large are a detectors, such as those used in positron emission mammography (PEM) and scintimammography, utilize arrays of discrete semtillator elements mounted on arrays of position sensitive photomultiplier tubes (PSPMT). Scintillator elements can be packed very densely (minimizing area between elements), allowing good detection sensitivity and spatial resolution. And, while new flat panel PSPMTS have minimal inactive edges, when they are placed in arrays significant dead spaces where scintillation light is undetectable are created. To address this problem, a light guide is often placed between the detector and PSPMT array to spread scintillation light so that these gaps can be bridged. In this investigation we studied the effect of light guides of various thickness on system performance. A 10×10 element array of LYSO detector elements was coupled to the center of a 2×2 array of PSPMTs through varying thicknesses (1 to 4 mm) of UV glass. The spot size of the imaged elements and distortions in the regular square pattern of the imaged scintillator arrays were evaluated. Energy resolution was measured by placing single elements of LYSO at several locations of the PSPMT array. Spatial distortions in the images of the array were reduced by using thicker light guides (3–4 mm). Use of thicker light guides, however, resulted in reduced pixel resolution and slight degradation of energy resolution. Therefore, some loss of pixel and energy resolution will accompany the use of thick light guides (minimum of 3 mm) required for optimum identification of detector elements.     

Real-time protein biosensor arrays based on surface plasmon resonance differential phase imaging

This paper reports the application of differential phase surface plasmon resonance (SPR) imaging in two-dimensional (2D) protein biosensor arrays. Our phase imaging approach offers a distinct advantage over the conventional angular SPR technique in terms of utilization efficiency of optical sensor elements in the imaging device. In the angular approach, each biosensor site in the biosensor array requires a linear array of optical detector elements to locate the SPR angular dip. The maximum biosensor density that a two-dimensional imaging device can offer is a one-dimensional SPR biosensor array. On the other hand, the phase-sensitive SPR approach captures data in the time domain instead of the spatial domain. It is possible that each pixel in the captured interferogram represents one sensor site, thus offering high-density two-dimensional biosensor arrays. In addition, our differential phase approach improves detection resolution through removing common-mode disturbances. Experimental results demonstrate a system resolution of 8.8 x 10(-7)RIU (refractive index unit). Real-time monitoring of bovine serum albumin (BSA)/anti-BSA binding interactions at various concentration levels was achieved using a biosensor array. The detection limit was 0.77 microg/ml. The reported two-dimensional SPR biosensor array offers a real-time and non-labeling detection tool for high-throughput protein array analysis. It may find promising applications in protein therapeutics, drug screening and clinical diagnostics.