Determinants for membrane association and permeabilization of the coxsackievirus 2B protein and the identification of the Golgi complex as the target organelle.

PubMed

de Jong, Arjan S; Wessels, Els; Dijkman, Henri B P M; Galama, Jochem M D; Melchers, Willem J G; Willems, Peter H G M; van Kuppeveld, Frank J M

2003-01-10

The 2B protein of enterovirus is responsible for the alterations in the permeability of secretory membranes and the plasma membrane in infected cells. The structural requirements for the membrane association and the subcellular localization of this essential virus protein, however, have not been defined. Here, we provide evidence that the 2B protein is an integral membrane protein in vivo that is predominantly localized at the Golgi complex upon individual expression. Addition of organelle-specific targeting signals to the 2B protein revealed that the Golgi localization is an absolute prerequisite for the ability of the protein to modify plasma membrane permeability. Expression of deletion mutants and heterologous proteins containing specific domains of the 2B protein demonstrated that each of the two hydrophobic regions could mediate membrane binding individually. However, the presence of both hydrophobic regions was required for the correct membrane association, efficient Golgi targeting, and the membrane-permeabilizing activity of the 2B protein, suggesting that the two hydrophobic regions are cooperatively involved in the formation of a membrane-integral complex. The formation of membrane-integral pores by the 2B protein in the Golgi complex and the possible mechanism by which a Golgi-localized virus protein modifies plasma membrane permeability are discussed.

Tandem neopentyl glycol maltosides (TNMs) for membrane protein stabilisation†

PubMed Central

Bae, Hyoung Eun; Mortensen, Jonas S.; Ribeiro, Orquidea; Du, Yang; Ehsan, Muhammad; Kobilka, Brian K.; Loland, Claus J.; Byrne, Bernadette

2017-01-01

A novel class of detergents, designated tandem neopentyl glycol maltosides (TNMs), were evaluated with four target membrane proteins. The best detergent varied depending on the target, but TNM-C12L and TNM-C11S were notable for their ability to confer increased membrane protein stability compared to DDM. These agents have potential for use in membrane protein research. PMID:27711401

Tandem neopentyl glycol maltosides (TNMs) for membrane protein stabilisation.

PubMed

Bae, Hyoung Eun; Mortensen, Jonas S; Ribeiro, Orquidea; Du, Yang; Ehsan, Muhammad; Kobilka, Brian K; Loland, Claus J; Byrne, Bernadette; Chae, Pil Seok

2016-10-04

A novel class of detergents, designated tandem neopentyl glycol maltosides (TNMs), were evaluated with four target membrane proteins. The best detergent varied depending on the target, but TNM-C12L and TNM-C11S were notable for their ability to confer increased membrane protein stability compared to DDM. These agents have potential for use in membrane protein research.

LeCPK1, a Calcium-Dependent Protein Kinase from Tomato. Plasma Membrane Targeting and Biochemical Characterization1

PubMed Central

Rutschmann, Frank; Stalder, Urs; Piotrowski, Markus; Oecking, Claudia; Schaller, Andreas

2002-01-01

The cDNA of LeCPK1, a calcium-dependent protein kinase, was cloned from tomato (Lycopersicon esculentum Mill.). LeCPK1 was expressed in Escherichia coli and purified from bacterial extracts. The recombinant protein was shown to be a functional protein kinase using a synthetic peptide as the substrate (syntide-2, Km = 85 μm). Autophosphorylation of LeCPK1 was observed on threonine and serine residues, one of which was identified as serine-439. Kinase activity was shown to be Ca2+ dependent and required the C-terminal, calmodulin-like domain of LeCPK1. Two classes of high- and low-affinity Ca2+-binding sites were observed, exhibiting dissociation constants of 0.6 and 55 μm, respectively. LeCPK1 was found to phosphorylate the regulatory C-terminal domain of the plasma membrane H+-ATPase in vitro. A potential role in the regulation of proton pump activity is corroborated by the apparent colocalization of the plasma membrane H+-ATPase and LeCPK1 in vivo. Upon transient expression in suspension-cultured cells, a C-terminal fusion of LeCPK1 with the green fluorescent protein was targeted to the plasma membrane. Myristoylation of the LeCPK1 N terminus was found to be required for plasma membrane targeting. PMID:12011347

Two-step membrane binding by the bacterial SRP receptor enable efficient and accurate Co-translational protein targeting.

PubMed

Hwang Fu, Yu-Hsien; Huang, William Y C; Shen, Kuang; Groves, Jay T; Miller, Thomas; Shan, Shu-Ou

2017-07-28

The signal recognition particle (SRP) delivers ~30% of the proteome to the eukaryotic endoplasmic reticulum, or the bacterial plasma membrane. The precise mechanism by which the bacterial SRP receptor, FtsY, interacts with and is regulated at the target membrane remain unclear. Here, quantitative analysis of FtsY-lipid interactions at single-molecule resolution revealed a two-step mechanism in which FtsY initially contacts membrane via a Dynamic mode, followed by an SRP-induced conformational transition to a Stable mode that activates FtsY for downstream steps. Importantly, mutational analyses revealed extensive auto-inhibitory mechanisms that prevent free FtsY from engaging membrane in the Stable mode; an engineered FtsY pre-organized into the Stable mode led to indiscriminate targeting in vitro and disrupted FtsY function in vivo. Our results show that the two-step lipid-binding mechanism uncouples the membrane association of FtsY from its conformational activation, thus optimizing the balance between the efficiency and fidelity of co-translational protein targeting.

Biogenesis of a Mitochondrial Outer Membrane Protein in Trypanosoma brucei: TARGETING SIGNAL AND DEPENDENCE ON A UNIQUE BIOGENESIS FACTOR.

PubMed

Bruggisser, Julia; Käser, Sandro; Mani, Jan; Schneider, André

2017-02-24

The mitochondrial outer membrane (OM) contains single and multiple membrane-spanning proteins that need to contain signals that ensure correct targeting and insertion into the OM. The biogenesis of such proteins has so far essentially only been studied in yeast and related organisms. Here we show that POMP10, an OM protein of the early diverging protozoan Trypanosoma brucei , is signal-anchored. Transgenic cells expressing variants of POMP10 fused to GFP demonstrate that the N-terminal membrane-spanning domain flanked by a few positively charged or neutral residues is both necessary and sufficient for mitochondrial targeting. Carbonate extraction experiments indicate that although the presence of neutral instead of positively charged residues did not interfere with POMP10 localization, it weakened its interaction with the OM. Expression of GFP-tagged POMP10 in inducible RNAi cell lines shows that its mitochondrial localization depends on pATOM36 but does not require Sam50 or ATOM40, the trypanosomal analogue of the Tom40 import pore. pATOM36 is a kinetoplastid-specific OM protein that has previously been implicated in the assembly of OM proteins and in mitochondrial DNA inheritance. In summary, our results show that although the features of the targeting signal in signal-anchored proteins are widely conserved, the protein machinery that mediates their biogenesis is not. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Essential Function of Protein 4.1G in Targeting of Membrane Protein Palmitoylated 6 into Schmidt-Lanterman Incisures in Myelinated Nerves

PubMed Central

Saitoh, Yurika; Ohno, Nobuhiko; Komada, Masayuki; Saitoh, Sei; Peles, Elior; Ohno, Shinichi

2012-01-01

Protein 4.1G is a membrane skeletal protein found in specific subcellular structures in myelinated Schwann cells and seminiferous tubules. Here, we show that in the mouse sciatic nerve, protein 4.1G colocalized at Schmidt-Lanterman incisures (SLI) and the paranodes with a member of the membrane-associated guanylate kinase (MAGUK) family, membrane protein palmitoylated 6 (MPP6). Coimmunoprecipitation experiments revealed that MPP6 was interacting with protein 4.1G. In contrast to wild-type nerves, in 4.1G knockout mice, MPP6 was found largely in the cytoplasm near Schwann cell nuclei, indicating an abnormal protein transport. Although the SLI remained in the 4.1G knockout sciatic nerves, as confirmed by E-cadherin immunostaining, their shape was altered in aged 4.1G knockout nerves compared to their shape in wild-type nerves. In the seminiferous tubules, MPP6 was localized similarly to protein 4.1G along cell membranes of the spermatogonium and early spermatocytes. However, in contrast to myelinated peripheral nerves, the specific localization of MPP6 in the seminiferous tubules was unaltered in the absence of protein 4.1G. These results indicate that 4.1G has a specific role in the targeting of MPP6 to the SLI and the assembly of these subcellular structures. PMID:22025680

Structural Insights into Membrane Targeting by the Flagellar Calcium-binding Protein (FCaBP) a Myristoylated and Palmitoylated Calcium Sensor in Trypanosoma cruzi

DOE Office of Scientific and Technical Information (OSTI.GOV)

J Wingard; J Ladner; M Vanarotti

2011-12-31

The flagellar calcium-binding protein (FCaBP) of the protozoan Trypanosoma cruzi is targeted to the flagellar membrane where it regulates flagellar function and assembly. As a first step toward understanding the Ca{sup 2+}-induced conformational changes important for membrane-targeting, we report here the x-ray crystal structure of FCaBP in the Ca{sup 2+}-free state determined at 2.2{angstrom} resolution. The first 17 residues from the N terminus appear unstructured and solvent-exposed. Residues implicated in membrane targeting (Lys-19, Lys-22, and Lys-25) are flanked by an exposed N-terminal helix (residues 26-37), forming a patch of positive charge on the protein surface that may interact electrostatically withmore » flagellar membrane targets. The four EF-hands in FCaBP each adopt a 'closed conformation' similar to that seen in Ca{sup 2+}-free calmodulin. The overall fold of FCaBP is closest to that of grancalcin and other members of the penta EF-hand superfamily. Unlike the dimeric penta EF-hand proteins, FCaBP lacks a fifth EF-hand and is monomeric. The unstructured N-terminal region of FCaBP suggests that its covalently attached myristoyl group at the N terminus may be solvent-exposed, in contrast to the highly sequestered myristoyl group seen in recoverin and GCAP1. NMR analysis demonstrates that the myristoyl group attached to FCaBP is indeed solvent-exposed in both the Ca{sup 2+}-free and Ca{sup 2+}-bound states, and myristoylation has no effect on protein structure and folding stability. We propose that exposed acyl groups at the N terminus may anchor FCaBP to the flagellar membrane and that Ca{sup 2+}-induced conformational changes may control its binding to membrane-bound protein targets..« less

A high-throughput assay of membrane protein stability.

PubMed

Postis, Vincent L G; Deacon, Sarah E; Roach, Peter C J; Wright, Gareth S A; Xia, Xiaobing; Ingram, Jean C; Hadden, Jonathan M; Henderson, Peter J F; Phillips, Simon E V; McPherson, Michael J; Baldwin, Stephen A

2008-12-01

The preparation of purified, detergent-solubilized membrane proteins in a monodisperse and stable form is usually a prerequisite for investigation not only of their function but also for structural studies by X-ray crystallography and other approaches. Typically, it is necessary to explore a wide range of conditions, including detergent type, buffer pH, and the presence of additives such as glycerol, in order to identify those optimal for stability. Given the difficulty of expressing and purifying membrane proteins in large amounts, such explorations must ideally be performed on as small a scale as practicable. To achieve this objective in the UK Membrane Protein Structure Initiative, we have developed a rapid, economical, light-scattering assay of membrane protein aggregation that allows the testing of 48 buffer conditions in parallel on 6 protein targets, requiring less than 2 mg protein for each target. Testing of the assay on a number of unrelated membrane transporters has shown that it is of generic applicability. Proteins of sufficient purity for this plate-based assay are first rapidly prepared using simple affinity purification procedures performed in batch mode. Samples are then transferred by microdialysis into each of the conditions to be tested. Finally, attenuance at 340 nm is monitored in a 384-well plate using a plate reader. Optimal conditions for protein stability identified in the assay can then be exploited for the tailored purification of individual targets in as stable a form as possible.

Membrane Transporters: Structure, Function and Targets for Drug Design

NASA Astrophysics Data System (ADS)

Ravna, Aina W.; Sager, Georg; Dahl, Svein G.; Sylte, Ingebrigt

Current therapeutic drugs act on four main types of molecular targets: enzymes, receptors, ion channels and transporters, among which a major part (60-70%) are membrane proteins. This review discusses the molecular structures and potential impact of membrane transporter proteins on new drug discovery. The three-dimensional (3D) molecular structure of a protein contains information about the active site and possible ligand binding, and about evolutionary relationships within the protein family. Transporters have a recognition site for a particular substrate, which may be used as a target for drugs inhibiting the transporter or acting as a false substrate. Three groups of transporters have particular interest as drug targets: the major facilitator superfamily, which includes almost 4000 different proteins transporting sugars, polyols, drugs, neurotransmitters, metabolites, amino acids, peptides, organic and inorganic anions and many other substrates; the ATP-binding cassette superfamily, which plays an important role in multidrug resistance in cancer chemotherapy; and the neurotransmitter:sodium symporter family, which includes the molecular targets for some of the most widely used psychotropic drugs. Recent technical advances have increased the number of known 3D structures of membrane transporters, and demonstrated that they form a divergent group of proteins with large conformational flexibility which facilitates transport of the substrate.

Application of Fragment Based Drug Discovery to Membrane Proteins: Biophysical Identification of Ligands of the Integral Membrane Enzyme DsbB

PubMed Central

Früh, Virginie; Zhou, Yunpeng; Chen, Dan; Loch, Caroline; Eiso, AB; Grinkova, Yelena N.; Verheij, Herman; Sligar, Stephen G; Bushweller, John H.; Siegal, Gregg

2014-01-01

Summary Membrane proteins are important pharmaceutical targets, but they pose significant challenges for fragment based drug discovery approaches. Here we present the first successful use of biophysical methods to screen for fragment ligands to an integral membrane protein. The E. coli inner membrane protein DsbB was solubilized in detergent micelles and lipid bilayer nanodiscs. The solubilized protein was immobilized with retention of functionality and used to screen 1,071 drug fragments for binding using Target Immobilized NMR Screening. Biochemical and biophysical validation of the 8 most potent hits revealed an IC50 range of 7 to 200 μM. The ability to insert a broad array of membrane proteins into nanodiscs, combined with the efficiency of TINS, demonstrates the feasibility of finding fragments targeting membrane proteins. PMID:20797617

Computational Approaches for Designing Protein/Inhibitor Complexes and Membrane Protein Variants

NASA Astrophysics Data System (ADS)

Vijayendran, Krishna Gajan

Drug discovery of small-molecule protein inhibitors is a vast enterprise that involves several scientific disciplines (i.e. genomics, cell biology, x-ray crystallography, chemistry, computer science, statistics), with each discipline focusing on a particular aspect of the process. In this thesis, I use computational and experimental approaches to explore the most fundamental aspect of drug discovery: the molecular interactions of small-molecules inhibitors with proteins. In Part I (Chapters I and II), I describe how computational docking approaches can be used to identify structurally diverse molecules that can inhibit multiple protein targets in the brain. I illustrate this approach using the examples of microtubule-stabilizing agents and inhibitors of cyclooxygenase(COX)-I and 5-lipoxygenase (5-LOX). In Part II (Chapters III and IV), I focus on membrane proteins, which are notoriously difficult to work with due to their low natural abundances, low yields for heterologous over expression, and propensities toward aggregation. I describe a general approach for designing water-soluble variants of membrane proteins, for the purpose of developing cell-free, label-free, detergent-free, solution-phase studies of protein structure and small-molecule binding. I illustrate this approach through the design of a water-soluble variant of the membrane protein Smoothened, wsSMO. This wsSMO stands to serve as a first-step towards developing membrane protein analogs of this important signaling protein and drug target.

High-throughput Cloning and Expression of Integral Membrane Proteins in Escherichia coli

PubMed Central

Bruni, Renato

2014-01-01

Recently, several structural genomics centers have been established and a remarkable number of three-dimensional structures of soluble proteins have been solved. For membrane proteins, the number of structures solved has been significantly trailing those for their soluble counterparts, not least because over-expression and purification of membrane proteins is a much more arduous process. By using high throughput technologies, a large number of membrane protein targets can be screened simultaneously and a greater number of expression and purification conditions can be employed, leading to a higher probability of successfully determining the structure of membrane proteins. This unit describes the cloning, expression and screening of membrane proteins using high throughput methodologies developed in our laboratory. Basic Protocol 1 deals with the cloning of inserts into expression vectors by ligation-independent cloning. Basic Protocol 2 describes the expression and purification of the target proteins on a miniscale. Lastly, for the targets that express at the miniscale, basic protocols 3 and 4 outline the methods employed for the expression and purification of targets at the midi-scale, as well as a procedure for detergent screening and identification of detergent(s) in which the target protein is stable. PMID:24510647

A Circular Dichroism Reference Database for Membrane Proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wallace,B.; Wien, F.; Stone, T.

2006-01-01

Membrane proteins are a major product of most genomes and the target of a large number of current pharmaceuticals, yet little information exists on their structures because of the difficulty of crystallising them; hence for the most part they have been excluded from structural genomics programme targets. Furthermore, even methods such as circular dichroism (CD) spectroscopy which seek to define secondary structure have not been fully exploited because of technical limitations to their interpretation for membrane embedded proteins. Empirical analyses of circular dichroism (CD) spectra are valuable for providing information on secondary structures of proteins. However, the accuracy of themore » results depends on the appropriateness of the reference databases used in the analyses. Membrane proteins have different spectral characteristics than do soluble proteins as a result of the low dielectric constants of membrane bilayers relative to those of aqueous solutions (Chen & Wallace (1997) Biophys. Chem. 65:65-74). To date, no CD reference database exists exclusively for the analysis of membrane proteins, and hence empirical analyses based on current reference databases derived from soluble proteins are not adequate for accurate analyses of membrane protein secondary structures (Wallace et al (2003) Prot. Sci. 12:875-884). We have therefore created a new reference database of CD spectra of integral membrane proteins whose crystal structures have been determined. To date it contains more than 20 proteins, and spans the range of secondary structures from mostly helical to mostly sheet proteins. This reference database should enable more accurate secondary structure determinations of membrane embedded proteins and will become one of the reference database options in the CD calculation server DICHROWEB (Whitmore & Wallace (2004) NAR 32:W668-673).« less

Biophysical EPR Studies Applied to Membrane Proteins

PubMed Central

Sahu, Indra D; Lorigan, Gary A

2015-01-01

Membrane proteins are very important in controlling bioenergetics, functional activity, and initializing signal pathways in a wide variety of complicated biological systems. They also represent approximately 50% of the potential drug targets. EPR spectroscopy is a very popular and powerful biophysical tool that is used to study the structural and dynamic properties of membrane proteins. In this article, a basic overview of the most commonly used EPR techniques and examples of recent applications to answer pertinent structural and dynamic related questions on membrane protein systems will be presented. PMID:26855825

Membrane Fusion Proteins as Nanomachines

NASA Astrophysics Data System (ADS)

Tamm, Lukas

2009-03-01

Membrane fusion is key to fertilization, virus infection, and neurotransmission. Specific proteins work like nanomachines to stitch together fluid, yet highly ordered lipid bilayers. The energy gained from large exothermic conformational changes of these proteins is utilized to fuse lipid bilayers that do not fuse spontaneously. Structural studies using x-ray crystallography and NMR spectroscopy have yielded detailed information about architecture and inner workings of these molecular machines. The question now is: how is mechanical energy gained from such protein transformations harnessed to transform membrane topology? To answer this question, we have determined that a boomerang-shaped structure of the influenza fusion peptide is critical to generate a high-energy binding intermediate in the target membrane and to return the ``boomerang'' to its place of release near the viral membrane for completion of the fusion cycle. In presynaptic exocytosis, receptor and acceptor SNAREs are zippered to form a helical bundle that is arrested shortly before the membrane. Ca binding to interlocked synaptotagmin releases the fusion block. Structural NMR and single molecule fluorescence data are combined to arrive at and further refine this picture.

Enhancing Membrane Protein Identification Using a Simplified Centrifugation and Detergent-Based Membrane Extraction Approach.

PubMed

Zhou, Yanting; Gao, Jing; Zhu, Hongwen; Xu, Jingjing; He, Han; Gu, Lei; Wang, Hui; Chen, Jie; Ma, Danjun; Zhou, Hu; Zheng, Jing

2018-02-20

Membrane proteins may act as transporters, receptors, enzymes, and adhesion-anchors, accounting for nearly 70% of pharmaceutical drug targets. Difficulties in efficient enrichment, extraction, and solubilization still exist because of their relatively low abundance and poor solubility. A simplified membrane protein extraction approach with advantages of user-friendly sample processing procedures, good repeatability and significant effectiveness was developed in the current research for enhancing enrichment and identification of membrane proteins. This approach combining centrifugation and detergent along with LC-MS/MS successfully identified higher proportion of membrane proteins, integral proteins and transmembrane proteins in membrane fraction (76.6%, 48.1%, and 40.6%) than in total cell lysate (41.6%, 16.4%, and 13.5%), respectively. Moreover, our method tended to capture membrane proteins with high degree of hydrophobicity and number of transmembrane domains as 486 out of 2106 (23.0%) had GRAVY > 0 in membrane fraction, 488 out of 2106 (23.1%) had TMs ≥ 2. It also provided for improved identification of membrane proteins as more than 60.6% of the commonly identified membrane proteins in two cell samples were better identified in membrane fraction with higher sequence coverage. Data are available via ProteomeXchange with identifier PXD008456.

Retention mechanisms for ER and Golgi membrane proteins.

PubMed

Gao, Caiji; Cai, Yi; Wang, Yejun; Kang, Byung-Ho; Aniento, Fernando; Robinson, David G; Jiang, Liwen

2014-08-01

Unless there are mechanisms to selectively retain membrane proteins in the endoplasmic reticulum (ER) or in the Golgi apparatus, they automatically proceed downstream to the plasma or vacuole membranes. Two types of coat protein complex I (COPI)-interacting motifs in the cytosolic tails of membrane proteins seem to facilitate membrane retention in the early secretory pathway of plants: a dilysine (KKXX) motif (which is typical of p24 proteins) for the ER and a KXE/D motif (which occurs in the Arabidopsis endomembrane protein EMP12) for the Golgi apparatus. The KXE/D motif is highly conserved in all eukaryotic EMPs and is additionally present in hundreds of other proteins of unknown subcellular localization and function. This novel signal may represent a new general mechanism for Golgi targeting and the retention of polytopic integral membrane proteins. Copyright © 2014 Elsevier Ltd. All rights reserved.

Membrane-targeted strategies for modulating APP and Aβ-mediated toxicity

PubMed Central

Price, Katherine A; Crouch, Peter J; Donnelly, Paul S; Masters, Colin L; White, Anthony R; Curtain, Cyril C

2009-01-01

Abstract Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by numerous pathological features including the accumulation of neurotoxic amyloid-β (Aβ) peptide. There is currently no effective therapy for AD, but the development of therapeutic strategies that target the cell membrane is gaining increased interest. The amyloid precursor protein (APP) from which Aβ is formed is a membrane-bound protein, and Aβ production and toxicity are both membrane mediated events. This review describes the critical role of cell membranes in AD with particular emphasis on how the composition and structure of the membrane and its specialized regions may influence toxic or benign Aβ/APP pathways in AD. The putative role of copper (Cu) in AD is also discussed, and we highlight how targeting the cell membrane with Cu complexes has therapeutic potential in AD. PMID:19278455

Analysis of Protein Interactions at Native Chloroplast Membranes by Ellipsometry

PubMed Central

Kriechbaumer, Verena; Nabok, Alexei; Mustafa, Mohd K.; Al-Ammar, Rukaiah; Tsargorodskaya, Anna; Smith, David P.; Abell, Ben M.

2012-01-01

Membrane bound receptors play vital roles in cell signaling, and are the target for many drugs, yet their interactions with ligands are difficult to study by conventional techniques due to the technical difficulty of monitoring these interactions in lipid environments. In particular, the ability to analyse the behaviour of membrane proteins in their native membrane environment is limited. Here, we have developed a quantitative approach to detect specific interactions between low-abundance chaperone receptors within native chloroplast membranes and their soluble chaperone partners. Langmuir-Schaefer film deposition was used to deposit native chloroplasts onto gold-coated glass slides, and interactions between the molecular chaperones Hsp70 and Hsp90 and their receptors in the chloroplast membranes were detected and quantified by total internal reflection ellipsometry (TIRE). We show that native chloroplast membranes deposited on gold-coated glass slides using Langmuir-Schaefer films retain functional receptors capable of binding chaperones with high specificity and affinity. Taking into account the low chaperone receptor abundance in native membranes, these binding properties are consistent with data generated using soluble forms of the chloroplast chaperone receptors, OEP61 and Toc64. Therefore, we conclude that chloroplasts have the capacity to selectively bind chaperones, consistent with the notion that chaperones play an important role in protein targeting to chloroplasts. Importantly, this method of monitoring by TIRE does not require any protein labelling. This novel combination of techniques should be applicable to a wide variety of membranes and membrane protein receptors, thus presenting the opportunity to quantify protein interactions involved in fundamental cellular processes, and to screen for drugs that target membrane proteins. PMID:22479632

Current strategies for protein production and purification enabling membrane protein structural biology.

PubMed

Pandey, Aditya; Shin, Kyungsoo; Patterson, Robin E; Liu, Xiang-Qin; Rainey, Jan K

2016-12-01

Membrane proteins are still heavily under-represented in the protein data bank (PDB), owing to multiple bottlenecks. The typical low abundance of membrane proteins in their natural hosts makes it necessary to overexpress these proteins either in heterologous systems or through in vitro translation/cell-free expression. Heterologous expression of proteins, in turn, leads to multiple obstacles, owing to the unpredictability of compatibility of the target protein for expression in a given host. The highly hydrophobic and (or) amphipathic nature of membrane proteins also leads to challenges in producing a homogeneous, stable, and pure sample for structural studies. Circumventing these hurdles has become possible through the introduction of novel protein production protocols; efficient protein isolation and sample preparation methods; and, improvement in hardware and software for structural characterization. Combined, these advances have made the past 10-15 years very exciting and eventful for the field of membrane protein structural biology, with an exponential growth in the number of solved membrane protein structures. In this review, we focus on both the advances and diversity of protein production and purification methods that have allowed this growth in structural knowledge of membrane proteins through X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cryo-electron microscopy (cryo-EM).

Current strategies for protein production and purification enabling membrane protein structural biology

PubMed Central

Pandey, Aditya; Shin, Kyungsoo; Patterson, Robin E.; Liu, Xiang-Qin; Rainey, Jan K.

2017-01-01

Membrane proteins are still heavily underrepresented in the protein data bank (PDB) due to multiple bottlenecks. The typical low abundance of membrane proteins in their natural hosts makes it necessary to overexpress these proteins either in heterologous systems or through in vitro translation/cell-free expression. Heterologous expression of proteins, in turn, leads to multiple obstacles due to the unpredictability of compatibility of the target protein for expression in a given host. The highly hydrophobic and/or amphipathic nature of membrane proteins also leads to challenges in producing a homogeneous, stable, and pure sample for structural studies. Circumventing these hurdles has become possible through introduction of novel protein production protocols; efficient protein isolation and sample preparation methods; and, improvement in hardware and software for structural characterization. Combined, these advances have made the past 10–15 years very exciting and eventful for the field of membrane protein structural biology, with an exponential growth in the number of solved membrane protein structures. In this review, we focus on both the advances and diversity of protein production and purification methods that have allowed this growth in structural knowledge of membrane proteins through X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cryo-electron microscopy (cryo-EM). PMID:27010607

Membrane Binding of HIV-1 Matrix Protein: Dependence on Bilayer Composition and Protein Lipidation

PubMed Central

Barros, Marilia; Nanda, Hirsh

2016-01-01

ABSTRACT By assembling in a protein lattice on the host's plasma membrane, the retroviral Gag polyprotein triggers formation of the viral protein/membrane shell. The MA domain of Gag employs multiple signals—electrostatic, hydrophobic, and lipid-specific—to bring the protein to the plasma membrane, thereby complementing protein-protein interactions, located in full-length Gag, in lattice formation. We report the interaction of myristoylated and unmyristoylated HIV-1 Gag MA domains with bilayers composed of purified lipid components to dissect these complex membrane signals and quantify their contributions to the overall interaction. Surface plasmon resonance on well-defined planar membrane models is used to quantify binding affinities and amounts of protein and yields free binding energy contributions, ΔG, of the various signals. Charge-charge interactions in the absence of the phosphatidylinositide PI(4,5)P2 attract the protein to acidic membrane surfaces, and myristoylation increases the affinity by a factor of 10; thus, our data do not provide evidence for a PI(4,5)P2 trigger of myristate exposure. Lipid-specific interactions with PI(4,5)P2, the major signal lipid in the inner plasma membrane, increase membrane attraction at a level similar to that of protein lipidation. While cholesterol does not directly engage in interactions, it augments protein affinity strongly by facilitating efficient myristate insertion and PI(4,5)P2 binding. We thus observe that the isolated MA protein, in the absence of protein-protein interaction conferred by the full-length Gag, binds the membrane with submicromolar affinities. IMPORTANCE Like other retroviral species, the Gag polyprotein of HIV-1 contains three major domains: the N-terminal, myristoylated MA domain that targets the protein to the plasma membrane of the host; a central capsid-forming domain; and the C-terminal, genome-binding nucleocapsid domain. These domains act in concert to condense Gag into a membrane

Outer Membrane Targeting of Passenger Proteins by the Vacuolating Cytotoxin Autotransporter of Helicobacter pylori

PubMed Central

Fischer, Wolfgang; Buhrdorf, Renate; Gerland, Elke; Haas, Rainer

2001-01-01

Helicobacter pylori produces a number of proteins associated with the outer membrane, including adhesins and the vacuolating cytotoxin. These proteins are supposed to integrate into the outer membrane by β-barrel structures, characteristic of the family of autotransporter proteins. By using the SOMPES (shuttle vector-based outer membrane protein expression) system for outer membrane protein production, we were able to functionally express in H. pylori the cholera toxin B subunit genetically fused to the C-terminal VacA domain. We demonstrate that the fusion protein is translocated to the H. pylori outer membrane and that the CtxB domain is exposed on the H. pylori surface. Thus, we provide the first experimental evidence that the C-terminal β-domain of VacA can transport a foreign passenger protein to the H. pylori surface and hence acts as a functional autotransporter. PMID:11598049

Get3 is a holdase chaperone and moves to deposition sites for aggregated proteins when membrane targeting is blocked

PubMed Central

Powis, Katie; Schrul, Bianca; Tienson, Heather; Gostimskaya, Irina; Breker, Michal; High, Stephen; Schuldiner, Maya; Jakob, Ursula; Schwappach, Blanche

2013-01-01

Summary The endomembrane system of yeast contains different tail-anchored proteins that are post-translationally targeted to membranes via their C-terminal transmembrane domain. This hydrophobic segment could be hazardous in the cytosol if membrane insertion fails, resulting in the need for energy-dependent chaperoning and the degradation of aggregated tail-anchored proteins. A cascade of GET proteins cooperates in a conserved pathway to accept newly synthesized tail-anchored proteins from ribosomes and guide them to a receptor at the endoplasmic reticulum, where membrane integration takes place. It is, however, unclear how the GET system reacts to conditions of energy depletion that might prevent membrane insertion and hence lead to the accumulation of hydrophobic proteins in the cytosol. Here we show that the ATPase Get3, which accommodates the hydrophobic tail anchor of clients, has a dual function: promoting tail-anchored protein insertion when glucose is abundant and serving as an ATP-independent holdase chaperone during energy depletion. Like the generic chaperones Hsp42, Ssa2, Sis1 and Hsp104, we found that Get3 moves reversibly to deposition sites for protein aggregates, hence supporting the sequestration of tail-anchored proteins under conditions that prevent tail-anchored protein insertion. Our findings support a ubiquitous role for the cytosolic GET complex as a triaging platform involved in cellular proteostasis. PMID:23203805

Living target of Ce(III) action on horseradish cells: proteins on/in cell membrane.

PubMed

Yang, Guangmei; Sun, Zhaoguo; Lv, Xiaofen; Deng, Yunyun; Zhou, Qing; Huang, Xiaohua

2012-12-01

Positive and negative effects of rare earth elements (REEs) in life have been reported in many papers, but the cellular mechanisms have not been answered, especially the action sites of REEs on plasma membrane are unknown. Proteins on/in the plasma membrane perform main functions of the plasma membrane. Cerium (Ce) is the richest REEs in crust. Thus, the interaction between Ce(III) and the proteins on/in the plasma membrane, the morphology of protoplast, and the contents of nutrient elements in protoplast of horseradish were investigated using the optimized combination of the fluorescence microscopy, fluorescence spectroscopy, circular dichroism, scanning electron microscopy, and X-ray energy dispersive spectroscopy. It was found that Ce(III) at the low concentrations (10, 30 μM) could interact with proteins on/in the plasma membrane of horseradish, leading to the improvement in the structure of membrane proteins and the plasma membrane, which accelerated the intra-/extra-cellular substance exchange and further promoted the development of cells. When horseradish was treated with Ce(III) at the high concentrations (60, 80 μM), Ce(III) also could interact with the proteins on/in the plasma membrane of horseradish, leading to the destruction in the structure of membrane proteins and the plasma membrane. These effects decelerated the intra-/extra-cellular substance exchange and further inhibited the development of cells. Thus, the interaction between Ce(III) and proteins on/in the plasma membrane in plants was an important reason of the positive and negative effects of Ce(III) on plants. The results would provide some references for understanding the cellular effect mechanisms of REEs on plants.

Evolved Escherichia coli strains for amplified, functional expression of membrane proteins.

PubMed

Gul, Nadia; Linares, Daniel M; Ho, Franz Y; Poolman, Bert

2014-01-09

The major barrier to the physical characterization and structure determination of membrane proteins is low protein yield and/or low functionality in recombinant expression. The enteric bacterium Escherichia coli is the most widely employed organism for producing recombinant proteins. Beside several advantages of this expression host, one major drawback is that the protein of interest does not always adopt its native conformation and may end up in large insoluble aggregates. We describe a robust strategy to increase the likelihood of overexpressing membrane proteins in a functional state. The method involves fusion in tandem of green fluorescent protein and the erythromycin resistance protein (23S ribosomal RNA adenine N-6 methyltransferase, ErmC) to the C-terminus of a target membrane protein. The fluorescence of green fluorescent protein is used to report the folding state of the target protein, whereas ErmC is used to select for increased expression. By gradually increasing the erythromycin concentration of the medium and testing different membrane protein targets, we obtained a number of evolved strains of which four (NG2, NG3, NG5 and NG6) were characterized and their genome was fully sequenced. Strikingly, each of the strains carried a mutation in the hns gene, whose product is involved in genome organization and transcriptional silencing. The degree of expression of (membrane) proteins correlates with the severity of the hns mutation, but cells in which hns was deleted showed an intermediate expression performance. We propose that (partial) removal of the transcriptional silencing mechanism changes the levels of proteins essential for the functional overexpression of membrane proteins. © 2013.

Binding and Fusion of Extracellular Vesicles to the Plasma Membrane of Their Cell Targets.

PubMed

Prada, Ilaria; Meldolesi, Jacopo

2016-08-09

Exosomes and ectosomes, extracellular vesicles of two types generated by all cells at multivesicular bodies and the plasma membrane, respectively, play critical roles in physiology and pathology. A key mechanism of their function, analogous for both types of vesicles, is the fusion of their membrane to the plasma membrane of specific target cells, followed by discharge to the cytoplasm of their luminal cargo containing proteins, RNAs, and DNA. Here we summarize the present knowledge about the interactions, binding and fusions of vesicles with the cell plasma membrane. The sequence initiates with dynamic interactions, during which vesicles roll over the plasma membrane, followed by the binding of specific membrane proteins to their cell receptors. Membrane binding is then converted rapidly into fusion by mechanisms analogous to those of retroviruses. Specifically, proteins of the extracellular vesicle membranes are structurally rearranged, and their hydrophobic sequences insert into the target cell plasma membrane which undergoes lipid reorganization, protein restructuring and membrane dimpling. Single fusions are not the only process of vesicle/cell interactions. Upon intracellular reassembly of their luminal cargoes, vesicles can be regenerated, released and fused horizontally to other target cells. Fusions of extracellular vesicles are relevant also for specific therapy processes, now intensely investigated.

Membrane Curvature Sensing by Amphipathic Helices Is Modulated by the Surrounding Protein Backbone.

PubMed

Doucet, Christine M; Esmery, Nina; de Saint-Jean, Maud; Antonny, Bruno

2015-01-01

Membrane curvature is involved in numerous biological pathways like vesicle trafficking, endocytosis or nuclear pore complex assembly. In addition to its topological role, membrane curvature is sensed by specific proteins, enabling the coordination of biological processes in space and time. Amongst membrane curvature sensors are the ALPS (Amphipathic Lipid Packing Sensors). ALPS motifs are short peptides with peculiar amphipathic properties. They are found in proteins targeted to distinct curved membranes, mostly in the early secretory pathway. For instance, the ALPS motif of the golgin GMAP210 binds trafficking vesicles, while the ALPS motif of Nup133 targets nuclear pores. It is not clear if, besides curvature sensitivity, ALPS motifs also provide target specificity, or if other domains in the surrounding protein backbone are involved. To elucidate this aspect, we studied the subcellular localization of ALPS motifs outside their natural protein context. The ALPS motifs of GMAP210 or Nup133 were grafted on artificial fluorescent probes. Importantly, ALPS motifs are held in different positions and these contrasting architectures were mimicked by the fluorescent probes. The resulting chimeras recapitulated the original proteins localization, indicating that ALPS motifs are sufficient to specifically localize proteins. Modulating the electrostatic or hydrophobic content of Nup133 ALPS motif modified its avidity for cellular membranes but did not change its organelle targeting properties. In contrast, the structure of the backbone surrounding the helix strongly influenced targeting. In particular, introducing an artificial coiled-coil between ALPS and the fluorescent protein increased membrane curvature sensitivity. This coiled-coil domain also provided membrane curvature sensitivity to the amphipathic helix of Sar1. The degree of curvature sensitivity within the coiled-coil context remains correlated to the natural curvature sensitivity of the helices. This suggests

A split ubiquitin system to reveal topology and released peptides of membrane proteins.

PubMed

Li, Qiu-Ping; Wang, Shuai; Gou, Jin-Ying

2017-09-02

Membrane proteins define biological functions of membranes in cells. Extracellular peptides of transmembrane proteins receive signals from pathogens or environments, and are the major targets of drug developments. Despite of their essential roles, membrane proteins remain elusive in topological studies due to technique difficulties in their expressions and purifications. First, the target gene is cloned into a destination vector to fuse with C terminal ubiquitin at the N or C terminus. Then, Cub vector with target gene and Nub WT or Nub G vectors are transformed into AP4 or AP5 yeast cells, respectively. After mating, the diploid cells are dipped onto selection medium to check the growth. Topology of the target protein is determined according to Table 1. We present a split ubiquitin topology (SUT) analysis system to study the topology and truncation peptide of membrane proteins in a simple yeast experiment. In the SUT system, transcription activator (TA) fused with a nucleo-cytoplasmic protein shows strong auto-activation with both positive and negative control vectors. TA fused with the cytoplasmic end of membrane proteins activates reporter genes only with positive control vector with a wild type N terminal ubiquitin (Nub WT ). However, TA fused with the extracellular termini of membrane proteins can't activate reporter genes even with Nub WT . Interestingly,TA fused with the released peptide of a membrane protein shows autoactivation in the SUT system. The SUT system is a simple and fast experimental procedure complementary to computational predictions and large scale proteomic techniques. The preliminary data from SUT are valuable for pathogen recognitions and new drug developments.

Reprogramming cellular functions with engineered membrane proteins.

PubMed

Arber, Caroline; Young, Melvin; Barth, Patrick

2017-10-01

Taking inspiration from Nature, synthetic biology utilizes and modifies biological components to expand the range of biological functions for engineering new practical devices and therapeutics. While early breakthroughs mainly concerned the design of gene circuits, recent efforts have focused on engineering signaling pathways to reprogram cellular functions. Since signal transduction across cell membranes initiates and controls intracellular signaling, membrane receptors have been targeted by diverse protein engineering approaches despite limited mechanistic understanding of their function. The modular architecture of several receptor families has enabled the empirical construction of chimeric receptors combining domains from distinct native receptors which have found successful immunotherapeutic applications. Meanwhile, progress in membrane protein structure determination, computational modeling and rational design promise to foster the engineering of a broader range of membrane receptor functions. Marrying empirical and rational membrane protein engineering approaches should enable the reprogramming of cells with widely diverse fine-tuned functions. Copyright © 2017 Elsevier Ltd. All rights reserved.

Challenges in the Development of Functional Assays of Membrane Proteins

PubMed Central

Tiefenauer, Louis; Demarche, Sophie

2012-01-01

Lipid bilayers are natural barriers of biological cells and cellular compartments. Membrane proteins integrated in biological membranes enable vital cell functions such as signal transduction and the transport of ions or small molecules. In order to determine the activity of a protein of interest at defined conditions, the membrane protein has to be integrated into artificial lipid bilayers immobilized on a surface. For the fabrication of such biosensors expertise is required in material science, surface and analytical chemistry, molecular biology and biotechnology. Specifically, techniques are needed for structuring surfaces in the micro- and nanometer scale, chemical modification and analysis, lipid bilayer formation, protein expression, purification and solubilization, and most importantly, protein integration into engineered lipid bilayers. Electrochemical and optical methods are suitable to detect membrane activity-related signals. The importance of structural knowledge to understand membrane protein function is obvious. Presently only a few structures of membrane proteins are solved at atomic resolution. Functional assays together with known structures of individual membrane proteins will contribute to a better understanding of vital biological processes occurring at biological membranes. Such assays will be utilized in the discovery of drugs, since membrane proteins are major drug targets.

Biopores/membrane proteins in synthetic polymer membranes.

PubMed

Garni, Martina; Thamboo, Sagana; Schoenenberger, Cora-Ann; Palivan, Cornelia G

2017-04-01

Mimicking cell membranes by simple models based on the reconstitution of membrane proteins in lipid bilayers represents a straightforward approach to understand biological function of these proteins. This biomimetic strategy has been extended to synthetic membranes that have advantages in terms of chemical and mechanical stability, thus providing more robust hybrid membranes. We present here how membrane proteins and biopores have been inserted both in the membrane of nanosized and microsized compartments, and in planar membranes under various conditions. Such bio-hybrid membranes have new properties (as for example, permeability to ions/molecules), and functionality depending on the specificity of the inserted biomolecules. Interestingly, membrane proteins can be functionally inserted in synthetic membranes provided these have appropriate properties to overcome the high hydrophobic mismatch between the size of the biomolecule and the membrane thickness. Functional insertion of membrane proteins and biopores in synthetic membranes of compartments or in planar membranes is possible by an appropriate selection of the amphiphilic copolymers, and conditions of the self-assembly process. These hybrid membranes have new properties and functionality based on the specificity of the biomolecules and the nature of the synthetic membranes. Bio-hybrid membranes represent new solutions for the development of nanoreactors, artificial organelles or active surfaces/membranes that, by further gaining in complexity and functionality, will promote translational applications. This article is part of a Special Issue entitled: Lipid order/lipid defects and lipid-control of protein activity edited by Dirk Schneider. Copyright © 2016. Published by Elsevier B.V.

Mutants in three novel complementation groups inhibit membrane protein insertion into and soluble protein translocation across the endoplasmic reticulum membrane of Saccharomyces cerevisiae

PubMed Central

1992-01-01

We have isolated mutants that inhibit membrane protein insertion into the ER membrane of Saccharomyces cerevisiae. The mutants were contained in three complementation groups, which we have named SEC70, SEC71, and SEC72. The mutants also inhibited the translocation of soluble proteins into the lumen of the ER, indicating that they pleiotropically affect protein transport across and insertion into the ER membrane. Surprisingly, the mutants inhibited the translocation and insertion of different proteins to drastically different degrees. We have also shown that mutations in SEC61 and SEC63, which were previously isolated as mutants inhibiting the translocation of soluble proteins, also affect the insertion of membrane proteins into the ER. Taken together our data indicate that the process of protein translocation across the ER membrane involves a much larger number of gene products than previously appreciated. Moreover, different translocation substrates appear to have different requirements for components of the cellular targeting and translocation apparatus. PMID:1730771

Membrane Bending by Protein Crowding

NASA Astrophysics Data System (ADS)

Stachowiak, Jeanne

2014-03-01

From endosomes and synaptic vesicles to the cristae of the mitochondria and the annulus of the nuclear pore, highly curved membranes are fundamental to the structure and physiology of living cells. The established view is that specific families of proteins are able to bend membranes by binding to them. For example, inherently curved proteins are thought to impose their structure on the membrane surface, while membrane-binding proteins with hydrophobic motifs are thought to insert into the membrane like wedges, driving curvature. However, computational models have recently revealed that these mechanisms would require specialized membrane-bending proteins to occupy nearly 100% of a curved membrane surface, an improbable physiological situation given the immense density and diversity of membrane-bound proteins, and the low expression levels of these specialized proteins within curved regions of the membrane. How then does curvature arise within the complex and crowded environment of cellular membranes? Our recent work using proteins involved in clathrin-mediated endocytosis, as well as engineered protein-lipid interactions, has suggested a new hypothesis - that lateral pressure generated by collisions between membrane-bound proteins can drive membrane bending. Specifically, by correlating membrane bending with quantitative optical measurements of protein density on synthetic membrane surfaces and simple physical models of collisions among membrane-bound proteins, we have demonstrated that protein-protein steric interactions can drive membrane curvature. These findings suggest that a simple imbalance in the concentration of membrane-bound proteins across a membrane surface can drive a membrane to bend, providing an efficient mechanism by which essentially any protein can contribute to shaping membranes.

