Modeling Membrane-Protein Interactions
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Membrane Protein Stabilization Strategies for Structural and Functional Studies
membranes Review Membrane Protein Stabilization Strategies for Structural and Functional Studies Ekaitz Errasti-Murugarren 1,2,*, Paola Bartoccioni 1,2 and Manuel Palacín 1,2,3,* 1 Laboratory of Amino acid Transporters and Disease, Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology (BIST), Baldiri Reixac 10, 08028 Barcelona, Spain; [email protected] 2 CIBERER (Centro Español en Red de Biomedicina de Enfermedades Raras), 28029 Barcelona, Spain 3 Department of Biochemistry and Molecular Biomedicine, Universitat de Barcelona, 08028 Barcelona, Spain * Correspondence: [email protected] (E.E.-M.); [email protected] (M.P.) Abstract: Accounting for nearly two-thirds of known druggable targets, membrane proteins are highly relevant for cell physiology and pharmacology. In this regard, the structural determination of pharmacologically relevant targets would facilitate the intelligent design of new drugs. The structural biology of membrane proteins is a field experiencing significant growth as a result of the development of new strategies for structure determination. However, membrane protein preparation for structural studies continues to be a limiting step in many cases due to the inherent instability of these molecules in non-native membrane environments. This review describes the approaches that have been developed to improve membrane protein stability. Membrane protein mutagenesis, detergent selection, lipid membrane mimics, antibodies, and ligands are described in this review as approaches to facilitate the production of purified and stable membrane proteins of interest for structural and functional studies. Keywords: membrane proteins; stability; mutagenesis; detergent; lipid; antibody; nanobody; ligand Citation: Errasti-Murugarren, E.; Bartoccioni, P.; Palacín, M. Membrane Protein Stabilization Strategies for Structural and Functional Studies. -
Snapshot: ER-Associated Protein Degradation Pathways Shinichi Kawaguchi and Davis T.W
SnapShot: ER-Associated Protein Degradation Pathways Shinichi Kawaguchi and Davis T.W. Ng Temasek Life Sciences Laboratory and Department of Biological Sciences, National University of Singapore, Singapore 117604 1230 Cell 129, June 15, 2007 ©2007 Elsevier Inc. DOI 10.1016/j.cell.2007.06.005 See online version for legend and references. SnapShot: ER-Associated Protein Degradation Pathways Shinichi Kawaguchi and Davis T.W. Ng Temasek Life Sciences Laboratory and Department of Biological Sciences, National University of Singapore, Singapore 117604 Arrows specify the routes of individual pathways. All pathways culminate in substrate degradation by the 26S proteasome. (Left) ERAD Pathways in Budding Yeast (A) Newly synthesized secretory and membrane proteins enter the ER through the Sec61 protein-conducting channel complex unfolded. Hsp70-related molecular chaperones (Kar2p) bind to nascent polypeptides in the ER lumen and to the cytosolic domains of membrane proteins (Hsp70, B). These factors assist in substrate folding and also assist in their disposal if they fail to fold. Mannose residues on misfolded glycoproteins are trimmed by the ER mannosidase Mns1p (E). Mannose trimming facilitates the recognition of misfolded glycoproteins by luminally oriented lectin factors Htm1p and Yos9p. (C, D, and F) At least two ER membrane-localized E3 ubiquitin ligases organize protein complexes that receive and process misfolded proteins. These complexes define three pathways that recognize lesions in the cytosolic (ERAD-C), transmembrane (ERAD-M), and luminal (ERAD-L) domains of substrates. Both ERAD-M and ERAD-L use the Hrd1 ubiquitin ligase but the luminal factor Yos9p is dispensable for ERAD-M. A Hrd1 complex lacking Yos9p has been observed suggesting dedicated complexes for all three pathways. -
Lipid Bilayer, with the Nonpolar Regions of the Lipids Facing Inward
