Spatial Arrangement Of Proteins Research Articles

Molecular pixelation: spatial proteomics of single cells by sequencing

The spatial distribution of cell surface proteins governs vital processes of the immune system such as intercellular communication and mobility. However, fluorescence microscopy has limited scalability in the multiplexing and throughput needed to drive spatial proteomics discoveries at subcellular level. We present Molecular Pixelation (MPX), an optics-free, DNA sequence-based method for spatial proteomics of single cells using antibody–oligonucleotide conjugates (AOCs) and DNA-based, nanometer-sized molecular pixels. The relative locations of AOCs are inferred by sequentially associating them into local neighborhoods using the sequence-unique DNA pixels, forming >1,000 spatially connected zones per cell in 3D. For each single cell, DNA-sequencing reads are computationally arranged into spatial proteomics networks for 76 proteins. By studying immune cell dynamics using spatial statistics on graph representations of the data, we identify known and new patterns of spatial organization of proteins on chemokine-stimulated T cells, highlighting the potential of MPX in defining cell states by the spatial arrangement of proteins.

Spatial arrangement of proteins in planar and curved membranes by PPM 3.0.

Cellular protrusions, invaginations, and many intracellular organelles have strongly curved membrane regions. Transmembrane and peripheral membrane proteins that induce, sense, or stabilize such regions cannot be properly fitted into a single flat bilayer. To treat such proteins, we developed a new method and a web tool, PPM 3.0, for positioning proteins in curved or planar, single or multiple membranes. This method determines the energetically optimal spatial position, the hydrophobic thickness, and the radius of intrinsic curvature of a membrane-deforming protein structure by arranging it in a single or several sphere-shaped or planar membrane sections. In addition, it can define the lipid-embedded regions of a protein that simultaneously spans several membranes or determine the optimal position of a peptide in a spherical micelle. The PPM 3.0 web server operates with 17 types of biological membranes and 4 types of artificial bilayers. It is publicly available at https://opm.phar.umich.edu/ppm_server3. PPM 3.0 was applied to identify and characterize arrangements in membranes of 128 proteins with a significant intrinsic curvature, such as BAR domains, annexins, Piezo, and MscS mechanosensitive channels, cation-chloride cotransporters, as well as mitochondrial ATP synthases, calcium uniporters, and TOM complexes. These proteins form large complexes that are mainly localized in mitochondria, plasma membranes, and endosomes. Structures of bacterial drug efflux pumps, AcrAB-TolC, MexAB-OrpM, and MacAB-TolC, were positioned in both membranes of the bacterial cell envelop, while structures of multimeric gap-junction channels were arranged in two opposed cellular membranes.

A Cross-linking Mass Spectrometry Approach Defines Protein Interactions in Yeast Mitochondria

Protein cross-linking and the analysis of cross-linked peptides by mass spectrometry is currently receiving much attention. Not only is this approach applied to isolated complexes to provide information about spatial arrangements of proteins, but it is also increasingly applied to entire cells and their organelles. As in quantitative proteomics, the application of isotopic labeling further makes it possible to monitor quantitative changes in the protein-protein interactions between different states of a system. Here, we cross-linked mitochondria from Saccharomyces cerevisiae grown on either glycerol- or glucose-containing medium to monitor protein-protein interactions under non-fermentative and fermentative conditions. We investigated qualitatively the protein-protein interactions of the 400 most abundant proteins applying stringent data-filtering criteria, i.e. a minimum of two cross-linked peptide spectrum matches and a cut-off in the spectrum scoring of the used search engine. The cross-linker BS3 proved to be equally suited for connecting proteins in all compartments of mitochondria when compared with its water-insoluble but membrane-permeable derivative DSS. We also applied quantitative cross-linking to mitochondria of both the growth conditions using stable-isotope labeled BS3. Significant differences of cross-linked proteins under glycerol and glucose conditions were detected, however, mainly because of the different copy numbers of these proteins in mitochondria under both the conditions. Results obtained from the glycerol condition indicate that the internal NADH:ubiquinone oxidoreductase Ndi1 is part of an electron transport chain supercomplex. We have also detected several hitherto uncharacterized proteins and identified their interaction partners. Among those, Min8 was found to be associated with cytochrome c oxidase. BN-PAGE analyses of min8Δ mitochondria suggest that Min8 promotes the incorporation of Cox12 into cytochrome c oxidase.

Spatial arrangement of proteins using scCro-tag: application for an in situ enzymatic microbead assay.