High-throughput Isolation and Characterization of Untagged Membrane Protein Complexes: Outer Membrane Complexes of Desulfovibrio vulgaris

PubMed Central

2012-01-01

Cell membranes represent the “front line” of cellular defense and the interface between a cell and its environment. To determine the range of proteins and protein complexes that are present in the cell membranes of a target organism, we have utilized a “tagless” process for the system-wide isolation and identification of native membrane protein complexes. As an initial subject for study, we have chosen the Gram-negative sulfate-reducing bacterium Desulfovibrio vulgaris. With this tagless methodology, we have identified about two-thirds of the outer membrane- associated proteins anticipated. Approximately three-fourths of these appear to form homomeric complexes. Statistical and machine-learning methods used to analyze data compiled over multiple experiments revealed networks of additional protein–protein interactions providing insight into heteromeric contacts made between proteins across this region of the cell. Taken together, these results establish a D. vulgaris outer membrane protein data set that will be essential for the detection and characterization of environment-driven changes in the outer membrane proteome and in the modeling of stress response pathways. The workflow utilized here should be effective for the global characterization of membrane protein complexes in a wide range of organisms. PMID:23098413

Production of okara and soy protein concentrates using membrane technology.

PubMed

Vishwanathan, K H; Govindaraju, K; Singh, Vasudeva; Subramanian, R

2011-01-01

Microfiltration (MF) membranes with pore sizes of 200 and 450 nm and ultrafiltration (UF) membranes with molecular weight cut off of 50, 100, and 500 kDa were assessed for their ability to eliminate nonprotein substances from okara protein extract in a laboratory cross-flow membrane system. Both MF and UF improved the protein content of okara extract to a similar extent from approximately 68% to approximately 81% owing to the presence of protein in the feed leading to the formation of dynamic layer controlling the performance rather than the actual pore size of membranes. Although normalized flux in MF-450 (117 LMH/MPa) was close to UF-500 (118 LMH/MPa), the latter was selected based on higher average flux (47 LMH) offering the advantage of reduced processing time. Membrane processing of soy extract improved the protein content from 62% to 85% much closer to the target value. However, the final protein content in okara (approximately 80%) did not reach the target value (90%) owing to the greater presence of soluble fibers that were retained by the membrane. Solubility curve of membrane okara protein concentrate (MOPC) showed lower solubility than soy protein concentrate and a commercial isolate in the entire pH range. However, water absorption and fat-binding capacities of MOPC were either superior or comparable while emulsifying properties were in accordance with its solubility. The results of this study showed that okara protein concentrate (80%) could be produced using membrane technology without loss of any true proteins, thus offering value addition to okara, hitherto underutilized. Practical Application: Okara, a byproduct obtained during processing soybean for soymilk, is either underutilized or unutilized in spite of the fact that its protein quality is as good as that of soy milk and tofu. Membrane-processed protein products have been shown to possess superior functional properties compared to conventionally produced protein products. However, the

Targeting malaria parasite proteins to the erythrocyte.

PubMed

Templeton, Thomas J; Deitsch, Kirk W

2005-09-01

The intraerythrocytic stages of the protozoan parasite Plasmodium falciparum reside within a parasitophorous vacuole (PV) and set up unique "extraparasite, intraerythrocyte" protein-trafficking pathways that target parasite-encoded proteins to the erythrocyte cytoplasm and cell surface. Two recent articles report the identification of trafficking motifs that regulate the transport of parasite-encoded proteins across the PV. These articles greatly aid the annotation of the parasite "secretome" catalog of proteins that are targeted to the erythrocyte cytoplasm or cell membrane.

Characterization of the targeting signal in mitochondrial β-barrel proteins

PubMed Central

Jores, Tobias; Klinger, Anna; Groß, Lucia E.; Kawano, Shin; Flinner, Nadine; Duchardt-Ferner, Elke; Wöhnert, Jens; Kalbacher, Hubert; Endo, Toshiya; Schleiff, Enrico; Rapaport, Doron

2016-01-01

Mitochondrial β-barrel proteins are synthesized on cytosolic ribosomes and must be specifically targeted to the organelle before their integration into the mitochondrial outer membrane. The signal that assures such precise targeting and its recognition by the organelle remained obscure. In the present study we show that a specialized β-hairpin motif is this long searched for signal. We demonstrate that a synthetic β-hairpin peptide competes with the import of mitochondrial β-barrel proteins and that proteins harbouring a β-hairpin peptide fused to passenger domains are targeted to mitochondria. Furthermore, a β-hairpin motif from mitochondrial proteins targets chloroplast β-barrel proteins to mitochondria. The mitochondrial targeting depends on the hydrophobicity of the β-hairpin motif. Finally, this motif interacts with the mitochondrial import receptor Tom20. Collectively, we reveal that β-barrel proteins are targeted to mitochondria by a dedicated β-hairpin element, and this motif is recognized at the organelle surface by the outer membrane translocase. PMID:27345737

Membrane Proteins Are Dramatically Less Conserved than Water-Soluble Proteins across the Tree of Life

PubMed Central

Sojo, Victor; Dessimoz, Christophe; Pomiankowski, Andrew; Lane, Nick

2016-01-01

Membrane proteins are crucial in transport, signaling, bioenergetics, catalysis, and as drug targets. Here, we show that membrane proteins have dramatically fewer detectable orthologs than water-soluble proteins, less than half in most species analyzed. This sparse distribution could reflect rapid divergence or gene loss. We find that both mechanisms operate. First, membrane proteins evolve faster than water-soluble proteins, particularly in their exterior-facing portions. Second, we demonstrate that predicted ancestral membrane proteins are preferentially lost compared with water-soluble proteins in closely related species of archaea and bacteria. These patterns are consistent across the whole tree of life, and in each of the three domains of archaea, bacteria, and eukaryotes. Our findings point to a fundamental evolutionary principle: membrane proteins evolve faster due to stronger adaptive selection in changing environments, whereas cytosolic proteins are under more stringent purifying selection in the homeostatic interior of the cell. This effect should be strongest in prokaryotes, weaker in unicellular eukaryotes (with intracellular membranes), and weakest in multicellular eukaryotes (with extracellular homeostasis). We demonstrate that this is indeed the case. Similarly, we show that extracellular water-soluble proteins exhibit an even stronger pattern of low homology than membrane proteins. These striking differences in conservation of membrane proteins versus water-soluble proteins have important implications for evolution and medicine. PMID:27501943

Membrane-Mediated Cooperativity of Proteins

NASA Astrophysics Data System (ADS)

Weikl, Thomas R.

2018-04-01

Besides direct protein-protein interactions, indirect interactions mediated by membranes play an important role for the assembly and cooperative function of proteins in membrane shaping and adhesion. The intricate shapes of biological membranes are generated by proteins that locally induce membrane curvature. Indirect curvature-mediated interactions between these proteins arise because the proteins jointly affect the bending energy of the membranes. These curvature-mediated interactions are attractive for crescent-shaped proteins and are a driving force in the assembly of the proteins during membrane tubulation. Membrane adhesion results from the binding of receptor and ligand proteins that are anchored in the apposing membranes. The binding of these proteins strongly depends on nanoscale shape fluctuations of the membranes, leading to a fluctuation-mediated binding cooperativity. A length mismatch between receptor-ligand complexes in membrane adhesion zones causes repulsive curvature-mediated interactions that are a driving force for the length-based segregation of proteins during membrane adhesion.

Direct Capture of Functional Proteins from Mammalian Plasma Membranes into Nanodiscs.

PubMed

Roy, Jahnabi; Pondenis, Holly; Fan, Timothy M; Das, Aditi

2015-10-20

Mammalian plasma membrane proteins make up the largest class of drug targets yet are difficult to study in a cell free system because of their intransigent nature. Herein, we perform direct encapsulation of plasma membrane proteins derived from mammalian cells into a functional nanodisc library. Peptide fingerprinting was used to analyze the proteome of the incorporated proteins in nanodiscs and to further demonstrate that the lipid composition of the nanodiscs directly affects the class of protein that is incorporated. Furthermore, the functionality of the incorporated membrane proteome was evaluated by measuring the activity of membrane proteins: Na(+)/K(+)-ATPase and receptor tyrosine kinases. This work is the first report of the successful establishment and characterization of a cell free functional library of mammalian membrane proteins into nanodiscs.

Vertebrate Membrane Proteins: Structure, Function, and Insights from Biophysical Approaches

PubMed Central

MÜLLER, DANIEL J.; WU, NAN; PALCZEWSKI, KRZYSZTOF

2008-01-01

Membrane proteins are key targets for pharmacological intervention because they are vital for cellular function. Here, we analyze recent progress made in the understanding of the structure and function of membrane proteins with a focus on rhodopsin and development of atomic force microscopy techniques to study biological membranes. Membrane proteins are compartmentalized to carry out extra- and intracellular processes. Biological membranes are densely populated with membrane proteins that occupy approximately 50% of their volume. In most cases membranes contain lipid rafts, protein patches, or paracrystalline formations that lack the higher-order symmetry that would allow them to be characterized by diffraction methods. Despite many technical difficulties, several crystal structures of membrane proteins that illustrate their internal structural organization have been determined. Moreover, high-resolution atomic force microscopy, near-field scanning optical microscopy, and other lower resolution techniques have been used to investigate these structures. Single-molecule force spectroscopy tracks interactions that stabilize membrane proteins and those that switch their functional state; this spectroscopy can be applied to locate a ligand-binding site. Recent development of this technique also reveals the energy landscape of a membrane protein, defining its folding, reaction pathways, and kinetics. Future development and application of novel approaches during the coming years should provide even greater insights to the understanding of biological membrane organization and function. PMID:18321962

On the targeting and membrane assembly of the Escherichia coli outer membrane porin, PhoE.

PubMed

Phoenix, D A

1996-12-01

Within gram-negative bacteria such as Escherichia coli, the outer membrane porins provide a relatively non-specific uptake route which is utilised by a wide range of solutes including many antibiotics. Understanding the targeting and membrane assembly of these proteins is therefore of importance and this mini review aims to discuss this process in light of present knowledge.

Rigid proteins and softening of biological membranes-with application to HIV-induced cell membrane softening.

PubMed

Agrawal, Himani; Zelisko, Matthew; Liu, Liping; Sharma, Pradeep

2016-05-06

A key step in the HIV-infection process is the fusion of the virion membrane with the target cell membrane and the concomitant transfer of the viral RNA. Experimental evidence suggests that the fusion is preceded by considerable elastic softening of the cell membranes due to the insertion of fusion peptide in the membrane. What are the mechanisms underpinning the elastic softening of the membrane upon peptide insertion? A broader question may be posed: insertion of rigid proteins in soft membranes ought to stiffen the membranes not soften them. However, experimental observations perplexingly appear to show that rigid proteins may either soften or harden membranes even though conventional wisdom only suggests stiffening. In this work, we argue that regarding proteins as merely non-specific rigid inclusions is flawed, and each protein has a unique mechanical signature dictated by its specific interfacial coupling to the surrounding membrane. Predicated on this hypothesis, we have carried out atomistic simulations to investigate peptide-membrane interactions. Together with a continuum model, we reconcile contrasting experimental data in the literature including the case of HIV-fusion peptide induced softening. We conclude that the structural rearrangements of the lipids around the inclusions cause the softening or stiffening of the biological membranes.

Cytosolic proteins can exploit membrane localization to trigger functional assembly

PubMed Central

2018-01-01

Cell division, endocytosis, and viral budding would not function without the localization and assembly of protein complexes on membranes. What is poorly appreciated, however, is that by localizing to membranes, proteins search in a reduced space that effectively drives up concentration. Here we derive an accurate and practical analytical theory to quantify the significance of this dimensionality reduction in regulating protein assembly on membranes. We define a simple metric, an effective equilibrium constant, that allows for quantitative comparison of protein-protein interactions with and without membrane present. To test the importance of membrane localization for driving protein assembly, we collected the protein-protein and protein-lipid affinities, protein and lipid concentrations, and volume-to-surface-area ratios for 46 interactions between 37 membrane-targeting proteins in human and yeast cells. We find that many of the protein-protein interactions between pairs of proteins involved in clathrin-mediated endocytosis in human and yeast cells can experience enormous increases in effective protein-protein affinity (10–1000 fold) due to membrane localization. Localization of binding partners thus triggers robust protein complexation, suggesting that it can play an important role in controlling the timing of endocytic protein coat formation. Our analysis shows that several other proteins involved in membrane remodeling at various organelles have similar potential to exploit localization. The theory highlights the master role of phosphoinositide lipid concentration, the volume-to-surface-area ratio, and the ratio of 3D to 2D equilibrium constants in triggering (or preventing) constitutive assembly on membranes. Our simple model provides a novel quantitative framework for interpreting or designing in vitro experiments of protein complexation influenced by membrane binding. PMID:29505559

Topological Transitions in Mitochondrial Membranes controlled by Apoptotic Proteins

NASA Astrophysics Data System (ADS)

Hwee Lai, Ghee; Sanders, Lori K.; Mishra, Abhijit; Schmidt, Nathan W.; Wong, Gerard C. L.; Ivashyna, Olena; Schlesinger, Paul H.

2010-03-01

The Bcl-2 family comprises pro-apoptotic proteins, capable of permeabilizing the mitochondrial membrane, and anti-apoptotic members interacting in an antagonistic fashion to regulate programmed cell death (apoptosis). They offer potential therapeutic targets to re-engage cellular suicide in tumor cells but the extensive network of implicated protein-protein interactions has impeded full understanding of the decision pathway. We show, using synchrotron x-ray diffraction, that pro-apoptotic proteins interact with mitochondrial-like model membranes to generate saddle-splay (negative Gaussian) curvature topologically required for pore formation, while anti-apoptotic proteins can deactivate curvature generation by molecules drastically different from Bcl-2 family members and offer evidence for membrane-curvature mediated interactions general enough to affect very disparate systems.

Host and viral RNA-binding proteins involved in membrane targeting, replication and intercellular movement of plant RNA virus genomes

PubMed Central

Hyodo, Kiwamu; Kaido, Masanori; Okuno, Tetsuro

2014-01-01

Many plant viruses have positive-strand RNA [(+)RNA] as their genome. Therefore, it is not surprising that RNA-binding proteins (RBPs) play important roles during (+)RNA virus infection in host plants. Increasing evidence demonstrates that viral and host RBPs play critical roles in multiple steps of the viral life cycle, including translation and replication of viral genomic RNAs, and their intra- and intercellular movement. Although studies focusing on the RNA-binding activities of viral and host proteins, and their associations with membrane targeting, and intercellular movement of viral genomes have been limited to a few viruses, these studies have provided important insights into the molecular mechanisms underlying the replication and movement of viral genomic RNAs. In this review, we briefly overview the currently defined roles of viral and host RBPs whose RNA-binding activity have been confirmed experimentally in association with their membrane targeting, and intercellular movement of plant RNA virus genomes. PMID:25071804

Interactions of Ras proteins with the plasma membrane and their roles in signaling.

PubMed

Eisenberg, Sharon; Henis, Yoav I

2008-01-01

The complex dynamic structure of the plasma membrane plays critical roles in cellular signaling; interactions with the membrane lipid milieu, spatial segregation within and between cellular membranes and/or targeting to specific membrane-associated scaffolds are intimately involved in many signal transduction pathways. In this review, we focus on the membrane interactions of Ras proteins. These small GTPases play central roles in the regulation of cell growth and proliferation, and their excessive activation is commonly encountered in human tumors. Ras proteins associate with the membrane continuously via C-terminal lipidation and additional interactions in both their inactive and active forms; this association, as well as the targeting of specific Ras isoforms to plasma membrane microdomains and to intracellular organelles, have recently been implicated in Ras signaling and oncogenic potential. We discuss biochemical and biophysical evidence for the roles of specific domains of Ras proteins in mediating their association with the plasma membrane, and consider the potential effects of lateral segregation and interactions with membrane-associated protein assemblies on the signaling outcomes.

Membrane Proteins Are Dramatically Less Conserved than Water-Soluble Proteins across the Tree of Life.

PubMed

Sojo, Victor; Dessimoz, Christophe; Pomiankowski, Andrew; Lane, Nick

2016-11-01

Membrane proteins are crucial in transport, signaling, bioenergetics, catalysis, and as drug targets. Here, we show that membrane proteins have dramatically fewer detectable orthologs than water-soluble proteins, less than half in most species analyzed. This sparse distribution could reflect rapid divergence or gene loss. We find that both mechanisms operate. First, membrane proteins evolve faster than water-soluble proteins, particularly in their exterior-facing portions. Second, we demonstrate that predicted ancestral membrane proteins are preferentially lost compared with water-soluble proteins in closely related species of archaea and bacteria. These patterns are consistent across the whole tree of life, and in each of the three domains of archaea, bacteria, and eukaryotes. Our findings point to a fundamental evolutionary principle: membrane proteins evolve faster due to stronger adaptive selection in changing environments, whereas cytosolic proteins are under more stringent purifying selection in the homeostatic interior of the cell. This effect should be strongest in prokaryotes, weaker in unicellular eukaryotes (with intracellular membranes), and weakest in multicellular eukaryotes (with extracellular homeostasis). We demonstrate that this is indeed the case. Similarly, we show that extracellular water-soluble proteins exhibit an even stronger pattern of low homology than membrane proteins. These striking differences in conservation of membrane proteins versus water-soluble proteins have important implications for evolution and medicine. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

The Charcot Marie Tooth disease protein LITAF is a zinc-binding monotopic membrane protein

PubMed Central

Qin, Wenxia; Wunderley, Lydia; Barrett, Anne L.; High, Stephen; Woodman, Philip G.

2016-01-01

LITAF (LPS-induced TNF-activating factor) is an endosome-associated integral membrane protein important for multivesicular body sorting. Several mutations in LITAF cause autosomal-dominant Charcot Marie Tooth disease type 1C. These mutations map to a highly conserved C-terminal region, termed the LITAF domain, which includes a 22 residue hydrophobic sequence and flanking cysteine-rich regions that contain peptide motifs found in zinc fingers. Although the LITAF domain is thought to be responsible for membrane integration, the membrane topology of LITAF has not been established. Here, we have investigated whether LITAF is a tail-anchored (TA) membrane-spanning protein or monotopic membrane protein. When translated in vitro, LITAF integrates poorly into ER-derived microsomes compared with Sec61β, a bona fide TA protein. Furthermore, introduction of N-linked glycosylation reporters shows that neither the N-terminal nor C-terminal domains of LITAF translocate into the ER lumen. Expression in cells of an LITAF construct containing C-terminal glycosylation sites confirms that LITAF is not a TA protein in cells. Finally, an immunofluorescence-based latency assay showed that both the N- and C-termini of LITAF are exposed to the cytoplasm. Recombinant LITAF contains 1 mol/mol zinc, while mutation of predicted zinc-binding residues disrupts LITAF membrane association. Hence, we conclude that LITAF is a monotopic membrane protein whose membrane integration is stabilised by a zinc finger. The related human protein, CDIP1 (cell death involved p53 target 1), displays identical membrane topology, suggesting that this mode of membrane integration is conserved in LITAF family proteins. PMID:27582497

The interactions of peripheral membrane proteins with biological membranes

DOE PAGES

Johs, Alexander; Whited, A. M.

2015-07-29

The interactions of peripheral proteins with membrane surfaces are critical to many biological processes, including signaling, recognition, membrane trafficking, cell division and cell structure. On a molecular level, peripheral membrane proteins can modulate lipid composition, membrane dynamics and protein-protein interactions. Biochemical and biophysical studies have shown that these interactions are in fact highly complex, dominated by several different types of interactions, and have an interdependent effect on both the protein and membrane. Here we examine three major mechanisms underlying the interactions between peripheral membrane proteins and membranes: electrostatic interactions, hydrophobic interactions, and fatty acid modification of proteins. While experimental approachesmore » continue to provide critical insights into specific interaction mechanisms, emerging bioinformatics resources and tools contribute to a systems-level picture of protein-lipid interactions. Through these recent advances, we begin to understand the pivotal role of protein-lipid interactions underlying complex biological functions at membrane interfaces.« less

Gibberellin DELLA signaling targets the retromer complex to redirect protein trafficking to the plasma membrane

PubMed Central

Salanenka, Yuliya; Verstraeten, Inge; Löfke, Christian; Tabata, Kaori; Naramoto, Satoshi; Glanc, Matouš; Friml, Jiří

2018-01-01

The plant hormone gibberellic acid (GA) is a crucial regulator of growth and development. The main paradigm of GA signaling puts forward transcriptional regulation via the degradation of DELLA transcriptional repressors. GA has also been shown to regulate tropic responses by modulation of the plasma membrane incidence of PIN auxin transporters by an unclear mechanism. Here we uncovered the cellular and molecular mechanisms by which GA redirects protein trafficking and thus regulates cell surface functionality. Photoconvertible reporters revealed that GA balances the protein traffic between the vacuole degradation route and recycling back to the cell surface. Low GA levels promote vacuolar delivery and degradation of multiple cargos, including PIN proteins, whereas high GA levels promote their recycling to the plasma membrane. This GA effect requires components of the retromer complex, such as Sorting Nexin 1 (SNX1) and its interacting, microtubule (MT)-associated protein, the Cytoplasmic Linker-Associated Protein (CLASP1). Accordingly, GA regulates the subcellular distribution of SNX1 and CLASP1, and the intact MT cytoskeleton is essential for the GA effect on trafficking. This GA cellular action occurs through DELLA proteins that regulate the MT and retromer presumably via their interaction partners Prefoldins (PFDs). Our study identified a branching of the GA signaling pathway at the level of DELLA proteins, which, in parallel to regulating transcription, also target by a nontranscriptional mechanism the retromer complex acting at the intersection of the degradation and recycling trafficking routes. By this mechanism, GA can redirect receptors and transporters to the cell surface, thus coregulating multiple processes, including PIN-dependent auxin fluxes during tropic responses. PMID:29463731

The synthesis of recombinant membrane proteins in yeast for structural studies.

PubMed

Routledge, Sarah J; Mikaliunaite, Lina; Patel, Anjana; Clare, Michelle; Cartwright, Stephanie P; Bawa, Zharain; Wilks, Martin D B; Low, Floren; Hardy, David; Rothnie, Alice J; Bill, Roslyn M

2016-02-15

Historically, recombinant membrane protein production has been a major challenge meaning that many fewer membrane protein structures have been published than those of soluble proteins. However, there has been a recent, almost exponential increase in the number of membrane protein structures being deposited in the Protein Data Bank. This suggests that empirical methods are now available that can ensure the required protein supply for these difficult targets. This review focuses on methods that are available for protein production in yeast, which is an important source of recombinant eukaryotic membrane proteins. We provide an overview of approaches to optimize the expression plasmid, host cell and culture conditions, as well as the extraction and purification of functional protein for crystallization trials in preparation for structural studies. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Membrane proteins from the cyanobacterium Synechocystis sp. PCC 6803 interacting with thioredoxin.

PubMed

Mata-Cabana, Alejandro; Florencio, Francisco J; Lindahl, Marika

2007-11-01

Cysteine dithiol/disulphide exchange forms the molecular basis for regulation of a wide variety of enzymatic activities and for transduction of cellular signals. Thus, the search for proteins with reactive, accessible cysteines is expected to contribute to the unravelling of new molecular mechanisms for enzyme regulation and signal transduction. Several methods have been designed for this purpose taking advantage of the interactions between thioredoxins and their protein substrates. Thioredoxins comprise a family of redox-active enzymes, which catalyse reduction of protein disulphides and sulphenic acids. Due to the inherent practical difficulties associated with studies of membrane proteins these have been largely overlooked in the many proteomic studies of thioredoxin-interacting proteins. In the present work, we have developed a procedure to isolate membrane proteins interacting with thioredoxin by binding in situ to a monocysteinic His-tagged thioredoxin added directly to the intact membranes. Following fractionation and solubilisation of the membranes, thioredoxin target proteins were isolated by Ni-affinity chromatography and 2-DE SDS-PAGE under nonreducing/reducing conditions. Applying this method to total membranes, including thylakoid and plasma membranes, from the cyanobacterium Synechocystis sp. PCC 6803 we have identified 50 thioredoxin-interacting proteins. Among the 38 newly identified thioredoxin targets are the ATP-binding subunits of several transporters and members of the AAA-family of ATPases.

Identification of a Membrane Targeting and Degradation Signal in the p42 Protein of Influenza C Virus

PubMed Central

Pekosz, Andrew; Lamb, Robert A.

2000-01-01

Two mRNA species are derived from the influenza C virus RNA segment six, (i) a colinear transcript containing a 374-amino-acid residue open reading frame (referred to herein as the seg 6 ORF) which is translated to yield the p42 protein, and (ii) a spliced mRNA which encodes the influenza C virus matrix (CM1) protein consisting of the first 242 amino acids of p42. The p42 protein undergoes proteolytic cleavage at a consensus signal peptidase cleavage site after residue 259, yielding the p31 and CM2 proteins. Translocation of p42 into the endoplasmic reticulum membrane occurs cotranslationally and requires the hydrophobic internal signal peptide (residues 239 to 259), as well as the predicted transmembrane domain of CM2 (residues 285 to 308). The p31 protein was found to undergo rapid degradation after cleavage from p42. Addition of the 26S proteasome inhibitor lactacystin to influenza C virus-infected or seg 6 ORF cDNA-transfected cells drastically reduced p31 degradation. Transfer of the 17-residue C-terminal region of p31 to heterologous proteins resulted in their rapid turnover. The hydrophobic nature, but not the specific amino acid sequence of the 17-amino-acid C terminus of p31 appears to act as the signal for targeting the protein to membranes and for degradation. PMID:11044092

The mitochondrial outer membrane protein MDI promotes local protein synthesis and mtDNA replication.

PubMed

Zhang, Yi; Chen, Yong; Gucek, Marjan; Xu, Hong

2016-05-17

Early embryonic development features rapid nuclear DNA replication cycles, but lacks mtDNA replication. To meet the high-energy demands of embryogenesis, mature oocytes are furnished with vast amounts of mitochondria and mtDNA However, the cellular machinery driving massive mtDNA replication in ovaries remains unknown. Here, we describe a Drosophila AKAP protein, MDI that recruits a translation stimulator, La-related protein (Larp), to the mitochondrial outer membrane in ovaries. The MDI-Larp complex promotes the synthesis of a subset of nuclear-encoded mitochondrial proteins by cytosolic ribosomes on the mitochondrial surface. MDI-Larp's targets include mtDNA replication factors, mitochondrial ribosomal proteins, and electron-transport chain subunits. Lack of MDI abolishes mtDNA replication in ovaries, which leads to mtDNA deficiency in mature eggs. Targeting Larp to the mitochondrial outer membrane independently of MDI restores local protein synthesis and rescues the phenotypes of mdi mutant flies. Our work suggests that a selective translational boost by the MDI-Larp complex on the outer mitochondrial membrane might be essential for mtDNA replication and mitochondrial biogenesis during oogenesis. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

A cDNA Immunization Strategy to Generate Nanobodies against Membrane Proteins in Native Conformation

PubMed Central

Eden, Thomas; Menzel, Stephan; Wesolowski, Janusz; Bergmann, Philine; Nissen, Marion; Dubberke, Gudrun; Seyfried, Fabienne; Albrecht, Birte; Haag, Friedrich; Koch-Nolte, Friedrich

2018-01-01

Nanobodies (Nbs) are soluble, versatile, single-domain binding modules derived from the VHH variable domain of heavy-chain antibodies naturally occurring in camelids. Nbs hold huge promise as novel therapeutic biologics. Membrane proteins are among the most interesting targets for therapeutic Nbs because they are accessible to systemically injected biologics. In order to be effective, therapeutic Nbs must recognize their target membrane protein in native conformation. However, raising Nbs against membrane proteins in native conformation can pose a formidable challenge since membrane proteins typically contain one or more hydrophobic transmembrane regions and, therefore, are difficult to purify in native conformation. Here, we describe a highly efficient genetic immunization strategy that circumvents these difficulties by driving expression of the target membrane protein in native conformation by cells of the immunized camelid. The strategy encompasses ballistic transfection of skin cells with cDNA expression plasmids encoding one or more orthologs of the membrane protein of interest and, optionally, other costimulatory proteins. The plasmid is coated onto 1 µm gold particles that are then injected into the shaved and depilated skin of the camelid. A gene gun delivers a helium pulse that accelerates the DNA-coated particles to a velocity sufficient to penetrate through multiple layers of cells in the skin. This results in the exposure of the extracellular domains of the membrane protein on the cell surface of transfected cells. Repeated immunization drives somatic hypermutation and affinity maturation of target-specific heavy-chain antibodies. The VHH/Nb coding region is PCR-amplified from B cells obtained from peripheral blood or a lymph node biopsy. Specific Nbs are selected by phage display or by screening of Nb-based heavy-chain antibodies expressed as secretory proteins in transfected HEK cells. Using this strategy, we have successfully generated agonistic

The cytoplasmic domain of the gamete membrane fusion protein HAP2 targets the protein to the fusion site in Chlamydomonas and regulates the fusion reaction.

PubMed

Liu, Yanjie; Pei, Jimin; Grishin, Nick; Snell, William J

2015-03-01

Cell-cell fusion between gametes is a defining step during development of eukaryotes, yet we know little about the cellular and molecular mechanisms of the gamete membrane fusion reaction. HAP2 is the sole gamete-specific protein in any system that is broadly conserved and shown by gene disruption to be essential for gamete fusion. The wide evolutionary distribution of HAP2 (also known as GCS1) indicates it was present in the last eukaryotic common ancestor and, therefore, dissecting its molecular properties should provide new insights into fundamental features of fertilization. HAP2 acts at a step after membrane adhesion, presumably directly in the merger of the lipid bilayers. Here, we use the unicellular alga Chlamydomonas to characterize contributions of key regions of HAP2 to protein location and function. We report that mutation of three strongly conserved residues in the ectodomain has no effect on targeting or fusion, although short deletions that include those residues block surface expression and fusion. Furthermore, HAP2 lacking a 237-residue segment of the cytoplasmic region is expressed at the cell surface, but fails to localize at the apical membrane patch specialized for fusion and fails to rescue fusion. Finally, we provide evidence that the ancient HAP2 contained a juxta-membrane, multi-cysteine motif in its cytoplasmic region, and that mutation of a cysteine dyad in this motif preserves protein localization, but substantially impairs HAP2 fusion activity. Thus, the ectodomain of HAP2 is essential for its surface expression, and the cytoplasmic region targets HAP2 to the site of fusion and regulates the fusion reaction. © 2015. Published by The Company of Biologists Ltd.

Protein-centric N-glycoproteomics analysis of membrane and plasma membrane proteins.

PubMed

Sun, Bingyun; Hood, Leroy

2014-06-06

The advent of proteomics technology has transformed our understanding of biological membranes. The challenges for studying membrane proteins have inspired the development of many analytical and bioanalytical tools, and the techniques of glycoproteomics have emerged as an effective means to enrich and characterize membrane and plasma-membrane proteomes. This Review summarizes the development of various glycoproteomics techniques to overcome the hurdles formed by the unique structures and behaviors of membrane proteins with a focus on N-glycoproteomics. Example contributions of N-glycoproteomics to the understanding of membrane biology are provided, and the areas that require future technical breakthroughs are discussed.

Expression of membrane targeted aequorin in Xenopus laevis oocytes.

PubMed

Daguzan, C; Nicolas, M T; Mazars, C; Leclerc, C; Moreau, M

1995-08-01

We described here a system for high level of expression of the calcium activated photoprotein aequorin. This protein has been targeted to the plasma membrane of Xenopus oocyte by nuclear microinjection of a plasmid containing a construction of a chimeric cDNA encoding a fusion protein composed of the photoprotein aequorin and the 5-HT1A receptor. The expression of this fusion protein is placed under the control of RSV promoter. Functional photoprotein was reconstituted in the oocyte by incubation with coelenterazine. The amount of photoprotein 24 h after nuclear microinjection of the plasmid was sufficient to trigger a detectable light emission following calcium entry. The efficiency of the expression is correlated with the dose of plasmid injected. Intracytoplasmic injection of the plasmid always failed in photoprotein expression. Targeting of the apoprotein was demonstrated by immunolocalization under confocal microscopy. In our experimental conditions, the apoprotein was always localized at the animal pole above the nucleus. We never observed expression and targeting to the plasma membrane of the vegetal pole. WE suggest that such expression might be of great interest for the study of numerous problems of developmental biology, in which calcium-dependent pathways are involved.

New Potent Membrane-Targeting Antibacterial Peptides from Viral Capsid Proteins

PubMed Central

Dias, Susana A.; Freire, João M.; Pérez-Peinado, Clara; Domingues, Marco M.; Gaspar, Diana; Vale, Nuno; Gomes, Paula; Andreu, David; Henriques, Sónia T.; Castanho, Miguel A. R. B.; Veiga, Ana S.

2017-01-01

The increasing prevalence of multidrug-resistant bacteria urges the development of new antibacterial agents. With a broad spectrum activity, antimicrobial peptides have been considered potential antibacterial drug leads. Using bioinformatic tools we have previously shown that viral structural proteins are a rich source for new bioactive peptide sequences, namely antimicrobial and cell-penetrating peptides. Here, we test the efficacy and mechanism of action of the most promising peptides among those previously identified against both Gram-positive and Gram-negative bacteria. Two cell-penetrating peptides, vCPP 0769 and vCPP 2319, have high antibacterial activity against Staphylococcus aureus, MRSA, Escherichia coli, and Pseudomonas aeruginosa, being thus multifunctional. The antibacterial mechanism of action of the two most active viral protein-derived peptides, vAMP 059 and vCPP 2319, was studied in detail. Both peptides act on both Gram-positive S. aureus and Gram-negative P. aeruginosa, with bacterial cell death occurring within minutes. Also, these peptides cause bacterial membrane permeabilization and damage of the bacterial envelope of P. aeruginosa cells. Overall, the results show that structural viral proteins are an abundant source for membrane-active peptides sequences with strong antibacterial properties. PMID:28522994

Protein secretion and membrane insertion systems in gram-negative bacteria.

PubMed

Saier, Milton H

2006-01-01

In contrast to other organisms, gram-negative bacteria have evolved numerous systems for protein export. Eight types are known that mediate export across or insertion into the cytoplasmic membrane, while eight specifically mediate export across or insertion into the outer membrane. Three of the former secretory pathway (SP) systems, type I SP (ISP, ABC), IIISP (Fla/Path) and IVSP (Conj/Vir), can export proteins across both membranes in a single energy-coupled step. A fourth generalized mechanism for exporting proteins across the two-membrane envelope in two distinct steps (which we here refer to as type II secretory pathways [IISP]) utilizes either the general secretory pathway (GSP or Sec) or the twin-arginine targeting translocase for translocation across the inner membrane, and either the main terminal branch or one of several protein-specific export systems for translocation across the outer membrane. We here survey the various well-characterized protein translocation systems found in living organisms and then focus on the systems present in gram-negative bacteria. Comparisons between these systems suggest specific biogenic, mechanistic and evolutionary similarities as well as major differences.

Conformationally Induced Off-On Cell Membrane Chemosensor Targeting Receptor Protein-Tyrosine Kinases for in Vivo and in Vitro Fluorescence Imaging of Cancers.

PubMed

Jiao, Yang; Yin, Jiqiu; He, Haiyang; Peng, Xiaojun; Gao, Qianmiao; Duan, Chunying

2018-05-09

Molecules capable of monitoring receptor protein-tyrosine kinase expression could potentially serve as useful tools for cancer diagnosis due to the overexpression of tyrosine kinases during tumor growth and metastasis. In this work, a conformationally induced "off-on" tyrosine kinase cell membrane fluorescent sensor (SP1) was designed and evaluated for the detection and imaging of receptor protein-tyrosine kinases in vivo and in vitro. SP1 consists of sunitinib and pyrene linked via hexamethylenediamine and displays quenched fluorescence as a dimer. The fluorescence of SP1 is restored in the presence of receptor protein-tyrosine kinases upon strong interaction with SP1 at the target terminal. The unique signal response mechanism enables SP1 use for fluorescence microscopy imaging of receptor protein-tyrosine kinases in the cell membranes of living cells, allowing for the rapid differentiation of cancer cells from normal cells. SP1 can be used to visualize the chick embryo chorioallantoic membrane and mouse model tumors, suggesting its possible application for early cancer diagnosis.

ESCRT-dependent degradation of ubiquitylated plasma membrane proteins in plants.

PubMed

Isono, Erika; Kalinowska, Kamila

2017-12-01

To control the abundance of plasma membrane receptors and transporters is crucial for proper perception and response to extracellular signals from surrounding cells and the environment. Posttranslational modification of plasma membrane proteins, especially ubiquitin conjugation or ubiquitylation, is key for the determination of stability for many transmembrane proteins localized on the cell surface. The targeted degradation is ensured by a complex network of proteins among which the endosomal sorting complex required for transport (ESCRT) plays a central role. This review focuses on progresses made in recent years on the understanding of the function of the ESCRT machinery in the degradation of ubiquitylated plasma membrane proteins in plants. Copyright © 2017 Elsevier Ltd. All rights reserved.

Transmembrane protein sorting driven by membrane curvature

NASA Astrophysics Data System (ADS)

Strahl, H.; Ronneau, S.; González, B. Solana; Klutsch, D.; Schaffner-Barbero, C.; Hamoen, L. W.

2015-11-01

The intricate structure of prokaryotic and eukaryotic cells depends on the ability to target proteins to specific cellular locations. In most cases, we have a poor understanding of the underlying mechanisms. A typical example is the assembly of bacterial chemoreceptors at cell poles. Here we show that the classical chemoreceptor TlpA of Bacillus subtilis does not localize according to the consensus stochastic nucleation mechanism but accumulates at strongly curved membrane areas generated during cell division. This preference was confirmed by accumulation at non-septal curved membranes. Localization appears to be an intrinsic property of the protein complex and does not rely on chemoreceptor clustering, as was previously shown for Escherichia coli. By constructing specific amino-acid substitutions, we demonstrate that the preference for strongly curved membranes arises from the curved shape of chemoreceptor trimer of dimers. These findings demonstrate that the intrinsic shape of transmembrane proteins can determine their cellular localization.

A proteomic screen reveals the mitochondrial outer membrane protein Mdm34p as an essential target of the F-box protein Mdm30p.

PubMed

Ota, Kazuhisa; Kito, Keiji; Okada, Satoshi; Ito, Takashi

2008-10-01

Ubiquitination plays various critical roles in eukaryotic cellular regulation and is mediated by a cascade of enzymes including ubiquitin protein ligase (E3). The Skp1-Cullin-F-box protein complex comprises the largest E3 family, in each member of which a unique F-box protein binds its targets to define substrate specificity. Although genome sequencing uncovers a growing number of F-box proteins, most of them have remained as "orphans" because of the difficulties in identification of their substrates. To address this issue, we tested a quantitative proteomic approach by combining the stable isotope labeling by amino acids in cell culture (SILAC), parallel affinity purification (PAP) that we had developed for efficient enrichment of ubiquitinated proteins, and mass spectrometry (MS). We applied this SILAC-PAP-MS approach to compare ubiquitinated proteins between yeast cells with and without over-expressed Mdm30p, an F-box protein implicated in mitochondrial morphology. Consequently, we identified the mitochondrial outer membrane protein Mdm34p as a target of Mdm30p. Furthermore, we found that mitochondrial defects induced by deletion of MDM30 are not only recapitulated by a mutant Mdm34p defective in interaction with Mdm30p but alleviated by ubiquitination-mimicking forms of Mdm34p. These results indicate that Mdm34p is a physiologically important target of Mdm30p.

Identification of new intrinsic proteins in Arabidopsis plasma membrane proteome.

PubMed

Marmagne, Anne; Rouet, Marie-Aude; Ferro, Myriam; Rolland, Norbert; Alcon, Carine; Joyard, Jacques; Garin, Jérome; Barbier-Brygoo, Hélène; Ephritikhine, Geneviève

2004-07-01

Identification and characterization of anion channel genes in plants represent a goal for a better understanding of their central role in cell signaling, osmoregulation, nutrition, and metabolism. Though channel activities have been well characterized in plasma membrane by electrophysiology, the corresponding molecular entities are little documented. Indeed, the hydrophobic protein equipment of plant plasma membrane still remains largely unknown, though several proteomic approaches have been reported. To identify new putative transport systems, we developed a new proteomic strategy based on mass spectrometry analyses of a plasma membrane fraction enriched in hydrophobic proteins. We produced from Arabidopsis cell suspensions a highly purified plasma membrane fraction and characterized it in detail by immunological and enzymatic tests. Using complementary methods for the extraction of hydrophobic proteins and mass spectrometry analyses on mono-dimensional gels, about 100 proteins have been identified, 95% of which had never been found in previous proteomic studies. The inventory of the plasma membrane proteome generated by this approach contains numerous plasma membrane integral proteins, one-third displaying at least four transmembrane segments. The plasma membrane localization was confirmed for several proteins, therefore validating such proteomic strategy. An in silico analysis shows a correlation between the putative functions of the identified proteins and the expected roles for plasma membrane in transport, signaling, cellular traffic, and metabolism. This analysis also reveals 10 proteins that display structural properties compatible with transport functions and will constitute interesting targets for further functional studies.

Properties of Protein Drug Target Classes

PubMed Central

Bull, Simon C.; Doig, Andrew J.

2015-01-01

Accurate identification of drug targets is a crucial part of any drug development program. We mined the human proteome to discover properties of proteins that may be important in determining their suitability for pharmaceutical modulation. Data was gathered concerning each protein’s sequence, post-translational modifications, secondary structure, germline variants, expression profile and drug target status. The data was then analysed to determine features for which the target and non-target proteins had significantly different values. This analysis was repeated for subsets of the proteome consisting of all G-protein coupled receptors, ion channels, kinases and proteases, as well as proteins that are implicated in cancer. Machine learning was used to quantify the proteins in each dataset in terms of their potential to serve as a drug target. This was accomplished by first inducing a random forest that could distinguish between its targets and non-targets, and then using the random forest to quantify the drug target likeness of the non-targets. The properties that can best differentiate targets from non-targets were primarily those that are directly related to a protein’s sequence (e.g. secondary structure). Germline variants, expression levels and interactions between proteins had minimal discriminative power. Overall, the best indicators of drug target likeness were found to be the proteins’ hydrophobicities, in vivo half-lives, propensity for being membrane bound and the fraction of non-polar amino acids in their sequences. In terms of predicting potential targets, datasets of proteases, ion channels and cancer proteins were able to induce random forests that were highly capable of distinguishing between targets and non-targets. The non-target proteins predicted to be targets by these random forests comprise the set of the most suitable potential future drug targets, and should therefore be prioritised when building a drug development programme. PMID

KHARON Is an Essential Cytoskeletal Protein Involved in the Trafficking of Flagellar Membrane Proteins and Cell Division in African Trypanosomes*

PubMed Central

Sanchez, Marco A.; Tran, Khoa D.; Valli, Jessica; Hobbs, Sam; Johnson, Errin; Gluenz, Eva; Landfear, Scott M.

2016-01-01

African trypanosomes and related kinetoplastid parasites selectively traffic specific membrane proteins to the flagellar membrane, but the mechanisms for this trafficking are poorly understood. We show here that KHARON, a protein originally identified in Leishmania parasites, interacts with a putative trypanosome calcium channel and is required for its targeting to the flagellar membrane. KHARON is located at the base of the flagellar axoneme, where it likely mediates targeting of flagellar membrane proteins, but is also on the subpellicular microtubules and the mitotic spindle. Hence, KHARON is probably a multifunctional protein that associates with several components of the trypanosome cytoskeleton. RNA interference-mediated knockdown of KHARON mRNA results in failure of the calcium channel to enter the flagellar membrane, detachment of the flagellum from the cell body, and disruption of mitotic spindles. Furthermore, knockdown of KHARON mRNA induces a lethal failure of cytokinesis in both bloodstream (mammalian host) and procyclic (insect vector) life cycle stages, and KHARON is thus critical for parasite viability. PMID:27489106

A Class of Rigid Linker-bearing Glucosides for Membrane Protein Structural Study.

