DOCSLIB.ORG

Immobilizing Strategies for Membranes to Screen Against Antibody Mimetics with Phage Display

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Immobilizing Strategies for Membranes to Screen Against Antibody Mimetics with Phage Display Immobilizing strategies for membranes to screen against antibody mimetics with phage display Vincent Ochejeni Agboh Submitted in accordance with the requirements for the degree of Doctor of Philosophy The University of Leeds School of Biomedical Sciences The Astbury centre of structural molecular biology November 2015 1 The candidate confirms that the work submitted is his own and that appropriate credit has been given where reference has been made to the work of others. This copy has been supplied on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement © 2013 The University of Leeds and Vincent Agboh The right of Vincent Agboh to be identified as Author of this work has been asserted by him in accordance with the Copyright, Designs and Patents Act 1988. 2 Acknowledgments I would like to thank my supervisor, Professor Stephen A. Baldwin, who remains instrumental to this PhD despite his unfortunate passing on 13th November 2014. I would also like thank my supervisors Dr. Lars Jeuken and Dr. Stephen Muench for allowing me to work on this project, as well as their critical input, constant advice, guidance and support throughout. I am also thankful to Dr. Trevor Wilkinson, Dr. David Bannister, Dr. Stacey Chin and Dr. Benjamin Kemp, as well as all members of the biologics and protein sciences teams from MedImmune who trained me in phage display and the subsequent validation experiments. I am also grateful to MedImmune for providing funding. Special thanks to all past and present members of the Baldwin, Henderson and Jeuken groups from the University of Leeds. Particularly Mrs Jocelyn Baldwin, Dr Cheng Ma, Dr Vincent Postis and Zhenyu Hao who trained me in stopped flow, provided samples of VcCNT and NupC SMALPs, as well as AcrB nanodiscs. Thanks also to David Sharples who performed 30 L fermentations of several E.coli host strains. Also, thank you to Ivona Petrache, for training me in the production of nanodiscs. Thank you to Professor Tim Daffron, Dr Sarah Lee and Mrs. Rosemary Parslow from the University of Birmingham for providing the styrene maleic acid polymer and training me in its use. I would finally like to thank my family, Dr. Chris Agboh, Mrs Regina Agboh, Dr. Kelvin Agboh, Mrs Katie Agboh-Davison, Mr. Michael Agboh-Davison, Frankie Agboh-Davison and Ms. Pat Huffer for all of their encouragement and support. 3 Abstract Antibody mimetics are a novel class of potential therapeutic agents which improve on several limitations which hinder antibodies. As a result, they are the subject of increasing interest from both the pharmaceutical and academic sectors. They are commonly selected via phage display against an immobilised target. However the presentation of these targets, particularly in the case of membrane proteins, is often a rate limiting step in their production. In this study, several commonly-used protein tags were compared in their ability to immobilise a model membrane protein - MPSIL0294, a metal ion transporter from Enterococcus faecalis, a streptavidin binding peptide tag, an Avitag which has undergone in vivo biotinylation and a chemically biotinylated C-terminal cysteine. A fourth method of immobilisation was also included, aspecific chemical biotinylation of lysine residues in AcrB, a component of an E.coli multidrug efflux pump. The tags were compared using a phage display assay, in which designed ankyrin repeat proteins (DARPins) from a naïve library were screened against both model membrane proteins, which were both solubilised in n-Dodecyl-β-D-maltopyranoside (DDM). With the exception of the C-terminal cysteine variant of MPSIL0294, no DARPins capable of binding could be selected by any of the other immobilisation techniques after two rounds of phage display. Furthermore, the C-terminal cysteine MPSIL0294 also yielded poor success rates and only two binding DARPins were identified. Despite showing a moderately high level of sequence similarity, these two DARPins showed different binding activities during validation experiments. The alternative tags under test however; do not appear to be beneficial in the selection of antibody mimetics against immobilised membrane protein targets. Different methods of protein solubilisation were also compared in an identical manner: detergent solubilisation (in DDM), lipid nanodiscs and styrene maleic acid lipid particles (SMALP). These solubilisation methods were compared using a panel of membrane proteins composed of MPSIL0294, AcrB, NupC, an E.coli concentrative nucleoside transporter and VcCNT, NupCs homologue from Vibrio Cholerae. SMALPs of AcrB, VcCNT and NupC displayed the highest success rate for selecting DARPins capable of binding the respective targets. Subsequent validation tests showed that the best DARPins selected against AcrB SMALPs and nanodiscs behaved in a similar manner. SMALPs are relatively easy to produce, especially compared to nanodiscs so this study concludes that SMALPs are the best format for phage display. 4 Table of Contents Acknowledgments ......................................................................................................................... 3 Abstract ......................................................................................................................................... 4 List of figures ............................................................................................................................... 10 List of tables ................................................................................................................................ 