Chapter 7 Membranes: Their Structure, Function, and Chemistry Lectures by Kathleen Fitzpatrick Simon Fraser University © 2012 Pearson Education, Inc. Membranes: Their Structure, Function, and Chemistry • Membranes define the boundaries of a cell, and its internal compartments • Membranes play multiple roles in the life of a cell © 2012 Pearson Education, Inc. Figure 7-1A © 2012 Pearson Education, Inc. Figure 7-1B © 2012 Pearson Education, Inc. The Functions of Membranes • 1. Define boundaries of a cell and organelles and act as permeability barriers • 2. Serve as sites for biological functions such as electron transport • 3. Possess transport proteins that regulate the movement of substances into and out of cells and organelles © 2012 Pearson Education, Inc. The Functions of Membranes (continued) • 4. Contain protein molecules that act as receptors to detect external signals • 5. Provide mechanisms for cell-to-cell contact, adhesion, and communication © 2012 Pearson Education, Inc. Figure 7-2 © 2012 Pearson Education, Inc. Models of Membrane Structure: An Experimental Approach • The development of electron microscopy in the 1950s was important for understanding membrane structure • The fluid mosaic model is thought to be descriptive of all biological membranes • The model envisions a membrane as two fluid layers of lipids with proteins within and on the layers © 2012 Pearson Education, Inc. Overton and Langmuir: Lipids Are Important Components of Membranes • In the 1890s Overton observed the easy penetration of lipid-soluble substances into cells and concluded that the cell surface had some kind of lipid “coat” on it • Langmuir studied phospholipids and found that they were amphipathic and reasoned that they must orient on water with the hydrophobic tails away from the water © 2012 Pearson Education, Inc. -
(4,5) Bisphosphate-Phospholipase C Resynthesis Cycle: Pitps Bridge the ER-PM GAP
View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by UCL Discovery Topological organisation of the phosphatidylinositol (4,5) bisphosphate-phospholipase C resynthesis cycle: PITPs bridge the ER-PM GAP Shamshad Cockcroft and Padinjat Raghu* Dept. of Neuroscience, Physiology and Pharmacology, Division of Biosciences, University College London, London WC1E 6JJ, UK; *National Centre for Biological Sciences, TIFR-GKVK Campus, Bellary Road, Bangalore 560065, India Address correspondence to: Shamshad Cockcroft, University College London UK; Phone: 0044-20-7679-6259; Email: [email protected] Abstract Phospholipase C (PLC) is a receptor-regulated enzyme that hydrolyses phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) at the plasma membrane (PM) triggering three biochemical consequences, the generation of soluble inositol 1,4,5-trisphosphate (IP3), membrane– associated diacylglycerol (DG) and the consumption of plasma membrane PI(4,5)P2. Each of these three signals triggers multiple molecular processes impacting key cellular properties. The activation of PLC also triggers a sequence of biochemical reactions, collectively referred to as the PI(4,5)P2 cycle that culminates in the resynthesis of this lipid. The biochemical intermediates of this cycle and the enzymes that mediate these reactions are topologically distributed across two membrane compartments, the PM and the endoplasmic reticulum (ER). At the plasma membrane, the DG formed during PLC activation is rapidly converted to phosphatidic acid (PA) that needs to be transported to the ER where the machinery for its conversion into PI is localised. Conversely, PI from the ER needs to be rapidly transferred to the plasma membrane where it can be phosphorylated by lipid kinases to regenerate PI(4,5)P2. -
Non-Canonical Regulation of Phosphatidylserine Metabolism by a Phosphatidylinositol Transfer Protein and a Phosphatidylinositol 4-OH Kinase
bioRxiv preprint doi: https://doi.org/10.1101/696336; this version posted July 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Non-Canonical Regulation of Phosphatidylserine Metabolism by a Phosphatidylinositol Transfer Protein and a Phosphatidylinositol 4-OH Kinase Yaxi Wang1,2, Peihua Yuan2, Ashutosh Tripathi2, Martin Rodriguez1, Max Lönnfors2, Michal Eisenberg-Bord3, Maya Schuldiner3, and Vytas A. Bankaitis1,2,4† 1Department of Biochemistry and Biophysics Texas A&M University College Station, Texas 77843-2128 USA 2Department of Molecular and Cellular Medicine Texas A&M Health Science Center College Station, Texas 77843-1114 USA 3Department of Molecular Genetics Weizmann Institute of Science, Rehovot 7610001, Israel 4Department of Chemistry Texas A&M University College Station, Texas 77840 USA Key Words: phosphoinositides/ PITPs/ lipid kinases/ lipi metabolism/ membrane contact site † -- Corresponding author TEL: 979-436-0757 Email: [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/696336; this version posted July 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. ABSTRACT The phosphatidylserine (PtdSer) decarboxylase Psd2 is proposed to engage in an endoplasmic reticulum (ER)-Golgi/endosome membrane contact site (MCS) that facilitates phosphatidylserine decarboxylation to phosphatidylethanomaine (PtdEtn) in Saccharomyces cerevisiae. -