PubMed

Sadaf, Aiman; Mortensen, Jonas S; Capaldi, Stefano; Tikhonova, Elena; Hariharan, Parameswaran; de Castro Ribeiro, Orquidea; Loland, Claus J; Guan, Lan; Byrne, Bernadette; Chae, Pil Seok

2016-03-01

Membrane proteins are amphipathic bio-macromolecules incompatible with the polar environments of aqueous media. Conventional detergents encapsulate the hydrophobic surfaces of membrane proteins allowing them to exist in aqueous solution. Membrane proteins stabilized by detergent micelles are used for structural and functional analysis. Despite the availability of a large number of detergents, only a few agents are sufficiently effective at maintaining the integrity of membrane proteins to allow successful crystallization. In the present study, we describe a novel class of synthetic amphiphiles with a branched tail group and a triglucoside head group. These head and tail groups were connected via an amide or ether linkage by using a tris(hydroxylmethyl)aminomethane (TRIS) or neopentyl glycol (NPG) linker to produce TRIS-derived triglucosides (TDTs) and NPG-derived triglucosides (NDTs), respectively. Members of this class conferred enhanced stability on target membrane proteins compared to conventional detergents. Because of straightforward synthesis of the novel agents and their favourable effects on a range of membrane proteins, these agents should be of wide applicability to membrane protein science.

A Class of Rigid Linker-bearing Glucosides for Membrane Protein Structural Study

PubMed Central

Sadaf, Aiman; Mortensen, Jonas S.; Capaldi, Stefano; Tikhonova, Elena; Hariharan, Parameswaran; de Castro Ribeiro, Orquidea; Loland, Claus J; Guan, Lan; Byrne, Bernadette

2015-01-01

Membrane proteins are amphipathic bio-macromolecules incompatible with the polar environments of aqueous media. Conventional detergents encapsulate the hydrophobic surfaces of membrane proteins allowing them to exist in aqueous solution. Membrane proteins stabilized by detergent micelles are used for structural and functional analysis. Despite the availability of a large number of detergents, only a few agents are sufficiently effective at maintaining the integrity of membrane proteins to allow successful crystallization. In the present study, we describe a novel class of synthetic amphiphiles with a branched tail group and a triglucoside head group. These head and tail groups were connected via an amide or ether linkage by using a tris(hydroxylmethyl)aminomethane (TRIS) or neopentyl glycol (NPG) linker to produce TRIS-derived triglucosides (TDTs) and NPG-derived triglucosides (NDTs), respectively. Members of this class conferred enhanced stability on target membrane proteins compared to conventional detergents. Because of straightforward synthesis of the novel agents and their favourable effects on a range of membrane proteins, these agents should be of wide applicability to membrane protein science. PMID:27110345

A cell death assay for assessing the mitochondrial targeting of proteins.

PubMed

Camara Teixeira, Daniel; Cordonier, Elizabeth L; Wijeratne, Subhashinee S K; Huebbe, Patricia; Jamin, Augusta; Jarecke, Sarah; Wiebe, Matthew; Zempleni, Janos

2018-06-01

The mitochondrial proteome comprises 1000 to 1500 proteins, in addition to proteins for which the mitochondrial localization is uncertain. About 800 diseases have been linked with mutations in mitochondrial proteins. We devised a cell survival assay for assessing the mitochondrial localization in a high-throughput format. This protocol allows us to assess the mitochondrial localization of proteins and their mutants, and to identify drugs and nutrients that modulate the mitochondrial targeting of proteins. The assay works equally well for proteins directed to the outer mitochondrial membrane, inner mitochondrial membrane mitochondrial and mitochondrial matrix, as demonstrated by assessing the mitochondrial targeting of the following proteins: carnitine palmitoyl transferase 1 (consensus sequence and R123C mutant), acetyl-CoA carboxylase 2, uncoupling protein 1 and holocarboxylase synthetase. Our screen may be useful for linking the mitochondrial proteome with rare diseases and for devising drug- and nutrition-based strategies for altering the mitochondrial targeting of proteins. Copyright © 2018 Elsevier Inc. All rights reserved.

Membrane-associated stress proteins: more than simply chaperones.

PubMed

Horváth, Ibolya; Multhoff, Gabriele; Sonnleitner, Alois; Vígh, László

2008-01-01

The protein- and/or lipid-mediated association of chaperone proteins to membranes is a widespread phenomenon and implicated in a number of physiological and pathological events that were earlier partially or completely overlooked. A temporary association of certain HSPs with membranes can re-establish the fluidity and bilayer stability and thereby restore the membrane functionality during stress conditions. The fluidity and microdomain organization of membranes are decisive factors in the perception and transduction of stresses into signals that trigger the activation of specific HS genes. Conversely, the membrane association of HSPs may result in the inactivation of membrane-perturbing signals, thereby switch off the heat shock response. Interactions between certain HSPs and specific lipid microdomains ("rafts") might be a previously unrecognized means for the compartmentalization of HSPs to specific signaling platforms, where key signaling proteins are known to be concentrated. Any modulations of the membranes, especially the raft-lipid composition of the cells can alter the extracellular release and thus the immuno-stimulatory activity of certain HSPs. Reliable techniques, allowing mapping of the composition and dynamics of lipid microdomains and simultaneously the spatio-temporal localization of HSPs in and near the plasma membrane can provide suitable means with which to address fundamental questions, such as how HSPs are transported to and translocated through the plasma membrane. The possession of such information is critical if we are to target the membrane association principles of HSPs for successful drug development in most various diseases.

Secretagogue-triggered Transfer of Membrane Proteins from Neuroendocrine Secretory Granules to Synaptic-like Microvesicles

PubMed Central

Strasser, Jane E.; Arribas, Monica; Blagoveshchenskaya, Anastasia D.; Cutler, Daniel F.

1999-01-01

The membrane proteins of all regulated secretory organelles (RSOs) recycle after exocytosis. However, the recycling of those membrane proteins that are targeted to both dense core granules (DCGs) and synaptic-like microvesicles (SLMVs) has not been addressed. Since neuroendocrine cells contain both RSOs, and the recycling routes that lead to either organelle overlap, transfer between the two pools of membrane proteins could occur during recycling. We have previously demonstrated that a chimeric protein containing the cytosolic and transmembrane domains of P-selectin coupled to horseradish peroxidase is targeted to both the DCG and the SLMV in PC12 cells. Using this chimera, we have characterized secretagogue-induced traffic in PC12 cells. After stimulation, this chimeric protein traffics from DCGs to the cell surface, internalizes into transferrin receptor (TFnR)-positive endosomes and thence to a population of secretagogue-responsive SLMVs. We therefore find a secretagogue-dependent rise in levels of HRP within SLMVs. In addition, the levels within SLMVs of the endogenous membrane protein, synaptotagmin, as well as a green fluorescent protein-tagged version of vesicle-associated membrane protein (VAMP)/synaptobrevin, also show a secretagogue-dependent increase. PMID:10436017

Detergent Optimized Membrane Protein Reconstitution in Liposomes for Solid State NMR

PubMed Central

2015-01-01

For small helical membrane proteins, their structures are highly sensitive to their environment, and solid state NMR is a structural technique that can characterize these membrane proteins in native-like lipid bilayers and proteoliposomes. To date, a systematic method by which to evaluate the effect of the solubilizing detergent on proteoliposome preparations for solid state NMR of membrane proteins has not been presented in the literature. A set of experiments are presented aimed at determining the conditions most amenable to dialysis mediated reconstitution sample preparation. A membrane protein from M. tuberculosis is used to illustrate the method. The results show that a detergent that stabilizes the most protein is not always ideal and sometimes cannot be removed by dialysis. By focusing on the lipid and protein binding properties of the detergent, proteoliposome preparations can be readily produced, which provide double the signal-to-noise ratios for both the oriented sample and magic angle spinning solid state NMR. The method will allow more membrane protein drug targets to be structurally characterized in lipid bilayer environments. PMID:24665863

Epithelial Membrane Protein-2 in Human Proliferative Vitreoretinopathy and Epiretinal Membranes.

PubMed

Telander, David G; Yu, Alfred K; Forward, Krisztina I; Morales, Shawn A; Morse, Lawrence S; Park, Susanna S; Gordon, Lynn K

2016-06-01

To determine the level of epithelial membrane protein-2 (EMP2) expression in preretinal membranes from surgical patients with proliferative vitreoretinopathy (PVR) or epiretinal membranes (ERMs). EMP2, an integrin regulator, is expressed in the retinal pigment epithelium and understanding EMP2 expression in human retinal disease may help determine whether EMP2 is a potential therapeutic target. Preretinal membranes were collected during surgical vitrectomies after obtaining consents. The membranes were fixed, processed, sectioned, and protein expression of EMP2 was evaluated by immunohistochemistry. The staining intensity (SI) and percentage of positive cells (PP) in membranes were compared by masked observers. Membranes were categorized by their cause and type including inflammatory and traumatic. All of the membranes stained positive for EMP2. Proliferative vitreoretinopathy-induced membranes (all causes) showed greater expression of EMP2 than ERMs with higher SI (1.81 vs. 1.38; P = 0.07) and PP (2.08 vs. 1.54; P = 0.09). However all the PVR subgroups had similar levels of EMP2 expression without statistically significant differences by Kruskal-Wallis test. Inflammatory PVR had higher expression of EMP2 than ERMs (SI of 2.58 vs. 1.38); however, this was not statistically significant. No correlation was found between duration of PVR membrane and EMP2 expression. EMP2 was detected by RT-PCR in all samples (n = 6) tested. All studied ERMs and PVR membranes express EMP2. Levels of EMP2 trended higher in all PVR subgroups than in ERMs, especially in inflammatory and traumatic PVR. Future studies are needed to determine the role of EMP2 in the pathogenesis and treatment of various retinal conditions including PVR.

Using Haloarcula marismortui Bacteriorhodopsin as a Fusion Tag for Enhancing and Visible Expression of Integral Membrane Proteins in Escherichia coli

PubMed Central

Hsu, Min-Feng; Yu, Tsung-Fu; Chou, Chia-Cheng; Fu, Hsu-Yuan; Yang, Chii-Shen; Wang, Andrew H. J.

2013-01-01

Membrane proteins are key targets for pharmacological intervention because of their vital functions. Structural and functional studies of membrane proteins have been severely hampered because of the difficulties in producing sufficient quantities of properly folded and biologically active proteins. Here we generate a high-level expression system of integral membrane proteins in Escherichia coli by using a mutated bacteriorhodopsin (BR) from Haloarcula marismortui (HmBRI/D94N) as a fusion partner. A purification strategy was designed by incorporating a His-tag on the target membrane protein for affinity purification and an appropriate protease cleavage site to generate the final products. The fusion system can be used to detect the intended target membrane proteins during overexpression and purification either with the naked eye or by directly monitoring their characteristic optical absorption. In this study, we applied this approach to produce two functional integral membrane proteins, undecaprenyl pyrophosphate phosphatase and carnitine/butyrobetaine antiporter with significant yield enhancement. This technology could facilitate the development of a high-throughput strategy to screen for conditions that improve the yield of correctly folded target membrane proteins. Other robust BRs can also be incorporated in this system. PMID:23457558

Functionalizing Microporous Membranes for Protein Purification and Protein Digestion

NASA Astrophysics Data System (ADS)

Dong, Jinlan; Bruening, Merlin L.

2015-07-01

This review examines advances in the functionalization of microporous membranes for protein purification and the development of protease-containing membranes for controlled protein digestion prior to mass spectrometry analysis. Recent studies confirm that membranes are superior to bead-based columns for rapid protein capture, presumably because convective mass transport in membrane pores rapidly brings proteins to binding sites. Modification of porous membranes with functional polymeric films or TiO2 nanoparticles yields materials that selectively capture species ranging from phosphopeptides to His-tagged proteins, and protein-binding capacities often exceed those of commercial beads. Thin membranes also provide a convenient framework for creating enzyme-containing reactors that afford control over residence times. With millisecond residence times, reactors with immobilized proteases limit protein digestion to increase sequence coverage in mass spectrometry analysis and facilitate elucidation of protein structures. This review emphasizes the advantages of membrane-based techniques and concludes with some challenges for their practical application.

Functionalizing Microporous Membranes for Protein Purification and Protein Digestion.

PubMed

Dong, Jinlan; Bruening, Merlin L

2015-01-01

This review examines advances in the functionalization of microporous membranes for protein purification and the development of protease-containing membranes for controlled protein digestion prior to mass spectrometry analysis. Recent studies confirm that membranes are superior to bead-based columns for rapid protein capture, presumably because convective mass transport in membrane pores rapidly brings proteins to binding sites. Modification of porous membranes with functional polymeric films or TiO₂ nanoparticles yields materials that selectively capture species ranging from phosphopeptides to His-tagged proteins, and protein-binding capacities often exceed those of commercial beads. Thin membranes also provide a convenient framework for creating enzyme-containing reactors that afford control over residence times. With millisecond residence times, reactors with immobilized proteases limit protein digestion to increase sequence coverage in mass spectrometry analysis and facilitate elucidation of protein structures. This review emphasizes the advantages of membrane-based techniques and concludes with some challenges for their practical application.

Membrane Topology and Insertion of Membrane Proteins: Search for Topogenic Signals

PubMed Central

van Geest, Marleen; Lolkema, Juke S.

2000-01-01

Integral membrane proteins are found in all cellular membranes and carry out many of the functions that are essential to life. The membrane-embedded domains of integral membrane proteins are structurally quite simple, allowing the use of various prediction methods and biochemical methods to obtain structural information about membrane proteins. A critical step in the biosynthetic pathway leading to the folded protein in the membrane is its insertion into the lipid bilayer. Understanding of the fundamentals of the insertion and folding processes will significantly improve the methods used to predict the three-dimensional membrane protein structure from the amino acid sequence. In the first part of this review, biochemical approaches to elucidate membrane protein topology are reviewed and evaluated, and in the second part, the use of similar techniques to study membrane protein insertion is discussed. The latter studies search for signals in the polypeptide chain that direct the insertion process. Knowledge of the topogenic signals in the nascent chain of a membrane protein is essential for the evaluation of membrane topology studies. PMID:10704472

Lipid-Mediated Targeting with Membrane Wrapped Nanoparticles in the Presence of Corona Formation

PubMed Central

Xu, Fangda; Reiser, Michael; Yu, Xinwei; Gummuluru, Suryaram; Wetzler, Lee; Reinhard, Björn M.

2016-01-01

Membrane wrapped nanoparticles represent a versatile platform for utilizing specific lipid-receptor interactions, such as siallyllactose-mediated binding of the ganglioside GM3 to Siglec1 (CD169), for targeting purposes. The membrane wrap around the nanoparticles does not only serve as a matrix to incorporate GM3 as targeting moiety for antigen presenting cells but also offers unique opportunities for constructing a biomimetic surface from lipids with potentially protein repellent properties. We characterize non-specific protein adsorption (corona formation) to membrane wrapped nanoparticles with core diameters of approx. 35 nm and 80 nm and its effect on the GM3-mediated targeting efficacy as function of surface charge through combined in vitro and in vivo studies. The stability and fate of the membrane wrap around the nanoparticles in a simulated biological fluid and after uptake in CD169 expressing antigen presenting cells is experimentally tested. Finally, we demonstrate in hock immunization studies in mice that GM3 decorated membrane wrapped nanoparticles achieve a selective enrichment in the peripheral regions of popliteal lymph nodes that contain high concentrations of CD169 expressing antigen presenting cells. PMID:26720275

Physico-Pathologic Mechanisms Involved in Neurodegeneration: Misfolded Protein-Plasma Membrane Interactions.

PubMed

Shrivastava, Amulya Nidhi; Aperia, Anita; Melki, Ronald; Triller, Antoine

2017-07-05

Several neurodegenerative disorders, such as Alzheimer's and Parkinson's disease, are characterized by prominent loss of synapses and neurons associated with the presence of abnormally structured or misfolded protein assemblies. Cell-to-cell transfer of misfolded proteins has been proposed for the intra-cerebral propagation of these diseases. When released, misfolded proteins diffuse in the 3D extracellular space before binding to the plasma membrane of neighboring cells, where they diffuse on a 2D plane. This reduction in diffusion dimension and the cell surface molecular crowding promote deleterious interactions with native membrane proteins, favoring clustering and further aggregation of misfolded protein assemblies. These processes open up new avenues for therapeutics development targeting the initial interactions of deleterious proteins with the plasma membrane or the subsequent pathological signaling. Copyright © 2017 Elsevier Inc. All rights reserved.

Protein receptor-independent plasma membrane remodeling by HAMLET: a tumoricidal protein-lipid complex.

PubMed

Nadeem, Aftab; Sanborn, Jeremy; Gettel, Douglas L; James, Ho C S; Rydström, Anna; Ngassam, Viviane N; Klausen, Thomas Kjær; Pedersen, Stine Falsig; Lam, Matti; Parikh, Atul N; Svanborg, Catharina

2015-11-12

A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses. This ''protein-centric" view is increasingly challenged by evidence for the involvement of specialized membrane domains in signal transduction. Here, we propose that membrane perturbation may serve as an alternative mechanism to activate a conserved cell-death program in cancer cells. This view emerges from the extraordinary manner in which HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range of tumor cells in vitro and demonstrates therapeutic efficacy and selectivity in cancer models and clinical studies. We identify a ''receptor independent" transformation of vesicular motifs in model membranes, which is paralleled by gross remodeling of tumor cell membranes. Furthermore, we find that HAMLET accumulates within these de novo membrane conformations and define membrane blebs as cellular compartments for direct interactions of HAMLET with essential target proteins such as the Ras family of GTPases. Finally, we demonstrate lower sensitivity of healthy cell membranes to HAMLET challenge. These features suggest that HAMLET-induced curvature-dependent membrane conformations serve as surrogate receptors for initiating signal transduction cascades, ultimately leading to cell death.

Lipid transfer proteins do their thing anchored at membrane contact sites… but what is their thing?

PubMed

Wong, Louise H; Levine, Tim P

2016-04-15

Membrane contact sites are structures where two organelles come close together to regulate flow of material and information between them. One type of inter-organelle communication is lipid exchange, which must occur for membrane maintenance and in response to environmental and cellular stimuli. Soluble lipid transfer proteins have been extensively studied, but additional families of transfer proteins have been identified that are anchored into membranes by transmembrane helices so that they cannot diffuse through the cytosol to deliver lipids. If such proteins target membrane contact sites they may be major players in lipid metabolism. The eukaryotic family of so-called Lipid transfer proteins Anchored at Membrane contact sites (LAMs) all contain both a sterol-specific lipid transfer domain in the StARkin superfamily (related to StART/Bet_v1), and one or more transmembrane helices anchoring them in the endoplasmic reticulum (ER), making them interesting subjects for study in relation to sterol metabolism. They target a variety of membrane contact sites, including newly described contacts between organelles that were already known to make contact by other means. Lam1-4p target punctate ER-plasma membrane contacts. Lam5p and Lam6p target multiple contacts including a new category: vacuolar non-NVJ cytoplasmic ER (VancE) contacts. These developments confirm previous observations on tubular lipid-binding proteins (TULIPs) that established the importance of membrane anchored proteins for lipid traffic. However, the question remaining to be solved is the most difficult of all: are LAMs transporters, or alternately are they regulators that affect traffic more indirectly? © 2016 Authors; published by Portland Press Limited.

Topologically Diverse Human Membrane Proteins Partition to Liquid-Disordered Domains in Phase-Separated Lipid Vesicles.

PubMed

Schlebach, Jonathan P; Barrett, Paul J; Day, Charles A; Kim, Ji Hun; Kenworthy, Anne K; Sanders, Charles R

2016-02-23

The integration of membrane proteins into "lipid raft" membrane domains influences many biochemical processes. The intrinsic structural properties of membrane proteins are thought to mediate their partitioning between membrane domains. However, whether membrane topology influences the targeting of proteins to rafts remains unclear. To address this question, we examined the domain preference of three putative raft-associated membrane proteins with widely different topologies: human caveolin-3, C99 (the 99 residue C-terminal domain of the amyloid precursor protein), and peripheral myelin protein 22. We find that each of these proteins are excluded from the ordered domains of giant unilamellar vesicles containing coexisting liquid-ordered and liquid-disordered phases. Thus, the intrinsic structural properties of these three topologically distinct disease-linked proteins are insufficient to confer affinity for synthetic raft-like domains.

Methods to study the biogenesis of membrane proteins in yeast mitochondria.

PubMed

Weckbecker, Daniel; Herrmann, Johannes M

2013-01-01

The biogenesis of mitochondrial membrane proteins is an intricate process that relies on the import and submitochondrial sorting of nuclear-encoded preproteins and on the synthesis of mitochondrial translation products in the matrix. Subsequently, these polypeptides need to be inserted into the outer and the inner membranes of the organelle where many of them assemble into multisubunit complexes. In this chapter we provide established protocols to study these different processes experimentally using mitochondria of budding yeast. In particular, methods are described in detail to purify mitochondria, to study mitochondrial protein synthesis, to follow the import of radiolabeled preproteins into isolated mitochondria, and to assess membrane association and the aggregation of mitochondrial proteins by fractionation. These protocols and a list of dos and don'ts shall enable beginners and experienced scientists to address the targeting and assembly of mitochondrial membrane proteins.

Tubby family proteins are adapters for ciliary trafficking of integral membrane proteins

PubMed Central

Shimada, Issei S.; Loriot, Evan

2017-01-01

The primary cilium is a paradigmatic organelle for studying compartmentalized signaling; however, unlike soluble protein trafficking, processes targeting integral membrane proteins to cilia are poorly understood. In this study, we determine that the tubby family protein TULP3 functions as a general adapter for ciliary trafficking of structurally diverse integral membrane cargo, including multiple reported and novel rhodopsin family G protein–coupled receptors (GPCRs) and the polycystic kidney disease–causing polycystin 1/2 complex. The founding tubby family member TUB also localizes to cilia similar to TULP3 and determines trafficking of a subset of these GPCRs to neuronal cilia. Using minimal ciliary localization sequences from GPCRs and fibrocystin (also implicated in polycystic kidney disease), we demonstrate these motifs to be sufficient and TULP3 dependent for ciliary trafficking. We propose a three-step model for TULP3/TUB-mediated ciliary trafficking, including the capture of diverse membrane cargo by the tubby domain in a phosphoinositide 4,5-bisphosphate (PI(4,5)P2)-dependent manner, ciliary delivery by intraflagellar transport complex A binding to the TULP3/TUB N terminus, and subsequent release into PI(4,5)P2-deficient ciliary membrane. PMID:28154160

Improvement of n-butanol tolerance in Escherichia coli by membrane-targeted tilapia metallothionein

PubMed Central

2013-01-01

Background Though n-butanol has been proposed as a potential transportation biofuel, its toxicity often causes oxidative stress in the host microorganism and is considered one of the bottlenecks preventing its efficient mass production. Results To relieve the oxidative stress in the host cell, metallothioneins (MTs), which are known as scavengers for reactive oxygen species (ROS), were engineered in E. coli hosts for both cytosolic and outer-membrane-targeted (osmoregulatory membrane protein OmpC fused) expression. Metallothioneins from human (HMT), mouse (MMT), and tilapia fish (TMT) were tested. The host strain expressing membrane-targeted TMT showed the greatest ability to reduce oxidative stresses induced by n-butanol, ethanol, furfural, hydroxymethylfurfural, and nickel. The same strain also allowed for an increased growth rate of recombinant E. coli under n-butanol stress. Further experiments indicated that the TMT-fused OmpC protein could not only function in ROS scavenging but also regulate either glycine betaine (GB) or glucose uptake via osmosis, and the dual functional fusion protein could contribute in an enhancement of the host microorganism’s growth rate. Conclusions The abilities of scavenging intracellular or extracellular ROS by these engineering E. coli were examined, and TMT show the best ability among three MTs. Additionally, the membrane-targeted fusion protein, OmpC-TMT, improved host tolerance up to 1.5% n-butanol above that of TMT which is only 1%. These results presented indicate potential novel approaches for engineering stress tolerant microorganism strains. PMID:24020941

Improvement of n-butanol tolerance in Escherichia coli by membrane-targeted tilapia metallothionein.

PubMed

Chin, Wei-Chih; Lin, Kuo-Hsing; Chang, Jui-Jen; Huang, Chieh-Chen

2013-09-11

Though n-butanol has been proposed as a potential transportation biofuel, its toxicity often causes oxidative stress in the host microorganism and is considered one of the bottlenecks preventing its efficient mass production. To relieve the oxidative stress in the host cell, metallothioneins (MTs), which are known as scavengers for reactive oxygen species (ROS), were engineered in E. coli hosts for both cytosolic and outer-membrane-targeted (osmoregulatory membrane protein OmpC fused) expression. Metallothioneins from human (HMT), mouse (MMT), and tilapia fish (TMT) were tested. The host strain expressing membrane-targeted TMT showed the greatest ability to reduce oxidative stresses induced by n-butanol, ethanol, furfural, hydroxymethylfurfural, and nickel. The same strain also allowed for an increased growth rate of recombinant E. coli under n-butanol stress. Further experiments indicated that the TMT-fused OmpC protein could not only function in ROS scavenging but also regulate either glycine betaine (GB) or glucose uptake via osmosis, and the dual functional fusion protein could contribute in an enhancement of the host microorganism's growth rate. The abilities of scavenging intracellular or extracellular ROS by these engineering E. coli were examined, and TMT show the best ability among three MTs. Additionally, the membrane-targeted fusion protein, OmpC-TMT, improved host tolerance up to 1.5% n-butanol above that of TMT which is only 1%. These results presented indicate potential novel approaches for engineering stress tolerant microorganism strains.

Present and future of membrane protein structure determination by electron crystallography.

PubMed

Ubarretxena-Belandia, Iban; Stokes, David L

2010-01-01

Membrane proteins are critical to cell physiology, playing roles in signaling, trafficking, transport, adhesion, and recognition. Despite their relative abundance in the proteome and their prevalence as targets of therapeutic drugs, structural information about membrane proteins is in short supply. This chapter describes the use of electron crystallography as a tool for determining membrane protein structures. Electron crystallography offers distinct advantages relative to the alternatives of X-ray crystallography and NMR spectroscopy. Namely, membrane proteins are placed in their native membranous environment, which is likely to favor a native conformation and allow changes in conformation in response to physiological ligands. Nevertheless, there are significant logistical challenges in finding appropriate conditions for inducing membrane proteins to form two-dimensional arrays within the membrane and in using electron cryo-microscopy to collect the data required for structure determination. A number of developments are described for high-throughput screening of crystallization trials and for automated imaging of crystals with the electron microscope. These tools are critical for exploring the necessary range of factors governing the crystallization process. There have also been recent software developments to facilitate the process of structure determination. However, further innovations in the algorithms used for processing images and electron diffraction are necessary to improve throughput and to make electron crystallography truly viable as a method for determining atomic structures of membrane proteins. Copyright © 2010 Elsevier Inc. All rights reserved.

Present and future of membrane protein structure determination by electron crystallography

PubMed Central

Ubarretxena-Belandia, Iban; Stokes, David L.

2011-01-01

Membrane proteins are critical to cell physiology, playing roles in signaling, trafficking, transport, adhesion, and recognition. Despite their relative abundance in the proteome and their prevalence as targets of therapeutic drugs, structural information about membrane proteins is in short supply. This review describes the use of electron crystallography as a tool for determining membrane protein structures. Electron crystallography offers distinct advantages relative to the alternatives of X-ray crystallography and NMR spectroscopy. Namely, membrane proteins are placed in their native membranous environment, which is likely to favor a native conformation and allow changes in conformation in response to physiological ligands. Nevertheless, there are significant logistical challenges in finding appropriate conditions for inducing membrane proteins to form two-dimensional arrays within the membrane and in using electron cryo-microscopy to collect the data required for structure determination. A number of developments are described for high-throughput screening of crystallization trials and for automated imaging of crystals with the electron microscope. These tools are critical for exploring the necessary range of factors governing the crystallization process. There have also been recent software developments to facilitate the process of structure determination. However, further innovations in the algorithms used for processing images and electron diffraction are necessary to improve throughput and to make electron crystallography truly viable as a method for determining atomic structures of membrane proteins. PMID:21115172

Intermolecular detergent-membrane protein noes for the characterization of the dynamics of membrane protein-detergent complexes.

PubMed

Eichmann, Cédric; Orts, Julien; Tzitzilonis, Christos; Vögeli, Beat; Smrt, Sean; Lorieau, Justin; Riek, Roland

2014-12-11

The interaction between membrane proteins and lipids or lipid mimetics such as detergents is key for the three-dimensional structure and dynamics of membrane proteins. In NMR-based structural studies of membrane proteins, qualitative analysis of intermolecular nuclear Overhauser enhancements (NOEs) or paramagnetic resonance enhancement are used in general to identify the transmembrane segments of a membrane protein. Here, we employed a quantitative characterization of intermolecular NOEs between (1)H of the detergent and (1)H(N) of (2)H-perdeuterated, (15)N-labeled α-helical membrane protein-detergent complexes following the exact NOE (eNOE) approach. Structural considerations suggest that these intermolecular NOEs should show a helical-wheel-type behavior along a transmembrane helix or a membrane-attached helix within a membrane protein as experimentally demonstrated for the complete influenza hemagglutinin fusion domain HAfp23. The partial absence of such a NOE pattern along the amino acid sequence as shown for a truncated variant of HAfp23 and for the Escherichia coli inner membrane protein YidH indicates the presence of large tertiary structure fluctuations such as an opening between helices or the presence of large rotational dynamics of the helices. Detergent-protein NOEs thus appear to be a straightforward probe for a qualitative characterization of structural and dynamical properties of membrane proteins embedded in detergent micelles.

Glasslike Membrane Protein Diffusion in a Crowded Membrane.

PubMed

Munguira, Ignacio; Casuso, Ignacio; Takahashi, Hirohide; Rico, Felix; Miyagi, Atsushi; Chami, Mohamed; Scheuring, Simon

2016-02-23

Many functions of the plasma membrane depend critically on its structure and dynamics. Observation of anomalous diffusion in vivo and in vitro using fluorescence microscopy and single particle tracking has advanced our concept of the membrane from a homogeneous fluid bilayer with freely diffusing proteins to a highly organized crowded and clustered mosaic of lipids and proteins. Unfortunately, anomalous diffusion could not be related to local molecular details given the lack of direct and unlabeled molecular observation capabilities. Here, we use high-speed atomic force microscopy and a novel analysis methodology to analyze the pore forming protein lysenin in a highly crowded environment and document coexistence of several diffusion regimes within one membrane. We show the formation of local glassy phases, where proteins are trapped in neighbor-formed cages for time scales up to 10 s, which had not been previously experimentally reported for biological membranes. Furthermore, around solid-like patches and immobile molecules a slower glass phase is detected leading to protein trapping and creating a perimeter of decreased membrane diffusion.

The Multifaceted Role of SNARE Proteins in Membrane Fusion

PubMed Central

Han, Jing; Pluhackova, Kristyna; Böckmann, Rainer A.

2017-01-01

Membrane fusion is a key process in all living organisms that contributes to a variety of biological processes including viral infection, cell fertilization, as well as intracellular transport, and neurotransmitter release. In particular, the various membrane-enclosed compartments in eukaryotic cells need to exchange their contents and communicate across membranes. Efficient and controllable fusion of biological membranes is known to be driven by cooperative action of SNARE proteins, which constitute the central components of the eukaryotic fusion machinery responsible for fusion of synaptic vesicles with the plasma membrane. During exocytosis, vesicle-associated v-SNARE (synaptobrevin) and target cell-associated t-SNAREs (syntaxin and SNAP-25) assemble into a core trans-SNARE complex. This complex plays a versatile role at various stages of exocytosis ranging from the priming to fusion pore formation and expansion, finally resulting in the release or exchange of the vesicle content. This review summarizes current knowledge on the intricate molecular mechanisms underlying exocytosis triggered and catalyzed by SNARE proteins. Particular attention is given to the function of the peptidic SNARE membrane anchors and the role of SNARE-lipid interactions in fusion. Moreover, the regulatory mechanisms by synaptic auxiliary proteins in SNARE-driven membrane fusion are briefly outlined. PMID:28163686

The Multifaceted Role of SNARE Proteins in Membrane Fusion.

PubMed

Han, Jing; Pluhackova, Kristyna; Böckmann, Rainer A

2017-01-01

Membrane fusion is a key process in all living organisms that contributes to a variety of biological processes including viral infection, cell fertilization, as well as intracellular transport, and neurotransmitter release. In particular, the various membrane-enclosed compartments in eukaryotic cells need to exchange their contents and communicate across membranes. Efficient and controllable fusion of biological membranes is known to be driven by cooperative action of SNARE proteins, which constitute the central components of the eukaryotic fusion machinery responsible for fusion of synaptic vesicles with the plasma membrane. During exocytosis, vesicle-associated v-SNARE (synaptobrevin) and target cell-associated t-SNAREs (syntaxin and SNAP-25) assemble into a core trans-SNARE complex. This complex plays a versatile role at various stages of exocytosis ranging from the priming to fusion pore formation and expansion, finally resulting in the release or exchange of the vesicle content. This review summarizes current knowledge on the intricate molecular mechanisms underlying exocytosis triggered and catalyzed by SNARE proteins. Particular attention is given to the function of the peptidic SNARE membrane anchors and the role of SNARE-lipid interactions in fusion. Moreover, the regulatory mechanisms by synaptic auxiliary proteins in SNARE-driven membrane fusion are briefly outlined.

Discovery of functional monoclonal antibodies targeting G-protein-coupled receptors and ion channels.

PubMed

Wilkinson, Trevor C I

2016-06-15

The development of recombinant antibody therapeutics is a significant area of growth in the pharmaceutical industry with almost 50 approved monoclonal antibodies on the market in the US and Europe. Despite this growth, however, certain classes of important molecular targets have remained intractable to therapeutic antibodies due to complexity of the target molecules. These complex target molecules include G-protein-coupled receptors and ion channels which represent a large potential target class for therapeutic intervention with monoclonal antibodies. Although these targets have typically been addressed by small molecule approaches, the exquisite specificity of antibodies provides a significant opportunity to provide selective modulation of these target proteins. Given this opportunity, substantial effort has been applied to address the technical challenges of targeting these complex membrane proteins with monoclonal antibodies. In this review recent progress made in the strategies for discovery of functional monoclonal antibodies for these challenging membrane protein targets is addressed. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

Interaction between La(III) and proteins on the plasma membrane of horseradish

NASA Astrophysics Data System (ADS)

Yang, Guang-Mei; Chu, Yun-Xia; Lv, Xiao-Fen; Zhou, Qing; Huang, Xiao-Hua

2012-06-01

Lanthanum (La) is an important rare earth element in the ecological environment of plant. The proteins on the plasma membrane control the transport of molecules into and out of cell. It is very important to investigate the effect of La(III) on the proteins on the plasma membrane in the plant cell. In the present work, the interaction between La(III) and proteins on the plasma membrane of horseradish was investigated using optimization of the fluorescence microscopy and fluorescence spectroscopy. It is found that the fluorescence of the complex system of protoplasts and 1-aniline Kenai-8-sulfonic acid in horseradish treated with the low concentration of La(III) is increased compared with that of the control horseradish. The opposite effect is observed in horseradish treated with the high concentration of La(III). These results indicated that the low concentration of La(III) can interact with the proteins on the plasma membrane of horseradish, causing the improvement in the structure of proteins on the plasma membrane. The high concentration of La(III) can also interact with the proteins on the plasma membrane of horseradish, leading to the destruction of the structure of proteins on the plasma membrane. We demonstrate that the proteins on the plasma membrane are the targets of La(III) action on plant cell.

Proteopolymersomes: in vitro production of a membrane protein in polymersome membranes.

PubMed

Nallani, Madhavan; Andreasson-Ochsner, Mirjam; Tan, Cherng-Wen Darren; Sinner, Eva-Kathrin; Wisantoso, Yudi; Geifman-Shochat, Susana; Hunziker, Walter

2011-12-01

Polymersomes are stable self-assembled architectures which mimic cell membranes. For characterization, membrane proteins can be incorporated into such bio-mimetic membranes by reconstitution methods, leading to so-called proteopolymersomes. In this work, we demonstrate the direct incorporation of a membrane protein into polymersome membranes by a cell-free expression system. Firstly, we demonstrate pore formation in the preformed polymersome membrane using α-hemolysin. Secondly, we use claudin-2, a protein involved in cell-cell interactions, to demonstrate the in vitro expression of a membrane protein into these polymersomes. Surface plasmon resonance (Biacore) binding studies with the claudin-2 proteopolymersomes and claudin-2 specific antibodies are performed to show the presence of the in vitro expressed protein in polymersome membranes.

Peripheral Protein Unfolding Drives Membrane Bending.

PubMed

Siaw, Hew Ming Helen; Raghunath, Gokul; Dyer, R Brian

2018-06-20

Dynamic modulation of lipid membrane curvature can be achieved by a number of peripheral protein binding mechanisms such as hy-drophobic insertion of amphipathic helices and membrane scaffolding. Recently, an alternative mechanism was proposed in which crowding of peripherally bound proteins induces membrane curvature through steric pressure generated by lateral collisions. This effect was enhanced using intrinsically disordered proteins that possess high hydrodynamic radii, prompting us to explore whether membrane bending can be triggered by the folding-unfolding transition of surface-bound proteins. We utilized histidine-tagged human serum albumin bound to Ni-NTA-DGS containing liposomes as our model system to test this hypothesis. We found that reduction of the disulfide bonds in the protein resulted in unfolding of HSA, which subsequently led to membrane tubule formation. The frequency of tubule formation was found to be significantly higher when the proteins were unfolded while being localized to a phase-separated domain as opposed to randomly distributed in fluid phase liposomes, indicating that the steric pressure generated from protein unfolding is directly responsible for membrane deformation. Our results are critical for the design of peripheral membrane protein-immobilization strategies and open new avenues for exploring mechanisms of membrane bending driven by conformational changes of peripheral membrane proteins.

Computational modeling of membrane proteins

PubMed Central

Leman, Julia Koehler; Ulmschneider, Martin B.; Gray, Jeffrey J.

2014-01-01

The determination of membrane protein (MP) structures has always trailed that of soluble proteins due to difficulties in their overexpression, reconstitution into membrane mimetics, and subsequent structure determination. The percentage of MP structures in the protein databank (PDB) has been at a constant 1-2% for the last decade. In contrast, over half of all drugs target MPs, only highlighting how little we understand about drug-specific effects in the human body. To reduce this gap, researchers have attempted to predict structural features of MPs even before the first structure was experimentally elucidated. In this review, we present current computational methods to predict MP structure, starting with secondary structure prediction, prediction of trans-membrane spans, and topology. Even though these methods generate reliable predictions, challenges such as predicting kinks or precise beginnings and ends of secondary structure elements are still waiting to be addressed. We describe recent developments in the prediction of 3D structures of both α-helical MPs as well as β-barrels using comparative modeling techniques, de novo methods, and molecular dynamics (MD) simulations. The increase of MP structures has (1) facilitated comparative modeling due to availability of more and better templates, and (2) improved the statistics for knowledge-based scoring functions. Moreover, de novo methods have benefitted from the use of correlated mutations as restraints. Finally, we outline current advances that will likely shape the field in the forthcoming decade. PMID:25355688

Structures of membrane proteins

PubMed Central

Vinothkumar, Kutti R.; Henderson, Richard

2010-01-01

In reviewing the structures of membrane proteins determined up to the end of 2009, we present in words and pictures the most informative examples from each family. We group the structures together according to their function and architecture to provide an overview of the major principles and variations on the most common themes. The first structures, determined 20 years ago, were those of naturally abundant proteins with limited conformational variability, and each membrane protein structure determined was a major landmark. With the advent of complete genome sequences and efficient expression systems, there has been an explosion in the rate of membrane protein structure determination, with many classes represented. New structures are published every month and more than 150 unique membrane protein structures have been determined. This review analyses the reasons for this success, discusses the challenges that still lie ahead, and presents a concise summary of the key achievements with illustrated examples selected from each class. PMID:20667175

Autoinhibition of ankyrin-B/G membrane target bindings by intrinsically disordered segments from the tail regions

PubMed Central

Wang, Chao; Wei, Zhiyi

2017-01-01

Ankyrins together with their spectrin partners are the master organizers of micron-scale membrane domains in diverse tissues. The 24 ankyrin (ANK) repeats of ankyrins bind to numerous membrane proteins, linking them to spectrin-based cytoskeletons at specific membrane microdomains. The accessibility of the target binding groove of ANK repeats must be regulated to achieve spatially defined functions of ankyrins/target complexes in different tissues, though little is known in this regard. Here we systemically investigated the autoinhibition mechanism of ankyrin-B/G by combined biochemical, biophysical and structural biology approaches. We discovered that the entire ANK repeats are inhibited by combinatorial and quasi-independent bindings of multiple disordered segments located in the ankyrin-B/G linkers and tails, suggesting a mechanistic basis for differential regulations of membrane target bindings by ankyrins. In addition to elucidating the autoinhibition mechanisms of ankyrins, our study may also shed light on regulations on target bindings by other long repeat-containing proteins. PMID:28841137

Quantification of Ligand Binding to G-Protein Coupled Receptors on Cell Membranes by Ellipsometry

PubMed Central

Kriechbaumer, Verena; Nabok, Alexei; Widdowson, Robert; Smith, David P.; Abell, Ben M.

2012-01-01

G-protein-coupled receptors (GPCRs) are prime drug targets and targeted by approximately 60% of current therapeutic drugs such as β-blockers, antipsychotics and analgesics. However, no biophysical methods are available to quantify their interactions with ligand binding in a native environment. Here, we use ellipsometry to quantify specific interactions of receptors within native cell membranes. As a model system, the GPCR-ligand CXCL12α and its receptor CXCR4 are used. Human-derived Ishikawa cells were deposited onto gold coated slides via Langmuir-Schaefer film deposition and interactions between the receptor CXCR4 on these cells and its ligand CXCL12α were detected via total internal reflection ellipsometry (TIRE). This interaction could be inhibited by application of the CXCR4-binding drug AMD3100. Advantages of this approach are that it allows measurement of interactions in a lipid environment without the need for labelling, protein purification or reconstitution of membrane proteins. This technique is potentially applicable to a wide variety of cell types and their membrane receptors, providing a novel method to determine ligand or drug interactions targeting GPCRs and other membrane proteins. PMID:23049983

Detection of Proteins on Blot Membranes

PubMed Central

Goldman, Aaron; Harper, Sandra; Speicher, David W.

2017-01-01

Staining of blot membranes enables the visualization of bound proteins. Proteins are usually transferred to blot membranes by electroblotting, by direct spotting of protein solutions, or by contact blots. Staining allows the efficiency of transfer to the membrane to be monitored. This unit describes protocols for staining proteins after electroblotting from polyacrylamide gels to blot membranes such as polyvinylidene difluoride (PVDF), nitrocellulose, or nylon membranes. The same methods can be used if proteins are directly spotted, either manually or using robotics. Protocols are included for seven general protein stains (amido black, Coomassie blue, Ponceau S, colloidal gold, colloidal silver, India ink, and MemCode) and three fluorescent protein stains (fluorescamine, IAEDANS, and SYPRO Ruby). Also included is an in-depth discussion of the different blot membrane types and the compatibility of different protein stains with downstream applications, such as immunoblotting or N-terminal Edman sequencing. PMID:27801518

Micro-scale NMR Screening of New Detergents for Membrane Protein Structural Biology

PubMed Central

Zhang, Qinghai; Horst, Reto; Geralt, Michael; Ma, Xingquan; Hong, Wen-Xu; Finn, M. G.; Stevens, Raymond C.; Wüthrich, Kurt

2008-01-01

The rate limiting step in biophysical characterization of membrane proteins is often the availability of suitable amounts of protein material. It was therefore of interest to demonstrate that micro-coil nuclear magnetic resonance (NMR) technology can be used to screen microscale quantities of membrane proteins for proper folding in samples destined for structural studies. Micoscale NMR was then used to screen a series of newly designed zwitterionic phosphocholine detergents for their ability to reconstitute membrane proteins, using the previously well characterized β-barrel E.coli outer membrane protein OmpX as a test case. Fold screening was thus achieved with μg-amounts of uniformly 2H,15N-labeld OmpX and affordable amounts of the detergents, and prescreening with SDS-gel electrophoresis ensured efficient selection of the targets for NMR studies. A systematic approach to optimize the phosphocholine motif for membrane protein refolding led to the identification of two new detergents, 138-Fos and 179-Fos, that yield 2D [15N,1H]-TROSY correlation NMR spectra of natively folded reconstituted OmpX. PMID:18479092

Subcellular targeting of nine calcium-dependent protein kinase isoforms from Arabidopsis

NASA Technical Reports Server (NTRS)

Dammann, Christian; Ichida, Audrey; Hong, Bimei; Romanowsky, Shawn M.; Hrabak, Estelle M.; Harmon, Alice C.; Pickard, Barbara G.; Harper, Jeffrey F.; Evans, M. L. (Principal Investigator)

2003-01-01

Calcium-dependent protein kinases (CDPKs) are specific to plants and some protists. Their activation by calcium makes them important switches for the transduction of intracellular calcium signals. Here, we identify the subcellular targeting potentials for nine CDPK isoforms from Arabidopsis, as determined by expression of green fluorescent protein (GFP) fusions in transgenic plants. Subcellular locations were determined by fluorescence microscopy in cells near the root tip. Isoforms AtCPK3-GFP and AtCPK4-GFP showed a nuclear and cytosolic distribution similar to that of free GFP. Membrane fractionation experiments confirmed that these isoforms were primarily soluble. A membrane association was observed for AtCPKs 1, 7, 8, 9, 16, 21, and 28, based on imaging and membrane fractionation experiments. This correlates with the presence of potential N-terminal acylation sites, consistent with acylation as an important factor in membrane association. All but one of the membrane-associated isoforms targeted exclusively to the plasma membrane. The exception was AtCPK1-GFP, which targeted to peroxisomes, as determined by covisualization with a peroxisome marker. Peroxisome targeting of AtCPK1-GFP was disrupted by a deletion of two potential N-terminal acylation sites. The observation of a peroxisome-located CDPK suggests a mechanism for calcium regulation of peroxisomal functions involved in oxidative stress and lipid metabolism.