13 Abbreviations .............................................................................................................................. 14 Chapter 1: Introduction .............................................................................................................. 16 1.1 Membrane proteins in modern research.............................................................................. 17 1.2 Solubilising membrane proteins ........................................................................................... 18 1.2.1 Detergent solubilisation ................................................................................................. 18 1.2.2 Nanodiscs ....................................................................................................................... 20 1.2.3 Polymer solubilisation .................................................................................................... 21 1.2.3.1 Styrene-maleic acid ................................................................................................. 22 1.2.3.2 Amphipols ............................................................................................................... 23 1.3 The membrane protein panel under test ............................................................................. 25 1.3.1 MPSIL0294 ..................................................................................................................... 25 1.3.1.1 NRAMP .................................................................................................................... 27 1.3.2 AcrB ................................................................................................................................ 30 1.3.3 NupC and VcCNT ............................................................................................................ 32 1.4 In vitro selection strategies ................................................................................................... 34 1.4.1 Phage display ................................................................................................................. 34 1.4.2 Bacterial display ............................................................................................................. 36 1.4.3 Yeast display................................................................................................................... 37 1.4.4 Ribosome display ........................................................................................................... 38 1.4.5 mRNA display ................................................................................................................. 39 1.5 Antibody mimetics ................................................................................................................ 41 1.5.1 Single chain variable fragments ..................................................................................... 42 1.5.2 Nanobodies .................................................................................................................... 43 1.5.3 Designed ankyrin repeat proteins and antibody fragments .......................................... 44 1.5.4 Adhirons ......................................................................................................................... 46 1.6 Project Aim ............................................................................................................................ 48 Chapter 2: Materials and Methods ............................................................................................ 49 2.1 Materials and Suppliers ........................................................................................................ 50 2.2 Bacterial strains, Growth and Expression Media .................................................................. 50 5 2.2.1 Bacterial strain genotypes ............................................................................................
Load more

Recommended publications
  • Cell-Free Expressed Bacteriorhodopsin in Different Soluble Membrane Mimetics: Biophysical Properties and NMR Accessibility
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Structure Article Cell-free Expressed Bacteriorhodopsin in Different Soluble Membrane Mimetics: Biophysical Properties and NMR Accessibility Manuel Etzkorn,1 Thomas Raschle,1 Franz Hagn,1 Vladimir Gelev,3 Amanda J. Rice,2 Thomas Walz,2 and Gerhard Wagner1,* 1Department of Biological Chemistry and Molecular Pharmacology 2Department of Cell Biology Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA 3FB Reagents, 267 Pearl Street, Cambridge, MA 02139, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.str.2013.01.005 SUMMARY polymers (amphipols) (Popot et al., 2011) or lipid bilayer nano- discs (Denisov et al., 2004) have lately received increased atten- Selecting a suitable membrane-mimicking environ- tion as promising tools for the investigation of membrane ment is of fundamental importance for the investiga- proteins (Gorzelle et al., 2002; Raschle et al., 2010; Zoonens tion of membrane proteins. Nonconventional surfac- et al., 2005). Advantages of using nonconventional membrane tants, such as amphipathic polymers (amphipols) mimetics include the exceptionally good refolding properties and lipid bilayer nanodiscs, have been introduced of amphipols for heptahelical membrane proteins such as as promising environments that may overcome G-protein-coupled receptors (GPCRs) (Dahmane et al., 2009) or the absence of detergent as well as the more native-like envi- intrinsic disadvantages of detergent micelle sys- ronment provided by nanodiscs. Additionally it has been demon- tems. However, structural insights into the effects strated that the use of nonconventional surfactants can increase of different environments on the embedded protein protein stability and improve the accessibility of the functional are limited.
