The BAM Complex: Methods and Protocols

297 Pages·2015·8.292 MB·English

by  Susan K. Buchanan, Nicholas Noinaj

Methods in
Molecular Biology 1329
Susan K. Buchanan
Nicholas Noinaj Editors
The BAM
Complex
Methods and Protocols
M M B
ETHODS IN OLECULAR IOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire , AL10 9AB, UK
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The BAM Complex
Methods and Protocols
Edited by
Susan K. Buchanan
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases,
National Institutes of Health, Bethesda, MD, USA
Nicholas Noinaj
Department of Biological Sciences, Markey Center for Structural Biology, Purdue University,
West Lafayette, IN, USA
Editors
Susan K. B uchanan Nicholas N oinaj
Laboratory of Molecular Biology Department of Biological Sciences
National Institute of Diabetes Markey Center for Structural Biology
and Digestive and Kidney Diseases Purdue University, West Lafayette
National Institutes of Health IN, USA
Bethesda, MD, USA
ISSN 1064-3745 ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-4939-2870-5 ISBN 978-1-4939-2871-2 (eBook)
DOI 10.1007/978-1-4939-2871-2
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Prefa ce
Cells are encapsulated by a single lipid bilayer called a membrane that forms the boundary
separating the inside of the cell from the outside. The membrane serves many essential
functions for the cell including nutrient import, signaling, motility, adhesion, endocytosis,
and replication. These functions are accomplished by a large family of proteins called mem-
brane proteins that are either partially or fully integrated into the membrane. Fully inte-
grated membrane proteins are embedded into the membrane by hydrophobic domains that
contain either an α-helical fold or a β-barrel fold. While α-helical membrane proteins can be
found in nearly all known membranes in nature, β-barrel membrane proteins can only be
found within the outermost membranes of mitochondria, chloroplasts, and Gram-negative
bacteria, all of which are unique in that they contain two concentric membranes (inner and
outer) and are related by their endosymbiotic lineage. The mechanism for how these
β-barrel membrane proteins are folded and inserted into the outer membrane remains
unknown. However, within the past 10 years, signifi cant advancements have been made to
understand this process, particularly in Gram-negative bacteria where genetic analyses,
mutagenesis studies, biochemical assays, in vitro assays, and structural biology techniques
have all contributed.
Early work identifi ed a multicomponent complex that we now refer to as the β-barrel
assembly machinery (BAM) complex, which is required in Gram-negative bacteria to inte-
grate newly synthesized β-barrel membrane proteins into the outer membrane. From the
initial identifi cation of the BAM complex and its individual components to the recent struc-
tural characterization of all individual proteins, much has been learned about the role the
BAM complex plays in the biogenesis of β-barrel membrane proteins. In this volume of the
Methods in Molecular Biology series, we have assembled a collection of experimental proto-
cols for common techniques and strategies used to study the biogenesis of β-barrel mem-
brane proteins in Gram-negative bacteria. This volume contains step-by-step methods
based on the protocols that were used during the research efforts performed in determining
what is currently known about the regulation and function of the BAM complex, the roles
played by each of the individual components, the expression and purifi cation of the com-
ponents, crystallization and structure determination of the components, and how the indi-
vidual Bam components may assemble into a functional complex. Given that several studies
have reported the folding of β-barrel membrane proteins from Gram-negative bacteria in
mitochondria and vice versa, one chapter focuses on methods used to study the evolution-
arily conserved system that exists in mitochondria.
The methods and protocols here will appeal to a wide variety of scientists in academia,
government, and industry including microbiologists, biochemists, bacteriologists, struc-
tural biologists, and those looking to target the BAM complex for therapeutic discovery
and development. It is our hope that this volume will serve as an invaluable reference for
those interested in studying the BAM complex and how it functions at the outer mem-
brane, as well as for those who may want to apply the protocols communicated here to
other interesting biological systems.
v
vi Preface
Last but certainly not least, this volume would not have been possible without the con-
tributions from the authors, to whom we are truly indebted.
Bethesda, MD, USA S usan K. Buchanan
West Lafayette, IN, USA Nicholas N oinaj
Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i x
1 The β-Barrel Assembly Machinery Complex . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Denisse L. Leyton , M atthew J . B elousoff , and T revor Lithgow
2 Y east Mitochondria as a Model System to Study the Biogenesis
of Bacterial β-Barrel Proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Thomas U lrich , Philipp O berhettinger , I ngo B. Autenrieth ,
and Doron Rapaport
3 E xperimental Methods for Studying the BAM Complex
in Neisseria meningitidis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 3
Martine P. Bos , Ria Tommassen-van Boxtel , and Jan T ommassen
4 H eat Modifiability of Outer Membrane Proteins
from Gram-Negative Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 1
Nicholas N oinaj , Adam J . K uszak , and Susan K. Buchanan
5 The Role of a Destabilized Membrane for OMP Insertion. . . . . . . . . . . . . . . . 5 7
Ashlee M. Plummer , Dennis G essmann , and Karen G. Fleming