Targeted Approach for Proteomic Analysis of a Hidden Membrane Protein.

PubMed

Martins-Marques, Tania; Anjo, Sandra I; Ribeiro-Rodrigues, Teresa; Manadas, Bruno; Girao, Henrique

2017-01-01

Given the properties of plasma membrane proteins, namely, their hydrophobicity, low solubility, and high resistance to digestion and extraction, their identification by traditional mass spectrometry (MS) has been a challenging task. Hence, proteomic studies involving the transmembrane protein connexin43 (Cx43) are scarce. Additionally, studies demonstrating the presence of proteins embedded in the lipid bilayer of extracellular vesicles (EVs) are difficult to perform and require specific changes and fine adjustments in the experimental and technical procedure to allow their detection by MS. In this review, we provide a detailed description of the protocol we have used to detect Cx43 in EVs of human peripheral blood. This includes some of the modifications that we have introduced in order to improve the detection of Cx43 in EVs, including an optimization of vesicle isolation, Cx43 purification, MS acquisition data, and further analysis.

Protein receptor-independent plasma membrane remodeling by HAMLET: A tumoricidal protein-lipid complex

DOE PAGES

Nadeem, Aftab; Sanborn, Jeremy; Gettel, Douglas L.; ...

2015-11-12

A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses. This ‘’protein-centric” view is increasingly challenged by evidence for the involvement of specialized membrane domains in signal transduction. Here, we propose that membrane perturbation may serve as an alternative mechanism to activate a conserved cell-death program in cancer cells. This view emerges from the extraordinary manner in which HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range of tumor cells in vitro and demonstrates therapeutic efficacy and selectivity in cancer models and clinical studies. Wemore » identify a ‘’receptor independent” transformation of vesicular motifs in model membranes, which is paralleled by gross remodeling of tumor cell membranes. Furthermore, we find that HAMLET accumulates within these de novo membrane conformations and define membrane blebs as cellular compartments for direct interactions of HAMLET with essential target proteins such as the Ras family of GTPases. In conclusion, we demonstrate lower sensitivity of healthy cell membranes to HAMLET challenge. These features suggest that HAMLET-induced curvature-dependent membrane conformations serve as surrogate receptors for initiating signal transduction cascades, ultimately leading to cell death.« less

Protein receptor-independent plasma membrane remodeling by HAMLET: a tumoricidal protein-lipid complex

PubMed Central

Nadeem, Aftab; Sanborn, Jeremy; Gettel, Douglas L.; James, Ho C. S.; Rydström, Anna; Ngassam, Viviane N.; Klausen, Thomas Kjær; Pedersen, Stine Falsig; Lam, Matti; Parikh, Atul N.; Svanborg, Catharina

2015-01-01

A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses. This ‘’protein-centric” view is increasingly challenged by evidence for the involvement of specialized membrane domains in signal transduction. Here, we propose that membrane perturbation may serve as an alternative mechanism to activate a conserved cell-death program in cancer cells. This view emerges from the extraordinary manner in which HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range of tumor cells in vitro and demonstrates therapeutic efficacy and selectivity in cancer models and clinical studies. We identify a ‘’receptor independent” transformation of vesicular motifs in model membranes, which is paralleled by gross remodeling of tumor cell membranes. Furthermore, we find that HAMLET accumulates within these de novo membrane conformations and define membrane blebs as cellular compartments for direct interactions of HAMLET with essential target proteins such as the Ras family of GTPases. Finally, we demonstrate lower sensitivity of healthy cell membranes to HAMLET challenge. These features suggest that HAMLET-induced curvature-dependent membrane conformations serve as surrogate receptors for initiating signal transduction cascades, ultimately leading to cell death. PMID:26561036

Detection of Proteins on Blot Membranes.

PubMed

Goldman, Aaron; Harper, Sandra; Speicher, David W

2016-11-01

Staining of blot membranes enables the visualization of bound proteins. Proteins are usually transferred to blot membranes by electroblotting, by direct spotting of protein solutions, or by contact blots. Staining allows the efficiency of transfer to the membrane to be monitored. This unit describes protocols for staining proteins after electroblotting from polyacrylamide gels to blot membranes such as polyvinylidene difluoride (PVDF), nitrocellulose, or nylon membranes. The same methods can be used if proteins are directly spotted, either manually or using robotics. Protocols are included for seven general protein stains (amido black, Coomassie blue, Ponceau S, colloidal gold, colloidal silver, India ink, and MemCode) and three fluorescent protein stains (fluorescamine, IAEDANS, and SYPRO Ruby). Also included is an in-depth discussion of the different blot membrane types and the compatibility of different protein stains with downstream applications, such as immunoblotting or N-terminal Edman sequencing. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

Sodium modulates opioid receptors through a membrane component different from G-proteins. Demonstration by target size analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ott, S.; Costa, T.; Herz, A.

1988-07-25

The target size for opioid receptor binding was studied after manipulations known to affect the interactions between receptor and GTP-binding regulatory proteins (G-proteins). Addition of GTP or its analogs to the binding reaction, exposure of intact cells to pertussis toxin prior to irradiation, or treatment of irradiated membranes with N-ethylmaleimide did not change the target size (approximately equal to 100 kDa) for opioid receptors in NG 108-15 cells and rat brain. These data suggest that the 100-kDa species does not include an active subunit of a G-protein or alternatively that GTP does not promote the dissociation of the receptor-G-protein complex.more » The presence of Na+ (100 mM) in the radioligand binding assay induced a biphasic decay curve for agonist binding and a flattening of the monoexponential decay curve for a partial agonist. In both cases the effect was explained by an irradiation-induced loss of the low affinity state of the opioid receptor produced by the addition of Na+. This suggests that an allosteric inhibitor that mediates the effect of sodium on the receptor is destroyed at low doses of irradiation, leaving receptors which are no longer regulated by sodium. The effect of Na+ on target size was slightly increased by the simultaneous addition of GTP but was not altered by pertussis toxin treatment. Thus, the sodium unit is distinct from G-proteins and may represent a new component of the opioid receptor complex. Assuming a simple bimolecular model of one Na+ unit/receptor, the size of this inhibitor can be measured as 168 kDa.« less

A protein interaction network analysis for yeast integral membrane protein.

PubMed

Shi, Ming-Guang; Huang, De-Shuang; Li, Xue-Ling

2008-01-01

Although the yeast Saccharomyces cerevisiae is the best exemplified single-celled eukaryote, the vast number of protein-protein interactions of integral membrane proteins of Saccharomyces cerevisiae have not been characterized by experiments. Here, based on the kernel method of Greedy Kernel Principal Component analysis plus Linear Discriminant Analysis, we identify 300 protein-protein interactions involving 189 membrane proteins and get the outcome of a highly connected protein-protein interactions network. Furthermore, we study the global topological features of integral membrane proteins network of Saccharomyces cerevisiae. These results give the comprehensive description of protein-protein interactions of integral membrane proteins and reveal global topological and robustness of the interactome network at a system level. This work represents an important step towards a comprehensive understanding of yeast protein interactions.

Plasma membrane-targeted PIN proteins drive shoot development in a moss.

PubMed

Bennett, Tom A; Liu, Maureen M; Aoyama, Tsuyoshi; Bierfreund, Nicole M; Braun, Marion; Coudert, Yoan; Dennis, Ross J; O'Connor, Devin; Wang, Xiao Y; White, Chris D; Decker, Eva L; Reski, Ralf; Harrison, C Jill

2014-12-01

Plant body plans arise by the activity of meristematic growing tips during development and radiated independently in the gametophyte (n) and sporophyte (2n) stages of the life cycle during evolution. Although auxin and its intercellular transport by PIN family efflux carriers are primary regulators of sporophytic shoot development in flowering plants, the extent of conservation in PIN function within the land plants and the mechanisms regulating bryophyte gametophytic shoot development are largely unknown. We have found that treating gametophytic shoots of the moss Physcomitrella patens with exogenous auxins and auxin transport inhibitors disrupts apical function and leaf development. Two plasma membrane-targeted PIN proteins are expressed in leafy shoots, and pin mutants resemble plants treated with auxins or auxin transport inhibitors. PIN-mediated auxin transport regulates apical cell function, leaf initiation, leaf shape, and shoot tropisms in moss gametophytes. pin mutant sporophytes are sometimes branched, reproducing a phenotype only previously seen in the fossil record and in rare natural moss variants. Our results show that PIN-mediated auxin transport is an ancient, conserved regulator of shoot development. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Targeted Degradation of Proteins Localized in Subcellular Compartments by Hybrid Small Molecules.

PubMed

Okuhira, Keiichiro; Shoda, Takuji; Omura, Risa; Ohoka, Nobumichi; Hattori, Takayuki; Shibata, Norihito; Demizu, Yosuke; Sugihara, Ryo; Ichino, Asato; Kawahara, Haruka; Itoh, Yukihiro; Ishikawa, Minoru; Hashimoto, Yuichi; Kurihara, Masaaki; Itoh, Susumu; Saito, Hiroyuki; Naito, Mikihiko

2017-03-01

Development of novel small molecules that selectively degrade pathogenic proteins would provide an important advance in targeted therapy. Recently, we have devised a series of hybrid small molecules named SNIPER (specific and nongenetic IAP-dependent protein ERaser) that induces the degradation of target proteins via the ubiquitin-proteasome system. To understand the localization of proteins that can be targeted by this protein knockdown technology, we examined whether SNIPER molecules are able to induce degradation of cellular retinoic acid binding protein II (CRABP-II) proteins localized in subcellular compartments of cells. CRABP-II is genetically fused with subcellular localization signals, and they are expressed in the cells. SNIPER(CRABP) with different IAP-ligands, SNIPER(CRABP)-4 with bestatin and SNIPER(CRABP)-11 with MV1 compound, induce the proteasomal degradation of wild-type (WT), cytosolic, nuclear, and membrane-localized CRABP-II proteins, whereas only SNIPER(CRABP)-11 displayed degradation activity toward the mitochondrial CRABP-II protein. The small interfering RNA-mediated silencing of cIAP1 expression attenuated the knockdown activity of SNIPER(CRABP) against WT and cytosolic CRABP-II proteins, indicating that cIAP1 is the E3 ligase responsible for degradation of these proteins. Against membrane-localized CRABP-II protein, cIAP1 is also a primary E3 ligase in the cells, but another E3 ligase distinct from cIAP2 and X-linked inhibitor of apoptosis protein (XIAP) could also be involved in the SNIPER(CRABP)-11-induced degradation. However, for the degradation of nuclear and mitochondrial CRABP-II proteins, E3 ligases other than cIAP1, cIAP2, and XIAP play a role in the SNIPER-mediated protein knockdown. These results indicate that SNIPER can target cytosolic, nuclear, membrane-localized, and mitochondrial proteins for degradation, but the responsible E3 ligase is different, depending on the localization of the target protein. Copyright © 2017 by

Deorphanizing the human transmembrane genome: A landscape of uncharacterized membrane proteins.

PubMed

Babcock, Joseph J; Li, Min

2014-01-01

The sequencing of the human genome has fueled the last decade of work to functionally characterize genome content. An important subset of genes encodes membrane proteins, which are the targets of many drugs. They reside in lipid bilayers, restricting their endogenous activity to a relatively specialized biochemical environment. Without a reference phenotype, the application of systematic screens to profile candidate membrane proteins is not immediately possible. Bioinformatics has begun to show its effectiveness in focusing the functional characterization of orphan proteins of a particular functional class, such as channels or receptors. Here we discuss integration of experimental and bioinformatics approaches for characterizing the orphan membrane proteome. By analyzing the human genome, a landscape reference for the human transmembrane genome is provided.

Membrane and luminal proteins reach the apicoplast by different trafficking pathways in the malaria parasite Plasmodium falciparum

PubMed Central

Chaudhari, Rahul; Dey, Vishakha; Narayan, Aishwarya; Sharma, Shobhona

2017-01-01

The secretory pathway in Plasmodium falciparum has evolved to transport proteins to the host cell membrane and to an endosymbiotic organelle, the apicoplast. The latter can occur via the ER or the ER-Golgi route. Here, we study these three routes using proteins Erythrocyte Membrane Protein-1 (PfEMP1), Acyl Carrier Protein (ACP) and glutathione peroxidase-like thioredoxin peroxidase (PfTPxGl) and inhibitors of vesicular transport. As expected, the G protein-dependent vesicular fusion inhibitor AlF4− and microtubule destabilizing drug vinblastine block the trafficking of PfEMP-1, a protein secreted to the host cell membrane. However, while both PfTPxGl and ACP are targeted to the apicoplast, only ACP trafficking remains unaffected by these treatments. This implies that G protein-dependent vesicles do not play a role in classical apicoplast protein targeting. Unlike the soluble protein ACP, we show that PfTPxGl is localized to the outermost membrane of the apicoplast. Thus, the parasite apicoplast acquires proteins via two different pathways: first, the vesicular trafficking pathway appears to handle not only secretory proteins, but an apicoplast membrane protein, PfTPxGl; second, trafficking of apicoplast luminal proteins appear to be independent of G protein-coupled vesicles. PMID:28462015

Membrane and luminal proteins reach the apicoplast by different trafficking pathways in the malaria parasite Plasmodium falciparum.

PubMed

Chaudhari, Rahul; Dey, Vishakha; Narayan, Aishwarya; Sharma, Shobhona; Patankar, Swati

2017-01-01

The secretory pathway in Plasmodium falciparum has evolved to transport proteins to the host cell membrane and to an endosymbiotic organelle, the apicoplast. The latter can occur via the ER or the ER-Golgi route. Here, we study these three routes using proteins Erythrocyte Membrane Protein-1 (PfEMP1), Acyl Carrier Protein (ACP) and glutathione peroxidase-like thioredoxin peroxidase (PfTPx Gl ) and inhibitors of vesicular transport. As expected, the G protein-dependent vesicular fusion inhibitor AlF 4 - and microtubule destabilizing drug vinblastine block the trafficking of PfEMP-1, a protein secreted to the host cell membrane. However, while both PfTPx Gl and ACP are targeted to the apicoplast, only ACP trafficking remains unaffected by these treatments. This implies that G protein-dependent vesicles do not play a role in classical apicoplast protein targeting. Unlike the soluble protein ACP, we show that PfTPx Gl is localized to the outermost membrane of the apicoplast. Thus, the parasite apicoplast acquires proteins via two different pathways: first, the vesicular trafficking pathway appears to handle not only secretory proteins, but an apicoplast membrane protein, PfTPx Gl ; second, trafficking of apicoplast luminal proteins appear to be independent of G protein-coupled vesicles.

Cooperative tumour cell membrane targeted phototherapy

NASA Astrophysics Data System (ADS)

Kim, Heegon; Lee, Junsung; Oh, Chanhee; Park, Ji-Ho

2017-06-01

The targeted delivery of therapeutics using antibodies or nanomaterials has improved the precision and safety of cancer therapy. However, the paucity and heterogeneity of identified molecular targets within tumours have resulted in poor and uneven distribution of targeted agents, thus compromising treatment outcomes. Here, we construct a cooperative targeting system in which synthetic and biological nanocomponents participate together in the tumour cell membrane-selective localization of synthetic receptor-lipid conjugates (SR-lipids) to amplify the subsequent targeting of therapeutics. The SR-lipids are first delivered selectively to tumour cell membranes in the perivascular region using fusogenic liposomes. By hitchhiking with extracellular vesicles secreted by the cells, the SR-lipids are transferred to neighbouring cells and further spread throughout the tumour tissues where the molecular targets are limited. We show that this tumour cell membrane-targeted delivery of SR-lipids leads to uniform distribution and enhanced phototherapeutic efficacy of the targeted photosensitizer.

Thioredoxin h regulates calcium dependent protein kinases in plasma membranes.

PubMed

Ueoka-Nakanishi, Hanayo; Sazuka, Takashi; Nakanishi, Yoichi; Maeshima, Masayoshi; Mori, Hitoshi; Hisabori, Toru

2013-07-01

Thioredoxin (Trx) is a key player in redox homeostasis in various cells, modulating the functions of target proteins by catalyzing a thiol-disulfide exchange reaction. Target proteins of cytosolic Trx-h of higher plants were studied, particularly in the plasma membrane, because plant plasma membranes include various functionally important protein molecules such as transporters and signal receptors. Plasma membrane proteins from Arabidopsis thaliana cell cultures were screened using a resin Trx-h1 mutant-immobilized, and a total of 48 candidate proteins obtained. These included two calcium-sensing proteins: a phosphoinositide-specific phospholipase 2 (AtPLC2) and a calcium-dependent protein kinase 21 (AtCPK21). A redox-dependent change in AtCPK21 kinase activity was demonstrated in vitro. Oxidation of AtCPK21 resulted in a decrease in kinase activity to 19% of that of untreated AtCPK21, but Trx-h1 effectively restored the activity to 90%. An intramolecular disulfide bond (Cys97-Cys108) that is responsible for this redox modulation was then identified. In addition, endogenous AtCPK21 was shown to be oxidized in vivo when the culture cells were treated with H2 O2 . These results suggest that redox regulation of AtCPK21 by Trx-h in response to external stimuli is important for appropriate cellular responses. The relationship between the redox regulation system and Ca(2+) signaling pathways is discussed. © 2013 The Authors. FEBS Journal published by John Wiley & Sons Ltd on behalf of FEBS.

High yield cell-free production of integral membrane proteins without refolding or detergents.

PubMed

Wuu, Jessica J; Swartz, James R

2008-05-01

Integral membrane proteins act as critical cellular components and are important drug targets. However, difficulties in producing membrane proteins have hampered investigations of structure and function. In vivo production systems are often limited by cell toxicity, and previous in vitro approaches have required unnatural folding pathways using detergents or lipid solutions. To overcome these limitations, we present an improved cell-free expression system which produces high yields of integral membrane proteins without the use of detergents or refolding steps. Our cell-free reaction activates an Escherichia coli-derived cell extract for transcription and translation. Purified E. coli inner membrane vesicles supply membrane-bound components and the lipid environment required for insertion and folding. Using this system, we demonstrated successful synthesis of two complex integral membrane transporters, the tetracycline pump (TetA) and mannitol permease (MtlA), in yields of 570+/-50 microg/mL and 130+/-30 microg/mL of vesicle-associated protein, respectively. These yields are up to 400 times typical in vivo concentrations. Insertion and folding of these proteins are verified by sucrose flotation, protease digestion, and activity assays. Whereas TetA incorporates efficiently into vesicle membranes with over two-thirds of the synthesized protein being inserted, MtlA yields appear to be limited by insufficient concentrations of a membrane-associated chaperone.

Size-dependent protein segregation at membrane interfaces

NASA Astrophysics Data System (ADS)

Schmid, Eva M.; Bakalar, Matthew H.; Choudhuri, Kaushik; Weichsel, Julian; Ann, Hyoung Sook; Geissler, Phillip L.; Dustin, Michael L.; Fletcher, Daniel A.

2016-07-01

Membrane interfaces formed at cell-cell junctions are associated with characteristic patterns of membrane proteins whose organization is critical for intracellular signalling. To isolate the role of membrane protein size in pattern formation, we reconstituted model membrane interfaces in vitro using giant unilamellar vesicles decorated with synthetic binding and non-binding proteins. We show that size differences between membrane proteins can drastically alter their organization at membrane interfaces, with as little as a ~5 nm increase in non-binding protein size driving its exclusion from the interface. Combining in vitro measurements with Monte Carlo simulations, we find that non-binding protein exclusion is also influenced by lateral crowding, binding protein affinity, and thermally driven membrane height fluctuations that transiently limit access to the interface. This sensitive and highly effective means of physically segregating proteins has implications for cell-cell contacts such as T-cell immunological synapses (for example, CD45 exclusion) and epithelial cell junctions (for example, E-cadherin enrichment), as well as for protein sorting at intracellular contact points between membrane-bound organelles.

Caveolin, sterol carrier protein-2, membrane cholesterol-rich microdomains and intracellular cholesterol trafficking.

PubMed

Schroeder, Friedhelm; Huang, Huan; McIntosh, Avery L; Atshaves, Barbara P; Martin, Gregory G; Kier, Ann B

2010-01-01

While the existence of membrane lateral microdomains has been known for over 30 years, interest in these structures accelerated in the past decade due to the discovery that cholesterol-rich microdomains serve important biological functions. It is increasingly appreciated that cholesterol-rich microdomains in the plasma membranes of eukaryotic cells represent an organizing nexus for multiple cellular proteins involved in transmembrane nutrient uptake (cholesterol, fatty acid, glucose, etc.), cell-signaling, immune recognition, pathogen entry, and many other roles. Despite these advances, however, relatively little is known regarding the organization of cholesterol itself in these plasma membrane microdomains. Although a variety of non-sterol markers indicate the presence of microdomains in the plasma membranes of living cells, none of these studies have demonstrated that cholesterol is enriched in these microdomains in living cells. Further, the role of cholesterol-rich membrane microdomains as targets for intracellular cholesterol trafficking proteins such as sterol carrier protein-2 (SCP-2) that facilitate cholesterol uptake and transcellular transport for targeting storage (cholesterol esters) or efflux is only beginning to be understood. Herein, we summarize the background as well as recent progress in this field that has advanced our understanding of these issues.

Shuttling of G protein subunits between the plasma membrane and intracellular membranes.

PubMed

Chisari, Mariangela; Saini, Deepak Kumar; Kalyanaraman, Vani; Gautam, Narasimhan

2007-08-17

Heterotrimeric G proteins (alphabetagamma) mediate the majority of signaling pathways in mammalian cells. It is long held that G protein function is localized to the plasma membrane. Here we examined the spatiotemporal dynamics of G protein localization using fluorescence recovery after photobleaching, fluorescence loss in photobleaching, and a photoswitchable fluorescent protein, Dronpa. Unexpectedly, G protein subunits shuttle rapidly (t1/2 < 1 min) between the plasma membrane and intracellular membranes. We show that consistent with such shuttling, G proteins constitutively reside in endomembranes. Furthermore, we show that shuttling is inhibited by 2-bromopalmitate. Thus, contrary to present thought, G proteins do not reside permanently on the plasma membrane but are constantly testing the cytoplasmic surfaces of the plasma membrane and endomembranes to maintain G protein pools in intracellular membranes to establish direct communication between receptors and endomembranes.

Heterologous Expression of Membrane Proteins: Choosing the Appropriate Host

PubMed Central

Pochon, Nathalie; Dementin, Sébastien; Hivin, Patrick; Boutigny, Sylvain; Rioux, Jean-Baptiste; Salvi, Daniel; Seigneurin-Berny, Daphné; Richaud, Pierre; Joyard, Jacques; Pignol, David; Sabaty, Monique; Desnos, Thierry; Pebay-Peyroula, Eva; Darrouzet, Elisabeth; Vernet, Thierry; Rolland, Norbert

2011-01-01

Background Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein. Methodology/Principal Findings The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides) and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells) hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals), functions (transporters, receptors, enzymes) and topologies (between 0 and 13 transmembrane segments). The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts. Conclusions/Significance Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein. PMID:22216205

Purification and Identification of Membrane Proteins from Urinary Extracellular Vesicles using Triton X-114 Phase Partitioning.

PubMed

Hu, Shuiwang; Musante, Luca; Tataruch, Dorota; Xu, Xiaomeng; Kretz, Oliver; Henry, Michael; Meleady, Paula; Luo, Haihua; Zou, Hequn; Jiang, Yong; Holthofer, Harry

2018-01-05

Urinary extracellular vesicles (uEVs) have become a promising source for biomarkers accurately reflecting biochemical changes in kidney and urogenital diseases. Characteristically, uEVs are rich in membrane proteins associated with several cellular functions like adhesion, transport, and signaling. Hence, membrane proteins of uEVs should represent an exciting protein class with unique biological properties. In this study, we utilized uEVs to optimize the Triton X-114 detergent partitioning protocol targeted for membrane proteins and proceeded to their subsequent characterization while eliminating effects of Tamm-Horsfall protein, the most abundant interfering protein in urine. This is the first report aiming to enrich and characterize the integral transmembrane proteins present in human urinary vesicles. First, uEVs were enriched using a "hydrostatic filtration dialysis'' appliance, and then the enriched uEVs and lysates were verified by transmission electron microscopy. After using Triton X-114 phase partitioning, we generated an insoluble pellet fraction and aqueous phase (AP) and detergent phase (DP) fractions and analyzed them with LC-MS/MS. Both in- and off-gel protein digestion methods were used to reveal an increased number of membrane proteins of uEVs. After comparing with the identified proteins without phase separation as in our earlier publication, 199 different proteins were detected in DP. Prediction of transmembrane domains (TMDs) from these protein fractions showed that DP had more TMDs than other groups. The analyses of hydrophobicity revealed that the GRAVY score of DP was much higher than those of the other fractions. Furthermore, the analysis of proteins with lipid anchor revealed that DP proteins had more lipid anchors than other fractions. Additionally, KEGG pathway analysis showed that the DP proteins detected participate in endocytosis and signaling, which is consistent with the expected biological functions of membrane proteins. Finally

Refractive-Index-Based Screening of Membrane-Protein-Mediated Transfer across Biological Membranes

PubMed Central

Brändén, Magnus; Tabaei, Seyed R.; Fischer, Gerhard; Neutze, Richard; Höök, Fredrik

2010-01-01

Abstract Numerous membrane-transport proteins are major drug targets, and therefore a key ingredient in pharmaceutical development is the availability of reliable, efficient tools for membrane transport characterization and inhibition. Here, we present the use of evanescent-wave sensing for screening of membrane-protein-mediated transport across lipid bilayer membranes. This method is based on a direct recording of the temporal variations in the refractive index that occur upon a transfer-dependent change in the solute concentration inside liposomes associated to a surface plasmon resonance (SPR) active sensor surface. The applicability of the method is demonstrated by a functional study of the aquaglyceroporin PfAQP from the malaria parasite Plasmodium falciparum. Assays of the temperature dependence of facilitated diffusion of sugar alcohols on a single set of PfAQP-reconstituted liposomes reveal that the activation energies for facilitated diffusion of xylitol and sorbitol are the same as that previously measured for glycerol transport in the aquaglyceroporin of Escherichia coli (5 kcal/mole). These findings indicate that the aquaglyceroporin selectivity filter does not discriminate sugar alcohols based on their length, and that the extra energy cost of dehydration of larger sugar alcohols, upon entering the pore, is compensated for by additional hydrogen-bond interactions within the aquaglyceroporin pore. PMID:20655840

Membrane Protein Crystallization Using Laser Irradiation

NASA Astrophysics Data System (ADS)

Adachi, Hiroaki; Murakami, Satoshi; Niino, Ai; Matsumura, Hiroyoshi; Takano, Kazufumi; Inoue, Tsuyoshi; Mori, Yusuke; Yamaguchi, Akihito; Sasaki, Takatomo

2004-10-01

We demonstrate the crystallization of a membrane protein using femtosecond laser irradiation. This method, which we call the laser irradiated growth technique (LIGHT), is useful for producing AcrB crystals in a solution of low supersaturation range. LIGHT is characterized by reduced nucleation times. This feature is important for crystallizing membrane proteins because of their labile properties when solubilized as protein-detergent micelles. Using LIGHT, high-quality crystals of a membrane transporter protein, AcrB, were obtained. The resulting crystals were found to be of sufficiently high resolution for X-ray diffraction. The results reported here indicate that LIGHT is a powerful tool for membrane protein crystallization, as well as for the growth of soluble proteins.

Biomimetic devices functionalized by membrane channel proteins

NASA Astrophysics Data System (ADS)

Schmidt, Jacob

2004-03-01

We are developing a new family of active materials which derive their functional properties from membrane proteins. These materials have two primary components: the proteins and the membranes themselves. I will discuss our recent work directed toward development of a generic platform for a "plug-and-play" philosophy of membrane protein engineering. By creating a stable biomimetic polymer membrane a single molecular monolayer thick, we will enable the exploitation of the function of any membrane protein, from pores and pumps to sensors and energy transducers. Our initial work has centered on the creation, study, and characterization of the biomimetic membranes. We are attempting to make large areas of membrane monolayers using Langmuir-Blodgett film formation as well as through arrays of microfabricated black lipid membrane-type septa. A number of techniques allow the insertion of protein into the membranes. As a benchmark, we have been employing a model system of voltage-gated pore proteins, which have electrically controllable porosities. I will report on the progress of this work, the characterization of the membranes, protein insertion processes, and the yield and functionality of the composite.

Z-membranes: artificial organelles for overexpressing recombinant integral membrane proteins.

PubMed Central

Gong, F C; Giddings, T H; Meehl, J B; Staehelin, L A; Galbraith, D W

1996-01-01

We have expressed a fusion protein formed between the avian infectious bronchitis virus M protein and the bacterial enzyme beta-glucuronidase in transgenic tobacco cells. Electron microscope images of such cells demonstrate that overexpression of this fusion protein gives rise to a type of endoplasmic reticulum membrane domain in which adjacent membranes become zippered together apparently as a consequence of the oligomerizing action of beta-glucuronidase. These zippered (Z-) membranes lack markers of the endoplasmic reticulum (NADH cytochrome c reductase and ribosomes) and accumulate in the cells in the form of multilayered scroll-like structures (up to 2 micrometers in diameter; 20-50 per cell) without affecting plant growth. The discovery of Z-membranes has broad implications for biology and biotechnology in that they provide a means for accumulating large quantities of recombinant membrane proteins within discrete domains of native membranes. Images Fig. 1 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:8700911

A cell-free translocation system using extracts of cultured insect cells to yield functional membrane proteins.

PubMed

Ezure, Toru; Nanatani, Kei; Sato, Yoko; Suzuki, Satomi; Aizawa, Keishi; Souma, Satoshi; Ito, Masaaki; Hohsaka, Takahiro; von Heijine, Gunnar; Utsumi, Toshihiko; Abe, Keietsu; Ando, Eiji; Uozumi, Nobuyuki

2014-01-01

Cell-free protein synthesis is a powerful method to explore the structure and function of membrane proteins and to analyze the targeting and translocation of proteins across the ER membrane. Developing a cell-free system based on cultured cells for the synthesis of membrane proteins could provide a highly reproducible alternative to the use of tissues from living animals. We isolated Sf21 microsomes from cultured insect cells by a simplified isolation procedure and evaluated the performance of the translocation system in combination with a cell-free translation system originating from the same source. The isolated microsomes contained the basic translocation machinery for polytopic membrane proteins including SRP-dependent targeting components, translocation channel (translocon)-dependent translocation, and the apparatus for signal peptide cleavage and N-linked glycosylation. A transporter protein synthesized with the cell-free system could be functionally reconstituted into a lipid bilayer. In addition, single and double labeling with non-natural amino acids could be achieved at both the lumen side and the cytosolic side in this system. Moreover, tail-anchored proteins, which are post-translationally integrated by the guided entry of tail-anchored proteins (GET) machinery, were inserted correctly into the microsomes. These results showed that the newly developed cell-free translocation system derived from cultured insect cells is a practical tool for the biogenesis of properly folded polytopic membrane proteins as well as tail-anchored proteins.

Gene cloning and prokaryotic expression of recombinant outer membrane protein from Vibrio parahaemolyticus

NASA Astrophysics Data System (ADS)

Yuan, Ye; Wang, Xiuli; Guo, Sheping; Qiu, Xuemei

2011-06-01

Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals. The outer membrane protein of bacteria plays an important role in the infection and pathogenicity to the host. Thus, the outer membrane proteins are an ideal target for vaccines. We amplified a complete outer membrane protein gene (ompW) from V. parahaemolyticus ATCC 17802. We then cloned and expressed the gene into Escherichia coli BL21 (DE3) cells. The gene coded for a protein that was 42.78 kDa. We purified the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting, respectively. Our results provide a basis for future application of the OmpW protein as a vaccine candidate against infection by V. parahaemolyticus. In addition, the purified OmpW protein can be used for further functional and structural studies.

Protein cleavage strategies for an improved analysis of the membrane proteome

PubMed Central

Fischer, Frank; Poetsch, Ansgar

2006-01-01

Background Membrane proteins still remain elusive in proteomic studies. This is in part due to the distribution of the amino acids lysine and arginine, which are less frequent in integral membrane proteins and almost absent in transmembrane helices. As these amino acids are cleavage targets for the commonly used protease trypsin, alternative cleavage conditions, which should improve membrane protein analysis, were tested by in silico digestion for the three organisms Saccharomyces cerevisiae, Halobacterium sp. NRC-1, and Corynebacterium glutamicum as hallmarks for eukaryotes, archea and eubacteria. Results For the membrane proteomes from all three analyzed organisms, we identified cleavage conditions that achieve better sequence and proteome coverage than trypsin. Greater improvement was obtained for bacteria than for yeast, which was attributed to differences in protein size and GRAVY. It was demonstrated for bacteriorhodopsin that the in silico predictions agree well with the experimental observations. Conclusion For all three examined organisms, it was found that a combination of chymotrypsin and staphylococcal peptidase I gave significantly better results than trypsin. As some of the improved cleavage conditions are not more elaborate than trypsin digestion and have been proven useful in practice, we suppose that the cleavage at both hydrophilic and hydrophobic amino acids should facilitate in general the analysis of membrane proteins for all organisms. PMID:16512920

Membrane organization of virus and target cell plays a role in HIV entry.

PubMed

Dumas, Fabrice; Preira, Pascal; Salomé, Laurence

2014-12-01

The initial steps of the Human Immunodeficiency Virus (HIV) replication cycle play a crucial role that arbitrates viral tropism and infection efficiency. Before the release of its genome into the host cell cytoplasm, viruses operate a complex sequence of events that take place at the plasma membrane of the target cell. The first step is the binding of the HIV protein envelope (Env) to the cellular receptor CD4. This triggers conformational changes of the gp120 viral protein that allow its interaction with a co-receptor that can be either CCR5 or CXCR4, defining the tropism of the virus entering the cell. This sequential interaction finally drives the fusion of the viral and host cell membrane or to the endocytosis of the viruses. Here, we discuss how the membrane composition and organization of both the virus and the target cell can affect these steps and thus influence the capability of the viruses to infect cells. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

A novel lipoprotein nanoparticle system for membrane proteins

PubMed Central

Frauenfeld, Jens; Löving, Robin; Armache, Jean-Paul; Sonnen, Andreas; Guettou, Fatma; Moberg, Per; Zhu, Lin; Jegerschöld, Caroline; Flayhan, Ali; Briggs, John A.G.; Garoff, Henrik; Löw, Christian; Cheng, Yifan; Nordlund, Pär

2016-01-01

Membrane proteins are of outstanding importance in biology, drug discovery and vaccination. A common limiting factor in research and applications involving membrane proteins is the ability to solubilize and stabilize membrane proteins. Although detergents represent the major means for solubilizing membrane proteins, they are often associated with protein instability and poor applicability in structural and biophysical studies. Here, we present a novel lipoprotein nanoparticle system that allows for the reconstitution of membrane proteins into a lipid environment that is stabilized by a scaffold of Saposin proteins. We showcase the applicability of the method on two purified membrane protein complexes as well as the direct solubilization and nanoparticle-incorporation of a viral membrane protein complex from the virus membrane. We also demonstrate that this lipid nanoparticle methodology facilitates high-resolution structural studies of membrane proteins in a lipid environment by single-particle electron cryo-microscopy (cryo-EM) and allows for the stabilization of the HIV-envelope glycoprotein in a functional state. PMID:26950744

Targeting RAS Membrane Association: Back to the Future for Anti-RAS Drug Discovery?

PubMed Central

Cox, Adrienne D.; Der, Channing J.; Philips, Mark R.

2015-01-01

RAS proteins require membrane association for their biological activity, making this association a logical target for anti-RAS therapeutics. Lipid modification of RAS proteins by a farnesyl isoprenoid is an obligate step in that association, and is an enzymatic process. Accordingly, farnesyltransferase inhibitors (FTIs) were developed as potential anti-RAS drugs. The lack of efficacy of FTIs as anti-cancer drugs was widely seen as indicating that blocking RAS membrane association was a flawed approach to cancer treatment. However, a deeper understanding of RAS modification and trafficking has revealed that this was an erroneous conclusion. In the presence of FTIs, KRAS and NRAS, which are the RAS isoforms most frequently mutated in cancer, become substrates for alternative modification, can still associate with membranes, and can still function. Thus, FTIs failed not because blocking RAS membrane association is an ineffective approach, but because FTIs failed to accomplish that task. Recent findings regarding RAS isoform trafficking and the regulation of RAS subcellular localization have rekindled interest in efforts to target these processes. In particular, improved understanding of the palmitoylation/depalmitoylation cycle that regulates RAS interaction with the plasma membrane, endomembranes and cytosol, and of the potential importance of RAS chaperones, have led to new approaches. Efforts to validate and target other enzymatically regulated post-translational modifications are also ongoing. In this review, we revisit lessons learned, describe the current state of the art, and highlight challenging but promising directions to achieve the goal of disrupting RAS membrane association and subcellular localization for anti-RAS drug development. PMID:25878363

Sensing Membrane Stresses by Protein Insertions

PubMed Central

Campelo, Felix; Kozlov, Michael M.

2014-01-01

Protein domains shallowly inserting into the membrane matrix are ubiquitous in peripheral membrane proteins involved in various processes of intracellular membrane shaping and remodeling. It has been suggested that these domains sense membrane curvature through their preferable binding to strongly curved membranes, the binding mechanism being mediated by lipid packing defects. Here we make an alternative statement that shallow protein insertions are universal sensors of the intra-membrane stresses existing in the region of the insertion embedding rather than sensors of the curvature per se. We substantiate this proposal computationally by considering different independent ways of the membrane stress generation among which some include changes of the membrane curvature whereas others do not alter the membrane shape. Our computations show that the membrane-binding coefficient of shallow protein insertions is determined by the resultant stress independently of the way this stress has been produced. By contrast, consideration of the correlation between the insertion binding and the membrane curvature demonstrates that the binding coefficient either increases or decreases with curvature depending on the factors leading to the curvature generation. To validate our computational model, we treat quantitatively the experimental results on membrane binding by ALPS1 and ALPS2 motifs of ArfGAP1. PMID:24722359

Identification and Molecular Characterization of the Chloroplast Targeting Domain of Turnip yellow mosaic virus Replication Proteins

PubMed Central

Moriceau, Lucille; Jomat, Lucile; Bressanelli, Stéphane; Alcaide-Loridan, Catherine; Jupin, Isabelle

2017-01-01

Turnip yellow mosaic virus (TYMV) is a positive-strand RNA virus infecting plants. The TYMV 140K replication protein is a key organizer of viral replication complex (VRC) assembly, being responsible for recruitment of the viral polymerase and for targeting the VRCs to the chloroplast envelope where viral replication takes place. However, the structural requirements determining the subcellular localization and membrane association of this essential viral protein have not yet been defined. In this study, we investigated determinants for the in vivo chloroplast targeting of the TYMV 140K replication protein. Subcellular localization studies of deletion mutants identified a 41-residue internal sequence as the chloroplast targeting domain (CTD) of TYMV 140K; this sequence is sufficient to target GFP to the chloroplast envelope. The CTD appears to be located in the C-terminal extension of the methyltransferase domain—a region shared by 140K and its mature cleavage product 98K, which behaves as an integral membrane protein during infection. We predicted the CTD to fold into two amphipathic α-helices—a folding that was confirmed in vitro by circular dichroism spectroscopy analyses of a synthetic peptide. The importance for subcellular localization of the integrity of these amphipathic helices, and the function of 140K/98K, was demonstrated by performing amino acid substitutions that affected chloroplast targeting, membrane association and viral replication. These results establish a short internal α-helical peptide as an unusual signal for targeting proteins to the chloroplast envelope membrane, and provide new insights into membrane targeting of viral replication proteins—a universal feature of positive-strand RNA viruses. PMID:29312393

Protein Targeting: ER Leads the Way to the Inner Nuclear Envelope.

PubMed

Blackstone, Craig

2017-12-04

Efficient targeting of newly synthesized membrane proteins from the endoplasmic reticulum to the inner nuclear membrane depends on nucleotide hydrolysis. A new study shows that this dependence reflects critical actions of the atlastin family of GTPases in maintaining the morphology of the endoplasmic reticulum network. Published by Elsevier Ltd.

Plant and yeast cornichon possess a conserved acidic motif required for correct targeting of plasma membrane cargos.

PubMed

Rosas-Santiago, Paul; Lagunas-Gomez, Daniel; Yáñez-Domínguez, Carolina; Vera-Estrella, Rosario; Zimmermannová, Olga; Sychrová, Hana; Pantoja, Omar

2017-10-01

The export of membrane proteins along the secretory pathway is initiated at the endoplasmic reticulum after proteins are folded and packaged inside this organelle by their recruiting into the coat complex COPII vesicles. It is proposed that cargo receptors are required for the correct transport of proteins to its target membrane, however, little is known about ER export signals for cargo receptors. Erv14/Cornichon belong to a well conserved protein family in Eukaryotes, and have been proposed to function as cargo receptors for many transmembrane proteins. Amino acid sequence alignment showed the presence of a conserved acidic motif in the C-terminal in homologues from plants and yeast. Here, we demonstrate that mutation of the C-terminal acidic motif from ScErv14 or OsCNIH1, did not alter the localization of these cargo receptors, however it modified the proper targeting of the plasma membrane transporters Nha1p, Pdr12p and Qdr2p. Our results suggest that mistargeting of these plasma membrane proteins is a consequence of a weaker interaction between the cargo receptor and cargo proteins caused by the mutation of the C-terminal acidic motif. Copyright © 2017 Elsevier B.V. All rights reserved.

IFITM Proteins Restrict Viral Membrane Hemifusion

PubMed Central

Golfetto, Ottavia; Bungart, Brittani; Li, Minghua; Ding, Shilei; He, Yuxian; Liang, Chen; Lee, James C.; Gratton, Enrico; Cohen, Fredric S.; Liu, Shan-Lu

2013-01-01

The interferon-inducible transmembrane (IFITM) protein family represents a new class of cellular restriction factors that block early stages of viral replication; the underlying mechanism is currently not known. Here we provide evidence that IFITM proteins restrict membrane fusion induced by representatives of all three classes of viral membrane fusion proteins. IFITM1 profoundly suppressed syncytia formation and cell-cell fusion induced by almost all viral fusion proteins examined; IFITM2 and IFITM3 also strongly inhibited their fusion, with efficiency somewhat dependent on cell types. Furthermore, treatment of cells with IFN also markedly inhibited viral membrane fusion and entry. By using the Jaagsiekte sheep retrovirus envelope and influenza A virus hemagglutinin as models for study, we showed that IFITM-mediated restriction on membrane fusion is not at the steps of receptor- and/or low pH-mediated triggering; instead, the creation of hemifusion was essentially blocked by IFITMs. Chlorpromazine (CPZ), a chemical known to promote the transition from hemifusion to full fusion, was unable to rescue the IFITM-mediated restriction on fusion. In contrast, oleic acid (OA), a lipid analog that generates negative spontaneous curvature and thereby promotes hemifusion, virtually overcame the restriction. To explore the possible effect of IFITM proteins on membrane molecular order and fluidity, we performed fluorescence labeling with Laurdan, in conjunction with two-photon laser scanning and fluorescence-lifetime imaging microscopy (FLIM). We observed that the generalized polarizations (GPs) and fluorescence lifetimes of cell membranes expressing IFITM proteins were greatly enhanced, indicating higher molecularly ordered and less fluidized membranes. Collectively, our data demonstrated that IFITM proteins suppress viral membrane fusion before the creation of hemifusion, and suggested that they may do so by reducing membrane fluidity and conferring a positive spontaneous

Atomistic models for free energy evaluation of drug binding to membrane proteins.

PubMed

Durdagi, S; Zhao, C; Cuervo, J E; Noskov, S Y

2011-01-01

The binding of various molecules to integral membrane proteins with optimal affinity and specificity is central to normal function of cell. While membrane proteins represent about one third of the whole cell proteome, they are a majority of common drug targets. The quest for the development of computational models capable of accurate evaluation of binding affinities, decomposition of the binding into its principal components and thus mapping molecular mechanisms of binding remains one of the main goals of modern computational biophysics and related drug development. The primary scope of this review will be on the recent extension of computational methods for the study of drug binding to membrane proteins. Several examples of such applications will be provided ranging from secondary transporters to voltage gated channels. In this mini-review, we will provide a short summary on the breadth of different methods for binding affinity evaluation. These methods include molecular docking with docking scoring functions, molecular dynamics (MD) simulations combined with post-processing analysis using Molecular Mechanics/Poisson Boltzmann (Generalized Born) Surface Area (MM/PB(GB)SA), as well as direct evaluation of free energies from Free Energy Perturbation (FEP) with constraining schemes, and Potential of Mean Force (PMF) computations. We will compare advantages and shortcomings of popular techniques and provide discussion on the integrative strategies for drug development aimed at targeting membrane proteins.