    [Show full text]
  • The Peptidisc, a Simple Method for Stabilizing Membrane Proteins In
    TOOLS AND RESOURCES The Peptidisc, a simple method for stabilizing membrane proteins in detergent-free solution Michael Luke Carlson1, John William Young1, Zhiyu Zhao1, Lucien Fabre1, Daniel Jun2,3, Jianing Li4, Jun Li4, Harveer Singh Dhupar1, Irvin Wason1, Allan T Mills1, J Thomas Beatty3, John S Klassen4, Isabelle Rouiller2, Franck Duong1* 1Department of Biochemistry and Molecular Biology, Faculty of Medicine, Life Sciences Institute, University of British Columbia, Vancouver, Canada; 2Department of Anatomy and Cell Biology, McGill University, Montreal, Canada; 3Department of Microbiology and Immunology, University of British Columbia, Vancouver, Canada; 4Glycomics Centre and Department of Chemistry, University of Alberta, Alberta, Canada Abstract Membrane proteins are difficult to work with due to their insolubility in aqueous solution and quite often their poor stability in detergent micelles. Here, we present the peptidisc for their facile capture into water-soluble particles. Unlike the nanodisc, which requires scaffold proteins of different lengths and precise amounts of matching lipids, reconstitution of detergent solubilized proteins in peptidisc only requires a short amphipathic bi-helical peptide (NSPr) and no extra lipids. Multiple copies of the peptide wrap around to shield the membrane-exposed part of the target protein. We demonstrate the effectiveness of this ‘one size fits all’ method using five different membrane protein assemblies (MalFGK2, FhuA, SecYEG, OmpF, BRC) during ‘on-column’, ‘in-gel’, and ‘on-bead’ reconstitution
    [Show full text]
  • Nanodiscs in Membrane Biochemistry and Biophysics
    HHS Public Access Author manuscript Author ManuscriptAuthor Manuscript Author Chem Rev Manuscript Author . Author manuscript; Manuscript Author available in PMC 2018 March 22. Published in final edited form as: Chem Rev. 2017 March 22; 117(6): 4669–4713. doi:10.1021/acs.chemrev.6b00690. NANODISCS IN MEMBRANE BIOCHEMISTRY AND BIOPHYSICS Ilia G. Denisov and Stephen G. Sligar Department of Biochemistry and Department of Chemistry, University of Illinois, Urbana, IL, 61801 Abstract Membrane proteins play a most important part in metabolism, signaling, cell motility, transport, development, and many other biochemical and biophysical processes which constitute fundamentals of life on molecular level. Detailed understanding of these processes is necessary for the progress of life sciences and biomedical applications. Nanodiscs provide a new and powerful tool for a broad spectrum of biochemical and biophysical studies of membrane proteins and are commonly acknowledged as an optimal membrane mimetic system which provides control over size, composition and specific functional modifications on nanometer scale. In this review we attempted to combine a comprehensive list of various applications of Nanodisc technology with systematic analysis of the most attractive features of this system and advantages provided by Nanodiscs for structural and mechanistic studies of membrane proteins. Table of Content Figure An Introduction to Nanodiscs Membrane proteins are represented by a tremendous variety of sizes, structures and functions, including complex supra-molecular
    [Show full text]
  • Actual Fusion Efficiency in the Lipid Mixing Assay
    www.nature.com/scientificreports OPEN Actual fusion efficiency in the lipid mixing assay - Comparison between nanodiscs and liposomes Received: 10 October 2016 Claire François-Martin1,2,3 & Frédéric Pincet1,2,3 Accepted: 31 January 2017 Lipid exchange occurs between membranes during fusion or active lipid transfer. These processes are Published: 07 March 2017 necessary in vivo for the homeostasis of the cell at the level of the membranes, the organelles and the cell itself. They are also used by the cell to interact with the surrounding medium. Several assays have been developed to characterize in vitro these processes on model systems. The most common one, relying on fluorescence dequenching, measures lipid mixing between small membranes such as liposomes or nanodiscs in bulk. Usually, relative comparisons of the rate of lipid exchange are made between measurements performed in parallel. Here, we establish a quantitative standardization of this assay to avoid any bias resulting from the temperatures, the chosen fluorescent lipid fractions and from the various detergents used to normalize the measurements. We used this standardization to quantitatively compare the efficiency of SNARE-induced fusion in liposome-liposome and liposome-nanodisc configurations having similar collision frequency. We found that the initial yield of fusion is comparable in both cases, 1 per 2–3 million collisions in spite of a much larger dequenching signal with nanodiscs. Also, the long-term actual fusion rate is slightly lower with nanodiscs than in the liposome-liposome assay. In vivo, lipid mixing and exchange constantly occur between cells and/or organelles. Fusion is a common process that merges two membranes thereby mixing their initially separated lipids1,2.