Nuclear Ubiquitin-Proteasome Pathways in Proteostasis Maintenance
biomolecules Review Nuclear Ubiquitin-Proteasome Pathways in Proteostasis Maintenance Dina Frani´c †, Klara Zubˇci´c † and Mirta Boban * Croatian Institute for Brain Research, School of Medicine, University of Zagreb, 10000 Zagreb, Croatia; [email protected] (D.F.); [email protected] (K.Z.) * Correspondence: [email protected] † Equal contribution. Abstract: Protein homeostasis, or proteostasis, is crucial for the functioning of a cell, as proteins that are mislocalized, present in excessive amounts, or aberrant due to misfolding or other type of damage can be harmful. Proteostasis includes attaining the correct protein structure, localization, and the for- mation of higher order complexes, and well as the appropriate protein concentrations. Consequences of proteostasis imbalance are evident in a range of neurodegenerative diseases characterized by protein misfolding and aggregation, such as Alzheimer’s, Parkinson’s, and amyotrophic lateral sclerosis. To protect the cell from the accumulation of aberrant proteins, a network of protein quality control (PQC) pathways identifies the substrates and direct them towards refolding or elimination via regulated protein degradation. The main pathway for degradation of misfolded proteins is the ubiquitin-proteasome system. PQC pathways have been first described in the cytoplasm and the endoplasmic reticulum, however, accumulating evidence indicates that the nucleus is an important PQC compartment for ubiquitination and proteasomal degradation of not only nuclear, but also cyto- plasmic proteins. In this review, we summarize the nuclear ubiquitin-proteasome pathways involved in proteostasis maintenance in yeast, focusing on inner nuclear membrane-associated degradation (INMAD) and San1-mediated protein quality control. Keywords: proteasome; ubiquitin; nucleus; inner nuclear membrane; yeast; proteostasis; protein quality control; protein misfolding Citation: Frani´c,D.; Zubˇci´c,K.; Boban, M. -
Predicting Protein-Membrane Interfaces of Peripheral Membrane
bioRxiv preprint doi: https://doi.org/10.1101/2021.06.28.450157; this version posted June 29, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Predicting protein-membrane interfaces of pe- ripheral membrane proteins using ensemble machine learning Alexios Chatzigoulas1,2,* and Zoe Cournia1,* 1Biomedical Research Foundation, Academy of Athens, 4 Soranou Ephessiou, 11527 Athens, Greece, 2Depart- ment of Informatics and Telecommunications, National and Kapodistrian University of Athens, 15784 Athens, Greece *To whom correspondence should be addressed. Abstract Motivation: Abnormal protein-membrane attachment is involved in deregulated cellular pathways and in disease. Therefore, the possibility to modulate protein-membrane interactions represents a new promising therapeutic strategy for peripheral membrane proteins that have been considered so far undruggable. A major obstacle in this drug design strategy is that the membrane binding domains of peripheral membrane proteins are usually not known. The development of fast and efficient algorithms predicting the protein-membrane interface would shed light into the accessibility of membrane-protein interfaces by drug-like molecules. Results: Herein, we describe an ensemble machine learning methodology and algorithm for predicting membrane-penetrating residues. We utilize available experimental data in the literature for training 21 machine learning classifiers and a voting classifier. Evaluation of the ensemble classifier accuracy pro- duced a macro-averaged F1 score = 0.92 and an MCC = 0.84 for predicting correctly membrane-pen- etrating residues on unknown proteins of an independent test set. -
(4,5)-Bisphosphate Destabilizes the Membrane of Giant Unilamellar Vesicles