Biomimetic interfaces based on S-layer proteins, lipid membranes and functional biomolecules

PubMed Central

Schuster, Bernhard; Sleytr, Uwe B.

2014-01-01

Designing and utilization of biomimetic membrane systems generated by bottom-up processes is a rapidly growing scientific and engineering field. Elucidation of the supramolecular construction principle of archaeal cell envelopes composed of S-layer stabilized lipid membranes led to new strategies for generating highly stable functional lipid membranes at meso- and macroscopic scale. In this review, we provide a state-of-the-art survey of how S-layer proteins, lipids and polymers may be used as basic building blocks for the assembly of S-layer-supported lipid membranes. These biomimetic membrane systems are distinguished by a nanopatterned fluidity, enhanced stability and longevity and, thus, provide a dedicated reconstitution matrix for membrane-active peptides and transmembrane proteins. Exciting areas in the (lab-on-a-) biochip technology are combining composite S-layer membrane systems involving specific membrane functions with the silicon world. Thus, it might become possible to create artificial noses or tongues, where many receptor proteins have to be exposed and read out simultaneously. Moreover, S-layer-coated liposomes and emulsomes copying virus envelopes constitute promising nanoformulations for the production of novel targeting, delivery, encapsulation and imaging systems. PMID:24812051

Shape matters in protein mobility within membranes

PubMed Central

Quemeneur, François; Sigurdsson, Jon K.; Renner, Marianne; Atzberger, Paul J.; Bassereau, Patricia; Lacoste, David

2014-01-01

The lateral mobility of proteins within cell membranes is usually thought to be dependent on their size and modulated by local heterogeneities of the membrane. Experiments using single-particle tracking on reconstituted membranes demonstrate that protein diffusion is significantly influenced by the interplay of membrane curvature, membrane tension, and protein shape. We find that the curvature-coupled voltage-gated potassium channel (KvAP) undergoes a significant increase in protein mobility under tension, whereas the mobility of the curvature-neutral water channel aquaporin 0 (AQP0) is insensitive to it. Such observations are well explained in terms of an effective friction coefficient of the protein induced by the local membrane deformation. PMID:24706877

Plasma Membrane Proteomics of Human Breast Cancer Cell Lines Identifies Potential Targets for Breast Cancer Diagnosis and Treatment

PubMed Central

Ziegler, Yvonne S.; Moresco, James J.; Tu, Patricia G.; Yates, John R.; Nardulli, Ann M.

2014-01-01

The use of broad spectrum chemotherapeutic agents to treat breast cancer results in substantial and debilitating side effects, necessitating the development of targeted therapies to limit tumor proliferation and prevent metastasis. In recent years, the list of approved targeted therapies has expanded, and it includes both monoclonal antibodies and small molecule inhibitors that interfere with key proteins involved in the uncontrolled growth and migration of cancer cells. The targeting of plasma membrane proteins has been most successful to date, and this is reflected in the large representation of these proteins as targets of newer therapies. In view of these facts, experiments were designed to investigate the plasma membrane proteome of a variety of human breast cancer cell lines representing hormone-responsive, ErbB2 over-expressing and triple negative cell types, as well as a benign control. Plasma membranes were isolated by using an aqueous two-phase system, and the resulting proteins were subjected to mass spectrometry analysis. Overall, each of the cell lines expressed some unique proteins, and a number of proteins were expressed in multiple cell lines, but in patterns that did not always follow traditional clinical definitions of breast cancer type. From our data, it can be deduced that most cancer cells possess multiple strategies to promote uncontrolled growth, reflected in aberrant expression of tyrosine kinases, cellular adhesion molecules, and structural proteins. Our data set provides a very rich and complex picture of plasma membrane proteins present on breast cancer cells, and the sorting and categorizing of this data provides interesting insights into the biology, classification, and potential treatment of this prevalent and debilitating disease. PMID:25029196

Synthetic membrane-targeted antibiotics.

PubMed

Vooturi, S K; Firestine, S M

2010-01-01

Antimicrobial resistance continues to evolve and presents serious challenges in the therapy of both nosocomial and community-acquired infections. The rise of resistant strains like methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Staphylococcus aureus (VRSA) and vancomycin-resistant enterococci (VRE) suggests that antimicrobial resistance is an inevitable evolutionary response to antimicrobial use. This highlights the tremendous need for antibiotics against new bacterial targets. Agents that target the integrity of bacterial membrane are relatively novel in the clinical armamentarium. Daptomycin, a lipopeptide is a classical example of membrane-bound antibiotic. Nature has also utilized this tactic. Antimicrobial peptides (AMPs), which are found in all kingdoms, function primarily by permeabilizing the bacterial membrane. AMPs have several advantages over existing antibiotics including a broad spectrum of activity, rapid bactericidal activity, no cross-resistance with the existing antibiotics and a low probability for developing resistance. Currently, a small number of peptides have been developed for clinical use but therapeutic applications are limited because of poor bioavailability and high manufacturing cost. However, their broad specificity, potent activity and lower probability for resistance have spurred the search for synthetic mimetics of antimicrobial peptides as membrane-active antibiotics. In this review, we will discuss the different classes of synthetic membrane-bound antibiotics published since 2004.

Systematically Ranking the Tightness of Membrane Association for Peripheral Membrane Proteins (PMPs)*

PubMed Central

Gao, Liyan; Ge, Haitao; Huang, Xiahe; Liu, Kehui; Zhang, Yuanya; Xu, Wu; Wang, Yingchun

2015-01-01

Large-scale quantitative evaluation of the tightness of membrane association for nontransmembrane proteins is important for identifying true peripheral membrane proteins with functional significance. Herein, we simultaneously ranked more than 1000 proteins of the photosynthetic model organism Synechocystis sp. PCC 6803 for their relative tightness of membrane association using a proteomic approach. Using multiple precisely ranked and experimentally verified peripheral subunits of photosynthetic protein complexes as the landmarks, we found that proteins involved in two-component signal transduction systems and transporters are overall tightly associated with the membranes, whereas the associations of ribosomal proteins are much weaker. Moreover, we found that hypothetical proteins containing the same domains generally have similar tightness. This work provided a global view of the structural organization of the membrane proteome with respect to divergent functions, and built the foundation for future investigation of the dynamic membrane proteome reorganization in response to different environmental or internal stimuli. PMID:25505158

Structural basis for host membrane remodeling induced by protein 2B of hepatitis A virus.

PubMed

Vives-Adrián, Laia; Garriga, Damià; Buxaderas, Mònica; Fraga, Joana; Pereira, Pedro José Barbosa; Macedo-Ribeiro, Sandra; Verdaguer, Núria

2015-04-01

The complexity of viral RNA synthesis and the numerous participating factors require a mechanism to topologically coordinate and concentrate these multiple viral and cellular components, ensuring a concerted function. Similarly to all other positive-strand RNA viruses, picornaviruses induce rearrangements of host intracellular membranes to create structures that act as functional scaffolds for genome replication. The membrane-targeting proteins 2B and 2C, their precursor 2BC, and protein 3A appear to be primarily involved in membrane remodeling. Little is known about the structure of these proteins and the mechanisms by which they induce massive membrane remodeling. Here we report the crystal structure of the soluble region of hepatitis A virus (HAV) protein 2B, consisting of two domains: a C-terminal helical bundle preceded by an N-terminally curved five-stranded antiparallel β-sheet that displays striking structural similarity to the β-barrel domain of enteroviral 2A proteins. Moreover, the helicoidal arrangement of the protein molecules in the crystal provides a model for 2B-induced host membrane remodeling during HAV infection. No structural information is currently available for the 2B protein of any picornavirus despite it being involved in a critical process in viral factory formation: the rearrangement of host intracellular membranes. Here we present the structure of the soluble domain of the 2B protein of hepatitis A virus (HAV). Its arrangement, both in crystals and in solution under physiological conditions, can help to understand its function and sheds some light on the membrane rearrangement process, a putative target of future antiviral drugs. Moreover, this first structure of a picornaviral 2B protein also unveils a closer evolutionary relationship between the hepatovirus and enterovirus genera within the Picornaviridae family. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Structural Basis for Host Membrane Remodeling Induced by Protein 2B of Hepatitis A Virus

PubMed Central

Vives-Adrián, Laia; Garriga, Damià; Buxaderas, Mònica; Fraga, Joana; Pereira, Pedro José Barbosa

2015-01-01

ABSTRACT The complexity of viral RNA synthesis and the numerous participating factors require a mechanism to topologically coordinate and concentrate these multiple viral and cellular components, ensuring a concerted function. Similarly to all other positive-strand RNA viruses, picornaviruses induce rearrangements of host intracellular membranes to create structures that act as functional scaffolds for genome replication. The membrane-targeting proteins 2B and 2C, their precursor 2BC, and protein 3A appear to be primarily involved in membrane remodeling. Little is known about the structure of these proteins and the mechanisms by which they induce massive membrane remodeling. Here we report the crystal structure of the soluble region of hepatitis A virus (HAV) protein 2B, consisting of two domains: a C-terminal helical bundle preceded by an N-terminally curved five-stranded antiparallel β-sheet that displays striking structural similarity to the β-barrel domain of enteroviral 2A proteins. Moreover, the helicoidal arrangement of the protein molecules in the crystal provides a model for 2B-induced host membrane remodeling during HAV infection. IMPORTANCE No structural information is currently available for the 2B protein of any picornavirus despite it being involved in a critical process in viral factory formation: the rearrangement of host intracellular membranes. Here we present the structure of the soluble domain of the 2B protein of hepatitis A virus (HAV). Its arrangement, both in crystals and in solution under physiological conditions, can help to understand its function and sheds some light on the membrane rearrangement process, a putative target of future antiviral drugs. Moreover, this first structure of a picornaviral 2B protein also unveils a closer evolutionary relationship between the hepatovirus and enterovirus genera within the Picornaviridae family. PMID:25589659

Membrane protein stoichiometry studied in intact mammalian cells using liquid-phase electron microscopy.

PubMed

DE Jonge, N

2018-02-01

Receptor membrane proteins in the plasma membranes of cells respond to extracellular chemical signals by conformational changes, spatial redistribution, and (re-)assembly into protein complexes, for example, into homodimers (pairs of the same protein type). The functional state of the proteins can be determined from information about how subunits are assembled into protein complexes. Stoichiometric information about the protein complex subunits, however, is generally not obtained from intact cells but from pooled material extracted from many cells, resulting in a lack of fundamental knowledge about the functioning of membrane proteins. First, functional states may dramatically differ from cell to cell on account of cell heterogeneity. Second, extracting the membrane proteins from the plasma membrane may lead to many artefacts. Liquid-phase scanning transmission electron microscopy (STEM), in short liquid STEM, is a new technique capable of determining the locations of individual membrane proteins within the intact plasma membranes of cells in liquid. Many tens of whole cells can readily be imaged. It is possible to analyse the stoichiometry of membrane proteins in single cells while accounting for heterogenic cell populations. Liquid STEM was used to image epidermal growth factor receptors in whole COS7 cells. A study of the dimerisation of the HER2 protein in breast cancer cells revealed the presence of rare cancer cells in which HER2 was in a different functional state than in the bulk cells. Stoichiometric information about receptors is essential not only for basic science but also for biomedical application because they present many important pharmaceutical targets. © 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.

The cubicon method for concentrating membrane proteins in the cubic mesophase.

PubMed

Ma, Pikyee; Weichert, Dietmar; Aleksandrov, Luba A; Jensen, Timothy J; Riordan, John R; Liu, Xiangyu; Kobilka, Brian K; Caffrey, Martin

2017-09-01

The lipid cubic phase (in meso) method is an important approach for generating crystals and high-resolution X-ray structures of integral membrane proteins. However, as a consequence of instability, it can be impossible-using traditional methods-to concentrate certain membrane proteins and complexes to values suitable for in meso crystallization and structure determination. The cubicon method described here exploits the amphiphilic nature of membrane proteins and their natural tendency to partition preferentially into lipid bilayers from aqueous solution. Using several rounds of reconstitution, the protein concentration in the bilayer of the cubic mesophase can be ramped up stepwise from less than a milligram per milliliter to tens of milligrams per milliliter for crystallogenesis. The general applicability of the method is demonstrated with five integral membrane proteins: the β 2 -adrenergic G protein-coupled receptor (β 2 AR), the peptide transporter (PepT St ), diacylglycerol kinase (DgkA), the alginate transporter (AlgE) and the cystic fibrosis transmembrane conductance regulator (CFTR). In the cases of β 2 AR, PepT St , DgkA and AlgE, an effective 20- to 45-fold concentration was realized, resulting in a protein-laden mesophase that allowed the formation of crystals using the in meso method and structure determination to resolutions ranging from 2.4 Å to 3.2 Å. In addition to opening up in meso crystallization to a broader range of integral membrane protein targets, the cubicon method should find application in situations that require membrane protein reconstitution in a lipid bilayer at high concentrations. These applications include functional and biophysical characterization studies for ligand screening, drug delivery, antibody production and protein complex formation. A typical cubicon experiment can be completed in 3-5 h.

Targeting of a Nicotiana plumbaginifolia H+ -ATPase to the plasma membrane is not by default and requires cytosolic structural determinants.

PubMed

Lefebvre, Benoit; Batoko, Henri; Duby, Geoffrey; Boutry, Marc

2004-07-01

The structural determinants involved in the targeting of multitransmembrane-span proteins to the plasma membrane (PM) remain poorly understood. The plasma membrane H+ -ATPase (PMA) from Nicotiana plumbaginifolia, a well-characterized 10 transmembrane-span enzyme, was used as a model to identify structural elements essential for targeting to the PM. When PMA2 and PMA4, representatives of the two main PMA subfamilies, were fused to green fluorescent protein (GFP), the chimeras were shown to be still functional and to be correctly and rapidly targeted to the PM in transgenic tobacco. By contrast, chimeric proteins containing various combinations of PMA transmembrane spanning domains accumulated in the Golgi apparatus and not in the PM and displayed slow traffic properties through the secretory pathway. Individual deletion of three of the four cytosolic domains did not prevent PM targeting, but deletion of the large loop or of its nucleotide binding domain resulted in GFP fluorescence accumulating exclusively in the endoplasmic reticulum. The results show that, at least for this polytopic protein, the PM is not the default pathway and that, in contrast with single-pass membrane proteins, cytosolic structural determinants are required for correct targeting.

Targeting of a Nicotiana plumbaginifolia H+-ATPase to the Plasma Membrane Is Not by Default and Requires Cytosolic Structural Determinants

PubMed Central

Lefebvre, Benoit; Batoko, Henri; Duby, Geoffrey; Boutry, Marc

2004-01-01

The structural determinants involved in the targeting of multitransmembrane-span proteins to the plasma membrane (PM) remain poorly understood. The plasma membrane H+-ATPase (PMA) from Nicotiana plumbaginifolia, a well-characterized 10 transmembrane–span enzyme, was used as a model to identify structural elements essential for targeting to the PM. When PMA2 and PMA4, representatives of the two main PMA subfamilies, were fused to green fluorescent protein (GFP), the chimeras were shown to be still functional and to be correctly and rapidly targeted to the PM in transgenic tobacco. By contrast, chimeric proteins containing various combinations of PMA transmembrane spanning domains accumulated in the Golgi apparatus and not in the PM and displayed slow traffic properties through the secretory pathway. Individual deletion of three of the four cytosolic domains did not prevent PM targeting, but deletion of the large loop or of its nucleotide binding domain resulted in GFP fluorescence accumulating exclusively in the endoplasmic reticulum. The results show that, at least for this polytopic protein, the PM is not the default pathway and that, in contrast with single-pass membrane proteins, cytosolic structural determinants are required for correct targeting. PMID:15208389

Mapping the yeast host cell response to recombinant membrane protein production: relieving the biological bottlenecks.

PubMed

Ashe, Mark P; Bill, Roslyn M

2011-06-01

Understanding the structures and functions of membrane proteins is an active area of research within bioscience. Membrane proteins are key players in essential cellular processes such as the uptake of nutrients, the export of waste products, and the way in which cells communicate with their environment. It is therefore not surprising that membrane proteins are targeted by over half of all prescription drugs. Since most membrane proteins are not abundant in their native membranes, it is necessary to produce them in recombinant host cells to enable further structural and functional studies. Unfortunately, achieving the required yields of functional recombinant membrane proteins is still a bottleneck in contemporary bioscience. This has highlighted the need for defined and rational optimization strategies based upon experimental observation rather than relying on trial and error. We have published a transcriptome and subsequent genetic analysis that has identified genes implicated in high-yielding yeast cells. These results have highlighted a role for alterations to a cell's protein synthetic capacity in the production of high yields of recombinant membrane protein: paradoxically, reduced protein synthesis favors higher yields. These results highlight a potential bottleneck at the protein folding or translocation stage of protein production. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Size-dependent protein segregation at membrane interfaces

PubMed Central

Schmid, Eva M; Bakalar, Matthew H; Choudhuri, Kaushik; Weichsel, Julian; Ann, HyoungSook; Geissler, Phillip L; Dustin, Michael L; Fletcher, Daniel A

2016-01-01

Membrane interfaces formed at cell-cell junctions are associated with characteristic patterns of membrane protein organization, such as E-cadherin enrichment in epithelial junctional complexes and CD45 exclusion from the signaling foci of immunological synapses. To isolate the role of protein size in these processes, we reconstituted membrane interfaces in vitro using giant unilamellar vesicles decorated with synthetic binding and non-binding proteins. We show that size differences between binding and non-binding proteins can dramatically alter their organization at membrane interfaces in the absence of active contributions from the cytoskeleton, with as little as a ~5 nm increase in non-binding protein size driving its exclusion from the interface. Combining in vitro measurements with Monte Carlo simulations, we find that non-binding protein exclusion is also influenced by lateral crowding, binding protein affinity, and thermally-driven membrane height fluctuations that transiently limit access to the interface. This simple, sensitive, and highly effective means of passively segregating proteins has implications for signaling at cell-cell junctions and protein sorting at intracellular contact points between membrane-bound organelles. PMID:27980602

Refractive-index-based screening of membrane-protein-mediated transfer across biological membranes.

PubMed

Brändén, Magnus; Tabaei, Seyed R; Fischer, Gerhard; Neutze, Richard; Höök, Fredrik

2010-07-07

Numerous membrane-transport proteins are major drug targets, and therefore a key ingredient in pharmaceutical development is the availability of reliable, efficient tools for membrane transport characterization and inhibition. Here, we present the use of evanescent-wave sensing for screening of membrane-protein-mediated transport across lipid bilayer membranes. This method is based on a direct recording of the temporal variations in the refractive index that occur upon a transfer-dependent change in the solute concentration inside liposomes associated to a surface plasmon resonance (SPR) active sensor surface. The applicability of the method is demonstrated by a functional study of the aquaglyceroporin PfAQP from the malaria parasite Plasmodium falciparum. Assays of the temperature dependence of facilitated diffusion of sugar alcohols on a single set of PfAQP-reconstituted liposomes reveal that the activation energies for facilitated diffusion of xylitol and sorbitol are the same as that previously measured for glycerol transport in the aquaglyceroporin of Escherichia coli (5 kcal/mole). These findings indicate that the aquaglyceroporin selectivity filter does not discriminate sugar alcohols based on their length, and that the extra energy cost of dehydration of larger sugar alcohols, upon entering the pore, is compensated for by additional hydrogen-bond interactions within the aquaglyceroporin pore. Copyright 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Hematopoietic Protein-1 Regulates the Actin Membrane Skeleton and Membrane Stability in Murine Erythrocytes

PubMed Central

Chan, Maia M.; Wooden, Jason M.; Tsang, Mark; Gilligan, Diana M.; Hirenallur-S, Dinesh K.; Finney, Greg L.; Rynes, Eric; MacCoss, Michael; Ramirez, Julita A.; Park, Heon; Iritani, Brian M.

2013-01-01

Hematopoietic protein-1 (Hem-1) is a hematopoietic cell specific member of the WAVE (Wiskott-Aldrich syndrome verprolin-homologous protein) complex, which regulates filamentous actin (F-actin) polymerization in many cell types including immune cells. However, the roles of Hem-1 and the WAVE complex in erythrocyte biology are not known. In this study, we utilized mice lacking Hem-1 expression due to a non-coding point mutation in the Hem1 gene to show that absence of Hem-1 results in microcytic, hypochromic anemia characterized by abnormally shaped erythrocytes with aberrant F-actin foci and decreased lifespan. We find that Hem-1 and members of the associated WAVE complex are normally expressed in wildtype erythrocyte progenitors and mature erythrocytes. Using mass spectrometry and global proteomics, Coomassie staining, and immunoblotting, we find that the absence of Hem-1 results in decreased representation of essential erythrocyte membrane skeletal proteins including α- and β- spectrin, dematin, p55, adducin, ankyrin, tropomodulin 1, band 3, and band 4.1. Hem1−/− erythrocytes exhibit increased protein kinase C-dependent phosphorylation of adducin at Ser724, which targets adducin family members for dissociation from spectrin and actin, and subsequent proteolysis. Increased adducin Ser724 phosphorylation in Hem1−/− erythrocytes correlates with decreased protein expression of the regulatory subunit of protein phosphatase 2A (PP2A), which is required for PP2A-dependent dephosphorylation of PKC targets. These results reveal a novel, critical role for Hem-1 in the homeostasis of structural proteins required for formation and stability of the actin membrane skeleton in erythrocytes. PMID:23424621

Fine tuning cellular recognition: The function of the leucine rich repeat (LRR) trans-membrane protein, LRT, in muscle targeting to tendon cells.

PubMed

Gilsohn, Eli; Volk, Talila

2010-01-01

The formation of complex tissues during embryonic development is often accompanied by directed cellular migration towards a target tissue. Specific mutual recognition between the migrating cell and its target tissue leads to the arrest of the cell migratory behavior and subsequent contact formation between the two interacting cell types. Recent studies implicated a novel family of surface proteins containing a trans-membrane domain and single leucine-rich repeat (LRR) domain in inter-cellular recognition and the arrest of cell migration. Here, we describe the involvement of a novel LRR surface protein, LRT, in targeting migrating muscles towards their corresponding tendon cells in the Drosophila embryo. LRT is specifically expressed by the target tendon cells and is essential for arresting the migratory behavior of the muscle cells. Additional studies in Drosophila S2 cultured cells suggest that LRT forms a protein complex with the Roundabout (Robo) receptor, essential for guiding muscles towards their tendon partners. Genetic analysis supports a model in which LRT performs its activity non-autonomously through its interaction with the Robo receptors expressed on the muscle surfaces. These results suggest a novel mechanism of intercellular recognition through interactions between LRR family members and Robo receptors.

The effectiveness of styrene-maleic acid (SMA) copolymers for solubilisation of integral membrane proteins from SMA-accessible and SMA-resistant membranes.

PubMed

Swainsbury, David J K; Scheidelaar, Stefan; Foster, Nicholas; van Grondelle, Rienk; Killian, J Antoinette; Jones, Michael R

2017-10-01

Solubilisation of biological lipid bilayer membranes for analysis of their protein complement has traditionally been carried out using detergents, but there is increasing interest in the use of amphiphilic copolymers such as styrene maleic acid (SMA) for the solubilisation, purification and characterisation of integral membrane proteins in the form of protein/lipid nanodiscs. Here we survey the effectiveness of various commercially-available formulations of the SMA copolymer in solubilising Rhodobacter sphaeroides reaction centres (RCs) from photosynthetic membranes. We find that formulations of SMA with a 2:1 or 3:1 ratio of styrene to maleic acid are almost as effective as detergent in solubilising RCs, with the best solubilisation by short chain variants (<30kDa weight average molecular weight). The effectiveness of 10kDa 2:1 and 3:1 formulations of SMA to solubilise RCs gradually declined when genetically-encoded coiled-coil bundles were used to artificially tether normally monomeric RCs into dimeric, trimeric and tetrameric multimers. The ability of SMA to solubilise reaction centre-light harvesting 1 (RC-LH1) complexes from densely packed and highly ordered photosynthetic membranes was uniformly low, but could be increased through a variety of treatments to increase the lipid:protein ratio. However, proteins isolated from such membranes comprised clusters of complexes in small membrane patches rather than individual proteins. We conclude that short-chain 2:1 and 3:1 formulations of SMA are the most effective in solubilising integral membrane proteins, but that solubilisation efficiencies are strongly influenced by the size of the target protein and the density of packing of proteins in the membrane. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Deciphering membrane-associated molecular processes in target tissue of autoimmune uveitis by label-free quantitative mass spectrometry.

PubMed

Hauck, Stefanie M; Dietter, Johannes; Kramer, Roxane L; Hofmaier, Florian; Zipplies, Johanna K; Amann, Barbara; Feuchtinger, Annette; Deeg, Cornelia A; Ueffing, Marius

2010-10-01

Autoimmune uveitis is a blinding disease presenting with autoantibodies against eye-specific proteins as well as autoagressive T cells invading and attacking the immune-privileged target tissue retina. The molecular events enabling T cells to invade and attack the tissue have remained elusive. Changes in membrane protein expression patterns between diseased and healthy stages are especially interesting because initiating events of disease will most likely occur at membranes. Since disease progression is accompanied with a break-down of the blood-retinal barrier, serum-derived proteins mask the potential target tissue-related changes. To overcome this limitation, we used membrane-enriched fractions derived from retinas of the only available spontaneous animal model for the disease equine recurrent uveitis, and compared expression levels by a label-free LC-MSMS-based strategy to healthy control samples. We could readily identify a total of 893 equine proteins with 57% attributed to the Gene Ontology project term "membrane." Of these, 179 proteins were found differentially expressed in equine recurrent uveitis tissue. Pathway enrichment analyses indicated an increase in proteins related to antigen processing and presentation, TNF receptor signaling, integrin cell surface interactions and focal adhesions. Additionally, loss of retina-specific proteins reflecting decrease of vision was observed as well as an increase in Müller glial cell-specific proteins indicating glial reactivity. Selected protein candidates (caveolin 1, integrin alpha 1 and focal adhesion kinase) were validated by immunohistochemistry and tissue staining pattern pointed to a significant increase of these proteins at the level of the outer limiting membrane which is part of the outer blood-retinal barrier. Taken together, the membrane enrichment in combination with LC-MSMS-based label-free quantification greatly increased the sensitivity of the comparative tissue profiling and resulted in detection

Identification of specific posttranslational O-mycoloylations mediating protein targeting to the mycomembrane.

PubMed

Carel, Clément; Marcoux, Julien; Réat, Valérie; Parra, Julien; Latgé, Guillaume; Laval, Françoise; Demange, Pascal; Burlet-Schiltz, Odile; Milon, Alain; Daffé, Mamadou; Tropis, Maryelle G; Renault, Marie A M

2017-04-18

The outer membranes (OMs) of members of the Corynebacteriales bacterial order, also called mycomembranes, harbor mycolic acids and unusual outer membrane proteins (OMPs), including those with α-helical structure. The signals that allow precursors of such proteins to be targeted to the mycomembrane remain uncharacterized. We report here the molecular features responsible for OMP targeting to the mycomembrane of Corynebacterium glutamicum , a nonpathogenic member of the Corynebacteriales order. To better understand the mechanisms by which OMP precursors were sorted in C. glutamicum , we first investigated the partitioning of endogenous and recombinant PorA, PorH, PorB, and PorC between bacterial compartments and showed that they were both imported into the mycomembrane and secreted into the extracellular medium. A detailed investigation of cell extracts and purified proteins by top-down MS, NMR spectroscopy, and site-directed mutagenesis revealed specific and well-conserved posttranslational modifications (PTMs), including O -mycoloylation, pyroglutamylation, and N -formylation, for mycomembrane-associated and -secreted OMPs. PTM site sequence analysis from C. glutamicum OMP and other O -acylated proteins in bacteria and eukaryotes revealed specific patterns. Furthermore, we found that such modifications were essential for targeting to the mycomembrane and sufficient for OMP assembly into mycolic acid-containing lipid bilayers. Collectively, it seems that these PTMs have evolved in the Corynebacteriales order and beyond to guide membrane proteins toward a specific cell compartment.

Identification of specific posttranslational O-mycoloylations mediating protein targeting to the mycomembrane

PubMed Central

Carel, Clément; Réat, Valérie; Parra, Julien; Latgé, Guillaume; Laval, Françoise; Burlet-Schiltz, Odile; Milon, Alain; Daffé, Mamadou; Tropis, Maryelle G.; Renault, Marie A. M.

2017-01-01

The outer membranes (OMs) of members of the Corynebacteriales bacterial order, also called mycomembranes, harbor mycolic acids and unusual outer membrane proteins (OMPs), including those with α-helical structure. The signals that allow precursors of such proteins to be targeted to the mycomembrane remain uncharacterized. We report here the molecular features responsible for OMP targeting to the mycomembrane of Corynebacterium glutamicum, a nonpathogenic member of the Corynebacteriales order. To better understand the mechanisms by which OMP precursors were sorted in C. glutamicum, we first investigated the partitioning of endogenous and recombinant PorA, PorH, PorB, and PorC between bacterial compartments and showed that they were both imported into the mycomembrane and secreted into the extracellular medium. A detailed investigation of cell extracts and purified proteins by top-down MS, NMR spectroscopy, and site-directed mutagenesis revealed specific and well-conserved posttranslational modifications (PTMs), including O-mycoloylation, pyroglutamylation, and N-formylation, for mycomembrane-associated and -secreted OMPs. PTM site sequence analysis from C. glutamicum OMP and other O-acylated proteins in bacteria and eukaryotes revealed specific patterns. Furthermore, we found that such modifications were essential for targeting to the mycomembrane and sufficient for OMP assembly into mycolic acid-containing lipid bilayers. Collectively, it seems that these PTMs have evolved in the Corynebacteriales order and beyond to guide membrane proteins toward a specific cell compartment. PMID:28373551

Membrane nanotubes facilitate long-distance interactions between natural killer cells and target cells

PubMed Central

Chauveau, Anne; Aucher, Anne; Eissmann, Philipp; Vivier, Eric; Davis, Daniel M.

2010-01-01

Membrane nanotubes are membranous tethers that physically link cell bodies over long distances. Here, we present evidence that nanotubes allow human natural killer (NK) cells to interact functionally with target cells over long distances. Nanotubes were formed when NK cells contacted target cells and moved apart. The frequency of nanotube formation was dependent on the number of receptor/ligand interactions and increased on NK cell activation. Most importantly, NK cell nanotubes contained a submicron scale junction where proteins accumulated, including DAP10, the signaling adaptor that associates with the activating receptor NKG2D, and MHC class I chain-related protein A (MICA), a cognate ligand for NKG2D, as occurs at close intercellular synapses between NK cells and target cells. Quantitative live-cell fluorescence imaging suggested that MICA accumulated at small nanotube synapses in sufficient numbers to trigger cell activation. In addition, tyrosine-phosphorylated proteins and Vav-1 accumulated at such junctions. Functionally, nanotubes could aid the lysis of distant target cells either directly or by moving target cells along the nanotube path into close contact for lysis via a conventional immune synapse. Target cells moving along the nanotube path were commonly polarized such that their uropods faced the direction of movement. This is the opposite polarization than for normal cell migration, implying that nanotubes can specifically drive target cell movement. Finally, target cells that remained connected to an NK cell by a nanotube were frequently lysed, whereas removing the nanotube using a micromanipulator reduced lysis of these target cells. PMID:20212116

Membrane Compartmentalization Reducing the Mobility of Lipids and Proteins within a Model Plasma Membrane.

PubMed

Koldsø, Heidi; Reddy, Tyler; Fowler, Philip W; Duncan, Anna L; Sansom, Mark S P

2016-09-01

The cytoskeleton underlying cell membranes may influence the dynamic organization of proteins and lipids within the bilayer by immobilizing certain transmembrane (TM) proteins and forming corrals within the membrane. Here, we present coarse-grained resolution simulations of a biologically realistic membrane model of asymmetrically organized lipids and TM proteins. We determine the effects of a model of cytoskeletal immobilization of selected membrane proteins using long time scale coarse-grained molecular dynamics simulations. By introducing compartments with varying degrees of restraints within the membrane models, we are able to reveal how compartmentalization caused by cytoskeletal immobilization leads to reduced and anomalous diffusional mobility of both proteins and lipids. This in turn results in a reduced rate of protein dimerization within the membrane and of hopping of membrane proteins between compartments. These simulations provide a molecular realization of hierarchical models often invoked to explain single-molecule imaging studies of membrane proteins.

X-ray Diffraction from Membrane Protein Nanocrystals

PubMed Central

Hunter, M.S.; DePonte, D.P.; Shapiro, D.A.; Kirian, R.A.; Wang, X.; Starodub, D.; Marchesini, S.; Weierstall, U.; Doak, R.B.; Spence, J.C.H.; Fromme, P.

2011-01-01

Membrane proteins constitute >30% of the proteins in an average cell, and yet the number of currently known structures of unique membrane proteins is <300. To develop new concepts for membrane protein structure determination, we have explored the serial nanocrystallography method, in which fully hydrated protein nanocrystals are delivered to an x-ray beam within a liquid jet at room temperature. As a model system, we have collected x-ray powder diffraction data from the integral membrane protein Photosystem I, which consists of 36 subunits and 381 cofactors. Data were collected from crystals ranging in size from 100 nm to 2 μm. The results demonstrate that there are membrane protein crystals that contain <100 unit cells (200 total molecules) and that 3D crystals of membrane proteins, which contain <200 molecules, may be suitable for structural investigation. Serial nanocrystallography overcomes the problem of x-ray damage, which is currently one of the major limitations for x-ray structure determination of small crystals. By combining serial nanocrystallography with x-ray free-electron laser sources in the future, it may be possible to produce molecular-resolution electron-density maps using membrane protein crystals that contain only a few hundred or thousand unit cells. PMID:21190672

Small cationic antimicrobial peptides delocalize peripheral membrane proteins

PubMed Central

Wenzel, Michaela; Chiriac, Alina Iulia; Otto, Andreas; Zweytick, Dagmar; May, Caroline; Schumacher, Catherine; Gust, Ronald; Albada, H. Bauke; Penkova, Maya; Krämer, Ute; Erdmann, Ralf; Metzler-Nolte, Nils; Straus, Suzana K.; Bremer, Erhard; Becher, Dörte; Brötz-Oesterhelt, Heike; Sahl, Hans-Georg; Bandow, Julia Elisabeth

2014-01-01

Short antimicrobial peptides rich in arginine (R) and tryptophan (W) interact with membranes. To learn how this interaction leads to bacterial death, we characterized the effects of the minimal pharmacophore RWRWRW-NH2. A ruthenium-substituted derivative of this peptide localized to the membrane in vivo, and the peptide also integrated readily into mixed phospholipid bilayers that resemble Gram-positive membranes. Proteome and Western blot analyses showed that integration of the peptide caused delocalization of peripheral membrane proteins essential for respiration and cell-wall biosynthesis, limiting cellular energy and undermining cell-wall integrity. This delocalization phenomenon also was observed with the cyclic peptide gramicidin S, indicating the generality of the mechanism. Exogenous glutamate increases tolerance to the peptide, indicating that osmotic destabilization also contributes to antibacterial efficacy. Bacillus subtilis responds to peptide stress by releasing osmoprotective amino acids, in part via mechanosensitive channels. This response is triggered by membrane-targeting bacteriolytic peptides of different structural classes as well as by hypoosmotic conditions. PMID:24706874

Deoxycholate-Based Glycosides (DCGs) for Membrane Protein Stabilisation.

PubMed

Bae, Hyoung Eun; Gotfryd, Kamil; Thomas, Jennifer; Hussain, Hazrat; Ehsan, Muhammad; Go, Juyeon; Loland, Claus J; Byrne, Bernadette; Chae, Pil Seok

2015-07-06

Detergents are an absolute requirement for studying the structure of membrane proteins. However, many conventional detergents fail to stabilise denaturation-sensitive membrane proteins, such as eukaryotic proteins and membrane protein complexes. New amphipathic agents with enhanced efficacy in stabilising membrane proteins will be helpful in overcoming the barriers to studying membrane protein structures. We have prepared a number of deoxycholate-based amphiphiles with carbohydrate head groups, designated deoxycholate-based glycosides (DCGs). These DCGs are the hydrophilic variants of previously reported deoxycholate-based N-oxides (DCAOs). Membrane proteins in these agents, particularly the branched diglucoside-bearing amphiphiles DCG-1 and DCG-2, displayed favourable behaviour compared to previously reported parent compounds (DCAOs) and conventional detergents (LDAO and DDM). Given their excellent properties, these agents should have significant potential for membrane protein studies. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Membrane curvature and its generation by BAR proteins

PubMed Central

Mim, Carsten; Unger, Vinzenz M

2012-01-01

Membranes are flexible barriers that surround the cell and its compartments. To execute vital functions such as locomotion or receptor turnover, cells need to control the shapes of their membranes. In part, this control is achieved through membrane-bending proteins, such as the bin/amphiphysin/rvs domain (BAR) proteins. Many open questions remain about the mechanisms by which membrane-bending proteins function. Addressing this shortfall, recent structures of BAR protein:membrane complexes support existing mechanistic models, but also produced novel insights into how BAR-domain proteins sense, stabilize and generate curvature. Here we review these recent findings, focusing on how BAR proteins interact with the membrane, and how the resulting scaffold structures might aid the recruitment of other proteins to the sites where membranes are bent. PMID:23058040

Chimera proteins with affinity for membranes and microtubule tips polarize in the membrane of fission yeast cells.

PubMed

Recouvreux, Pierre; Sokolowski, Thomas R; Grammoustianou, Aristea; ten Wolde, Pieter Rein; Dogterom, Marileen

2016-02-16

Cell polarity refers to a functional spatial organization of proteins that is crucial for the control of essential cellular processes such as growth and division. To establish polarity, cells rely on elaborate regulation networks that control the distribution of proteins at the cell membrane. In fission yeast cells, a microtubule-dependent network has been identified that polarizes the distribution of signaling proteins that restricts growth to cell ends and targets the cytokinetic machinery to the middle of the cell. Although many molecular components have been shown to play a role in this network, it remains unknown which molecular functionalities are minimally required to establish a polarized protein distribution in this system. Here we show that a membrane-binding protein fragment, which distributes homogeneously in wild-type fission yeast cells, can be made to concentrate at cell ends by attaching it to a cytoplasmic microtubule end-binding protein. This concentration results in a polarized pattern of chimera proteins with a spatial extension that is very reminiscent of natural polarity patterns in fission yeast. However, chimera levels fluctuate in response to microtubule dynamics, and disruption of microtubules leads to disappearance of the pattern. Numerical simulations confirm that the combined functionality of membrane anchoring and microtubule tip affinity is in principle sufficient to create polarized patterns. Our chimera protein may thus represent a simple molecular functionality that is able to polarize the membrane, onto which additional layers of molecular complexity may be built to provide the temporal robustness that is typical of natural polarity patterns.

Anomalous Dynamics of a Lipid Recognition Protein on a Membrane Surface

PubMed Central

Yamamoto, Eiji; Kalli, Antreas C.; Akimoto, Takuma; Yasuoka, Kenji; Sansom, Mark S. P.

2015-01-01

Pleckstrin homology (PH) domains are lipid-binding modules present in peripheral membrane proteins which interact with phosphatidyl-inositol phosphates (PIPs) in cell membranes. We use multiscale molecular dynamics simulations to characterize the localization and anomalous dynamics of the DAPP1 PH domain on the surface of a PIP-containing lipid bilayer. Both translational and rotational diffusion of the PH domain on the lipid membrane surface exhibit transient subdiffusion, with an exponent α ≈ 0.5 for times of less than 10 ns. In addition to a PIP3 molecule at the canonical binding site of the PH domain, we observe additional PIP molecules in contact with the protein. Fluctuations in the number of PIPs associated with the PH domain exhibit 1/f noise. We suggest that the anomalous diffusion and long-term correlated interaction of the PH domain with the membrane may contribute to an enhanced probability of encounter with target complexes on cell membrane surfaces. PMID:26657413

New insights into the targeting of a sub-set of tail-anchored proteins to the outer mitochondrial membrane

USDA-ARS?s Scientific Manuscript database

Tail-anchored (TA) proteins are a unique class of functionally diverse membrane proteins that are defined by their single C-terminal membrane-spanning domain and their ability to insert post-translationally into specific organelles with an Nout-Cin orientation. The molecular mechanisms by which TA p...

Solubilization of a membrane protein by combinatorial supercharging.

PubMed

Hajduczki, Agnes; Majumdar, Sudipta; Fricke, Marie; Brown, Isola A M; Weiss, Gregory A

2011-04-15

Hydrophobic and aggregation-prone, membrane proteins often prove too insoluble for conventional in vitro biochemical studies. To engineer soluble variants of human caveolin-1, a phage-displayed library of caveolin variants targeted the hydrophobic intramembrane domain with substitutions to charged residues. Anti-selections for insolubility removed hydrophobic variants, and positive selections for binding to the known caveolin ligand HIV gp41 isolated functional, folded variants. Assays with several caveolin binding partners demonstrated the successful folding and functionality by a solubilized, full-length caveolin variant selected from the library. This caveolin variant allowed assay of the direct interaction between caveolin and cavin. Clustered along one face of a putative helix, the solubilizing mutations suggest a structural model for the intramembrane domain of caveolin. The approach provides a potentially general method for solubilization and engineering of membrane-associated proteins by phage display.

Microfluidics for the analysis of membrane proteins: how do we get there?

PubMed

Battle, Katrina N; Uba, Franklin I; Soper, Steven A

2014-08-01

The development of fully automated and high-throughput systems for proteomics is now in demand because of the need to generate new protein-based disease biomarkers. Unfortunately, it is difficult to identify protein biomarkers that are low abundant when in the presence of highly abundant proteins, especially in complex biological samples such as serum, cell lysates, and other biological fluids. Membrane proteins, which are in many cases of low abundance compared to the cytosolic proteins, have various functions and can provide insight into the state of a disease and serve as targets for new drugs making them attractive biomarker candidates. Traditionally, proteins are identified through the use of gel electrophoretic techniques, which are not always suitable for particular protein samples such as membrane proteins. Microfluidics offers the potential as a fully automated platform for the efficient and high-throughput analysis of complex samples, such as membrane proteins, and do so with performance metrics that exceed their bench-top counterparts. In recent years, there have been various improvements to microfluidics and their use for proteomic analysis as reported in the literature. Consequently, this review presents an overview of the traditional proteomic-processing pipelines for membrane proteins and insights into new technological developments with a focus on the applicability of microfluidics for the analysis of membrane proteins. Sample preparation techniques will be discussed in detail and novel interfacing strategies as it relates to MS will be highlighted. Lastly, some general conclusions and future perspectives are presented. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Membrane-targeted WAVE mediates photoreceptor axon targeting in the absence of the WAVE complex in Drosophila

PubMed Central

Stephan, Raiko; Gohl, Christina; Fleige, Astrid; Klämbt, Christian; Bogdan, Sven

2011-01-01

A tight spatial-temporal coordination of F-actin dynamics is crucial for a large variety of cellular processes that shape cells. The Abelson interactor (Abi) has a conserved role in Arp2/3-dependent actin polymerization, regulating Wiskott-Aldrich syndrome protein (WASP) and WASP family verprolin-homologous protein (WAVE). In this paper, we report that Abi exerts nonautonomous control of photoreceptor axon targeting in the Drosophila visual system through WAVE. In abi mutants, WAVE is unstable but restored by reexpression of Abi, confirming that Abi controls the integrity of the WAVE complex in vivo. Remarkably, expression of a membrane-tethered WAVE protein rescues the axonal projection defects of abi mutants in the absence of the other subunits of the WAVE complex, whereas cytoplasmic WAVE only slightly affects the abi mutant phenotype. Thus complex formation not only stabilizes WAVE, but also provides further membrane-recruiting signals, resulting in an activation of WAVE. PMID:21900504

Membrane bending: the power of protein imbalance.

PubMed

Derganc, Jure; Antonny, Bruno; Copič, Alenka

2013-11-01

Many cellular processes require membrane deformation, which is driven by specialized protein machinery and can often be recapitulated using pure lipid bilayers. However, biological membranes contain a large amount of embedded proteins. Recent research suggests that membrane-bound proteins with asymmetric distribution of mass across the bilayer can influence membrane bending in a nonspecific manner due to molecular crowding. This mechanism is physical in nature and arises from collisions between such 'mushroom-shaped' proteins. It can either facilitate or impede the action of protein coats, for example COPII, during vesicle budding. We describe the physics of how molecular crowding can influence membrane bending and discuss the implications for other cellular processes, such as sorting of glycosylphosphatidylinositol-anchored proteins (GPI-APs) and production of intraluminal vesicles. Copyright © 2013 Elsevier Ltd. All rights reserved.

Protein Composition of Trypanosoma brucei Mitochondrial Membranes

PubMed Central

Acestor, Nathalie; Panigrahi, Aswini K.; Ogata, Yuko; Anupama, Atashi; Stuart, Kenneth D.