    [Show full text]
  • The Power, Pitfalls and Potential of the Nanodisc System for NMR-Based Studies
    Biol. Chem. 2016; 397(12): 1335–1354 Review Aldino Viegasa, Thibault Vienneta and Manuel Etzkorn* The power, pitfalls and potential of the nanodisc system for NMR-based studies DOI 10.1515/hsz-2016-0224 influence of the environment on the protein structure as Received June 6, 2016; accepted July 19, 2016; previously published well as on the used experimental technique has to be con- online July 23, 2016 sidered. This is particularly true for the NMR-based inves- tigation of membrane proteins, which require a suitable Abstract: The choice of a suitable membrane mimicking membrane mimicking environment to promote a protein environment is of fundamental importance for the char- conformation that is, on the one hand, representative for acterization of structure and function of membrane pro- its native behavior and, on the other, compatible with the teins. In this respect, usage of the lipid bilayer nanodisc requirements of NMR spectroscopy. While a variety of dif- technology provides a unique potential for nuclear mag- ferent membrane mimetics for solution and/or solid-state netic resonance (NMR)-based studies. This review sum- NMR have been established (Popot, 2010; Raschle et al., marizes the recent advances in this field, focusing on (i) 2010; Ding et al., 2013; Catoire et al., 2014; Liang and the strengths of the system, (ii) the bottlenecks that may Tamm, 2016), the lipid bilayer nanodisc system distinc- be faced, and (iii) promising capabilities that may be tively combines various potentially advantageous proper- explored in future studies. ties including a native-like lipid surrounding, the absence Keywords: membrane proteins; nanodiscs; NMR.
    [Show full text]
  • Developing Nanodisc-ID for Label-Free Characterizations of Membrane Proteins ✉ Huan Bao 1
    ARTICLE https://doi.org/10.1038/s42003-021-02043-y OPEN Developing Nanodisc-ID for label-free characterizations of membrane proteins ✉ Huan Bao 1 Membrane proteins (MPs) influence all aspects of life, such as tumorigenesis, immune response, and neural transmission. However, characterization of MPs is challenging, as it often needs highly specialized techniques inaccessible to many labs. We herein introduce nanodisc-ID that enables quantitative analysis of membrane proteins using a gel electro- phoresis readout. By leveraging the power of nanodiscs and proximity labeling, nanodisc-ID serves both as scaffolds for encasing biochemical reactions and as sensitive reagents for 1234567890():,; detecting membrane protein-lipid and protein-protein interactions. We demonstrate this label-free and low-cost tool by characterizing a wide range of integral and peripheral membrane proteins from prokaryotes and eukaryotes. 1 Department of Molecular Medicine, The Scripps Research Institute, Jupiter, FL, USA. ✉email: [email protected] COMMUNICATIONS BIOLOGY | (2021) 4:514 | https://doi.org/10.1038/s42003-021-02043-y | www.nature.com/commsbio 1 ARTICLE COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-021-02043-y ignal transduction across cellular membranes is crucial for Developing Nanodisc-ID for protein–lipid interactions. Despite life and often involves the action of membrane proteins the robust efficiency in detecting syt1-PS interaction, we also S 1,2 (MPs) . The importance of these membrane-embedded observed moderate levels of labeled syt1 by PL enzymes using NDs molecules is underscored by the fact that they constitute over 60% containing only PC lipids (Fig. 1B). This result is surprising as syt1 of drug targets for numerous human diseases3.
    [Show full text]
  • Probing Membrane Protein Assembly Into Nanodiscs by in Situ Dynamic Light Scattering: A2A Receptor As a Case Study
    biology Article Probing Membrane Protein Assembly into Nanodiscs by In Situ Dynamic Light Scattering: A2A Receptor as a Case Study Rosana I. Reis 1,2 and Isabel Moraes 1,2,* 1 National Physical Laboratory, Hampton Road, Teddington TW11 0LW, UK; [email protected] 2 Research Complex at Harwell Rutherford, Appleton Laboratory, Harwell, Oxford, Didcot OX11 0FA, UK * Correspondence: [email protected] Received: 4 October 2020; Accepted: 12 November 2020; Published: 13 November 2020 Simple Summary: Located within the biological cell membranes, integral membrane proteins are responsible for a large variety of vital cellular processes. In humans, nearly a quarter of the genome codes integral membrane proteins, therefore malfunction of these proteins is associated with a variety of symptoms and diseases such as obesity, cancer and Parkinson’s disease. Clearly, knowledge of membrane proteins behaviour, in both structural and functional terms, is important not only in medicine but also in the design of better drugs with improved pharmaceutical properties. Nevertheless, much still remains unknown about these proteins, mainly because of the technical challenges associated with their production and stability in vitro once removed from their native lipidic environment. Recently, several membrane mimetic systems have been developed including nanodisc lipid particles. Nanodiscs are self-assembled lipidic structures that “trap” membrane proteins into a disc shaped phospholipid bilayer that is stabilised by a belt made of a protein know as membrane scaffold protein (MSP). Membrane proteins assembled into lipidic nanodiscs can maintain their structural and functional integrity and are compatible with most biophysical methods. Here we demonstrate the use of in situ dynamic light scattering as a high-throughput screening tool to assess the best conditions for nanodisc assembly and protein incorporation.