In natural systems, various metabolic reactions are often spatially organized to increase enzyme activity and specificity. Thus, by spatially arranging enzyme molecules in synthetic systems to imitate these natural systems, it is possible to promote a high rate of enzymatic turnover. In this present study, a normal and mutant form of the scCro DNA-binding protein were shown to bind orthogonally to specific recognition sequences under appropriate conditions. Furthermore, these DNA-binding tags were used to establish an enzyme assay system based on the spatial arrangement of transglutaminase and its substrate at the molecular level. Together, the results of the present study suggest that the scCro-tag may be a powerful tool to facilitate the synthetic spatial arrangement of proteins on a DNA ligand.

Mass spectrometry imaging: Towards mapping the elemental and molecular composition of the rhizosphere

This short review provides perspective regarding the use of mass spectrometry imaging (MSI) to study the rhizosphere. It also serves to complement the multi-omic-focused review by White et al. (in press). MSI is capable of elucidating chemical distributions within samples of interest in situ, and thus can provide spatial context to MS omics data in complementary experimental endeavors. Most MSI-based studies of plant-microbe interactions have focused on the phyllosphere and on the “associated rhizosphere” (our term for material that is not removed during harvesting). Sample preparation for these in situ analyses tends to be a limiting factor. These studies, however, have provided valuable insights into the spatial arrangement of proteins, peptides, lipids, and other metabolites within these systems. We intend this short review to be a primer on the fundamentals of MSI and its role in plant-microbe analysis. Lastly, we offer a perspective on the future of MSI and its use in understanding the molecular transformations beyond what we call the associated rhizosphere, one which extends to the rest of rhizosphere and into the bulk soil.

Protein patterning by a DNA origami framework.

A spatial arrangement of proteins provides structural and functional advantages in vast technological applications as well as fundamental research. Most protein patterning procedures employ complicated, time consuming and very costly nanofabrication techniques. As an alternative route, we developed a fully biomolecular self-assembly method using DNA Origami Frames (DOF) as a template for both small and large scale protein patterning. We employed a triangular DOF (tDOF) to arrange the Bovine Serum Albumin (BSA) protein. Our in situ protein patterning strategy provides a novel, fully organic platform using a fast and low-cost surface approach with possible utilization in fundamental science and technological applications.

Protein localisation by electron microscopy reveals the architecture of the yeast spliceosomal B complex.

The spliceosome assembles on a pre-mRNA intron by binding of five snRNPs and numerous proteins, leading to the formation of the pre-catalytic B complex. While the general morphology of the B complex is known, the spatial arrangement of proteins and snRNP subunits within it remain to be elucidated. To shed light on the architecture of the yeast B complex, we immuno-labelled selected proteins and located them by negative-stain electron microscopy. The B complex exhibited a triangular shape with main body, head and neck domains. We located the U5 snRNP components Brr2 at the top and Prp8 and Snu114 in the centre of the main body. We found several U2 SF3a (Prp9 and Prp11) and SF3b (Hsh155 and Cus1) proteins in the head domain and two U4/U6 snRNP proteins (Prp3 and Lsm4) in the neck domain that connects the main body with the head. Thus, we could assign distinct domains of the B complex to the respective snRNPs and provide the first detailed picture of the subunit architecture and protein arrangements of the B complex.

Life at the border: adaptation of proteins to anisotropic membrane environment.

This review discusses main features of transmembrane (TM) proteins which distinguish them from water-soluble proteins and allow their adaptation to the anisotropic membrane environment. We overview the structural limitations on membrane protein architecture, spatial arrangement of proteins in membranes and their intrinsic hydrophobic thickness, co-translational and post-translational folding and insertion into lipid bilayers, topogenesis, high propensity to form oligomers, and large-scale conformational transitions during membrane insertion and transport function. Special attention is paid to the polarity of TM protein surfaces described by profiles of dipolarity/polarizability and hydrogen-bonding capacity parameters that match polarity of the lipid environment. Analysis of distributions of Trp resides on surfaces of TM proteins from different biological membranes indicates that interfacial membrane regions with preferential accumulation of Trp indole rings correspond to the outer part of the lipid acyl chain region-between double bonds and carbonyl groups of lipids. These "midpolar" regions are not always symmetric in proteins from natural membranes. We also examined the hydrophobic effect that drives insertion of proteins into lipid bilayer and different free energy contributions to TM protein stability, including attractive van der Waals forces and hydrogen bonds, side-chain conformational entropy, the hydrophobic mismatch, membrane deformations, and specific protein-lipid binding.