5112 Biophysical Journal Volume 96 June 2009 5112–5121 Profilin Interaction with Phosphatidylinositol (4,5)-Bisphosphate Destabilizes the Membrane of Giant Unilamellar Vesicles Kannan Krishnan,† Oliver Holub,‡ Enrico Gratton,‡ Andrew H. A. Clayton,§ Stephen Cody,§ and Pierre D. J. Moens†* †Centre for Bioactive Discovery in Health and Ageing, School of Science and Technology, University of New England, Armidale, Australia; ‡Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, California; and §Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Victoria, Australia ABSTRACT Profilin, a small cytoskeletal protein, and phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] have been implicated in cellular events that alter the cell morphology, such as endocytosis, cell motility, and formation of the cleavage furrow during cytokinesis. Profilin has been shown to interact with PI(4,5)P2, but the role of this interaction is still poorly understood. Using giant unilamellar vesicles (GUVs) as a simple model of the cell membrane, we investigated the interaction between profilin and PI(4,5)P2. A number and brightness analysis demonstrated that in the absence of profilin, molar ratios of PI(4,5)P2 above 4% result in lipid demixing and cluster formations. Furthermore, adding profilin to GUVs made with 1% PI(4,5)P2 leads to the forma- tion of clusters of both profilin and PI(4,5)P2. However, due to the self-quenching of the dipyrrometheneboron difluoride-labeled PI(4,5)P2, we were unable to determine the size of these clusters. Finally, we show that the formation of these clusters results in the destabilization and deformation of the GUV membrane. -
How Does Protein Zero Assemble Compact Myelin?
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 13 May 2020 doi:10.20944/preprints202005.0222.v1 Peer-reviewed version available at Cells 2020, 9, 1832; doi:10.3390/cells9081832 Perspective How Does Protein Zero Assemble Compact Myelin? Arne Raasakka 1,* and Petri Kursula 1,2 1 Department of Biomedicine, University of Bergen, Jonas Lies vei 91, NO-5009 Bergen, Norway 2 Faculty of Biochemistry and Molecular Medicine & Biocenter Oulu, University of Oulu, Aapistie 7A, FI-90220 Oulu, Finland; [email protected] * Correspondence: [email protected] Abstract: Myelin protein zero (P0), a type I transmembrane protein, is the most abundant protein in peripheral nervous system (PNS) myelin – the lipid-rich, periodic structure that concentrically encloses long axonal segments. Schwann cells, the myelinating glia of the PNS, express P0 throughout their development until the formation of mature myelin. In the intramyelinic compartment, the immunoglobulin-like domain of P0 bridges apposing membranes together via homophilic adhesion, forming a dense, macroscopic ultrastructure known as the intraperiod line. The C-terminal tail of P0 adheres apposing membranes together in the narrow cytoplasmic compartment of compact myelin, much like myelin basic protein (MBP). In mouse models, the absence of P0, unlike that of MBP or P2, severely disturbs the formation of myelin. Therefore, P0 is the executive molecule of PNS myelin maturation. How and when is P0 trafficked and modified to enable myelin compaction, and how disease mutations that give rise to incurable peripheral neuropathies alter the function of P0, are currently open questions. The potential mechanisms of P0 function in myelination are discussed, providing a foundation for the understanding of mature myelin development and how it derails in peripheral neuropathies. -
Structural Insights Into Membrane Fusion Mediated by Convergent Small Fusogens
cells Review Structural Insights into Membrane Fusion Mediated by Convergent Small Fusogens Yiming Yang * and Nandini Nagarajan Margam Department of Microbiology and Immunology, Dalhousie University, Halifax, NS B3H 4R2, Canada; [email protected] * Correspondence: [email protected] Abstract: From lifeless viral particles to complex multicellular organisms, membrane fusion is inarguably the important fundamental biological phenomena. Sitting at the heart of membrane fusion are protein mediators known as fusogens. Despite the extensive functional and structural characterization of these proteins in recent years, scientists are still grappling with the fundamental mechanisms underlying membrane fusion. From an evolutionary perspective, fusogens follow divergent evolutionary principles in that they are functionally independent and do not share any sequence identity; however, they possess structural similarity, raising the possibility that membrane fusion is mediated by essential motifs ubiquitous to all. In this review, we particularly emphasize structural characteristics of small-molecular-weight fusogens in the hope of uncovering the most fundamental aspects mediating membrane–membrane interactions. By identifying and elucidating fusion-dependent functional domains, this review paves the way for future research exploring novel fusogens in health and disease. Keywords: fusogen; SNARE; FAST; atlastin; spanin; myomaker; myomerger; membrane fusion 1. Introduction Citation: Yang, Y.; Margam, N.N. Structural Insights into Membrane Membrane fusion -