    [Show full text]
  • Developing Nanodiscs As a Tool for Low Resolution Studies Of
    FACU LTY OF SCIENCE UNIV ERSI TY O F C O PENHAGE N PhD thesis Nicholas Skar-Gislinge Developing Nanodiscs as a Tool for Low Resolution Studies of Membrane Proteins Ac ademic advisor: Lise Arleth Submitted: 28/02/2014 ii Abstract Phospholipid nanodiscs are 10 nm sized disc shaped particles consisting of roughly 150 phos- pholipids arranged in a central⇠ bilayer, stabilized by two amphipathic protein ”belts” that wrap around the rim of the bilayer. Because they contain a small bilayer leaflet, in which membrane proteins can be incorporated, they can be used as a tool for solution studies of membrane pro- teins. So far most of the studies of membrane proteins using nanodiscs have been concerning the function of the incorporated membrane protein. However, due to the good control of the size and lipid composition of the nanodisc system, they seem an ideal tool for expanding the use of small angle scattering from studying water soluble proteins, to also include membrane proteins inserted into nanodiscs. This has been the main goal of this thesis. In order to reach this goal it is necessary to have a detailed understanding of the nanodisc system, without the membrane protein, as well at the self-assembly process in general, and in particular in relation to incorporation of membrane proteins. This was the aim of work done early in this thesis. Here a detailed model for the small angle x-ray and neutron scattering from the empty nanodisc system was derived and used to describe the nanodisc system with great detail. The high level of detail was achieved by combining data from both small-angle x-ray and neutron experiments performed on the same samples as well as the incorporation of so-called molecular constraints.
    [Show full text]
  • The Development of Nanodisc Technology As a Model Membrane System in Membrane Protein Research
    The development of Nanodisc technology as a model membrane system in membrane protein research. A literature research by Hessel van der Weide, supervised by Ilja Küsters and Arnold J M Driessen at the Department of Microbiology, University of Groningen. ABSTRACT This literature research focuses on the recent development of a new artificial system in which membrane proteins can be reconstituted: Nanodiscs. It will introduce with a general overview of the model membrane systems used for membrane protein reconstitution in biochemical research. A short review of early research into the high density lipoprotein complex is then followed by in-depth reviews of the article describing the engineering of the original membrane scaffold protein that makes up the Nanodisc and two following studies that further developed the membrane scaffold protein. Another three in-depth reviews cover the first membrane protein to be reconstituted into Nanodiscs, the demonstration of the use for Nanodiscs in single- molecule studies and the development of oligomeric Nanodisc reconstitution, followed by recent examples of the Nanodisc technology applied to membrane protein research. INTRODUCTION Proteins associated or attached to the membrane of a cell or organelle are vital to all cellular life. Such membrane proteins are responsible for the transport of chemicals and information across the membrane, and as such they perform a variety of essential biological functions in cellular metabolism, communication and transport and the maintenance of membrane gradients and ion concentrations. Cellular structure, movement and enzymatic activity are also dependant on membrane proteins. The importance of membrane proteins to cellular life is reflected by several estimates. The protein content of membranes is generally 50%, ranging between 18% in myelinated neurons and 75% in mitochondrial membranes1.
