This volume is comprised of a collection of experimental protocols for common techniques and strategies used to study the biogenesis of b-barrel outer membrane proteins in Gram-negative bacteria. The BAM Complex: Methods and Protocols guides readers through methods on the function of the BAM complex, the roles played by each of the individual components, the expression and purification of the components, crystallization and structure determination of the components, and how the individual Bam components may assemble into a functional complex. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and cutting-edge, The BAM Complex: Methods and Protocols will serve as an invaluable reference for those interested in studying the BAM complex.
1. The β-barrel Assembly Machinery Complex
Denisse L. Leyton, Matthew J. Belousoff, and Trevor Lithgow
2. Yeast Mitochondria as a Model System to Study the Biogenesis of Bacterial β-barrel Proteins
Thomas Ulrich, Philipp Oberhettinger, Ingo B. Autenrieth, and Doron Rapaport
3. Experimental Methods for Studying the BAM Complex in Neisseria meningitidis
Martine P. Bos, Ria Tommassen-van Boxtel, and Jan Tommassen
4. Heat Modifiability of Outer Membrane Proteins from Gram-Negative Bacteria
Nicholas Noinaj, Adam J. Kuszak, and Susan K. Buchanan
5. The Role of a Destabilized Membrane for OMP Insertion
Ashlee M. Plummer, Dennis Gessmann, 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
Amit Luthra, Arvind Anand, and Justin D. Radolf
7. Analyzing the Role of Periplasmic Folding Factors in the Biogenesis of OMPs and Members of the Type V Secretion System
Gustavo Bodelón, Elvira Marín, and Luis Ángel Fernández
8. An In Vitro Assay for Substrate Translocation by FhaC in Liposomes
Enguo Fan, Derrick Norell, and Matthias Müller
9. Measuring Cell-cell Binding Using Flow-Cytometry
Zachary C. Ruhe, Christopher S. Hayes, and David A. Low
10. Methods to Characterize Folding and Function of BamA Crosslink Mutants
Adam J. Kuszak, Nicholas Noinaj, and Susan K. Buchanan
11. Small Angle X-ray Scattering (SAXS) Characterization of the POTRA Domains of BamA
Pamela Arden Doerner and Marcelo Carlos Sousa
12. Assessing the Outer Membrane Insertion and Folding of Multimeric Transmembrane β-barrel Proteins
Jack C. Leo, Philipp Oberhettinger, and Dirk Linke
13. The Expression, Purification, and Structure Determination of BamA from E. coli
Dongchun Ni and Yihua Huang
14. Expression and Purification of the Individual Bam Components BamB-E
Suraaj Aulakh, Kelly H. Kim, and Mark Paetzel
15. Structure Determination of the BAM Complex Accessory Lipoproteins BamB-E
Kornelius Zeth
16. An In Vitro Assay for Outer Membrane Protein Assembly by the BAM complex
Giselle Roman-Hernandez and Harris D. Bernstein
17. Identification of BamC on the Surface of E. coli
Chaille T. Webb and Trevor Lithgow
18. Construction and Characterization of an E. coli bamD Depletion Strain
Dante P. Ricci
19. Expression, Purification and Screening of BamE, a Component of the BAM Complex, for Structural Characterization
Mark Jeeves, Pooja Sridhar, and Timothy J. Knowles
20. Purification and Bicelle Crystallization for Structure Determination of the E. coli Outer Membrane Protein TamA
Fabian Gruss, Sebastian Hiller, and Timm Maier
21. Strategies for the Analysis of Bam Recognition Motifs in Outer Membrane Proteins
Nagarajan Paramasivam and Dirk Linke
22. Summary and Future Directions
Nicholas Noinaj and Susan K. Buchanan