2010-01-01

Mitochondria consist of four compartments, outer membrane, intermembrane space, inner membrane and matrix; each harboring specific functions and structures. In this study, we used mass spectrometry (LC-MS/MS) to characterize the protein composition of Trypanosoma brucei mitochondrial membranes, which were enriched by different biochemical fractionation techniques. The analyses identified 202 proteins that contain one or more transmembrane domain(s) and/or positive GRAVY scores. Of these, various criteria were used to assign 72 proteins to mitochondrial membranes with high confidence, and 106 with moderate to low confidence. The sub-cellular localization of a selected subset of 13 membrane assigned proteins was confirmed by tagging and immunofluorescence analysis. While most proteins assigned to mitochondrial membrane have putative roles in metabolic, energy generating, and transport processes, ~50% have no known function. These studies result in a comprehensive profile of the composition and sub-organellar location of proteins in the T. brucei mitochondrion thus, providing useful information on mitochondrial functions. PMID:19834910

Getting Across the Cell Membrane: An Overview for Small Molecules, Peptides, and Proteins

PubMed Central

Yang, Nicole J.; Hinner, Marlon J.

2016-01-01

The ability to efficiently access cytosolic proteins is desired in both biological research and medicine. However, targeting intracellular proteins is often challenging, because to reach the cytosol, exogenous molecules must first traverse the cell membrane. This review provides a broad overview of how certain molecules are thought to cross this barrier, and what kinds of approaches are being made to enhance the intracellular delivery of those that are impermeable. We first discuss rules that govern the passive permeability of small molecules across the lipid membrane, and mechanisms of membrane transport that have evolved in nature for certain metabolites, peptides, and proteins. Then, we introduce design strategies that have emerged in the development of small molecules and peptides with improved permeability. Finally, intracellular delivery systems that have been engineered for protein payloads are surveyed. Viewpoints from varying disciplines have been brought together to provide a cohesive overview of how the membrane barrier is being overcome. PMID:25560066

Epithelial Membrane Protein-2 is a Novel Therapeutic Target in Ovarian Cancer

PubMed Central

Fu, Maoyong; Maresh, Erin L.; Soslow, Robert A.; Alavi, Mohammad; Mah, Vei; Zhou, Qin; Iasonos, Alexia; Goodglick, Lee; Gordon, Lynn K.; Braun, Jonathan; Wadehra, Madhuri

2010-01-01

Purpose The tetraspan protein epithelial membrane protein-2 (EMP2) has been shown to regulate the surface display and signaling from select integrin pairs, and it was recently identified as a prognostic biomarker in human endometrial cancer. In this study, we assessed the role of EMP2 in human ovarian cancer. Experimental Design We examined the expression of EMP2 within a population of women with ovarian cancer using tissue microarray assay technology. We evaluated the efficacy of EMP2-directed antibody therapy using a fully human recombinant bivalent antibody fragment (diabody) in vitro and ovarian cancer xenograft models in vivo. Results EMP2 was found to be highly expressed in over 70% of serous and endometrioid ovarian tumors compared to non-malignant ovarian epithelium using a human ovarian cancer tissue microarray. Using anti-EMP2 diabody, we evaluated the in vitro response of 9 human ovarian cancer cell lines with detectable EMP2 expression. Treatment of human ovarian cancer cell lines with anti-EMP2 diabodies induced cell death and retarded cell growth, and these response rates correlated with cellular EMP2 expression. We next assessed the effects of anti-EMP2 diabodies in mice bearing xenografts from the ovarian endometrioid carcinoma cell line OVCAR5. Anti-EMP2 diabodies significantly suppressed tumor growth and induced cell death in OVCAR5 xenografts. Conclusions These findings indicate that EMP2 is expressed in the majority of ovarian tumors and it may be a feasible target in vivo. PMID:20670949

Membrane protein synthesis in cell-free systems: from bio-mimetic systems to bio-membranes.

PubMed

Sachse, Rita; Dondapati, Srujan K; Fenz, Susanne F; Schmidt, Thomas; Kubick, Stefan

2014-08-25

When taking up the gauntlet of studying membrane protein functionality, scientists are provided with a plethora of advantages, which can be exploited for the synthesis of these difficult-to-express proteins by utilizing cell-free protein synthesis systems. Due to their hydrophobicity, membrane proteins have exceptional demands regarding their environment to ensure correct functionality. Thus, the challenge is to find the appropriate hydrophobic support that facilitates proper membrane protein folding. So far, various modes of membrane protein synthesis have been presented. Here, we summarize current state-of-the-art methodologies of membrane protein synthesis in biomimetic-supported systems. The correct folding and functionality of membrane proteins depend in many cases on their integration into a lipid bilayer and subsequent posttranslational modification. We highlight cell-free systems utilizing the advantages of biological membranes. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

Membrane and envelope virus proteins co-expressed as lysosome associated membrane protein (LAMP) fused antigens: a potential tool to develop DNA vaccines against flaviviruses.

PubMed

Dhalia, Rafael; Maciel, Milton; Cruz, Fábia S P; Viana, Isabelle F T; Palma, Mariana L; August, Thomas; Marques, Ernesto T A

2009-12-01

Vaccination is the most practical and cost-effective strategy to prevent the majority of the flavivirus infection to which there is an available vaccine. However, vaccines based on attenuated virus can potentially promote collateral side effects and even rare fatal reactions. Given this scenario, the development of alternative vaccination strategies such as DNA-based vaccines encoding specific flavivirus sequences are being considered. Endogenous cytoplasmic antigens, characteristically plasmid DNA-vaccine encoded, are mainly presented to the immune system through Major Histocompatibility Complex class I - MHC I molecules. The MHC I presentation via is mostly associated with a cellular cytotoxic response and often do not elicit a satisfactory humoral response. One of the main strategies to target DNA-encoded antigens to the MHC II compartment is expressing the antigen within the Lysosome-Associated Membrane Protein (LAMP). The flavivirus envelope protein is recognized as the major virus surface protein and the main target for neutralizing antibodies. Different groups have demonstrated that co-expression of flavivirus membrane and envelope proteins in mammalian cells, fused with the carboxyl-terminal of LAMP, is able to induce satisfactory levels of neutralizing antibodies. Here we reviewed the use of the envelope flavivirus protein co-expression strategy as LAMP chimeras with the aim of developing DNA vaccines for dengue, West Nile and yellow fever viruses.

Recent Progress in the Structure Determination of GPCRs, a Membrane Protein Family with High Potential as Pharmaceutical Targets

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cherezov, Vadim; Abola, Enrique; Stevens, Raymond C.

2015-11-30

G protein-coupled receptors (GPCRs) constitute a highly diverse and ubiquitous family of integral membrane proteins, transmitting signals inside the cells in response to an assortment of disparate extra-cellular stimuli. Their strategic location on the cell surface and their involvement in crucial cellular and physiological processes turn these receptors into highly important pharmaceutical targets. Recent technological developments aimed at stabilization and crystallization of these receptors have led to significant breakthroughs in GPCR structure determination efforts. One of the successful approaches involved receptor stabilization with the help of a fusion partner combined with crystallization in lipidic cubic phase (LCP). The success ofmore » using an LCP matrix for crystallization is generally attributed to the creation of a more native, membrane-like stabilizing environment for GPCRs just prior to nucleation and to the formation of type I crystal lattices, thus generating highly ordered and strongly diffracting crystals. Here they describe protocols for reconstituting purified GPCRs in LCP, performing pre-crystallization assays, setting up crystallization trials in manual mode, detecting crystallization hits, optimizing crystallization conditions, harvesting, and collecting crystallographic data. The protocols provide a sensible framework for approaching crystallization of stabilized GPCRs in LCP, however, as in any crystallization experiment, extensive screening and optimization of crystallization conditions as well as optimization of protein construct and purification steps are required. The process remains risky and these protocols do not necessarily guarantee success.« less

Cytoskeletal Components Define Protein Location to Membrane Microdomains*

PubMed Central

Szymanski, Witold G.; Zauber, Henrik; Erban, Alexander; Gorka, Michal; Wu, Xu Na; Schulze, Waltraud X.

2015-01-01

The plasma membrane is an important compartment that undergoes dynamic changes in composition upon external or internal stimuli. The dynamic subcompartmentation of proteins in ordered low-density (DRM) and disordered high-density (DSM) membrane phases is hypothesized to require interactions with cytoskeletal components. Here, we systematically analyzed the effects of actin or tubulin disruption on the distribution of proteins between membrane density phases. We used a proteomic screen to identify candidate proteins with altered submembrane location, followed by biochemical or cell biological characterization in Arabidopsis thaliana. We found that several proteins, such as plasma membrane ATPases, receptor kinases, or remorins resulted in a differential distribution between membrane density phases upon cytoskeletal disruption. Moreover, in most cases, contrasting effects were observed: Disruption of actin filaments largely led to a redistribution of proteins from DRM to DSM membrane fractions while disruption of tubulins resulted in general depletion of proteins from the membranes. We conclude that actin filaments are necessary for dynamic movement of proteins between different membrane phases and that microtubules are not necessarily important for formation of microdomains as such, but rather they may control the protein amount present in the membrane phases. PMID:26091700

ATP-independent reversal of a membrane protein aggregate by a chloroplast SRP

PubMed Central

Jaru-Ampornpan, Peera; Shen, Kuang; Lam, Vinh Q.; Ali, Mona; Doniach, Sebastian; Jia, Tony Z.; Shan, Shu-ou

2010-01-01

Membrane proteins impose enormous challenges to cellular protein homeostasis during their post-translational targeting, and require chaperones to keep them soluble and translocation-competent. Here we show that a novel targeting factor in the chloroplast Signal Recognition Particle (cpSRP), cpSRP43, is a highly specific molecular chaperone that efficiently reverses the aggregation of its substrate proteins. In contrast to AAA+-chaperones, cpSRP43 utilizes specific binding interactions with its substrate to mediate its disaggregase activity. This ‘disaggregase’ capability can allow targeting machineries to more effectively capture their protein substrates, and emphasizes a close connection between protein folding and trafficking processes. Moreover, cpSRP43 provides the first example of an ATP-independent disaggregase, and demonstrates that efficient reversal of protein aggregation can be attained by specific binding interactions between a chaperone and its substrate. PMID:20424608

Protein Solvation in Membranes and at Water-Membrane Interfaces

NASA Technical Reports Server (NTRS)

Pohorille, Andrew; Chipot, Christophe; Wilson, Michael A.

2002-01-01

Different salvation properties of water and membranes mediate a host of biologically important processes, such as folding, insertion into a lipid bilayer, associations and functions of membrane proteins. These processes will be discussed in several examples involving synthetic and natural peptides. In particular, a mechanism by which a helical peptide becomes inserted into a model membrane will be described. Further, the molecular mechanism of recognition and association of protein helical segments in membranes will be discussed. These processes are crucial for proper functioning of a cell. A membrane-spanning domain of glycophorin A, which exists as a helical dimer, serves as the model system. For this system, the free energy of dissociation of the helices is being determined for both the wild type and a mutant, in which dimerization is disrupted.

A Single Peroxisomal Targeting Signal Mediates Matrix Protein Import in Diatoms

PubMed Central

Gonzalez, Nicola H.; Felsner, Gregor; Schramm, Frederic D.; Klingl, Andreas; Maier, Uwe-G.; Bolte, Kathrin

2011-01-01

Peroxisomes are single membrane bound compartments. They are thought to be present in almost all eukaryotic cells, although the bulk of our knowledge about peroxisomes has been generated from only a handful of model organisms. Peroxisomal matrix proteins are synthesized cytosolically and posttranslationally imported into the peroxisomal matrix. The import is generally thought to be mediated by two different targeting signals. These are respectively recognized by the two import receptor proteins Pex5 and Pex7, which facilitate transport across the peroxisomal membrane. Here, we show the first in vivo localization studies of peroxisomes in a representative organism of the ecologically relevant group of diatoms using fluorescence and transmission electron microscopy. By expression of various homologous and heterologous fusion proteins we demonstrate that targeting of Phaeodactylum tricornutum peroxisomal matrix proteins is mediated only by PTS1 targeting signals, also for proteins that are in other systems imported via a PTS2 mode of action. Additional in silico analyses suggest this surprising finding may also apply to further diatoms. Our data suggest that loss of the PTS2 peroxisomal import signal is not reserved to Caenorhabditis elegans as a single exception, but has also occurred in evolutionary divergent organisms. Obviously, targeting switching from PTS2 to PTS1 across different major eukaryotic groups might have occurred for different reasons. Thus, our findings question the widespread assumption that import of peroxisomal matrix proteins is generally mediated by two different targeting signals. Our results implicate that there apparently must have been an event causing the loss of one targeting signal even in the group of diatoms. Different possibilities are discussed that indicate multiple reasons for the detected targeting switching from PTS2 to PTS1. PMID:21966495

Active Nuclear Import of Membrane Proteins Revisited

PubMed Central

Laba, Justyna K.; Steen, Anton; Popken, Petra; Chernova, Alina; Poolman, Bert; Veenhoff, Liesbeth M.

2015-01-01

It is poorly understood how membrane proteins destined for the inner nuclear membrane pass the crowded environment of the Nuclear Pore Complex (NPC). For the Saccharomyces cerevisiae proteins Src1/Heh1 and Heh2, a transport mechanism was proposed where the transmembrane domains diffuse through the membrane while the extralumenal domains encoding a nuclear localization signal (NLS) and intrinsically disordered linker (L) are accompanied by transport factors and travel through the NPC. Here, we validate the proposed mechanism and explore and discuss alternative interpretations of the data. First, to disprove an interpretation where the membrane proteins become membrane embedded only after nuclear import, we present biochemical and localization data to support that the previously used, as well as newly designed reporter proteins are membrane-embedded irrespective of the presence of the sorting signals, the specific transmembrane domain (multipass or tail anchored), independent of GET, and also under conditions that the proteins are trapped in the NPC. Second, using the recently established size limit for passive diffusion of membrane proteins in yeast, and using an improved assay, we confirm active import of polytopic membrane protein with extralumenal soluble domains larger than those that can pass by diffusion on similar timescales. This reinforces that NLS-L dependent active transport is distinct from passive diffusion. Thirdly, we revisit the proposed route through the center of the NPC and conclude that the previously used trapping assay is, unfortunately, poorly suited to address the route through the NPC, and the route thus remains unresolved. Apart from the uncertainty about the route through the NPC, the data confirm active, transport factor dependent, nuclear transport of membrane-embedded mono- and polytopic membrane proteins in baker’s yeast. PMID:26473931

Prostate-specific membrane antigen targeted protein contrast agents for molecular imaging of prostate cancer by MRI

NASA Astrophysics Data System (ADS)

Pu, Fan; Salarian, Mani; Xue, Shenghui; Qiao, Jingjuan; Feng, Jie; Tan, Shanshan; Patel, Anvi; Li, Xin; Mamouni, Kenza; Hekmatyar, Khan; Zou, Juan; Wu, Daqing; Yang, Jenny J.

2016-06-01

Prostate-specific membrane antigen (PSMA) is one of the most specific cell surface markers for prostate cancer diagnosis and targeted treatment. However, achieving molecular imaging using non-invasive MRI with high resolution has yet to be achieved due to the lack of contrast agents with significantly improved relaxivity for sensitivity, targeting capabilities and metal selectivity. We have previously reported our creation of a novel class of protein Gd3+ contrast agents, ProCA32, which displayed significantly improved relaxivity while exhibiting strong Gd3+ binding selectivity over physiological metal ions. In this study, we report our effort in further developing biomarker-targeted protein MRI contrast agents for molecular imaging of PSMA. Among three PSMA targeted contrast agents engineered with addition of different molecular recognition sequences, ProCA32.PSMA exhibits a binding affinity of 1.1 +/- 0.1 μM for PSMA while the metal binding affinity is maintained at 0.9 +/- 0.1 × 10-22 M. In addition, ProCA32.PSMA exhibits r1 of 27.6 mM-1 s-1 and r2 of 37.9 mM-1 s-1 per Gd (55.2 and 75.8 mM-1 s-1 per molecule r1 and r2, respectively) at 1.4 T. At 7 T, ProCA32.PSMA also has r2 of 94.0 mM-1 s-1 per Gd (188.0 mM-1 s-1 per molecule) and r1 of 18.6 mM-1 s-1 per Gd (37.2 mM-1 s-1 per molecule). This contrast capability enables the first MRI enhancement dependent on PSMA expression levels in tumor bearing mice using both T1 and T2-weighted MRI at 7 T. Further development of these PSMA-targeted contrast agents are expected to be used for the precision imaging of prostate cancer at an early stage and to monitor disease progression and staging, as well as determine the effect of therapeutic treatment by non-invasive evaluation of the PSMA level using MRI.Prostate-specific membrane antigen (PSMA) is one of the most specific cell surface markers for prostate cancer diagnosis and targeted treatment. However, achieving molecular imaging using non-invasive MRI with high

Characterization of Plasma Membrane Proteins from Ovarian Cancer Cells Using Mass Spectrometry

DOE PAGES

Springer, David L.; Auberry, Deanna L.; Ahram, Mamoun; ...

2004-01-01

To determine how the repertoire of plasma membrane proteins change with disease state, specifically related to cancer, several methods for preparation of plasma membrane proteins were evaluated. Cultured cells derived from stage IV ovarian tumors were grown to 90% confluence and harvested in buffer containing CHAPS detergent. This preparation was centrifuged at low speed to remove insoluble cellular debris resulting in a crude homogenate. Glycosylated proteins in the crude homogenate were selectively enriched using lectin affinity chromatography. The crude homogenate and the lectin purified sample were prepared for mass spectrometric evaluation. The general procedure for protein identification began with trypsinmore » digestion of protein fractions followed by separation by reversed phase liquid chromatography that was coupled directly to a conventional tandem mass spectrometer (i.e. LCQ ion trap). Mass and fragmentation data for the peptides were searched against a human proteome data base using the informatics program SEQUEST. Using this procedure 398 proteins were identified with high confidence, including receptors, membrane-associated ligands, proteases, phosphatases, as well as structural and adhesion proteins. Results indicate that lectin chromatography provides a select subset of proteins and that the number and quality of the identifications improve as does the confidence of the protein identifications for this subset. These results represent the first step in development of methods to separate and successfully identify plasma membrane proteins from advanced ovarian cancer cells. Further characterization of plasma membrane proteins will contribute to our understanding of the mechanisms underlying progression of this deadly disease and may lead to new targeted interventions as well as new biomarkers for diagnosis.« less

Production of membrane proteins without cells or detergents.

PubMed

Rajesh, Sundaresan; Knowles, Timothy; Overduin, Michael

2011-04-30

The production of membrane proteins in cellular systems is besieged by several problems due to their hydrophobic nature which often causes misfolding, protein aggregation and cytotoxicity, resulting in poor yields of stable proteins. Cell-free expression has emerged as one of the most versatile alternatives for circumventing these obstacles by producing membrane proteins directly into designed hydrophobic environments. Efficient optimisation of expression and solubilisation conditions using a variety of detergents, membrane mimetics and lipids has yielded structurally and functionally intact membrane proteins, with yields several fold above the levels possible from cell-based systems. Here we review recently developed techniques available to produce functional membrane proteins, and discuss amphipols, nanodisc and styrene maleic acid lipid particle (SMALP) technologies that can be exploited alongside cell-free expression of membrane proteins. Copyright © 2010 Elsevier B.V. All rights reserved.

Synthetic single domain antibodies for the conformational trapping of membrane proteins

PubMed Central

Arnold, Fabian M; Stohler, Peter; Bocquet, Nicolas; Hug, Melanie N; Huber, Sylwia; Siegrist, Martin; Hetemann, Lisa; Gera, Jennifer; Gmür, Samira; Spies, Peter; Gygax, Daniel

2018-01-01

Mechanistic and structural studies of membrane proteins require their stabilization in specific conformations. Single domain antibodies are potent reagents for this purpose, but their generation relies on immunizations, which impedes selections in the presence of ligands typically needed to populate defined conformational states. To overcome this key limitation, we developed an in vitro selection platform based on synthetic single domain antibodies named sybodies. To target the limited hydrophilic surfaces of membrane proteins, we designed three sybody libraries that exhibit different shapes and moderate hydrophobicity of the randomized surface. A robust binder selection cascade combining ribosome and phage display enabled the generation of conformation-selective, high affinity sybodies against an ABC transporter and two previously intractable human SLC transporters, GlyT1 and ENT1. The platform does not require access to animal facilities and builds exclusively on commercially available reagents, thus enabling every lab to rapidly generate binders against challenging membrane proteins. PMID:29792401

Applications of solid-state NMR to membrane proteins.

PubMed

Ladizhansky, Vladimir

2017-11-01

Membrane proteins mediate flow of molecules, signals, and energy between cells and intracellular compartments. Understanding membrane protein function requires a detailed understanding of the structural and dynamic properties involved. Lipid bilayers provide a native-like environment for structure-function investigations of membrane proteins. In this review we give a general discourse on the recent progress in the field of solid-state NMR of membrane proteins. Solid-state NMR is a variation of NMR spectroscopy that is applicable to molecular systems with restricted mobility, such as high molecular weight proteins and protein complexes, supramolecular assemblies, or membrane proteins in a phospholipid environment. We highlight recent advances in applications of solid-state NMR to membrane proteins, specifically focusing on the recent developments in the field of Dynamic Nuclear Polarization, proton detection, and solid-state NMR applications in situ (in cell membranes). This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John Baenziger, Albert Berghuis and Peter Tieleman. Copyright © 2017 Elsevier B.V. All rights reserved.

Dynamic shaping of cellular membranes by phospholipids and membrane-deforming proteins.

PubMed

Suetsugu, Shiro; Kurisu, Shusaku; Takenawa, Tadaomi

2014-10-01

All cellular compartments are separated from the external environment by a membrane, which consists of a lipid bilayer. Subcellular structures, including clathrin-coated pits, caveolae, filopodia, lamellipodia, podosomes, and other intracellular membrane systems, are molded into their specific submicron-scale shapes through various mechanisms. Cells construct their micro-structures on plasma membrane and execute vital functions for life, such as cell migration, cell division, endocytosis, exocytosis, and cytoskeletal regulation. The plasma membrane, rich in anionic phospholipids, utilizes the electrostatic nature of the lipids, specifically the phosphoinositides, to form interactions with cytosolic proteins. These cytosolic proteins have three modes of interaction: 1) electrostatic interaction through unstructured polycationic regions, 2) through structured phosphoinositide-specific binding domains, and 3) through structured domains that bind the membrane without specificity for particular phospholipid. Among the structured domains, there are several that have membrane-deforming activity, which is essential for the formation of concave or convex membrane curvature. These domains include the amphipathic helix, which deforms the membrane by hemi-insertion of the helix with both hydrophobic and electrostatic interactions, and/or the BAR domain superfamily, known to use their positively charged, curved structural surface to deform membranes. Below the membrane, actin filaments support the micro-structures through interactions with several BAR proteins as well as other scaffold proteins, resulting in outward and inward membrane micro-structure formation. Here, we describe the characteristics of phospholipids, and the mechanisms utilized by phosphoinositides to regulate cellular events. We then summarize the precise mechanisms underlying the construction of membrane micro-structures and their involvements in physiological and pathological processes. Copyright © 2014 the

Rigid proteins and softening of biological membranes—with application to HIV-induced cell membrane softening

NASA Astrophysics Data System (ADS)

Agrawal, Himani; Zelisko, Matthew; Liu, Liping; Sharma, Pradeep

2016-05-01

A key step in the HIV-infection process is the fusion of the virion membrane with the target cell membrane and the concomitant transfer of the viral RNA. Experimental evidence suggests that the fusion is preceded by considerable elastic softening of the cell membranes due to the insertion of fusion peptide in the membrane. What are the mechanisms underpinning the elastic softening of the membrane upon peptide insertion? A broader question may be posed: insertion of rigid proteins in soft membranes ought to stiffen the membranes not soften them. However, experimental observations perplexingly appear to show that rigid proteins may either soften or harden membranes even though conventional wisdom only suggests stiffening. In this work, we argue that regarding proteins as merely non-specific rigid inclusions is flawed, and each protein has a unique mechanical signature dictated by its specific interfacial coupling to the surrounding membrane. Predicated on this hypothesis, we have carried out atomistic simulations to investigate peptide-membrane interactions. Together with a continuum model, we reconcile contrasting experimental data in the literature including the case of HIV-fusion peptide induced softening. We conclude that the structural rearrangements of the lipids around the inclusions cause the softening or stiffening of the biological membranes.

The role of phosphatidylinositol-transfer proteins at membrane contact sites.

PubMed

Selitrennik, Michael; Lev, Sima

2016-04-15

Phosphatidylinositol-transfer proteins (PITPs) have been initially identified as soluble factors that accelerate the monomeric exchange of either phosphatidylinositol (PI) or phosphatidylcholine (PC) between membrane bilayersin vitro They are highly conserved in eukaryotes and have been implicated in different cellular processes, including vesicular trafficking, signal transduction, and lipid metabolism. Recent studies suggest that PITPs function at membrane contact sites (MCSs) to facilitate the transport of PI from its synthesis site at the endoplasmic reticulum (ER) to various membrane compartments. In this review, we describe the underlying mechanism of PITPs targeting to MCSs, discuss their cellular roles and potential mode of action. © 2016 Authors; published by Portland Press Limited.

Characterization of membrane association of Rinderpest virus matrix protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Subhashri, R.; Shaila, M.S.

2007-04-20

Paramyxovirus matrix protein is believed to play a crucial role in the assembly and maturation of the virus particle by bringing the major viral components together at the budding site in the host cell. The membrane association capability of many enveloped virus matrix proteins has been characterized to be their intrinsic property. In this work, we have characterized the membrane association of Rinderpest virus matrix (M) protein. The M protein of Rinderpest virus when expressed in the absence of other viral proteins is present both in the cytoplasm and plasma membrane. When expressed as GFP fusion protein, the M proteinmore » gets localized into plasma membrane protrusions. High salt and alkaline conditions resulted in partial dissociation of M protein from cell membrane. Thus, M protein behaves like an integral membrane protein although its primary structure suggests it to be a peripheral membrane protein.« less

Expression of a plant virus non-structural protein in Saccharomyces cerevisiae causes membrane proliferation and altered mitochondrial morphology.

PubMed

Rubino, L; Di Franco, A; Russo, M

2000-01-01

Carnation Italian ringspot tombusvirus encodes a protein, referred to as 36K, that possesses a mitochondrial targeting signal and two transmembrane segments which are thought to anchor this protein to the outer membrane of the mitochondrial envelope of infected plant cells. To determine the topology of the virus protein inserted in the cell membrane, as well as the sequence requirements for targeting and insertion, an in vivo system was set up in which this could be analysed in the absence of productive virus infection. The 36K protein was expressed in the yeast Saccharomyces cerevisiae in native form or fused to the green fluorescent protein. Using a fluorescence microscope, large green-fluorescing cytoplasmic aggregates were visible which stained red when cells were treated with the vital stain MitoTracker, which is specific for mitochondria. These aggregates were shown by electron microscopy to be composed of either mitochondria or membranes. The latter type was particularly abundant for the construct in which the green fluorescent protein was fused at the N terminus of the 36K protein. Immunoelectron microscopy demonstrated that the viral protein is present in the anomalous aggregates and Western blot analysis of protein extracts showed 36K to be resistant to alkaline, urea or salt extraction, a property of integral membrane proteins.

Pharmacoperone drugs: targeting misfolded proteins causing lysosomal storage-, ion channels-, and G protein-coupled receptors-associated conformational disorders.

PubMed

Hou, Zhi-Shuai; Ulloa-Aguirre, Alfredo; Tao, Ya-Xiong

2018-06-01

Conformational diseases are caused by structurally abnormal proteins that cannot fold properly and achieve their native conformation. Misfolded proteins frequently originate from genetic mutations that may lead to loss-of-function diseases involving a variety of structurally diverse proteins including enzymes, ion channels, and membrane receptors. Pharmacoperones are small molecules that cross the cell surface plasma membrane and reach their target proteins within the cell, serving as molecular scaffolds to stabilize the native conformation of misfolded or well-folded but destabilized proteins, to prevent their degradation and promote correct trafficking to their functional site of action. Because of their high specificity toward the target protein, pharmacoperones are currently the focus of intense investigation as therapy for several conformational diseases. Areas covered: This review summarizes data on the mechanisms leading to protein misfolding and the use of pharmacoperone drugs as an experimental approach to rescue function of distinct misfolded/misrouted proteins associated with a variety of diseases, such as lysosomal storage diseases, channelopathies, and G protein-coupled receptor misfolding diseases. Expert commentary: The fact that many misfolded proteins may retain function, offers a unique therapeutic opportunity to cure disease by directly correcting misrouting through administering pharmacoperone drugs thereby rescuing function of disease-causing, conformationally abnormal proteins.

Overcoming barriers to membrane protein structure determination.

PubMed

Bill, Roslyn M; Henderson, Peter J F; Iwata, So; Kunji, Edmund R S; Michel, Hartmut; Neutze, Richard; Newstead, Simon; Poolman, Bert; Tate, Christopher G; Vogel, Horst

2011-04-01

After decades of slow progress, the pace of research on membrane protein structures is beginning to quicken thanks to various improvements in technology, including protein engineering and microfocus X-ray diffraction. Here we review these developments and, where possible, highlight generic new approaches to solving membrane protein structures based on recent technological advances. Rational approaches to overcoming the bottlenecks in the field are urgently required as membrane proteins, which typically comprise ~30% of the proteomes of organisms, are dramatically under-represented in the structural database of the Protein Data Bank.

Synaptotagmin-mediated bending of the target membrane is a critical step in Ca2+-regulated fusion

PubMed Central

Hui, Enfu; Johnson, Colin P.; Yao, Jun; Dunning, F. Mark; Chapman, Edwin R.

2009-01-01

Summary Decades ago it was proposed that exocytosis involves invagination of the target membrane, resulting in a highly localized site of contact between the bilayers destined to fuse. The vesicle protein synaptotagmin-I (syt) bends membranes in response to Ca2+, but whether this drives localized invagination of the target membrane to accelerate fusion has not been determined; previous studies relied on reconstituted vesicles that were already highly curved and used mutations in syt that were not selective for membrane-bending activity. Here, we directly address this question by utilizing vesicles with different degrees of curvature. A tubulation-defective syt mutant was able to promote fusion between highly curved SNARE-bearing liposomes, but exhibited a marked loss of activity when the membranes were relatively flat. Moreover, bending of flat membranes by adding an N-BAR domain rescued the function of the tubulation-deficient syt mutant. Hence, syt-mediated membrane bending is a critical step in membrane fusion. PMID:19703397

Outer membrane vesicles from Neisseria gonorrhoeae target PorB to mitochondria and induce apoptosis

PubMed Central

Elgass, Kirstin D.; Gabriel, Kipros; Dougan, Gordon; Lithgow, Trevor; Heinz, Eva

2018-01-01

Neisseria gonorrhoeae causes the sexually transmitted disease gonorrhoea by evading innate immunity. Colonizing the mucosa of the reproductive tract depends on the bacterial outer membrane porin, PorB, which is essential for ion and nutrient uptake. PorB is also targeted to host mitochondria and regulates apoptosis pathways to promote infections. How PorB traffics from the outer membrane of N. gonorrhoeae to mitochondria and whether it modulates innate immune cells, such as macrophages, remains unclear. Here, we show that N. gonorrhoeae secretes PorB via outer membrane vesicles (OMVs). Purified OMVs contained primarily outer membrane proteins including oligomeric PorB. The porin was targeted to mitochondria of macrophages after exposure to purified OMVs and wild type N. gonorrhoeae. This was associated with loss of mitochondrial membrane potential, release of cytochrome c, activation of apoptotic caspases and cell death in a time-dependent manner. Consistent with this, OMV-induced macrophage death was prevented with the pan-caspase inhibitor, Q-VD-PH. This shows that N. gonorrhoeae utilizes OMVs to target PorB to mitochondria and to induce apoptosis in macrophages, thus affecting innate immunity. PMID:29601598

Surface plasmon resonance spectroscopy for characterisation of membrane protein-ligand interactions and its potential for drug discovery.

PubMed

Patching, Simon G

2014-01-01

Surface plasmon resonance (SPR) spectroscopy is a rapidly developing technique for the study of ligand binding interactions with membrane proteins, which are the major molecular targets for validated drugs and for current and foreseeable drug discovery. SPR is label-free and capable of measuring real-time quantitative binding affinities and kinetics for membrane proteins interacting with ligand molecules using relatively small quantities of materials and has potential to be medium-throughput. The conventional SPR technique requires one binding component to be immobilised on a sensor chip whilst the other binding component in solution is flowed over the sensor surface; a binding interaction is detected using an optical method that measures small changes in refractive index at the sensor surface. This review first describes the basic SPR experiment and the challenges that have to be considered for performing SPR experiments that measure membrane protein-ligand binding interactions, most importantly having the membrane protein in a lipid or detergent environment that retains its native structure and activity. It then describes a wide-range of membrane protein systems for which ligand binding interactions have been characterised using SPR, including the major drug targets G protein-coupled receptors, and how challenges have been overcome for achieving this. Finally it describes some recent advances in SPR-based technology and future potential of the technique to screen ligand binding in the discovery of drugs. This article is part of a Special Issue entitled: Structural and biophysical characterisation of membrane protein-ligand binding. Copyright © 2013 Elsevier B.V. All rights reserved.

Improving membrane protein expression by optimizing integration efficiency

PubMed Central

2017-01-01

The heterologous overexpression of integral membrane proteins in Escherichia coli often yields insufficient quantities of purifiable protein for applications of interest. The current study leverages a recently demonstrated link between co-translational membrane integration efficiency and protein expression levels to predict protein sequence modifications that improve expression. Membrane integration efficiencies, obtained using a coarse-grained simulation approach, robustly predicted effects on expression of the integral membrane protein TatC for a set of 140 sequence modifications, including loop-swap chimeras and single-residue mutations distributed throughout the protein sequence. Mutations that improve simulated integration efficiency were 4-fold enriched with respect to improved experimentally observed expression levels. Furthermore, the effects of double mutations on both simulated integration efficiency and experimentally observed expression levels were cumulative and largely independent, suggesting that multiple mutations can be introduced to yield higher levels of purifiable protein. This work provides a foundation for a general method for the rational overexpression of integral membrane proteins based on computationally simulated membrane integration efficiencies. PMID:28918393

Neuron membrane trafficking and protein kinases involved in autism and ADHD.

PubMed

Kitagishi, Yasuko; Minami, Akari; Nakanishi, Atsuko; Ogura, Yasunori; Matsuda, Satoru

2015-01-30

A brain-enriched multi-domain scaffolding protein, neurobeachin has been identified as a candidate gene for autism patients. Mutations in the synaptic adhesion protein cell adhesion molecule 1 (CADM1) are also associated with autism spectrum disorder, a neurodevelopmental disorder of uncertain molecular origin. Potential roles of neurobeachin and CADM1 have been suggested to a function of vesicle transport in endosomal trafficking. It seems that protein kinase B (AKT) and cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) have key roles in the neuron membrane trafficking involved in the pathogenesis of autism. Attention deficit hyperactivity disorder (ADHD) is documented to dopaminergic insufficiencies, which is attributed to synaptic dysfunction of dopamine transporter (DAT). AKT is also essential for the DAT cell-surface redistribution. In the present paper, we summarize and discuss the importance of several protein kinases that regulate the membrane trafficking involved in autism and ADHD, suggesting new targets for therapeutic intervention.

Liver plasma membranes: an effective method to analyze membrane proteome.

PubMed

Cao, Rui; Liang, Songping

2012-01-01

Plasma membrane proteins are critical for the maintenance of biological systems and represent important targets for the treatment of disease. The hydrophobicity and low abundance of plasma membrane proteins make them difficult to analyze. The protocols given here are the efficient isolation/digestion procedures for liver plasma membrane proteomic analysis. Both protocol for the isolation of plasma membranes and protocol for the in-gel digestion of gel-embedded plasma membrane proteins are presented. The later method allows the use of a high detergent concentration to achieve efficient solubilization of hydrophobic plasma membrane proteins while avoiding interference with the subsequent LC-MS/MS analysis.

Altered Escherichia coli membrane protein assembly machinery allows proper membrane assembly of eukaryotic protein vitamin K epoxide reductase.

PubMed

Hatahet, Feras; Blazyk, Jessica L; Martineau, Eugenie; Mandela, Eric; Zhao, Yongxin; Campbell, Robert E; Beckwith, Jonathan; Boyd, Dana

2015-12-08

Functional overexpression of polytopic membrane proteins, particularly when in a foreign host, is often a challenging task. Factors that negatively affect such processes are poorly understood. Using the mammalian membrane protein vitamin K epoxide reductase (VKORc1) as a reporter, we describe a genetic selection approach allowing the isolation of Escherichia coli mutants capable of functionally expressing this blood-coagulation enzyme. The isolated mutants map to components of membrane protein assembly and quality control proteins YidC and HslV. We show that changes in the VKORc1 sequence and in the YidC hydrophilic groove along with the inactivation of HslV promote VKORc1 activity and dramatically increase its expression level. We hypothesize that such changes correct for mismatches in the membrane topogenic signals between E. coli and eukaryotic cells guiding proper membrane integration. Furthermore, the obtained mutants allow the study of VKORc1 reaction mechanisms, inhibition by warfarin, and the high-throughput screening for potential anticoagulants.

Altered Escherichia coli membrane protein assembly machinery allows proper membrane assembly of eukaryotic protein vitamin K epoxide reductase

PubMed Central

Hatahet, Feras; Blazyk, Jessica L.; Martineau, Eugenie; Mandela, Eric; Zhao, Yongxin; Campbell, Robert E.; Beckwith, Jonathan; Boyd, Dana

2015-01-01

Functional overexpression of polytopic membrane proteins, particularly when in a foreign host, is often a challenging task. Factors that negatively affect such processes are poorly understood. Using the mammalian membrane protein vitamin K epoxide reductase (VKORc1) as a reporter, we describe a genetic selection approach allowing the isolation of Escherichia coli mutants capable of functionally expressing this blood-coagulation enzyme. The isolated mutants map to components of membrane protein assembly and quality control proteins YidC and HslV. We show that changes in the VKORc1 sequence and in the YidC hydrophilic groove along with the inactivation of HslV promote VKORc1 activity and dramatically increase its expression level. We hypothesize that such changes correct for mismatches in the membrane topogenic signals between E. coli and eukaryotic cells guiding proper membrane integration. Furthermore, the obtained mutants allow the study of VKORc1 reaction mechanisms, inhibition by warfarin, and the high-throughput screening for potential anticoagulants. PMID:26598701

Concepts of Protein Sorting or Targeting Signals and Membrane Topology in Undergraduate Teaching

ERIC Educational Resources Information Center

Tang, Bor Luen; Teng, Felicia Yu Hsuan

2005-01-01

The process of protein biogenesis culminates in its correct targeting to specific subcellular locations where it serves a function. Contemporary molecular and cell biology investigations often involve the exogenous expression of epitope- or fluorescent protein-tagged recombinant molecules as well as subsequent analysis of protein-protein…

X11/Mint Genes Control Polarized Localization of Axonal Membrane Proteins in Vivo

PubMed Central

Gross, Garrett G.; Lone, G. Mohiddin; Leung, Lok Kwan; Hartenstein, Volker

2013-01-01

Mislocalization of axonal proteins can result in misassembly and/or miswiring of neural circuits, causing disease. To date, only a handful of genes that control polarized localization of axonal membrane proteins have been identified. Here we report that Drosophila X11/Mint proteins are required for targeting several proteins, including human amyloid precursor protein (APP) and Drosophila APP-like protein (APPL), to axonal membranes and for their exclusion from dendrites of the mushroom body in Drosophila, a brain structure involved in learning and memory. Axonal localization of APP is mediated by an endocytic motif, and loss of X11/Mint results in a dramatic increase in cell-surface levels of APPL, especially on dendrites. Mutations in genes required for endocytosis show similar mislocalization of these proteins to dendrites, and strongly enhance defects seen in X11/Mint mutants. These results suggest that X11/Mint-dependent endocytosis in dendrites may serve to promote the axonal localization of membrane proteins. Since X11/Mint binds to APP, and abnormal trafficking of APP contributes to Alzheimer's disease, deregulation of X11/Mint may be important for Alzheimer's disease pathogenesis. PMID:23658195

Protein profiles of hatchery egg shell membrane.

PubMed

Rath, N C; Liyanage, R; Makkar, S K; Lay, J O

2016-01-01

Eggshells which consist largely of calcareous outer shell and shell membranes, constitute a significant part of poultry hatchery waste. The shell membranes (ESM) not only contain proteins that originate from egg whites but also from the developing embryos and different contaminants of microbial and environmental origins. As feed supplements, during post hatch growth, the hatchery egg shell membranes (HESM) have shown potential for imparting resistance of chickens to endotoxin stress and exert positive health effects. Considering that these effects are mediated by the bioactive proteins and peptides present in the membrane, the objective of the study was to identify the protein profiles of hatchery eggshell membranes (HESM). Hatchery egg shell membranes were extracted with acidified methanol and a guanidine hydrochloride buffer then subjected to reduction/alkylation, and trypsin digestion. The methanol extract was additionally analyzed by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). The tryptic digests were analyzed by liquid chromatography and tandem mass spectrometry (LC-MS-MS) to identify the proteins. Our results showed the presence of several proteins that are inherent and abundant in egg white such as, ovalbumin, ovotransferrin, ovocleidin-116, and lysozyme, and several proteins associated with cytoskeletal, cell signaling, antimicrobial, and catalytic functions involving carbohydrate, nucleic acid, and protein metabolisms. There were some blood derived proteins most likely originating from the embryos and several other proteins identified with different aerobic, anaerobic, gram positive, gram negative, soil, and marine bacterial species some commensals and others zoonotic. The variety of bioactive proteins, particularly the cell signaling and enzymatic proteins along with the diverse microbial proteins, make the HESM suitable for nutritional and biological application to improve post hatch immunity of poultry.

A Continuum Poisson-Boltzmann Model for Membrane Channel Proteins

PubMed Central

Xiao, Li; Diao, Jianxiong; Greene, D'Artagnan; Wang, Junmei; Luo, Ray

2017-01-01

Membrane proteins constitute a large portion of the human proteome and perform a variety of important functions as membrane receptors, transport proteins, enzymes, signaling proteins, and more. Computational studies of membrane proteins are usually much more complicated than those of globular proteins. Here we propose a new continuum model for Poisson-Boltzmann calculations of membrane channel proteins. Major improvements over the existing continuum slab model are as follows:1) The location and thickness of the slab model are fine-tuned based on explicit-solvent MD simulations. 2) The highly different accessibility in the membrane and water regions are addressed with a two-step, two-probe grid labeling procedure, and 3) The water pores/channels are automatically identified. The new continuum membrane model is optimized (by adjusting the membrane probe, as well as the slab thickness and center) to best reproduce the distributions of buried water molecules in the membrane region as sampled in explicit water simulations. Our optimization also shows that the widely adopted water probe of 1.4 Å for globular proteins is a very reasonable default value for membrane protein simulations. It gives the best compromise in reproducing the explicit water distributions in membrane channel proteins, at least in the water accessible pore/channel regions that we focus on. Finally, we validate the new membrane model by carrying out binding affinity calculations for a potassium channel, and we observe a good agreement with experiment results. PMID:28564540

Membrane tension controls the assembly of curvature-generating proteins

PubMed Central

Simunovic, Mijo; Voth, Gregory A.

2015-01-01

Proteins containing a Bin/Amphiphysin/Rvs (BAR) domain regulate membrane curvature in the cell. Recent simulations have revealed that BAR proteins assemble into linear aggregates, strongly affecting membrane curvature and its in-plane stress profile. Here, we explore the opposite question: do mechanical properties of the membrane impact protein association? By using coarse-grained molecular dynamics simulations, we show that increased surface tension significantly impacts the dynamics of protein assembly. While tensionless membranes promote a rapid formation of long-living linear aggregates of N-BAR proteins, increase in tension alters the geometry of protein association. At high tension, protein interactions are strongly inhibited. Increasing surface density of proteins leads to a wider range of protein association geometries, promoting the formation of meshes, which can be broken apart with membrane tension. Our work indicates that surface tension may play a key role in recruiting proteins to membrane-remodelling sites in the cell. PMID:26008710

Crystallizing Membrane Proteins in the Lipidic Mesophase. Experience with Human Prostaglandin E2 Synthase 1 and an Evolving Strategy.