An Overview of Lipid Membrane Models for Biophysical Studies
biomimetics Review Mimicking the Mammalian Plasma Membrane: An Overview of Lipid Membrane Models for Biophysical Studies Alessandra Luchini 1 and Giuseppe Vitiello 2,3,* 1 Niels Bohr Institute, University of Copenhagen, Universitetsparken 5, 2100 Copenhagen, Denmark; [email protected] 2 Department of Chemical, Materials and Production Engineering, University of Naples Federico II, Piazzale Tecchio 80, 80125 Naples, Italy 3 CSGI-Center for Colloid and Surface Science, via della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy * Correspondence: [email protected] Abstract: Cell membranes are very complex biological systems including a large variety of lipids and proteins. Therefore, they are difficult to extract and directly investigate with biophysical methods. For many decades, the characterization of simpler biomimetic lipid membranes, which contain only a few lipid species, provided important physico-chemical information on the most abundant lipid species in cell membranes. These studies described physical and chemical properties that are most likely similar to those of real cell membranes. Indeed, biomimetic lipid membranes can be easily prepared in the lab and are compatible with multiple biophysical techniques. Lipid phase transitions, the bilayer structure, the impact of cholesterol on the structure and dynamics of lipid bilayers, and the selective recognition of target lipids by proteins, peptides, and drugs are all examples of the detailed information about cell membranes obtained by the investigation of biomimetic lipid membranes. This review focuses specifically on the advances that were achieved during the last decade in the field of biomimetic lipid membranes mimicking the mammalian plasma membrane. In particular, we provide a description of the most common types of lipid membrane models used for biophysical characterization, i.e., lipid membranes in solution and on surfaces, as well as recent examples of their Citation: Luchini, A.; Vitiello, G. -
Membrane Proteins Are Associated with the Membrane of a Cell Or Particular Organelle and Are Generally More Problematic to Purify Than Water-Soluble Proteins
Strategies for the Purification of Membrane Proteins Sinéad Marian Smith Department of Clinical Medicine, School of Medicine, Trinity College Dublin, Ireland. Email: [email protected] Abstract Although membrane proteins account for approximately 30 % of the coding regions of all sequenced genomes and play crucial roles in many fundamental cell processes, there are relatively few membranes with known 3D structure. This is likely due to technical challenges associated with membrane protein extraction, solubilization and purification. Membrane proteins are classified based on the level of interaction with membrane lipid bilayers, with peripheral membrane proteins associating non- covalently with the membrane, and integral membrane proteins associating more strongly by means of hydrophobic interactions. Generally speaking, peripheral membrane proteins can be purified by milder techniques than integral membrane proteins, whose extraction require phospholipid bilayer disruption by detergents. Here, important criteria for strategies of membrane protein purification are addressed, with a focus on the initial stages of membrane protein solublilization, where problems are most frequently are encountered. Protocols are outlined for the successful extraction of peripheral membrane proteins, solubilization of integral membrane proteins, and detergent removal which is important not only for retaining native protein stability and biological functions, but also for the efficiency of downstream purification techniques. Key Words: peripheral membrane protein, integral membrane protein, detergent, protein purification, protein solubilization. 1. Introduction Membrane proteins are associated with the membrane of a cell or particular organelle and are generally more problematic to purify than water-soluble proteins. Membrane proteins represent approximately 30 % of the open-reading frames of an organism’s genome (1-4), and play crucial roles in basic cell functions including signal transduction, energy production, nutrient uptake and cell-cell communication.