6 Treponema pallidum in Gel Microdroplets: A Method for
Topological Analysis of BamA (TP0326) and Localization of Rare
Outer Membrane Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Amit L uthra , Arvind A nand , and Justin D. Radolf
7 A nalyzing the Role of Periplasmic Folding Factors in the Biogenesis
of OMPs and Members of the Type V Secretion System . . . . . . . . . . . . . . . . . 77
Gustavo Bodelón , Elvira Marín , and Luis Ángel Fernández
8 A n In Vitro Assay for Substrate Translocation by FhaC in Liposomes . . . . . . . 1 11
Enguo Fan , D errick N orell , and Matthias Müller
9 Measuring Cell–Cell Binding Using Flow-Cytometry . . . . . . . . . . . . . . . . . . . 1 27
Zachary C. R uhe , C hristopher S. H ayes , and David A. L ow
10 Methods to Characterize Folding and Function of BamA
Cross-Link Mutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 37
Adam J. K uszak , Nicholas Noinaj , and Susan K. B uchanan
11 Small Angle X-ray Scattering (SAXS) Characterization of the POTRA
Domains of BamA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
Pamela A rden D oerner and Marcelo Carlos Sousa
12 A ssessing the Outer Membrane Insertion and Folding of Multimeric
Transmembrane β-Barrel Proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
Jack C. Leo , P hilipp O berhettinger , and Dirk Linke
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viii Contents
13 The Expression, Purification, and Structure Determination
of BamA from E. coli. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
Dongchun N i and Yihua Huang
14 E xpression and Purification of the Individual Bam Components BamB–E . . . . 179
Suraaj A ulakh , Kelly H. K im , and Mark Paetzel
15 Structure Determination of the BAM Complex Accessory
Lipoproteins BamB–E. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
Kornelius Zeth
16 An In Vitro Assay for Outer Membrane Protein Assembly
by the BAM Complex. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
Giselle R oman-Hernandez and Harris D . B ernstein
17 Identification of BamC on the Surface of E. coli . . . . . . . . . . . . . . . . . . . . . . . 2 15
Chaille T. W ebb and Trevor L ithgow
18 C onstruction and Characterization of an E. coli bamD Depletion Strain. . . . . . 227
D ante P . R icci
19 E xpression, Purification, and Screening of BamE, a Component
of the BAM Complex, for Structural Characterization. . . . . . . . . . . . . . . . . . . 245
Mark J eeves , P ooja S ridhar , and Timothy J . Knowles
20 Purification and Bicelle Crystallization for Structure Determination
of the E. coli Outer Membrane Protein TamA. . . . . . . . . . . . . . . . . . . . . . . . . 259
Fabian Gruss , Sebastian H iller , and Timm Maier
21 S trategies for the Analysis of Bam Recognition Motifs in Outer
Membrane Proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271
Nagarajan P aramasivam and Dirk Linke
22 Summary and Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 79
Nicholas N oinaj and Susan K. Buchanan
Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281
Contributors
ARVIND ANAND • Department of Medicine, University of Connecticut Health , F armington,
CT , U SA
SURAAJ AULAKH • Department of Molecular Biology and Biochemistry ,
Simon Fraser University , Burnaby, BC, Canada
INGO B . AUTENRIETH • Interfaculty Institute of Microbiology and Infection Medicine ,
University of Tübingen , T übingen, Germany
MATTHEW J . B ELOUSOFF • Department of Microbiology, Monash University , M elbourne,
VIC, A ustralia
HARRIS D . BERNSTEIN • Genetics and Biochemistry Branch, National Institute of Diabetes
and Digestive and Kidney Diseases, National Institutes of Health , B ethesda, MD , U SA
GUSTAVO B ODELÓN • Department of Microbial Biotechnology, C entro Nacional de
Biotecnología, Consejo Superior de Investigaciones Científi cas , M adrid , S pain
MARTINE P . BOS • Department of Molecular Microbiology and Institute of Biomembranes,
Utrecht University, Utrecht, The Netherlands; Department of Medical Microbiology and
Infection Control , V U University Medical Center , A msterdam , T he Netherlands
RIA TOMMASSEN-VAN BOXTEL • Department of Molecular Microbiology and Institute of
Biomembranes, U trecht University , U trecht , T he Netherlands
SUSAN K. BUCHANAN • Laboratory of Molecular Biology , National Institute of Diabetes
and Digestive and Kidney Diseases, National Institutes of Health , B ethesda, MD, USA
PAMELA ARDEN DOERNER • Department of Chemistry and Biochemistry , University of
Colorado, Boulder , Boulder, CO , U SA
ENGUO F AN • Institute of Biochemistry and Molecular Biology, ZBMZ,
University of Freiburg , F reiburg , G ermany
LUIS ÁNGEL FERNÁNDEZ • Department of Microbial Biotechnology, C entro Nacional de
Biotecnología, Consejo Superior de Investigaciones Científi cas , M adrid , S pain
KAREN G . F LEMING • T.C. Jenkins Department of Biophysics, Johns Hopkins University ,
Baltimore, M D, USA
DENNIS G ESSMANN • T.C. Jenkins Department of Biophysics, J ohns Hopkins University ,
Baltimore, M D, USA
FABIAN GRUSS • Biozentrum, U niversity of Basel , B asel , S witzerland
CHRISTOPHER S. HAYES • Department of Molecular, Cellular and Developmental Biology,
University of California , Santa Barbara, C A , U SA ; B iomolecular Science
and Engineering Program , University of California , S anta Barbara, C A , U SA
SEBASTIAN H ILLER • Biozentrum, U niversity of Basel , B asel, Switzerland
YIHUA HUANG • National Laboratory of Biomacromolecules, National Center of Protein
Science-Beijing, Institute of Biophysics, C hinese Academy of Sciences , B eijing, C hina
MARK JEEVES • School of Cancer Sciences, U niversity of Birmingham , E dgbaston,
Birmingham, U K
KELLY H . KIM • Department of Molecular Biology and Biochemistry , S imon Fraser
University , B urnaby, B C, Canada
ix