PubMed

Li, Dianfan; Howe, Nicole; Dukkipati, Abhiram; Shah, Syed T A; Bax, Benjamin D; Edge, Colin; Bridges, Angela; Hardwicke, Phil; Singh, Onkar M P; Giblin, Ged; Pautsch, Alexander; Pfau, Roland; Schnapp, Gisela; Wang, Meitian; Olieric, Vincent; Caffrey, Martin

2014-04-02

The lipidic mesophase or in meso method for crystallizing membrane proteins has several high profile targets to its credit and is growing in popularity. Despite its success, the method is in its infancy as far as rational crystallogenesis is concerned. Consequently, significant time, effort, and resources are still required to generate structure-grade crystals, especially with a new target type. Therefore, a need exists for crystallogenesis protocols that are effective with a broad range of membrane protein types. Recently, a strategy for crystallizing a prokaryotic α-helical membrane protein, diacylglycerol kinase (DgkA), by the in meso method was reported (Cryst. Growth. Des.2013, 14, 2846-2857). Here, we describe its application to the human α-helical microsomal prostaglandin E2 synthase 1 (mPGES1). While the DgkA strategy proved useful, significant modifications were needed to generate structure-quality crystals of this important therapeutic target. These included protein engineering, using an additive phospholipid in the hosting mesophase, performing multiple rounds of salt screening, and carrying out trials at 4 °C in the presence of a tight binding ligand. The crystallization strategy detailed here should prove useful for generating structures of other integral membrane proteins by the in meso method.

Selective Targeting of Proteins within Secretory Pathway for Endoplasmic Reticulum-associated Degradation

PubMed Central

Vecchi, Lara; Petris, Gianluca; Bestagno, Marco; Burrone, Oscar R.

2012-01-01

The endoplasmic reticulum-associated degradation (ERAD) is a cellular quality control mechanism to dispose of misfolded proteins of the secretory pathway via proteasomal degradation. SEL1L is an ER-resident protein that participates in identification of misfolded molecules as ERAD substrates, therefore inducing their ER-to-cytosol retrotranslocation and degradation. We have developed a novel class of fusion proteins, termed degradins, composed of a fragment of SEL1L fused to a target-specific binding moiety located on the luminal side of the ER. The target-binding moiety can be a ligand of the target or derived from specific mAbs. Here, we describe the ability of degradins with two different recognition moieties to promote degradation of a model target. Degradins recognize the target protein within the ER both in secretory and membrane-bound forms, inducing their degradation following retrotranslocation to the cytosol. Thus, degradins represent an effective technique to knock-out proteins within the secretory pathway with high specificity. PMID:22523070

Msp1 Is a Membrane Protein Dislocase for Tail-Anchored Proteins.

PubMed

Wohlever, Matthew L; Mateja, Agnieszka; McGilvray, Philip T; Day, Kasey J; Keenan, Robert J

2017-07-20

Mislocalized tail-anchored (TA) proteins of the outer mitochondrial membrane are cleared by a newly identified quality control pathway involving the conserved eukaryotic protein Msp1 (ATAD1 in humans). Msp1 is a transmembrane AAA-ATPase, but its role in TA protein clearance is not known. Here, using purified components reconstituted into proteoliposomes, we show that Msp1 is both necessary and sufficient to drive the ATP-dependent extraction of TA proteins from the membrane. A crystal structure of the Msp1 cytosolic region modeled into a ring hexamer suggests that active Msp1 contains a conserved membrane-facing surface adjacent to a central pore. Structure-guided mutagenesis of the pore residues shows that they are critical for TA protein extraction in vitro and for functional complementation of an msp1 deletion in yeast. Together, these data provide a molecular framework for Msp1-dependent extraction of mislocalized TA proteins from the outer mitochondrial membrane. Copyright © 2017 Elsevier Inc. All rights reserved.

Major intrinsic proteins in biomimetic membranes.

PubMed

Nielsen, Claus Hélix

2010-01-01

Biological membranes define the structural and functional boundaries in living cells and their organelles. The integrity of the cell depends on its ability to separate inside from outside and yet at the same time allow massive transport of matter in and out the cell. Nature has elegantly met this challenge by developing membranes in the form of lipid bilayers in which specialized transport proteins are incorporated. This raises the question: is it possible to mimic biological membranes and create a membrane based sensor and/or separation device? In the development of a biomimetic sensor/separation technology, a unique class of membrane transport proteins is especially interesting-the major intrinsic proteins (MIPs). Generally, MIPs conduct water molecules and selected solutes in and out of the cell while preventing the passage of other solutes, a property critical for the conservation of the cells internal pH and salt concentration. Also known as water channels or aquaporins they are highly efficient membrane pore proteins some of which are capable of transporting water at very high rates up to 10(9) molecules per second. Some MIPs transport other small, uncharged solutes, such as glycerol and other permeants such as carbon dioxide, nitric oxide, ammonia, hydrogen peroxide and the metalloids antimonite, arsenite, silicic and boric acid depending on the effective restriction mechanism of the protein. The flux properties of MIPs thus lead to the question ifMIPs can be used in separation devices or as sensor devices based on, e.g., the selective permeation of metalloids. In principle a MIP based membrane sensor/separation device requires the supporting biomimetic matrix to be virtually impermeable to anything but water or the solute in question. In practice, however, a biomimetic support matrix will generally have finite permeabilities to both electrolytes and non-electrolytes. The feasibility of a biomimetic MIP device thus depends on the relative transport

Improving membrane protein expression and function using genomic edits

DOE PAGES

Jensen, Heather M.; Eng, Thomas; Chubukov, Victor; ...

2017-10-12

Expression of membrane proteins often leads to growth inhibition and perturbs central metabolism and this burden varies with the protein being overexpressed. There are also known strain backgrounds that allow greater expression of membrane proteins but that differ in efficacy across proteins. Here, we hypothesized that for any membrane protein, it may be possible to identify a modified strain background where its expression can be accommodated with less burden. To directly test this hypothesis, we used a bar-coded transposon insertion library in tandem with cell sorting to assess genome-wide impact of gene deletions on membrane protein expression. The expression ofmore » five membrane proteins (CyoB, CydB, MdlB, YidC, and LepI) and one soluble protein (GST), each fused to GFP, was examined. We identified Escherichia coli mutants that demonstrated increased membrane protein expression relative to that in wild type. For two of the proteins (CyoB and CydB), we conducted functional assays to confirm that the increase in protein expression also led to phenotypic improvement in function. This study represents a systematic approach to broadly identify genetic loci that can be used to improve membrane protein expression, and our method can be used to improve expression of any protein that poses a cellular burden.« less

Improving membrane protein expression and function using genomic edits

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jensen, Heather M.; Eng, Thomas; Chubukov, Victor

Expression of membrane proteins often leads to growth inhibition and perturbs central metabolism and this burden varies with the protein being overexpressed. There are also known strain backgrounds that allow greater expression of membrane proteins but that differ in efficacy across proteins. Here, we hypothesized that for any membrane protein, it may be possible to identify a modified strain background where its expression can be accommodated with less burden. To directly test this hypothesis, we used a bar-coded transposon insertion library in tandem with cell sorting to assess genome-wide impact of gene deletions on membrane protein expression. The expression ofmore » five membrane proteins (CyoB, CydB, MdlB, YidC, and LepI) and one soluble protein (GST), each fused to GFP, was examined. We identified Escherichia coli mutants that demonstrated increased membrane protein expression relative to that in wild type. For two of the proteins (CyoB and CydB), we conducted functional assays to confirm that the increase in protein expression also led to phenotypic improvement in function. This study represents a systematic approach to broadly identify genetic loci that can be used to improve membrane protein expression, and our method can be used to improve expression of any protein that poses a cellular burden.« less

Changes in topography and function of thylakoid membranes following membrane protein phosphorylation.

PubMed

Black, M T; Lee, P; Horton, P

1986-09-01

Changes in topography and function of pea (Pisum sativum L.) thylakoid membrane fractions following membrane protein phosphorylation have been studied. After protein phosphorylation the stromal membrane fraction had a higher chlorophyll a/b ratio, an increased content of light-harvesting chlorophyll protein and a higher ratio of chlorophyll to cytochrome f. This indicates that a pool of light-harvesting chlorophyll protein migrates from the photosystem II-enriched grana regions to the photosystem I-enriched stroma lamellae, in agreement with Kyle et al. (1984, Biochim. Biophys. Acta 765, 89-96) and Larsson et al. (1983, Eur. J. Biochem. 136, 25-29). Phosphorylation caused a stimulation in the rate of light-limited photosystem-I electron transfer in the unappressed membrane fraction, indicating that the translocated LHC-II becomes functionally associated with photosystem I.

Functional dynamics of cell surface membrane proteins

NASA Astrophysics Data System (ADS)

Nishida, Noritaka; Osawa, Masanori; Takeuchi, Koh; Imai, Shunsuke; Stampoulis, Pavlos; Kofuku, Yutaka; Ueda, Takumi; Shimada, Ichio

2014-04-01

Cell surface receptors are integral membrane proteins that receive external stimuli, and transmit signals across plasma membranes. In the conventional view of receptor activation, ligand binding to the extracellular side of the receptor induces conformational changes, which convert the structure of the receptor into an active conformation. However, recent NMR studies of cell surface membrane proteins have revealed that their structures are more dynamic than previously envisioned, and they fluctuate between multiple conformations in an equilibrium on various timescales. In addition, NMR analyses, along with biochemical and cell biological experiments indicated that such dynamical properties are critical for the proper functions of the receptors. In this review, we will describe several NMR studies that revealed direct linkage between the structural dynamics and the functions of the cell surface membrane proteins, such as G-protein coupled receptors (GPCRs), ion channels, membrane transporters, and cell adhesion molecules.

Hydrophobic mismatch sorts SNARE proteins into distinct membrane domains

PubMed Central

Milovanovic, Dragomir; Honigmann, Alf; Koike, Seiichi; Göttfert, Fabian; Pähler, Gesa; Junius, Meike; Müllar, Stefan; Diederichsen, Ulf; Janshoff, Andreas; Grubmüller, Helmut; Risselada, Herre J.; Eggeling, Christian; Hell, Stefan W.; van den Bogaart, Geert; Jahn, Reinhard

2015-01-01

The clustering of proteins and lipids in distinct microdomains is emerging as an important principle for the spatial patterning of biological membranes. Such domain formation can be the result of hydrophobic and ionic interactions with membrane lipids as well as of specific protein–protein interactions. Here using plasma membrane-resident SNARE proteins as model, we show that hydrophobic mismatch between the length of transmembrane domains (TMDs) and the thickness of the lipid membrane suffices to induce clustering of proteins. Even when the TMDs differ in length by only a single residue, hydrophobic mismatch can segregate structurally closely homologous membrane proteins in distinct membrane domains. Domain formation is further fine-tuned by interactions with polyanionic phosphoinositides and homo and heterotypic protein interactions. Our findings demonstrate that hydrophobic mismatch contributes to the structural organization of membranes. PMID:25635869

Protein-membrane interaction and fatty acid transfer from intestinal fatty acid-binding protein to membranes. Support for a multistep process.

PubMed

Falomir-Lockhart, Lisandro J; Laborde, Lisandro; Kahn, Peter C; Storch, Judith; Córsico, Betina

2006-05-19

Fatty acid transfer from intestinal fatty acid-binding protein (IFABP) to phospholipid membranes occurs during protein-membrane collisions. Electrostatic interactions involving the alpha-helical "portal" region of the protein have been shown to be of great importance. In the present study, the role of specific lysine residues in the alpha-helical region of IFABP was directly examined. A series of point mutants in rat IFABP was engineered in which the lysine positive charges in this domain were eliminated or reversed. Using a fluorescence resonance energy transfer assay, we analyzed the rates and mechanism of fatty acid transfer from wild type and mutant proteins to acceptor membranes. Most of the alpha-helical domain mutants showed slower absolute fatty acid transfer rates to zwitterionic membranes, with substitution of one of the lysines of the alpha2 helix, Lys27, resulting in a particularly dramatic decrease in the fatty acid transfer rate. Sensitivity to negatively charged phospholipid membranes was also reduced, with charge reversal mutants in the alpha2 helix the most affected. The results support the hypothesis that the portal region undergoes a conformational change during protein-membrane interaction, which leads to release of the bound fatty acid to the membrane and that the alpha2 segment is of particular importance in the establishment of charge-charge interactions between IFABP and membranes. Cross-linking experiments with a phospholipid-photoactivable reagent underscored the importance of charge-charge interactions, showing that the physical interaction between wild-type intestinal fatty acid-binding protein and phospholipid membranes is enhanced by electrostatic interactions. Protein-membrane interactions were also found to be enhanced by the presence of ligand, suggesting different collisional complex structures for holo- and apo-IFABP.

The fine art of integral membrane protein crystallisation.

PubMed

Birch, James; Axford, Danny; Foadi, James; Meyer, Arne; Eckhardt, Annette; Thielmann, Yvonne; Moraes, Isabel

2018-05-18

Integral membrane proteins are among the most fascinating and important biomolecules as they play a vital role in many biological functions. Knowledge of their atomic structures is fundamental to the understanding of their biochemical function and key in many drug discovery programs. However, over the years, structure determination of integral membrane proteins has proven to be far from trivial, hence they are underrepresented in the protein data bank. Low expression levels, insolubility and instability are just a few of the many hurdles one faces when studying these proteins. X-ray crystallography has been the most used method to determine atomic structures of membrane proteins. However, the production of high quality membrane protein crystals is always very challenging, often seen more as art than a rational experiment. Here we review valuable approaches, methods and techniques to successful membrane protein crystallisation. Copyright © 2018 Diamond Light Source LTD. Published by Elsevier Inc. All rights reserved.

Rupturing Giant Plasma Membrane Vesicles to Form Micron-sized Supported Cell Plasma Membranes with Native Transmembrane Proteins.

PubMed

Chiang, Po-Chieh; Tanady, Kevin; Huang, Ling-Ting; Chao, Ling

2017-11-09

Being able to directly obtain micron-sized cell blebs, giant plasma membrane vesicles (GPMVs), with native membrane proteins and deposit them on a planar support to form supported plasma membranes could allow the membrane proteins to be studied by various surface analytical tools in native-like bilayer environments. However, GPMVs do not easily rupture on conventional supports because of their high protein and cholesterol contents. Here, we demonstrate the possibility of using compression generated by the air-water interface to efficiently rupture GPMVs to form micron-sized supported membranes with native plasma membrane proteins. We demonstrated that not only lipid but also a native transmembrane protein in HeLa cells, Aquaporin 3 (AQP3), is mobile in the supported membrane platform. This convenient method for generating micron-sized supported membrane patches with mobile native transmembrane proteins could not only facilitate the study of membrane proteins by surface analytical tools, but could also enable us to use native membrane proteins for bio-sensing applications.

Disturbed vesicular trafficking of membrane proteins in prion disease.

PubMed

Uchiyama, Keiji; Miyata, Hironori; Sakaguchi, Suehiro

2013-01-01

The pathogenic mechanism of prion diseases remains unknown. We recently reported that prion infection disturbs post-Golgi trafficking of certain types of membrane proteins to the cell surface, resulting in reduced surface expression of membrane proteins and abrogating the signal from the proteins. The surface expression of the membrane proteins was reduced in the brains of mice inoculated with prions, well before abnormal symptoms became evident. Prions or pathogenic prion proteins were mainly detected in endosomal compartments, being particularly abundant in recycling endosomes. Some newly synthesized membrane proteins are delivered to the surface from the Golgi apparatus through recycling endosomes, and some endocytosed membrane proteins are delivered back to the surface through recycling endosomes. These results suggest that prions might cause neuronal dysfunctions and cell loss by disturbing post-Golgi trafficking of membrane proteins via accumulation in recycling endosomes. Interestingly, it was recently shown that delivery of a calcium channel protein to the cell surface was impaired and its function was abrogated in a mouse model of hereditary prion disease. Taken together, these results suggest that impaired delivery of membrane proteins to the cell surface is a common pathogenic event in acquired and hereditary prion diseases.

Protein quality control at the inner nuclear membrane

PubMed Central

Khmelinskii, Anton; Blaszczak, Ewa; Pantazopoulou, Marina; Fischer, Bernd; Omnus, Deike J.; Le Dez, Gaëlle; Brossard, Audrey; Gunnarsson, Alexander; Barry, Joseph D.; Meurer, Matthias; Kirrmaier, Daniel; Boone, Charles; Huber, Wolfgang; Rabut, Gwenaël; Ljungdahl, Per O.; Knop, Michael

2015-01-01

The nuclear envelope is a double membrane that separates the nucleus from the cytoplasm. The inner nuclear membrane (INM) functions in essential nuclear processes including chromatin organization and regulation of gene expression1. The outer nuclear membrane is continuous with the endoplasmic reticulum (ER) and is the site of membrane protein synthesis. Protein homeostasis in this compartment is ensured by ER-associated protein degradation (ERAD) pathways that in yeast involve the integral membrane E3 ubiquitin ligases Hrd1 and Doa10 operating with the E2 ubiquitin-conjugating enzymes Ubc6 and Ubc72,3. However, little is known regarding protein quality control at the INM. Here we describe a protein degradation pathway at the INM mediated by the Asi complex consisting of the RING domain proteins Asi1 and Asi34. We report that the As complex functions together with the ubiquitin conjugating enzymes Ubc6andUbc7to degrade soluble and integral membrane proteins. Genetic evidence suggest that the Asi ubiquitin ligase defines a pathway distinct from but complementary to ERAD. Using unbiased screening with a novel genome-wide yeast library based on a tandem fluorescent protein timer (tFT)5, we identify more than 50 substrates of the Asi, Hrd1 and Doa10 E3 ubiquity ligases. We show that the Asi ubiquitin ligase is involved in degradation of mislocalised integral membrane proteins, thus acting to maintain and safeguard the identity of the INM. PMID:25519137

Metaproteomic analysis of biocake proteins to understand membrane fouling in a submerged membrane bioreactor.

PubMed

Zhou, Zhongbo; Meng, Fangang; He, Xiang; Chae, So-Ryong; An, Yujia; Jia, Xiaoshan

2015-01-20

Metaproteomic analyses, including two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) separation and matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF)/TOF mass spectrometer (MS) detection, were used to trace and identify biocake proteins on membranes in a bench-scale submerged membrane bioreactor (MBR). 2D-PAGE images showed that proteins in the biocake (S3) at a low transmembrane pressure (TMP) level (i.e., before the TMP jump) had larger gray intensities in the pH 5.5–7.0 region regardless of the membrane flux, similar to soluble microbial product (SMP) proteins. However, the biocake (S2 and S4) at a high TMP level (i.e., after the TMP jump) had many more proteins in the pH range of 4.0–5.5, similar to extracellular polymeric substance (EPS) proteins. Such similarities between biocake proteins and SMP or EPS proteins can be useful for tracing the sources of proteins resulting in membrane fouling. In total, 183 differentially abundant protein spots were marked in the three biocakes (S2, S3, and S4). However, only 32 protein spots co-occurred in the 2D gels of the three biocakes, indicating that membrane fluxes and TMP evolution levels had significant effects on the abundance of biocake proteins. On the basis of the MS and MS/MS data, 23 of 71 protein spots were successfully identified. Of the 23 proteins, outer membrane proteins (Omp) were a major contributor (60.87%). These Omps were mainly from potential surface colonizers such as Aeromonas, Enterobacter, Pseudomonas, and Thauera. Generally, the metaproteomic analysis is a useful alternative to trace the sources and compositions of biocake proteins on the levels of molecules and bacteria species that can provide new insight into membrane fouling.

An Integrated Framework Advancing Membrane Protein Modeling and Design

PubMed Central

Weitzner, Brian D.; Duran, Amanda M.; Tilley, Drew C.; Elazar, Assaf; Gray, Jeffrey J.

2015-01-01

Membrane proteins are critical functional molecules in the human body, constituting more than 30% of open reading frames in the human genome. Unfortunately, a myriad of difficulties in overexpression and reconstitution into membrane mimetics severely limit our ability to determine their structures. Computational tools are therefore instrumental to membrane protein structure prediction, consequently increasing our understanding of membrane protein function and their role in disease. Here, we describe a general framework facilitating membrane protein modeling and design that combines the scientific principles for membrane protein modeling with the flexible software architecture of Rosetta3. This new framework, called RosettaMP, provides a general membrane representation that interfaces with scoring, conformational sampling, and mutation routines that can be easily combined to create new protocols. To demonstrate the capabilities of this implementation, we developed four proof-of-concept applications for (1) prediction of free energy changes upon mutation; (2) high-resolution structural refinement; (3) protein-protein docking; and (4) assembly of symmetric protein complexes, all in the membrane environment. Preliminary data show that these algorithms can produce meaningful scores and structures. The data also suggest needed improvements to both sampling routines and score functions. Importantly, the applications collectively demonstrate the potential of combining the flexible nature of RosettaMP with the power of Rosetta algorithms to facilitate membrane protein modeling and design. PMID:26325167

Phosphatidic acid binding proteins display differential binding as a function of membrane curvature stress and chemical properties.

PubMed

Putta, Priya; Rankenberg, Johanna; Korver, Ruud A; van Wijk, Ringo; Munnik, Teun; Testerink, Christa; Kooijman, Edgar E

2016-11-01

Phosphatidic acid (PA) is a crucial membrane phospholipid involved in de novo lipid synthesis and numerous intracellular signaling cascades. The signaling function of PA is mediated by peripheral membrane proteins that specifically recognize PA. While numerous PA-binding proteins are known, much less is known about what drives specificity of PA-protein binding. Previously, we have described the ionization properties of PA, summarized in the electrostatic-hydrogen bond switch, as one aspect that drives the specific binding of PA by PA-binding proteins. Here we focus on membrane curvature stress induced by phosphatidylethanolamine and show that many PA-binding proteins display enhanced binding as a function of negative curvature stress. This result is corroborated by the observation that positive curvature stress, induced by lyso phosphatidylcholine, abolishes PA binding of target proteins. We show, for the first time, that a novel plant PA-binding protein, Arabidopsis Epsin-like Clathrin Adaptor 1 (ECA1) displays curvature-dependence in its binding to PA. Other established PA targets examined in this study include, the plant proteins TGD2, and PDK1, the yeast proteins Opi1 and Spo20, and, the mammalian protein Raf-1 kinase and the C2 domain of the mammalian phosphatidylserine binding protein Lact as control. Based on our observations, we propose that liposome binding assays are the preferred method to investigate lipid binding compared to the popular lipid overlay assays where membrane environment is lost. The use of complex lipid mixtures is important to elucidate further aspects of PA binding proteins. Copyright © 2016. Published by Elsevier B.V.

Dynamic nuclear polarization methods in solids and solutions to explore membrane proteins and membrane systems.

PubMed

Cheng, Chi-Yuan; Han, Songi

2013-01-01

Membrane proteins regulate vital cellular processes, including signaling, ion transport, and vesicular trafficking. Obtaining experimental access to their structures, conformational fluctuations, orientations, locations, and hydration in membrane environments, as well as the lipid membrane properties, is critical to understanding their functions. Dynamic nuclear polarization (DNP) of frozen solids can dramatically boost the sensitivity of current solid-state nuclear magnetic resonance tools to enhance access to membrane protein structures in native membrane environments. Overhauser DNP in the solution state can map out the local and site-specific hydration dynamics landscape of membrane proteins and lipid membranes, critically complementing the structural and dynamics information obtained by electron paramagnetic resonance spectroscopy. Here, we provide an overview of how DNP methods in solids and solutions can significantly increase our understanding of membrane protein structures, dynamics, functions, and hydration in complex biological membrane environments.

Conditions that allow for effective transfer of membrane proteins onto nitrocellulose membrane in Western blots.

PubMed

Abeyrathne, Priyanka D; Lam, Joseph S

2007-04-01

A major hurdle in characterizing bacterial membrane proteins by Western blotting is the ineffectiveness of transferring these proteins from sodium dodecyl sulfate -- polyacrylamide gel electrophoresis (SDS-PAGE) gel onto nitrocellulose membrane, using standard Western blot buffers and electrophoretic conditions. In this study, we compared a number of modified Western blotting buffers and arrived at a composition designated as the SDS-PAGE-Urea Lysis buffer. The use of this buffer and specific conditions allowed the reproducible transfer of highly hydrophobic bacterial membrane proteins with 2-12 transmembrane-spanning segments as well as soluble proteins onto nitrocellulose membranes. This method should be broadly applicable for immunochemical studies of other membrane proteins.

Bacterial Origin of a Mitochondrial Outer Membrane Protein Translocase

PubMed Central

Harsman, Anke; Niemann, Moritz; Pusnik, Mascha; Schmidt, Oliver; Burmann, Björn M.; Hiller, Sebastian; Meisinger, Chris; Schneider, André; Wagner, Richard

2012-01-01

Mitochondria are of bacterial ancestry and have to import most of their proteins from the cytosol. This process is mediated by Tom40, an essential protein that forms the protein-translocating pore in the outer mitochondrial membrane. Tom40 is conserved in virtually all eukaryotes, but its evolutionary origin is unclear because bacterial orthologues have not been identified so far. Recently, it was shown that the parasitic protozoon Trypanosoma brucei lacks a conventional Tom40 and instead employs the archaic translocase of the outer mitochondrial membrane (ATOM), a protein that shows similarities to both eukaryotic Tom40 and bacterial protein translocases of the Omp85 family. Here we present electrophysiological single channel data showing that ATOM forms a hydrophilic pore of large conductance and high open probability. Moreover, ATOM channels exhibit a preference for the passage of cationic molecules consistent with the idea that it may translocate unfolded proteins targeted by positively charged N-terminal presequences. This is further supported by the fact that the addition of a presequence peptide induces transient pore closure. An in-depth comparison of these single channel properties with those of other protein translocases reveals that ATOM closely resembles bacterial-type protein export channels rather than eukaryotic Tom40. Our results support the idea that ATOM represents an evolutionary intermediate between a bacterial Omp85-like protein export machinery and the conventional Tom40 that is found in mitochondria of other eukaryotes. PMID:22778261

Overcoming bottlenecks in the membrane protein structural biology pipeline.

PubMed

Hardy, David; Bill, Roslyn M; Jawhari, Anass; Rothnie, Alice J

2016-06-15

Membrane proteins account for a third of the eukaryotic proteome, but are greatly under-represented in the Protein Data Bank. Unfortunately, recent technological advances in X-ray crystallography and EM cannot account for the poor solubility and stability of membrane protein samples. A limitation of conventional detergent-based methods is that detergent molecules destabilize membrane proteins, leading to their aggregation. The use of orthologues, mutants and fusion tags has helped improve protein stability, but at the expense of not working with the sequence of interest. Novel detergents such as glucose neopentyl glycol (GNG), maltose neopentyl glycol (MNG) and calixarene-based detergents can improve protein stability without compromising their solubilizing properties. Styrene maleic acid lipid particles (SMALPs) focus on retaining the native lipid bilayer of a membrane protein during purification and biophysical analysis. Overcoming bottlenecks in the membrane protein structural biology pipeline, primarily by maintaining protein stability, will facilitate the elucidation of many more membrane protein structures in the near future. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

Functional dynamics of cell surface membrane proteins.

PubMed

Nishida, Noritaka; Osawa, Masanori; Takeuchi, Koh; Imai, Shunsuke; Stampoulis, Pavlos; Kofuku, Yutaka; Ueda, Takumi; Shimada, Ichio

2014-04-01

Cell surface receptors are integral membrane proteins that receive external stimuli, and transmit signals across plasma membranes. In the conventional view of receptor activation, ligand binding to the extracellular side of the receptor induces conformational changes, which convert the structure of the receptor into an active conformation. However, recent NMR studies of cell surface membrane proteins have revealed that their structures are more dynamic than previously envisioned, and they fluctuate between multiple conformations in an equilibrium on various timescales. In addition, NMR analyses, along with biochemical and cell biological experiments indicated that such dynamical properties are critical for the proper functions of the receptors. In this review, we will describe several NMR studies that revealed direct linkage between the structural dynamics and the functions of the cell surface membrane proteins, such as G-protein coupled receptors (GPCRs), ion channels, membrane transporters, and cell adhesion molecules. Copyright © 2013 Elsevier Inc. All rights reserved.

The Origin and Early Evolution of Membrane Proteins

NASA Technical Reports Server (NTRS)

Pohorille, Andrew; Schweighofer, Karl; Wilson, Michael A.

2005-01-01

Membrane proteins mediate functions that are essential to all cells. These functions include transport of ions, nutrients and waste products across cell walls, capture of energy and its transduction into the form usable in chemical reactions, transmission of environmental signals to the interior of the cell, cellular growth and cell volume regulation. In the absence of membrane proteins, ancestors of cell (protocells), would have had only very limited capabilities to communicate with their environment. Thus, it is not surprising that membrane proteins are quite common even in simplest prokaryotic cells. Considering that contemporary membrane channels are large and complex, both structurally and functionally, a question arises how their presumably much simpler ancestors could have emerged, perform functions and diversify in early protobiological evolution. Remarkably, despite their overall complexity, structural motifs in membrane proteins are quite simple, with a-helices being most common. This suggests that these proteins might have evolved from simple building blocks. To explain how these blocks could have organized into functional structures, we performed large-scale, accurate computer simulations of folding peptides at a water-membrane interface, their insertion into the membrane, self-assembly into higher-order structures and function. The results of these simulations, combined with analysis of structural and functional experimental data led to the first integrated view of the origin and early evolution of membrane proteins.

Targeted Protein Degradation of Outer Membrane Decaheme Cytochrome MtrC Metal Reductase in Shewanella oneidensis MR-1 Measured Using Biarsenical Probe CrAsH-EDT2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xiong, Yijia; Chen, Baowei; Shi, Liang

2011-10-14

consistent with the inherent complexity associated with correct heme insertion and acylation of MtrC that occurs in the periplasm prior to its targeting to the outer membrane. These latter results suggest that MtrC protein trafficking to the outer membrane and its subsequent degradation are tightly regulated, which is consistent with cellular processing pathways that target MtrC to extracellular structures and their possible role in promoting electron transfer from Shewanella to extracellular acceptors.« less

Durable vesicles for reconstitution of membrane proteins in biotechnology.

PubMed

Beales, Paul A; Khan, Sanobar; Muench, Stephen P; Jeuken, Lars J C

2017-02-08

The application of membrane proteins in biotechnology requires robust, durable reconstitution systems that enhance their stability and support their functionality in a range of working environments. Vesicular architectures are highly desirable to provide the compartmentalisation to utilise the functional transmembrane transport and signalling properties of membrane proteins. Proteoliposomes provide a native-like membrane environment to support membrane protein function, but can lack the required chemical and physical stability. Amphiphilic block copolymers can also self-assemble into polymersomes: tough vesicles with improved stability compared with liposomes. This review discusses the reconstitution of membrane proteins into polymersomes and the more recent development of hybrid vesicles, which blend the robust nature of block copolymers with the biofunctionality of lipids. These novel synthetic vesicles hold great promise for enabling membrane proteins within biotechnologies by supporting their enhanced in vitro performance and could also contribute to fundamental biochemical and biophysical research by improving the stability of membrane proteins that are challenging to work with. © 2017 The Author(s).

Tracking single membrane targets of human autoantibodies using single nanoparticle imaging.

PubMed

Jézéquel, Julie; Dupuis, Julien P; Maingret, François; Groc, Laurent

2018-04-21

Over the past decade, an increasing number of neurological and neuropsychiatric diseases have been associated with the expression of autoantibodies directed against neuronal targets, including neurotransmitter receptors. Although cell-based assays are routinely used in clinics to detect the presence of immunoglobulins, such tests often provide heterogeneous outcomes due to their limited sensitivity, especially at low titers. Thus, there is an urging need for new methods allowing the detection of autoantibodies in seropositive patients that cannot always be clinically distinguished from seronegative ones. Here we make a case for single nanoparticle imaging approaches as a highly sensitive antibody detection assay. Through high-affinity interactions between functionalized nanoparticles and autoantibodies that recognize extracellular domains of membrane neuronal targets, single nanoparticle imaging allows a live surface staining of transmembrane proteins and gives access to their surface dynamics. We show here that this method is well-suited to detect low titers of purified immunoglobulin G (IgG) from first-episode psychotic patients and demonstrate that these IgG target glutamatergic N-Methyl-d-Aspartate receptors (NMDAR) in live hippocampal neurons. The molecular behaviors of targeted membrane receptors were indistinguishable from those of endogenous GluN1 NMDAR subunit and were virtually independent of the IgG concentration present in the sample contrary to classical cell-based assays. Single nanoparticle imaging emerges as a real-time sensitive method to detect IgG directed against neuronal surface proteins, which could be used as an additional step to rule out ambiguous seropositivity diagnoses. Copyright © 2018 Elsevier B.V. All rights reserved.

Prostate-specific membrane antigen targeted protein contrast agents for molecular imaging of prostate cancer by MRI†

PubMed Central

Pu, Fan; Salarian, Mani; Xue, Shenghui; Qiao, Jingjuan; Feng, Jie; Tan, Shanshan; Patel, Anvi; Li, Xin; Mamouni, Kenza; Hekmatyar, Khan; Zou, Juan; Wu, Daqing

2017-01-01

Prostate-specific membrane antigen (PSMA) is one of the most specific cell surface markers for prostate cancer diagnosis and targeted treatment. However, achieving molecular imaging using non-invasive MRI with high resolution has yet to be achieved due to the lack of contrast agents with significantly improved relaxivity for sensitivity, targeting capabilities and metal selectivity. We have previously reported our creation of a novel class of protein Gd3+ contrast agents, ProCA32, which displayed significantly improved relaxivity while exhibiting strong Gd3+ binding selectivity over physiological metal ions. In this study, we report our effort in further developing biomarker-targeted protein MRI contrast agents for molecular imaging of PSMA. Among three PSMA targeted contrast agents engineered with addition of different molecular recognition sequences, ProCA32.PSMA exhibits a binding affinity of 1.1 ± 0.1 μM for PSMA while the metal binding affinity is maintained at 0.9 ± 0.1 × 10−22 M. In addition, ProCA32.PSMA exhibits r1 of 27.6 mM−1 s−1 and r2 of 37.9 mM−1 s−1 per Gd (55.2 and 75.8 mM−1 s−1 per molecule r1 and r2, respectively) at 1.4 T. At 7 T, ProCA32.PSMA also has r2 of 94.0 mM−1 s−1 per Gd (188.0 mM−1 s−1 per molecule) and r1 of 18.6 mM−1 s−1 per Gd (37.2 mM−1 s−1 per molecule). This contrast capability enables the first MRI enhancement dependent on PSMA expression levels in tumor bearing mice using both T1 and T2-weighted MRI at 7 T. Further development of these PSMA-targeted contrast agents are expected to be used for the precision imaging of prostate cancer at an early stage and to monitor disease progression and staging, as well as determine the effect of therapeutic treatment by non-invasive evaluation of the PSMA level using MRI. PMID:26961235

[Interaction of FABP4 with plasma membrane proteins of endothelial cells].

PubMed

Saavedra, Paula; Girona, Josefa; Aragonès, Gemma; Cabré, Anna; Guaita, Sandra; Heras, Mercedes; Masana, Lluís

2015-01-01

Fatty acid binding protein (FABP4) is an adipose tissue-secreted adipokine implicated in the regulation of the energetic metabolism and inflammation. High levels of circulating FABP4 have been described in people with obesity, atherogenic dyslipidemia, diabetes and metabolic syndrome. Recent studies have demonstrated that FABP4 could have a direct effect on peripheral tissues and, specifically, on vascular function. It is still unknown how the interaction between FABP4 and the endothelial cells is produced to prompt these effects on vascular function. The objective of this work is studying the interaction between FABP4 and the plasma membrane proteins of endothelial cells. HUVEC cells were incubated with and without FABP4 (100 ng/ml) for 5 minutes. Immunolocalization of FABP4 was studied by confocal microscopy. The results showed that FABP4 colocalizates with CD31, a membrane protein marker. A strategy which combines 6XHistidine-tag FABP4 (FABP4-His), incubations with or without FABP4-His (100 ng/ml), formaldehyde cross-linking, cellular membrane protein extraction and western blot, was designed to study the FABP4 interactions with membrane proteins of HUVECs. The results showed different western blot profiles depending of the incubation with or without FABP4-His. The immunoblot revelead three covalent protein complexes of about 108, 77 and 33 kDa containing FAPB4 and its putative receptor. The existence of a specific binding protein complex able to bind FABP4 to endothelial cells is supported by these results. The obtained results will permit us advance in the molecular knowledge of FABP4 effects as well as use this protein and its receptor as therapeutic target to prevent cardiovascular. Copyright © 2014 Sociedad Española de Arteriosclerosis. Published by Elsevier España. All rights reserved.

Thermostabilisation of membrane proteins for structural studies

PubMed Central

Magnani, Francesca; Serrano-Vega, Maria J.; Shibata, Yoko; Abdul-Hussein, Saba; Lebon, Guillaume; Miller-Gallacher, Jennifer; Singhal, Ankita; Strege, Annette; Thomas, Jennifer A.; Tate, Christopher G.

2017-01-01

The thermostability of an integral membrane protein in detergent solution is a key parameter that dictates the likelihood of obtaining well-diffracting crystals suitable for structure determination. However, many mammalian membrane proteins are too unstable for crystallisation. We developed a thermostabilisation strategy based on systematic mutagenesis coupled to a radioligand-binding thermostability assay that can be applied to receptors, ion channels and transporters. It takes approximately 6-12 months to thermostabilise a G protein-coupled receptor (GPCR) containing 300 amino acid residues. The resulting thermostabilised membrane proteins are more easily crystallised and result in high-quality structures. This methodology has facilitated structure-based drug design applied to GPCRs, because it is possible to determine multiple structures of the thermostabilised receptors bound to low affinity ligands. Protocols and advice are given on how to develop thermostability assays for membrane proteins and how to combine mutations to make an optimally stable mutant suitable for structural studies. PMID:27466713

Detection of proteins on blot transfer membranes.

PubMed

Sasse, Joachim; Gallagher, Sean R

2003-11-01

In the basic and alternate protocols of this unit, proteins are stained after electroblotting from polyacrylamide gels to blot transfer membranes. If the samples of interest are electrophoresed in duplicate and transferred to a blot transfer membrane, half of the membrane can be stained to determine the efficiency of transfer to the membrane and the other half can be used for immunoblotting (i.e., western blotting). Detection limits of each staining method are given along with a list of compatible blot transfer membranes and gels. A support protocol describes a method for alkali treatment that enhances subsequent staining of bound proteins.

Mechanism of Membrane Curvature Sensing by Amphipathic Helix Containing Proteins

PubMed Central

Cui, Haosheng; Lyman, Edward; Voth, Gregory A.

2011-01-01

There are several examples of membrane-associated protein domains that target curved membranes. This behavior is believed to have functional significance in a number of essential pathways, such as clathrin-mediated endocytosis, which involve dramatic membrane remodeling and require the recruitment of various cofactors at different stages of the process. This work is motivated in part by recent experiments that demonstrated that the amphipathic N-terminal helix of endophilin (H0) targets curved membranes by binding to hydrophobic lipid bilayer packing defects which increase in number with increasing membrane curvature. Here we use state-of-the-art atomistic simulation to explore the packing defect structure of curved membranes, and the effect of this structure on the folding of H0. We find that not only are packing defects increased in number with increasing membrane curvature, but also that their size distribution depends nontrivially on the curvature, falling off exponentially with a decay constant that depends on the curvature, and crucially that even on highly curved membranes defects large enough to accommodate the hydrophobic face of H0 are never observed. We furthermore find that a percolation model for the defects explains the defect size distribution, which implies that larger defects are formed by coalescence of noninteracting smaller defects. We also use the recently developed metadynamics algorithm to study in detail the effect of such defects on H0 folding. It is found that the comparatively larger defects found on a convex membrane promote H0 folding by several kcal/mol, while the smaller defects found on flat and concave membrane surfaces inhibit folding by kinetically trapping the peptide. Together, these observations suggest H0 folding is a cooperative process in which the folding peptide changes the defect structure relative to an unperturbed membrane. PMID:21354400

Analysis of Membrane Protein Topology in the Plant Secretory Pathway.

PubMed

Guo, Jinya; Miao, Yansong; Cai, Yi

2017-01-01

Topology of membrane proteins provides important information for the understanding of protein function and intermolecular associations. Integrate membrane proteins are generally transported from endoplasmic reticulum (ER) to Golgi and downstream compartments in the plant secretory pathway. Here, we describe a simple method to study membrane protein topology along the plant secretory pathway by transiently coexpressing a fluorescent protein (XFP)-tagged membrane protein and an ER export inhibitor protein, ARF1 (T31N), in tobacco BY-2 protoplast. By fractionation, microsome isolation, and trypsin digestion, membrane protein topology could be easily detected by either direct confocal microscopy imaging or western-blot analysis using specific XFP antibodies. A similar strategy in determining membrane protein topology could be widely adopted and applied to protein analysis in a broad range of eukaryotic systems, including yeast cells and mammalian cells.

Early and late HIV-1 membrane fusion events are impaired by sphinganine lipidated peptides that target the fusion site.

PubMed

Klug, Yoel A; Ashkenazi, Avraham; Viard, Mathias; Porat, Ziv; Blumenthal, Robert; Shai, Yechiel

2014-07-15

Lipid-conjugated peptides have advanced the understanding of membrane protein functions and the roles of lipids in the membrane milieu. These lipopeptides modulate various biological systems such as viral fusion. A single function has been suggested for the lipid, binding to the membrane and thus elevating the local concentration of the peptide at the target site. In the present paper, we challenged this argument by exploring in-depth the antiviral mechanism of lipopeptides, which comprise sphinganine, the lipid backbone of DHSM (dihydrosphingomyelin), and an HIV-1 envelope-derived peptide. Surprisingly, we discovered a partnership between the lipid and the peptide that impaired early membrane fusion events by reducing CD4 receptor lateral diffusion and HIV-1 fusion peptide-mediated lipid mixing. Moreover, only the joint function of sphinganine and its conjugate peptide disrupted HIV-1 fusion protein assembly and folding at the later fusion steps. Via imaging techniques we revealed for the first time the direct localization of these lipopeptides to the virus-cell and cell-cell contact sites. Overall, the findings of the present study may suggest lipid-protein interactions in various biological systems and may help uncover a role for elevated DHSM in HIV-1 and its target cell membranes.

Curvature Forces in Membrane Lipid-Protein Interactions

PubMed Central

Brown, Michael F.

2012-01-01

Membrane biochemists are becoming increasingly aware of the role of lipid-protein interactions in diverse cellular functions. This review describes how conformational changes of membrane proteins—involving folding, stability, and membrane shape transitions—potentially involve elastic remodeling of the lipid bilayer. Evidence suggests that membrane lipids affect proteins through interactions of a relatively long-range nature, extending beyond a single annulus of next-neighbor boundary lipids. It is assumed the distance scale of the forces is large compared to the molecular range of action. Application of the theory of elasticity to flexible soft surfaces derives from classical physics, and explains the polymorphism of both detergents and membrane phospholipids. A flexible surface model (FSM) describes the balance of curvature and hydrophobic forces in lipid-protein interactions. Chemically nonspecific properties of the lipid bilayer modulate the conformational energetics of membrane proteins. The new biomembrane model challenges the standard model (the fluid mosaic model) found in biochemistry texts. The idea of a curvature force field based on data first introduced for rhodopsin gives a bridge between theory and experiment. Influences of bilayer thickness, nonlamellar-forming lipids, detergents, and osmotic stress are all explained by the FSM. An increased awareness of curvature forces suggests that research will accelerate as structural biology becomes more closely entwined with the physical chemistry of lipids in explaining membrane structure and function. PMID:23163284

Membrane Protein Mobility and Orientation Preserved in Supported Bilayers Created Directly from Cell Plasma Membrane Blebs.

PubMed

Richards, Mark J; Hsia, Chih-Yun; Singh, Rohit R; Haider, Huma; Kumpf, Julia; Kawate, Toshimitsu; Daniel, Susan

2016-03-29

Membrane protein interactions with lipids are crucial for their native biological behavior, yet traditional characterization methods are often carried out on purified protein in the absence of lipids. We present a simple method to transfer membrane proteins expressed in mammalian cells to an assay-friendly, cushioned, supported lipid bilayer platform using cell blebs as an intermediate. Cell blebs, expressing either GPI-linked yellow fluorescent proteins or neon-green fused transmembrane P2X2 receptors, were induced to rupture on glass surfaces using PEGylated lipid vesicles, which resulted in planar supported membranes with over 50% mobility for multipass transmembrane proteins and over 90% for GPI-linked proteins. Fluorescent proteins were tracked, and their diffusion in supported bilayers characterized, using single molecule tracking and moment scaling spectrum (MSS) analysis. Diffusion was characterized for individual proteins as either free or confined, revealing details of the local lipid membrane heterogeneity surrounding the protein. A particularly useful result of our bilayer formation process is the protein orientation in the supported planar bilayer. For both the GPI-linked and transmembrane proteins used here, an enzymatic assay revealed that protein orientation in the planar bilayer results in the extracellular domains facing toward the bulk, and that the dominant mode of bleb rupture is via the "parachute" mechanism. Mobility, orientation, and preservation of the native lipid environment of the proteins using cell blebs offers advantages over proteoliposome reconstitution or disrupted cell membrane preparations, which necessarily result in significant scrambling of protein orientation and typically immobilized membrane proteins in SLBs. The bleb-based bilayer platform presented here is an important step toward integrating membrane proteomic studies on chip, especially for future studies aimed at understanding fundamental effects of lipid interactions

Receptor-Targeted Nipah Virus Glycoproteins Improve Cell-Type Selective Gene Delivery and Reveal a Preference for Membrane-Proximal Cell Attachment.