H-Ras forms dimers on membrane surfaces via a protein–protein interface

The lipid-anchored small GTPase Ras is an important signaling node in mammalian cells. A number of observations suggest that Ras is laterally organized within the cell membrane, and this may play a regulatory role in its activation. Lipid anchors composed of palmitoyl and farnesyl moieties in H-, N-, and K-Ras are widely suspected to be responsible for guiding protein organization in membranes. Here, we report that H-Ras forms a dimer on membrane surfaces through a protein-protein binding interface. A Y64A point mutation in the switch II region, known to prevent Son of sevenless and PI3K effector interactions, abolishes dimer formation. This suggests that the switch II region, near the nucleotide binding cleft, is either part of, or allosterically coupled to, the dimer interface. By tethering H-Ras to bilayers via a membrane-miscible lipid tail, we show that dimer formation is mediated by protein interactions and does not require lipid anchor clustering. We quantitatively characterize H-Ras dimerization in supported membranes using a combination of fluorescence correlation spectroscopy, photon counting histogram analysis, time-resolved fluorescence anisotropy, single-molecule tracking, and step photobleaching analysis. The 2D dimerization Kd is measured to be ∼1 × 10(3) molecules/µm(2), and no higher-order oligomers were observed. Dimerization only occurs on the membrane surface; H-Ras is strictly monomeric at comparable densities in solution. Analysis of a number of H-Ras constructs, including key changes to the lipidation pattern of the hypervariable region, suggest that dimerization is a general property of native H-Ras on membrane surfaces.

Mechanics and Function of Protein Clustering and Spatial Sorting at Cell Membranes

Cell signalling regulated by the spatial arrangement of proteins into clusters ranging in size from a few molecules up to micron-scale assemblies is now heavily studied. Although the timing, placement, and even the molecular details of the proteins involved may be well known, their exact mechanism of formation and function still elude researchers. We seek to answer these questions in the context of the immunological synapse (IS) using a live T cell-supported membrane system. The IS is a T cell-antigen presenting cell (APC) interaction wherein membrane receptor-ligand pairs are reorganized into concentric patterns. In its simplest form, T cell receptors (TCR) bound to peptide-presenting major histocompatibility complex (pMHC) form a central cluster surrounded by a ring of leukocyte function associated antigen-1 (LFA-1) bound to intercellular adhesion molecule-1 (ICAM-1). Both ligand-bound TCR and LFA-1 are known to be centripetally transported by the actin cytoskeleton, but how they are differentially sorted is unknown. We hypothesize that protein cluster size prior to transport regulates sorting and probe this by increasing the LFA-1 cluster size. To do this, we use a bivalent or tetravalent crosslinker against LFA-1 or its ICAM-1 ligand and compare the distribution to non-crosslinked counterparts in the same cell. Peak fluorescence intensities across the IS averaged for whole cell populations reveal increased centripetal LFA-1 transport with increased size until LFA-1 forms a central cluster like TCR. Based on our results, we propose a frictional force coupling model as the mechanism of IS formation. Moreover, using fluorescence correlation spectroscopy, we discover a gradient of LFA-1:ICAM-1 cluster sizes across the synapse, as predicted by our model.

The Role of Cluster Size and Protein Spatial Pattern in the Immunological Synapse

During antigen recognition by T cells, signaling molecules on the T cell engage ligands on the antigen-presenting cell and organize into spatially distinctive patterns collectively known as the immunological synapse (IS). The spatial arrangement of proteins into well-defined zones within the IS is known to regulate T cell activation and signal transduction. The mechanisms by which this complex organization arises remain unclear. Here we alter the clustering state of the T cell costimulatory molecule, LFA-1, either by direct antibody crosslinking or by crosslinking its ligand, ICAM-1, displayed on the supported bilayer. Changes in receptor clustering lead to progressively more central localization of LFA-1 until it colocalizes with T cell receptors (TCR) at the center. The number of LFA-1 molecules within the resulting clusters is obtained by fluorescence correlation spectroscopy. Our results demonstrate that cluster size is a critical parameter in determining protein spatial positioning in the IS. We discuss a sorting mechanism, based on frictional coupling to the cytoskeleton, which is consistent with these observations and is, in principle, extendable to all cell surface proteins in the synapse. Furthermore, by presenting patterns of immobilized ICAM-1 within a fluid bilayer displaying the TCR ligand, peptide-loaded MHC, we investigate the importance of LFA-1 ring formation to T cell function and signaling.