    [Show full text]
  • Development of Lipid Nanodisc Technology for the Formulation of Poorly Water Soluble Drugs
    Development of Lipid Nanodisc Technology for the Formulation of Poorly Water Soluble Drugs Andreea Ivona Petrache Submitted in accordance with requirements for the degree of Doctor of Philosophy The University of Leeds School of Chemistry April 2016 i The candidate confirms that the work submitted is her own, except where work which has formed part of jointly-authored publication has been included. The contribution of the candidate and the other authors to this work has been explicitly indicated below. The candidate confirms that appropriate credit has been given within the thesis where reference has been made to the work of others. Chapter 5 – Modified nanodiscs for traceable cellular delivery includes content from the publication: “Sortase-mediated labelling of lipid nanodiscs for cellular tracing", Mol. BioSyst., 2016, Accepted Manuscript, A. I. Petrache, D. C. Machin, D. J. Williamson, M. E. Webb and P. A. Beales. The candidate reconstituted the nanodiscs and conducted the cleavage of His6 tag attached to the N-terminus of MSP1D1, incorporated in nanodiscs. The candidate also conducted the Sortase A mediated reaction. TEV protease, Sortase A and fluorescein depsipeptide were prepared in HEPES buffer by Dr Daniel Williamson. General passage procedure, preparation of HeLa cells before transfection, the protein transfection procedure and cell fixation were conducted by Mr Darren Machin. The candidate immobilised the cells and performed the measurements on the confocal fluorescence microscopy. M. E. Webb, P. A. Beales and the candidate composed the manuscript. This copy has been supplied on the understanding that it is a copyright material and that no quotation from the thesis may be published without proper acknowledgment.
    [Show full text]
  • Micelles, Bicelles, Amphipols, Nanodiscs, Liposomes, Or Intact Cells: the Hitchhiker’S Guide to the Study of Membrane Proteins by NMR
    Chapter 12 Micelles, Bicelles, Amphipols, Nanodiscs, Liposomes, or Intact Cells: The Hitchhiker’s Guide to the Study of Membrane Proteins by NMR Laurent J. Catoire, Xavier L. Warnet and Dror E. Warschawski 12.1 Introduction The vast majority of biophysical studies of membrane proteins (MPs) at the atomic scale are performed in vitro with preparations as homogeneous as possible, where the protein is isolated in a nonnative environment. MP samples for nuclear magnetic resonance (NMR) spectroscopy are no exception to the rule, in particular because purification helps to clearly detect and unambiguously identify signals from the protein of interest. By native environment, we consider the original membrane(s) where the protein exerts its biological role. It is difficult to place artificial systems on a scale defining how well they mimic native membranes, especially when a functional test is difficult or impossible to set up. Indeed, liposomes or nanomet- ric lipid bilayers still represent artificial environments, and, on the contrary, exotic surfactants like amphipols, which could be thought to be inappropriate given their chemical structures, have proven to keep numerous MPs stable and active in so- lution. Over the past decades, various membrane mimetics have been developed, chosen on the basis of the compatibility with the technique of investigation used, sometimes at the expense of the functionality of the protein. Paradoxically, after so many efforts to improve membrane substitutes, in-cell NMR has known significant advances during the past few years (Selenko and Wagner 2007; Ito and Selenko 2010), which represents a very attractive potential for future NMR studies of MPs in situ (Renault et al.
    [Show full text]
  • Membrane Protein Assembly Into Nanodiscs
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector FEBS Letters 584 (2010) 1721–1727 journal homepage: www.FEBSLetters.org Review Membrane protein assembly into Nanodiscs Timothy H. Bayburt, Stephen G. Sligar * Department of Biochemistry, School of Molecular and Cellular Biology, University of Illinois, Urbana-Champaign, Urbana, IL 61801, United States article info abstract Article history: Nanodiscs are soluble nanoscale phospholipid bilayers which can self-assemble integral membrane Received 1 September 2009 proteins for biophysical, enzymatic or structural investigations. This means for rendering mem- Accepted 9 October 2009 brane proteins soluble at the single molecule level offers advantages over liposomes or detergent Available online 16 October 2009 micelles in terms of size, stability, ability to add genetically modifiable features to the Nanodisc structure and ready access to both sides of the phospholipid bilayer domain. Thus the Nanodisc sys- Edited by Wilhelm Just tem provides a novel platform for understanding membrane protein function. We provide an over- view of the Nanodisc approach and document through several examples many of the applications to Keywords: the study of the structure and function of integral membrane proteins. Nanodisc Membrane protein Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Self-assembly 1. Introduction protein systems require specific types of phospholipids to maintain active function, a requirement which is not mimicked by detergent Membrane proteins have been difficult to study from the mech- micelles. Liposome preparations have been used to incorporate anistic perspective as many of the biophysical and chemical tech- membrane proteins and this approach has been found to be useful niques applicable to soluble enzymes fail to deal with insoluble when compartmentalization of each side of the bilayer is needed, aggregates.
    [Show full text]