PubMed

Bender, Ruben R; Muth, Anke; Schneider, Irene C; Friedel, Thorsten; Hartmann, Jessica; Plückthun, Andreas; Maisner, Andrea; Buchholz, Christian J

2016-06-01

Receptor-targeted lentiviral vectors (LVs) can be an effective tool for selective transfer of genes into distinct cell types of choice. Moreover, they can be used to determine the molecular properties that cell surface proteins must fulfill to act as receptors for viral glycoproteins. Here we show that LVs pseudotyped with receptor-targeted Nipah virus (NiV) glycoproteins effectively enter into cells when they use cell surface proteins as receptors that bring them closely enough to the cell membrane (less than 100 Å distance). Then, they were flexible in receptor usage as demonstrated by successful targeting of EpCAM, CD20, and CD8, and as selective as LVs pseudotyped with receptor-targeted measles virus (MV) glycoproteins, the current standard for cell-type specific gene delivery. Remarkably, NiV-LVs could be produced at up to two orders of magnitude higher titers compared to their MV-based counterparts and were at least 10,000-fold less effectively neutralized than MV glycoprotein pseudotyped LVs by pooled human intravenous immunoglobulin. An important finding for NiV-LVs targeted to Her2/neu was an about 100-fold higher gene transfer activity when particles were targeted to membrane-proximal regions as compared to particles binding to a more membrane-distal epitope. Likewise, the low gene transfer activity mediated by NiV-LV particles bound to the membrane distal domains of CD117 or the glutamate receptor subunit 4 (GluA4) was substantially enhanced by reducing receptor size to below 100 Å. Overall, the data suggest that the NiV glycoproteins are optimally suited for cell-type specific gene delivery with LVs and, in addition, for the first time define which parts of a cell surface protein should be targeted to achieve optimal gene transfer rates with receptor-targeted LVs.

Novel Mechanisms in the Regulation of G Protein-coupled Receptor Trafficking to the Plasma Membrane*

PubMed Central

Tholanikunnel, Baby G.; Joseph, Kusumam; Kandasamy, Karthikeyan; Baldys, Aleksander; Raymond, John R.; Luttrell, Louis M.; McDermott, Paul J.; Fernandes, Daniel J.

2010-01-01

β2-Adrenergic receptors (β2-AR) are low abundance, integral membrane proteins that mediate the effects of catecholamines at the cell surface. Whereas the processes governing desensitization of activated β2-ARs and their subsequent removal from the cell surface have been characterized in considerable detail, little is known about the mechanisms controlling trafficking of neo-synthesized receptors to the cell surface. Since the discovery of the signal peptide, the targeting of the integral membrane proteins to plasma membrane has been thought to be determined by structural features of the amino acid sequence alone. Here we report that localization of translationally silenced β2-AR mRNA to the peripheral cytoplasmic regions is critical for receptor localization to the plasma membrane. β2-AR mRNA is recognized by the nucleocytoplasmic shuttling RNA-binding protein HuR, which silences translational initiation while chaperoning the mRNA-protein complex to the cell periphery. When HuR expression is down-regulated, β2-AR mRNA translation is initiated prematurely in perinuclear polyribosomes, leading to overproduction of receptors but defective trafficking to the plasma membrane. Our results underscore the importance of the spatiotemporal relationship between β2-AR mRNA localization, translation, and trafficking to the plasma membrane, and establish a novel mechanism whereby G protein-coupled receptor (GPCR) responsiveness is regulated by RNA-based signals. PMID:20739277

Choroid plexus epithelial cells express the adhesion protein P-cadherin at cell-cell contacts and syntaxin-4 in the luminal membrane domain.

PubMed

Christensen, Inga Baasch; Mogensen, Esben Nees; Damkier, Helle Hasager; Praetorius, Jeppe

2018-05-01

The choroid plexus epithelial cells (CPECs) belong to a small group of polarized cells, where the Na + -K + -ATPase is expressed in the luminal membrane. The basic polarity of the cells is, therefore, still debated. We investigated the subcellular distribution of an array of proteins known to play fundamental roles either in establishing and maintaining basic cell polarity or in the polarized delivery and recycling of plasma membrane proteins. Immunofluorescence histochemical analysis was applied to determine the subcellular localization of apical and basolateral membrane determinants. Mass spectrometry analysis of CPECs isolated by fluorescence-activated cell sorting was applied to determine the expression of specific forms of the proteins. CPECs mainly express the cell-adhesive P-cadherin, which is localized to the lateral membranes. Proteins belonging to the Crumbs and partitioning defective (Par) protein complexes were all localized to the luminal membrane domain. Par-1 and the Scribble complex were localized to the basolateral membrane domain. Lethal(2) giant larvae homolog 2 (Lgl2) labeling was preferentially observed in the luminal membrane domain. Phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) was immunolocalized to the basolateral membrane domain, while phosphatidylinositol 4,5-bisphosphate (PIP 2 ) staining was most prominent in the luminal membrane domain along with the PIP 3 phosphatase, Pten. The apical target-SNARE syntaxin-3 and the basolateral target-SNARE syntaxin-4 were both localized to the apical membrane domain in CPECs, which lack cellular expression of the clathrin adaptor protein AP-1B for basolateral protein recycling. In conclusion, the CPECs are conventionally polarized, but express P-cadherin at cell-cell contacts, and Lgl2 and syntaxin-4 in the luminal plasma membrane domain.

Cluster Formation of Anchored Proteins Induced by Membrane-Mediated Interaction

PubMed Central

Li, Shuangyang; Zhang, Xianren; Wang, Wenchuan

2010-01-01

Abstract Computer simulations were used to study the cluster formation of anchored proteins in a membrane. The rate and extent of clustering was found to be dependent upon the hydrophobic length of the anchored proteins embedded in the membrane. The cluster formation mechanism of anchored proteins in our work was ascribed to the different local perturbations on the upper and lower monolayers of the membrane and the intermonolayer coupling. Simulation results demonstrated that only when the penetration depth of anchored proteins was larger than half the membrane thickness, could the structure of the lower monolayer be significantly deformed. Additionally, studies on the local structures of membranes indicated weak perturbation of bilayer thickness for a shallowly inserted protein, while there was significant perturbation for a more deeply inserted protein. The origin of membrane-mediated protein-protein interaction is therefore due to the local perturbation of the membrane thickness, and the entropy loss—both of which are caused by the conformation restriction on the lipid chains and the enhanced intermonolayer coupling for a deeply inserted protein. Finally, in this study we addressed the difference of cluster formation mechanisms between anchored proteins and transmembrane proteins. PMID:20513399

Phytochemicals perturb membranes and promiscuously alter protein function.

PubMed

Ingólfsson, Helgi I; Thakur, Pratima; Herold, Karl F; Hobart, E Ashley; Ramsey, Nicole B; Periole, Xavier; de Jong, Djurre H; Zwama, Martijn; Yilmaz, Duygu; Hall, Katherine; Maretzky, Thorsten; Hemmings, Hugh C; Blobel, Carl; Marrink, Siewert J; Koçer, Armağan; Sack, Jon T; Andersen, Olaf S

2014-08-15

A wide variety of phytochemicals are consumed for their perceived health benefits. Many of these phytochemicals have been found to alter numerous cell functions, but the mechanisms underlying their biological activity tend to be poorly understood. Phenolic phytochemicals are particularly promiscuous modifiers of membrane protein function, suggesting that some of their actions may be due to a common, membrane bilayer-mediated mechanism. To test whether bilayer perturbation may underlie this diversity of actions, we examined five bioactive phenols reported to have medicinal value: capsaicin from chili peppers, curcumin from turmeric, EGCG from green tea, genistein from soybeans, and resveratrol from grapes. We find that each of these widely consumed phytochemicals alters lipid bilayer properties and the function of diverse membrane proteins. Molecular dynamics simulations show that these phytochemicals modify bilayer properties by localizing to the bilayer/solution interface. Bilayer-modifying propensity was verified using a gramicidin-based assay, and indiscriminate modulation of membrane protein function was demonstrated using four proteins: membrane-anchored metalloproteases, mechanosensitive ion channels, and voltage-dependent potassium and sodium channels. Each protein exhibited similar responses to multiple phytochemicals, consistent with a common, bilayer-mediated mechanism. Our results suggest that many effects of amphiphilic phytochemicals are due to cell membrane perturbations, rather than specific protein binding.

Phytochemicals Perturb Membranes and Promiscuously Alter Protein Function

PubMed Central

2015-01-01

A wide variety of phytochemicals are consumed for their perceived health benefits. Many of these phytochemicals have been found to alter numerous cell functions, but the mechanisms underlying their biological activity tend to be poorly understood. Phenolic phytochemicals are particularly promiscuous modifiers of membrane protein function, suggesting that some of their actions may be due to a common, membrane bilayer-mediated mechanism. To test whether bilayer perturbation may underlie this diversity of actions, we examined five bioactive phenols reported to have medicinal value: capsaicin from chili peppers, curcumin from turmeric, EGCG from green tea, genistein from soybeans, and resveratrol from grapes. We find that each of these widely consumed phytochemicals alters lipid bilayer properties and the function of diverse membrane proteins. Molecular dynamics simulations show that these phytochemicals modify bilayer properties by localizing to the bilayer/solution interface. Bilayer-modifying propensity was verified using a gramicidin-based assay, and indiscriminate modulation of membrane protein function was demonstrated using four proteins: membrane-anchored metalloproteases, mechanosensitive ion channels, and voltage-dependent potassium and sodium channels. Each protein exhibited similar responses to multiple phytochemicals, consistent with a common, bilayer-mediated mechanism. Our results suggest that many effects of amphiphilic phytochemicals are due to cell membrane perturbations, rather than specific protein binding. PMID:24901212

Identification of membrane-associated proteins with pathogenic potential expressed by Corynebacterium pseudotuberculosis grown in animal serum.

PubMed

Raynal, José Tadeu; Bastos, Bruno Lopes; Vilas-Boas, Priscilla Carolinne Bagano; Sousa, Thiago de Jesus; Costa-Silva, Marcos; de Sá, Maria da Conceição Aquino; Portela, Ricardo Wagner; Moura-Costa, Lília Ferreira; Azevedo, Vasco; Meyer, Roberto

2018-01-25

Previous works defining antigens that might be used as vaccine targets against Corynebacterium pseudotuberculosis, which is the causative agent of sheep and goat caseous lymphadenitis, have focused on secreted proteins produced in a chemically defined culture media. Considering that such antigens might not reflect the repertoire of proteins expressed during infection conditions, this experiment aimed to investigate the membrane-associated proteins with pathogenic potential expressed by C. pseudotuberculosis grown directly in animal serum. Its membrane-associated proteins have been extracted using an organic solvent enrichment methodology, followed by LC-MS/MS and bioinformatics analysis for protein identification and classification. The results revealed 22 membrane-associated proteins characterized as potentially pathogenic. An interaction network analysis indicated that the four potentially pathogenic proteins ciuA, fagA, OppA4 and OppCD were biologically connected within two distinct network pathways, which were both associated with the ABC Transporters KEGG pathway. These results suggest that C. pseudotuberculosis pathogenesis might be associated with the transport and uptake of nutrients; other seven identified potentially pathogenic membrane proteins also suggest that pathogenesis might involve events of bacterial resistance and adhesion. The proteins herein reported potentially reflect part of the protein repertoire expressed during real infection conditions and might be tested as vaccine antigens.

Hijacked then lost in translation: the plight of the recombinant host cell in membrane protein structural biology projects.

PubMed

Bill, Roslyn M; von der Haar, Tobias

2015-06-01

Membrane protein structural biology is critically dependent upon the supply of high-quality protein. Over the last few years, the value of crystallising biochemically characterised, recombinant targets that incorporate stabilising mutations has been established. Nonetheless, obtaining sufficient yields of many recombinant membrane proteins is still a major challenge. Solutions are now emerging based on an improved understanding of recombinant host cells; as a 'cell factory' each cell is tasked with managing limited resources to simultaneously balance its own growth demands with those imposed by an expression plasmid. This review examines emerging insights into the role of translation and protein folding in defining high-yielding recombinant membrane protein production in a range of host cells. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

How curvature-generating proteins build scaffolds on membrane nanotubes

PubMed Central

Evergren, Emma; Golushko, Ivan; Prévost, Coline; Renard, Henri-François; Johannes, Ludger; McMahon, Harvey T.; Lorman, Vladimir; Voth, Gregory A.; Bassereau, Patricia

2016-01-01

Bin/Amphiphysin/Rvs (BAR) domain proteins control the curvature of lipid membranes in endocytosis, trafficking, cell motility, the formation of complex subcellular structures, and many other cellular phenomena. They form 3D assemblies that act as molecular scaffolds to reshape the membrane and alter its mechanical properties. It is unknown, however, how a protein scaffold forms and how BAR domains interact in these assemblies at protein densities relevant for a cell. In this work, we use various experimental, theoretical, and simulation approaches to explore how BAR proteins organize to form a scaffold on a membrane nanotube. By combining quantitative microscopy with analytical modeling, we demonstrate that a highly curving BAR protein endophilin nucleates its scaffolds at the ends of a membrane tube, contrary to a weaker curving protein centaurin, which binds evenly along the tube’s length. Our work implies that the nature of local protein–membrane interactions can affect the specific localization of proteins on membrane-remodeling sites. Furthermore, we show that amphipathic helices are dispensable in forming protein scaffolds. Finally, we explore a possible molecular structure of a BAR-domain scaffold using coarse-grained molecular dynamics simulations. Together with fluorescence microscopy, the simulations show that proteins need only to cover 30–40% of a tube’s surface to form a rigid assembly. Our work provides mechanical and structural insights into the way BAR proteins may sculpt the membrane as a high-order cooperative assembly in important biological processes. PMID:27655892

Pattern formation by curvature-inducing proteins on spherical membranes

NASA Astrophysics Data System (ADS)

Agudo-Canalejo, Jaime; Golestanian, Ramin

2017-12-01

Spatial organisation is a hallmark of all living cells, and recreating it in model systems is a necessary step in the creation of synthetic cells. It is therefore of both fundamental and practical interest to better understand the basic mechanisms underlying spatial organisation in cells. In this work, we use a continuum model of membrane and protein dynamics to study the behaviour of curvature-inducing proteins on membranes of spherical shape, such as living cells or lipid vesicles. We show that the interplay between curvature energy, entropic forces, and the geometric constraints on the membrane can result in the formation of patterns of highly-curved/protein-rich and weakly-curved/protein-poor domains on the membrane. The spontaneous formation of such patterns can be triggered either by an increase in the average density of curvature-inducing proteins, or by a relaxation of the geometric constraints on the membrane imposed by the membrane tension or by the tethering of the membrane to a rigid cell wall or cortex. These parameters can also be tuned to select the size and number of the protein-rich domains that arise upon pattern formation. The very general mechanism presented here could be related to protein self-organisation in many biological processes, ranging from (proto)cell division to the formation of membrane rafts.

Plasma membrane proteome analysis identifies a role of barley membrane steroid binding protein in root architecture response to salinity.

PubMed

Witzel, Katja; Matros, Andrea; Møller, Anders L B; Ramireddy, Eswarayya; Finnie, Christine; Peukert, Manuela; Rutten, Twan; Herzog, Andreas; Kunze, Gotthard; Melzer, Michael; Kaspar-Schoenefeld, Stephanie; Schmülling, Thomas; Svensson, Birte; Mock, Hans-Peter

2018-06-01

Although the physiological consequences of plant growth under saline conditions have been well described, understanding the core mechanisms conferring plant salt adaptation has only started. We target the root plasma membrane proteomes of two barley varieties, cvs. Steptoe and Morex, with contrasting salinity tolerance. In total, 588 plasma membrane proteins were identified by mass spectrometry, of which 182 were either cultivar or salinity stress responsive. Three candidate proteins with increased abundance in the tolerant cv. Morex were involved either in sterol binding (a GTPase-activating protein for the adenosine diphosphate ribosylation factor [ZIGA2], and a membrane steroid binding protein [MSBP]) or in phospholipid synthesis (phosphoethanolamine methyltransferase [PEAMT]). Overexpression of barley MSBP conferred salinity tolerance to yeast cells, whereas the knock-out of the heterologous AtMSBP1 increased salt sensitivity in Arabidopsis. Atmsbp1 plants showed a reduced number of lateral roots under salinity, and root-tip-specific expression of barley MSBP in Atmsbp1 complemented this phenotype. In barley, an increased abundance of MSBP correlates with reduced root length and lateral root formation as well as increased levels of auxin under salinity being stronger in the tolerant cv. Morex. Hence, we concluded the involvement of MSBP in phytohormone-directed adaptation of root architecture in response to salinity. © 2018 John Wiley & Sons Ltd.

Dynamic, electronically switchable surfaces for membrane protein microarrays.

PubMed

Tang, C S; Dusseiller, M; Makohliso, S; Heuschkel, M; Sharma, S; Keller, B; Vörös, J

2006-02-01

Microarray technology is a powerful tool that provides a high throughput of bioanalytical information within a single experiment. These miniaturized and parallelized binding assays are highly sensitive and have found widespread popularity especially during the genomic era. However, as drug diagnostics studies are often targeted at membrane proteins, the current arraying technologies are ill-equipped to handle the fragile nature of the protein molecules. In addition, to understand the complex structure and functions of proteins, different strategies to immobilize the probe molecules selectively onto a platform for protein microarray are required. We propose a novel approach to create a (membrane) protein microarray by using an indium tin oxide (ITO) microelectrode array with an electronic multiplexing capability. A polycationic, protein- and vesicle-resistant copolymer, poly(l-lysine)-grafted-poly(ethylene glycol) (PLL-g-PEG), is exposed to and adsorbed uniformly onto the microelectrode array, as a passivating adlayer. An electronic stimulation is then applied onto the individual ITO microelectrodes resulting in the localized release of the polymer thus revealing a bare ITO surface. Different polymer and biological moieties are specifically immobilized onto the activated ITO microelectrodes while the other regions remain protein-resistant as they are unaffected by the induced electrical potential. The desorption process of the PLL-g-PEG is observed to be highly selective, rapid, and reversible without compromising on the integrity and performance of the conductive ITO microelectrodes. As such, we have successfully created a stable and heterogeneous microarray of biomolecules by using selective electronic addressing on ITO microelectrodes. Both pharmaceutical diagnostics and biomedical technology are expected to benefit directly from this unique method.

Protein Stains to Detect Antigen on Membranes.

PubMed

Dsouza, Anil; Scofield, R Hal

2015-01-01

Western blotting (protein blotting/electroblotting) is the gold standard in the analysis of complex protein mixtures. Electroblotting drives protein molecules from a polyacrylamide (or less commonly, of an agarose) gel to the surface of a binding membrane, thereby facilitating an increased availability of the sites with affinity for both general and specific protein reagents. The analysis of these complex protein mixtures is achieved by the detection of specific protein bands on a membrane, which in turn is made possible by the visualization of protein bands either by chemical staining or by reaction with an antibody of a conjugated ligand. Chemical methods employ staining with organic dyes, metal chelates, autoradiography, fluorescent dyes, complexing with silver, or prelabeling with fluorophores. All of these methods have differing sensitivities and quantitative determinations vary significantly. This review will describe the various protein staining methods applied to membranes after western blotting. "Detection" precedes and is a prerequisite to obtaining qualitative and quantitative data on the proteins in a sample, as much as to comparing the protein composition of different samples. "Detection" is often synonymous to staining, i.e., the reversible or irreversible binding by the proteins of a colored organic or inorganic chemical.

Protein stains to detect antigen on membranes.

PubMed

D'souza, Anil; Scofield, R Hal

2009-01-01

Western blotting (protein blotting/electroblotting) is the gold standard in the analysis of complex protein mixtures. Electroblotting drives protein molecules from a polyacrylamide (or less commonly, of an agarose) gel to the surface of a binding membrane, thereby facilitating an increased availability of the sites with affinity for both general and specific protein reagents. The analysis of these complex protein mixtures is achieved by the detection of specific protein bands on a membrane, which in turn is made possible by the visualization of protein bands either by chemical staining or by reaction with an antibody of a conjugated ligand. Chemical methods employ staining with organic dyes, metal chelates, autoradiography, fluorescent dyes, complexing with silver, or prelabeling with fluorophores. All of these methods have differing sensitivities and quantitative determinations vary significantly. This review will describe the various protein staining methods applied to membranes after electrophoresis. "Detection" precedes and is a prerequisite to obtaining qualitative and quantitative data on the proteins in a sample, as much as to comparing the protein composition of different samples. Detection is often synonymous to staining, i.e., the reversible or irreversible binding by the proteins of a colored organic or inorganic chemical.

Maltose neopentyl glycol-3 (MNG-3) analogues for membrane protein study.

PubMed

Cho, Kyung Ho; Husri, Mohd; Amin, Anowarul; Gotfryd, Kamil; Lee, Ho Jin; Go, Juyeon; Kim, Jin Woong; Loland, Claus J; Guan, Lan; Byrne, Bernadette; Chae, Pil Seok

2015-05-07

Detergents are typically used to both extract membrane proteins (MPs) from the lipid bilayers and maintain them in solution. However, MPs encapsulated in detergent micelles are often prone to denaturation and aggregation. Thus, the development of novel agents with enhanced stabilization characteristics is necessary to advance MP research. Maltose neopentyl glycol-3 (MNG-3) has contributed to >10 crystal structures including G-protein coupled receptors. Here, we prepared MNG-3 analogues and characterised their properties using selected MPs. Most MNGs were superior to a conventional detergent, n-dodecyl-β-D-maltopyranoside (DDM), in terms of membrane protein stabilization efficacy. Interestingly, optimal stabilization was achieved with different MNG-3 analogues depending on the target MP. The origin for such detergent specificity could be explained by a novel concept: compatibility between detergent hydrophobicity and MP tendency to denature and aggregate. This set of MNGs represents viable alternatives to currently available detergents for handling MPs, and can be also used as tools to estimate MP sensitivity to denaturation and aggregation.

Role of membrane contact sites in protein import into mitochondria

PubMed Central

Horvath, Susanne E; Rampelt, Heike; Oeljeklaus, Silke; Warscheid, Bettina; van der Laan, Martin; Pfanner, Nikolaus

2015-01-01

Mitochondria import more than 1,000 different proteins from the cytosol. The proteins are synthesized as precursors on cytosolic ribosomes and are translocated by protein transport machineries of the mitochondrial membranes. Five main pathways for protein import into mitochondria have been identified. Most pathways use the translocase of the outer mitochondrial membrane (TOM) as the entry gate into mitochondria. Depending on specific signals contained in the precursors, the proteins are subsequently transferred to different intramitochondrial translocases. In this article, we discuss the connection between protein import and mitochondrial membrane architecture. Mitochondria possess two membranes. It is a long-standing question how contact sites between outer and inner membranes are formed and which role the contact sites play in the translocation of precursor proteins. A major translocation contact site is formed between the TOM complex and the presequence translocase of the inner membrane (TIM23 complex), promoting transfer of presequence-carrying preproteins to the mitochondrial inner membrane and matrix. Recent findings led to the identification of contact sites that involve the mitochondrial contact site and cristae organizing system (MICOS) of the inner membrane. MICOS plays a dual role. It is crucial for maintaining the inner membrane cristae architecture and forms contacts sites to the outer membrane that promote translocation of precursor proteins into the intermembrane space and outer membrane of mitochondria. The view is emerging that the mitochondrial protein translocases do not function as independent units, but are embedded in a network of interactions with machineries that control mitochondrial activity and architecture. PMID:25514890

Enhanced Membrane Pore Formation through High-Affinity Targeted Antimicrobial Peptides

PubMed Central

Arnusch, Christopher J.; Pieters, Roland J.; Breukink, Eefjan

2012-01-01

Many cationic antimicrobial peptides (AMPs) target the unique lipid composition of the prokaryotic cell membrane. However, the micromolar activities common for these peptides are considered weak in comparison to nisin, which follows a targeted, pore-forming mode of action. Here we show that AMPs can be modified with a high-affinity targeting module, which enables membrane permeabilization at low concentration. Magainin 2 and a truncated peptide analog were conjugated to vancomycin using click chemistry, and could be directed towards specific membrane embedded receptors both in model membrane systems and whole cells. Compared with untargeted vesicles, a gain in permeabilization efficacy of two orders of magnitude was reached with large unilamellar vesicles that included lipid II, the target of vancomycin. The truncated vancomycin-peptide conjugate showed an increased activity against vancomycin resistant Enterococci, whereas the full-length conjugate was more active against a targeted eukaryotic cell model: lipid II containing erythrocytes. This study highlights that AMPs can be made more selective and more potent against biological membranes that contain structures that can be targeted. PMID:22768121

Monitoring the function of membrane transport proteins in detergent-solubilized form

PubMed Central

Quick, Matthias; Javitch, Jonathan A.

2007-01-01

Transport proteins constitute ≈10% of most proteomes and play vital roles in the translocation of solutes across membranes of all organisms. Their (dys)function is implicated in many disorders, making them frequent targets for pharmacotherapy. The identification of substrates for members of this large protein family, still replete with many orphans of unknown function, has proven difficult, in part because high-throughput screening is greatly complicated by endogenous transporters present in many expression systems. In addition, direct structural studies require that transporters be extracted from the membrane with detergent, thereby precluding transport measurements because of the lack of a vectorial environment and necessitating reconstitution into proteoliposomes for activity measurements. Here, we describe a direct scintillation proximity-based radioligand-binding assay for determining transport protein function in crude cell extracts and in purified form. This rapid and universally applicable assay with advantages over cell-based platforms will greatly facilitate the identification of substrates for many orphan transporters and allows monitoring the function of transport proteins in a nonmembranous environment. PMID:17360689

Predictive energy landscapes for folding membrane protein assemblies

NASA Astrophysics Data System (ADS)

Truong, Ha H.; Kim, Bobby L.; Schafer, Nicholas P.; Wolynes, Peter G.

2015-12-01

We study the energy landscapes for membrane protein oligomerization using the Associative memory, Water mediated, Structure and Energy Model with an implicit membrane potential (AWSEM-membrane), a coarse-grained molecular dynamics model previously optimized under the assumption that the energy landscapes for folding α-helical membrane protein monomers are funneled once their native topology within the membrane is established. In this study we show that the AWSEM-membrane force field is able to sample near native binding interfaces of several oligomeric systems. By predicting candidate structures using simulated annealing, we further show that degeneracies in predicting structures of membrane protein monomers are generally resolved in the folding of the higher order assemblies as is the case in the assemblies of both nicotinic acetylcholine receptor and V-type Na+-ATPase dimers. The physics of the phenomenon resembles domain swapping, which is consistent with the landscape following the principle of minimal frustration. We revisit also the classic Khorana study of the reconstitution of bacteriorhodopsin from its fragments, which is the close analogue of the early Anfinsen experiment on globular proteins. Here, we show the retinal cofactor likely plays a major role in selecting the final functional assembly.

Structure refinement of membrane proteins via molecular dynamics simulations.

PubMed

Dutagaci, Bercem; Heo, Lim; Feig, Michael

2018-07-01

A refinement protocol based on physics-based techniques established for water soluble proteins is tested for membrane protein structures. Initial structures were generated by homology modeling and sampled via molecular dynamics simulations in explicit lipid bilayer and aqueous solvent systems. Snapshots from the simulations were selected based on scoring with either knowledge-based or implicit membrane-based scoring functions and averaged to obtain refined models. The protocol resulted in consistent and significant refinement of the membrane protein structures similar to the performance of refinement methods for soluble proteins. Refinement success was similar between sampling in the presence of lipid bilayers and aqueous solvent but the presence of lipid bilayers may benefit the improvement of lipid-facing residues. Scoring with knowledge-based functions (DFIRE and RWplus) was found to be as good as scoring using implicit membrane-based scoring functions suggesting that differences in internal packing is more important than orientations relative to the membrane during the refinement of membrane protein homology models. © 2018 Wiley Periodicals, Inc.

Structural adaptations of proteins to different biological membranes

PubMed Central

Pogozheva, Irina D.; Tristram-Nagle, Stephanie; Mosberg, Henry I.; Lomize, Andrei L.

2013-01-01

To gain insight into adaptations of proteins to their membranes, intrinsic hydrophobic thicknesses, distributions of different chemical groups and profiles of hydrogen-bonding capacities (α and β) and the dipolarity/polarizability parameter (π*) were calculated for lipid-facing surfaces of 460 integral α-helical, β-barrel and peripheral proteins from eight types of biomembranes. For comparison, polarity profiles were also calculated for ten artificial lipid bilayers that have been previously studied by neutron and X-ray scattering. Estimated hydrophobic thicknesses are 30-31 Å for proteins from endoplasmic reticulum, thylakoid, and various bacterial plasma membranes, but differ for proteins from outer bacterial, inner mitochondrial and eukaryotic plasma membranes (23.9, 28.6 and 33.5 Å, respectively). Protein and lipid polarity parameters abruptly change in the lipid carbonyl zone that matches the calculated hydrophobic boundaries. Maxima of positively charged protein groups correspond to the location of lipid phosphates at 20-22 Å distances from the membrane center. Locations of Tyr atoms coincide with hydrophobic boundaries, while distributions maxima of Trp rings are shifted by 3-4 Å toward the membrane center. Distributions of Trp atoms indicate the presence of two 5-8 Å-wide midpolar regions with intermediate π* values within the hydrocarbon core, whose size and symmetry depend on the lipid composition of membrane leaflets. Midpolar regions are especially asymmetric in outer bacterial membranes and cell membranes of mesophilic but not hyperthermophilic archaebacteria, indicating the larger width of the central nonpolar region in the later case. In artificial lipid bilayers, midpolar regions are observed up to the level of acyl chain double bonds. PMID:23811361

Plasma membrane domains enriched in cortical endoplasmic reticulum function as membrane protein trafficking hubs.

PubMed

Fox, Philip D; Haberkorn, Christopher J; Weigel, Aubrey V; Higgins, Jenny L; Akin, Elizabeth J; Kennedy, Matthew J; Krapf, Diego; Tamkun, Michael M

2013-09-01

In mammalian cells, the cortical endoplasmic reticulum (cER) is a network of tubules and cisterns that lie in close apposition to the plasma membrane (PM). We provide evidence that PM domains enriched in underlying cER function as trafficking hubs for insertion and removal of PM proteins in HEK 293 cells. By simultaneously visualizing cER and various transmembrane protein cargoes with total internal reflectance fluorescence microscopy, we demonstrate that the majority of exocytotic delivery events for a recycled membrane protein or for a membrane protein being delivered to the PM for the first time occur at regions enriched in cER. Likewise, we observed recurring clathrin clusters and functional endocytosis of PM proteins preferentially at the cER-enriched regions. Thus the cER network serves to organize the molecular machinery for both insertion and removal of cell surface proteins, highlighting a novel role for these unique cellular microdomains in membrane trafficking.

Plasma membrane domains enriched in cortical endoplasmic reticulum function as membrane protein trafficking hubs

PubMed Central

Fox, Philip D.; Haberkorn, Christopher J.; Weigel, Aubrey V.; Higgins, Jenny L.; Akin, Elizabeth J.; Kennedy, Matthew J.; Krapf, Diego; Tamkun, Michael M.

2013-01-01

In mammalian cells, the cortical endoplasmic reticulum (cER) is a network of tubules and cisterns that lie in close apposition to the plasma membrane (PM). We provide evidence that PM domains enriched in underlying cER function as trafficking hubs for insertion and removal of PM proteins in HEK 293 cells. By simultaneously visualizing cER and various transmembrane protein cargoes with total internal reflectance fluorescence microscopy, we demonstrate that the majority of exocytotic delivery events for a recycled membrane protein or for a membrane protein being delivered to the PM for the first time occur at regions enriched in cER. Likewise, we observed recurring clathrin clusters and functional endocytosis of PM proteins preferentially at the cER-enriched regions. Thus the cER network serves to organize the molecular machinery for both insertion and removal of cell surface proteins, highlighting a novel role for these unique cellular microdomains in membrane trafficking. PMID:23864710

Activated GTPase movement on an RNA scaffold drives cotranslational protein targeting

PubMed Central

Shen, Kuang; Arslan, Sinan; Akopian, David; Ha, Taekjip; Shan, Shu-ou

2012-01-01

Roughly one third of the proteome is initially destined for the eukaryotic endoplasmic reticulum or the bacterial plasma membrane1. The proper localization of these proteins is mediated by a universally conserved protein targeting machinery, the signal recognition particle (SRP), which recognizes ribosomes carrying signal sequences2–4 and, via interactions with the SRP receptor5,6, delivers them to the protein translocation machinery on the target membrane7. The SRP is an ancient ribonucleoprotein particle containing an essential, elongated SRP RNA whose precise functions have remained elusive. Here, we used single molecule fluorescence microscopy to demonstrate that the SRP-receptor GTPase complex, after initial assembly at the tetraloop end of SRP RNA, travels over 100 Å to the distal end of this RNA where rapid GTP hydrolysis occurs. This movement is negatively regulated by the translating ribosome and, at a later stage, positively regulated by the SecYEG translocon, providing an attractive mechanism to ensure the productive exchange of the targeting and translocation machineries at the ribosome exit site with exquisite spatial and temporal accuracy. Our results show that large RNAs can act as molecular scaffolds that enable the facile exchange of distinct factors and precise timing of molecular events in a complex cellular process; this concept may be extended to similar phenomena in other ribonucleoprotein complexes. PMID:23235881

Membrane protein properties revealed through data-rich electrostatics calculations

PubMed Central

Guerriero, Christopher J.; Brodsky, Jeffrey L.; Grabe, Michael

2015-01-01

SUMMARY The electrostatic properties of membrane proteins often reveal many of their key biophysical characteristics, such as ion channel selectivity and the stability of charged membrane-spanning segments. The Poisson-Boltzmann (PB) equation is the gold standard for calculating protein electrostatics, and the software APBSmem enables the solution of the PB equation in the presence of a membrane. Here, we describe significant advances to APBSmem including: full automation of system setup, per-residue energy decomposition, incorporation of PDB2PQR, calculation of membrane induced pKa shifts, calculation of non-polar energies, and command-line scripting for large scale calculations. We highlight these new features with calculations carried out on a number of membrane proteins, including the recently solved structure of the ion channel TRPV1 and a large survey of 1,614 membrane proteins of known structure. This survey provides a comprehensive list of residues with large electrostatic penalties for being embedded in the membrane potentially revealing interesting functional information. PMID:26118532

Protein translocation channel of mitochondrial inner membrane and matrix-exposed import motor communicate via two-domain coupling protein.

PubMed

Banerjee, Rupa; Gladkova, Christina; Mapa, Koyeli; Witte, Gregor; Mokranjac, Dejana

2015-12-29

The majority of mitochondrial proteins are targeted to mitochondria by N-terminal presequences and use the TIM23 complex for their translocation across the mitochondrial inner membrane. During import, translocation through the channel in the inner membrane is coupled to the ATP-dependent action of an Hsp70-based import motor at the matrix face. How these two processes are coordinated remained unclear. We show here that the two domain structure of Tim44 plays a central role in this process. The N-terminal domain of Tim44 interacts with the components of the import motor, whereas its C-terminal domain interacts with the translocation channel and is in contact with translocating proteins. Our data suggest that the translocation channel and the import motor of the TIM23 complex communicate through rearrangements of the two domains of Tim44 that are stimulated by translocating proteins.

Differential plasma membrane targeting of voltage-dependent calcium channel subunits expressed in a polarized epithelial cell line

PubMed Central

Brice, Nicola L; Dolphin, Annette C

1999-01-01

Voltage-dependent calcium channels (VDCCs) show a highly non-uniform distribution in many cell types, including neurons and other polarized secretory cells. We have examined whether this can be mimicked in a polarized epithelial cell line (Madin-Darby canine kidney), which has been used extensively to study the targeting of proteins. We expressed the VDCC α1A, α1B or α1C subunits either alone or in combination with accessory subunits α2-δ and the different β subunits, and examined their localization immunocytochemically. An α1 subunit was only targeted to the plasma membrane if co-expressed with the accessory subunits. The combination α1C/α2-δ and all β subunits was always localized predominantly to the basolateral membrane. It has been suggested that this is equivalent to somatodendritic targeting in neurons. In contrast, the α1B subunit was expressed at the apical membrane with all the accessory subunit combinations, by 24 h after microinjection. This membrane destination shows some parallels with axonal targeting in neurons. The α1A subunit was consistently observed at the apical membrane in the combinations α1A/α2-δ/β1b or β4. In contrast, when co-expressed with α2-δ/β2a, α1A was clearly targeted to the basolateral membrane. In conclusion, the VDCC α1 subunit appears to be the primary determinant for targeting the VDCC complex, but the β subunit can modify this destination, particularly for α1A. PMID:10066897

A discrete pathway for the transfer of intermembrane space proteins across the outer membrane of mitochondria.

PubMed

Gornicka, Agnieszka; Bragoszewski, Piotr; Chroscicki, Piotr; Wenz, Lena-Sophie; Schulz, Christian; Rehling, Peter; Chacinska, Agnieszka

2014-12-15

Mitochondrial proteins are synthesized on cytosolic ribosomes and imported into mitochondria with the help of protein translocases. For the majority of precursor proteins, the role of the translocase of the outer membrane (TOM) and mechanisms of their transport across the outer mitochondrial membrane are well recognized. However, little is known about the mode of membrane translocation for proteins that are targeted to the intermembrane space via the redox-driven mitochondrial intermembrane space import and assembly (MIA) pathway. On the basis of the results obtained from an in organello competition import assay, we hypothesized that MIA-dependent precursor proteins use an alternative pathway to cross the outer mitochondrial membrane. Here we demonstrate that this alternative pathway involves the protein channel formed by Tom40. We sought a translocation intermediate by expressing tagged versions of MIA-dependent proteins in vivo. We identified a transient interaction between our model substrates and Tom40. Of interest, outer membrane translocation did not directly involve other core components of the TOM complex, including Tom22. Thus MIA-dependent proteins take another route across the outer mitochondrial membrane that involves Tom40 in a form that is different from the canonical TOM complex. © 2014 Gornicka et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Directional control of WAVE2 membrane targeting by EB1 and phosphatidylinositol 3,4,5-triphosphate.

PubMed

Takahashi, Kazuhide; Tanaka, Tacu; Suzuki, Katsuo

2010-03-01

Membrane targeting of WAVE2 along microtubules is mediated by a motor protein kinesin and requires Pak1, a downstream effector of Rac1. However, the mechanism by which WAVE2 targeting to the leading edge is directionally controlled remains largely unknown. Here we demonstrate that EB1, a microtubule plus-end-binding protein, constitutively associates with stathmin, a microtubule-destabilizing protein, in human breast cancer cells. Stimulation of the cells with insulin-like growth factor I (IGF-I) induced Pak1-dependent binding of the EB1-stathmin complex to microtubules that bear WAVE2 and colocalization of the complex with WAVE2 at the leading edge. Depletion of EB1 by small interfering RNA (siRNA) abrogated the IGF-I-induced WAVE2 targeting and stathmin binding to microtubules. On the other hand, chemotaxis chamber assays indicated that the IGF-I receptor (IGF-IR) was locally activated in the region facing toward IGF-I. In addition, IGF-I caused phosphatidylinositol 3-kinase (PI 3-kinase)-dependent production of phosphatidylinositol 3,4,5-triphosphate (PIP3) near activated IGF-IR and WAVE2 colocalization with it. Collectively, WAVE2-membrane targeting is directionally controlled by binding of the EB1-stathmin complex to WAVE2-bearing microtubules and by the interaction between WAVE2 and PIP3 produced near IGF-IR that is locally activated by IGF-I.

Red Bell Pepper Chromoplasts Exhibit in Vitro Import Competency and Membrane Targeting of Passenger Proteins from the Thylakoidal Sec and ΔpH Pathways but Not the Chloroplast Signal Recognition Particle Pathway1

PubMed Central

Summer, Elizabeth J.; Cline, Kenneth

1999-01-01

Chloroplast to chromoplast development involves new synthesis and plastid localization of nuclear-encoded proteins, as well as changes in the organization of internal plastid membrane compartments. We have demonstrated that isolated red bell pepper (Capsicum annuum) chromoplasts contain the 75-kD component of the chloroplast outer envelope translocon (Toc75) and are capable of importing chloroplast precursors in an ATP-dependent fashion, indicating a functional general import apparatus. The isolated chromoplasts were able to further localize the 33- and 17-kD subunits of the photosystem II O2-evolution complex (OE33 and OE17, respectively), lumen-targeted precursors that utilize the thylakoidal Sec and ΔpH pathways, respectively, to the lumen of an internal membrane compartment. Chromoplasts contained the thylakoid Sec component protein, cpSecA, at levels comparable to chloroplasts. Routing of OE17 to the lumen was abolished by ionophores, suggesting that routing is dependent on a transmembrane ΔpH. The chloroplast signal recognition particle pathway precursor major photosystem II light-harvesting chlorophyll a/b protein failed to associate with chromoplast membranes and instead accumulated in the stroma following import. The Pftf (plastid fusion/translocation factor), a chromoplast protein, integrated into the internal membranes of chromoplasts during in vitro assays, and immunoblot analysis indicated that endogenous plastid fusion/translocation factor was also an integral membrane protein of chromoplasts. These data demonstrate that the internal membranes of chromoplasts are functional with respect to protein translocation on the thylakoid Sec and ΔpH pathways. PMID:9952453

[Adsorption characteristics of proteins on membrane surface and effect of protein solution environment on permeation behavior of berberine].

PubMed

Li, Yi-Qun; Xu, Li; Zhu, Hua-Xu; Tang, Zhi-Shu; Li, Bo; Pan, Yong-Lan; Yao, Wei-Wei; Fu, Ting-Ming; Guo, Li-Wei

2017-10-01

In order to explore the adsorption characteristics of proteins on the membrane surface and the effect of protein solution environment on the permeation behavior of berberine, berberine and proteins were used as the research object to prepare simulated solution. Low field NMR, static adsorption experiment and membrane separation experiment were used to study the interaction between the proteins and ceramic membrane or between the proteins and berberine. The static adsorption capacity of proteins, membrane relative flux, rejection rate of proteins, transmittance rate of berberine and the adsorption rate of proteins and berberine were used as the evaluation index. Meanwhile, the membrane resistance distribution, the particle size distribution and the scanning electron microscope (SEM) were determined to investigate the adsorption characteristics of proteins on ceramic membrane and the effect on membrane separation process of berberine. The results showed that the ceramic membrane could adsorb the proteins and the adsorption model was consistent with Langmuir adsorption model. In simulating the membrane separation process, proteins were the main factor to cause membrane fouling. However, when the concentration of proteins was 1 g•L⁻¹, the proteins had no significant effect on membrane separation process of berberine. Copyright© by the Chinese Pharmaceutical Association.

Customizing model membranes and samples for NMR spectroscopic studies of complex membrane proteins.

PubMed

Sanders, C R; Oxenoid, K

2000-11-23

Both solution and solid state nuclear magnetic resonance (NMR) techniques for structural determination are advancing rapidly such that it is possible to contemplate bringing these techniques to bear upon integral membrane proteins having multiple transmembrane segments. This review outlines existing and emerging options for model membrane media for use in such studies and surveys the special considerations which must be taken into account when preparing larger membrane proteins for NMR spectroscopic studies.

Role for ribosome-associated complex and stress-seventy subfamily B (RAC-Ssb) in integral membrane protein translation.

PubMed

Acosta-Sampson, Ligia; Döring, Kristina; Lin, Yuping; Yu, Vivian Y; Bukau, Bernd; Kramer, Günter; Cate, Jamie H D

2017-12-01

Targeting of most integral membrane proteins to the endoplasmic reticulum is controlled by the signal recognition particle, which recognizes a hydrophobic signal sequence near the protein N terminus. Proper folding of these proteins is monitored by the unfolded protein response and involves protein degradation pathways to ensure quality control. Here, we identify a new pathway for quality control of major facilitator superfamily transporters that occurs before the first transmembrane helix, the signal sequence recognized by the signal recognition particle, is made by the ribosome. Increased rates of translation elongation of the N-terminal sequence of these integral membrane proteins can divert the nascent protein chains to the ribosome-associated complex and stress-seventy subfamily B chaperones. We also show that quality control of integral membrane proteins by ribosome-associated complex-stress-seventy subfamily B couples translation rate to the unfolded protein response, which has implications for understanding mechanisms underlying human disease and protein production in biotechnology. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Organization and Dynamics of Receptor Proteins in a Plasma Membrane.

PubMed

Koldsø, Heidi; Sansom, Mark S P

2015-11-25

The interactions of membrane proteins are influenced by their lipid environment, with key lipid species able to regulate membrane protein function. Advances in high-resolution microscopy can reveal the organization and dynamics of proteins and lipids within living cells at resolutions <200 nm. Parallel advances in molecular simulations provide near-atomic-resolution models of the dynamics of the organization of membranes of in vivo-like complexity. We explore the dynamics of proteins and lipids in crowded and complex plasma membrane models, thereby closing the gap in length and complexity between computations and experiments. Our simulations provide insights into the mutual interplay between lipids and proteins in determining mesoscale (20-100 nm) fluctuations of the bilayer, and in enabling oligomerization and clustering of membrane proteins.