Deconvolution Combined with Digital Colocalization Analysis to Study the Spatial Distribution of Tight and Adherens Junction Proteins

The relative spatial distribution of proteins was investigated with immunofluorescent methods by confocal laser scanning microscopy and digital image restoration. For confocal data sets recorded with a voxel dimension of 50 × 50 × 150 nm noise and blur can be decreased and the resolution in the z-axis increased by applying the maximum likelihood estimation algorithm of the Huygens software. This approach was successfully applied to the study of tight and adherens junctions in relation to the actin cytoskeleton in Madin Darby Canine Kidney cells. Colocalization analysis was done for pairs of probes using a histogram-based method. F-actin, occludin, zonula occludens 1, and E-cadherin were included in the study. Double-labeled preparations were used. The combination of deconvolution with the colocalization of confocal data sets offers a powerful tool to investigate the spatial arrangement of proteins.

Proximity of 5.8 S RNA-binding proteins and A-site proteins in yeast ribosomes inferred from cross-linking.

In order to probe the spatial arrangement of proteins within the 5.8 S rRNA domain of the intact ribosomal subunit of Saccharomyces cerevisiae, 60 S ribosomal subunits were treated with 2-iminothiolane. Proteins were extracted from the cross-linked ribosomes, fractionated by Sephadex G-150 column chromatography, and analyzed by diagonal polyacrylamide-sodium dodecyl sulfate gel electrophoresis. Constituent proteins of cross-linked pairs were identified by two-dimensional polyacrylamide gel electrophoresis. Nine cross-links involving five of the 11 5.8 S rRNA-binding proteins were analyzed. A model showing the network of cross-links is presented. Several of the 5.8 S rRNA-binding proteins are in sufficiently close proximity of the ribosomal A-site proteins to be cross-linked.

Spatial arrangement of proteins within the small subunit of rat liver ribosomes studied by cross-linking.

Cross-linking of proteins within the small subunit of rat liver ribosomes by the bifunctional reagent dimethyl 4,7-dioxo-5,6-dihydroxy-3,8-diazadecanbisimidate produced numerous covalently linked protein dimers which could be separated by a combination of ion-exchange chromatography on carboxymethyl cellulose and polyacrylamide gel electrophoresis. The protein components of the dimers were identified electrophoretically after periodate cleavage of the cross-link(s). The analysis revealed 42 cross-linked dimers involving 25 different proteins. Among these, proteins S3, S4 and S20 occurred in combinations with six, eight and seven different proteins, respectively. For proteins S13, S14 and S17 five protein neighbours could be identified, while 13 of the remaining proteins were linked to three or four different protein partners. The involvement of the majority of proteins in the formation of multiple cross-linked dimers implies that a large number of protein-protein interaction sites exist within the ribosomal subunit. A preliminary model illustrating the arrangement of 16 proteins in the small ribosomal subunit is presented and discussed with respect to possible functions, especially in the event of translation initiation.

The effect of lead on the red cell membrane.

Summary Conformational changes have been shown in the proteins of the red blood cell membrane, induced by lead poisoning. Evidence that the change in spatial arrangement of proteins is believed to be responsible for inhibition of the sodium (Na+)/potassium (K+) ATPase of the RBC membrane is presented.

Conformation of Ribosomes from Escherichia Coli

The present knowledge of the three-dimensional structure of ribosomes is far too limited to enable a complete understanding of the various roles which ribosomes play in protein biosynthesis. The spatial arrangement of proteins and ribonuclec acids in ribosomes can be analysed in many ways. Determination of binding sites for individual proteins on ribonuclec acid and locations of the mutual positions of proteins on the ribosome using labeling with fluorescent dyes, cross-linking reagents, neutron-diffraction or antibodies against ribosomal proteins seem to be most successful approaches. Structure and function of ribosomes can be correlated be depleting the complete ribosomes of some proteins to the functionally inactive core and by subsequent partial reconstitution in order to regain active ribosomal particles.

Protein synthesis and cell surface proteins in molluscum contagiosum virus infected cells.

The proteins synthesized in molluscum contagiosum virus (MCV) infected cells during one hour pulse-labelling experiments with14C amino acids were separated by sodium dodecyl sulphate acrylamide gel electrophoresis and analysed by autoradiography. In addition, the surface proteins of infected cells were labelled with125I and then analysed with the same procedure in order to detect differences in the spatial arrangement of proteins during the cytopathic effect (c.p.e.) and compared with the controls. In both types of experiments the electrophoretic pattern of human embryonic cells did not show qualitative differences between infected cells and control cells. This indicated that MCV fails to express, among the “early” events, the function(s) necessary to switch off host protein synthesis. The iodination of the cell surface showed that the cytopathic cell alteration did not correspond with a variation in the protein distribution on the cell surface.