Main description:

This detailed volume utilizes our current understanding of the structural basis of multidrug recognition and multidrug efflux mechanisms to provide protocols involving these vital intrinsic membrane proteins widely distributed in bacteria. Beginning with protocols for the structural analysis of bacterial multidrug exporters, the book continues with sections on biochemical and bioengineering analysis, computational analysis, biomedical approaches, as well as advanced technologies expected to be applied to multidrug efflux transport studies. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective chapters, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Authoritative and practical, Bacterial Multidrug Exporters: Methods and Protocols serves as an ideal guide to

this fast moving field of study.

Contents:

Part I: Structural Analysis of Bacterial Multidrug Exporters

1. High Resolution Crystallographic Analysis of AcrB Using Designed Ankyrin Repeat Proteins (DARPins)

Heng Keat Tam, Viveka Nand Malviya, and Klaas M. Pos

2. Crystallographic Analysis of Drug and Inhibitor-Binding Structure of RND-Type Multidrug Exporter AcrB in Physiologically-Relevant Asymmetric Crystals

Ryosuke Nakashima, Keisuke Sakurai, and Akihito Yamaguchi

3. Crystallographic Analysis of MATE-Type Multidrug Exporter with Its Inhibitors

Tsukasa Kusakizako, Yoshiki Tanaka, Christopher J. Hipolito, Hiroaki Suga, and Osamu Nureki

4. Crystallographic Analysis of the CusBA Heavy-Me

tal Efflux Complex of Escherichia coli

Jared A. Delmar and Edward W. Yu

5. Purification of AcrAB-TolC Multidrug Efflux Pump for Cryo-EM Analysis

Dijun Du, Zhao Wang, Wah Chiu, and Ben F. Luisi

6. NMR Spectroscopy Approach to Study the Structure, Orientation, and Mechanism of the Multidrug Exporter EmrE

Maureen Leninger and Nathaniel J. Traaseth

7. Generation of Conformation-Specific Antibody Fragments for Crystallization of the Multidrug Resistance Transporter MdfA

Frank Jaenecke, Yoshiko Nakada-Nakura, Kumar Nagarathinam, Satoshi Ogasawara, Kehong Liu, Yunhon Hotta, So Iwata, Nor

imichi Nomura, and Mikio Tanabe

Part II: Biochemical and Bioengineering Analysis of Bacterial Multidrug Exporters

8. Biochemical Reconstitution and Characterization of Multi-Component Drug Efflux Transporters

Martin Picard, Elena B. Tikhonova, Isabelle Broutin, Shuo Lu, Alice Verchere, and Helen I. Zgurskaya

9. Covalently Linked Trimers of RND (Resistance-Nodulation-Division) Efflux Transporters to Study Their Mechanism of Action: Escherichia coli AcrB Multidrug Exporter as an Example

Hiroshi Nikaido

10. Determining Ligand Path through a Major Drug Transporter, AcrB, in Escherichia coli

Fasahath Husain and Hiroshi Nikaido

Part III: Computational Analysis of Bacterial Multidrug Exporters

11. Molecular Modeling of Multi-Drug Properties of Resistance Nodulation Division (RND) Transporters

Pierpaolo Cacciotto, V. K. Ramaswamy, Giuliano Malloci, Paolo Ruggerone, and Attilio V. Vargiu

12. A Transcriptomic Approach to Identify Novel Drug Efflux Pumps in Bacteria

Liping Li, Sasha G. Tetu, Ian T. Paulsen, and Karl A. Hassan

Part IV: Biomedical Approach for Bacterial Multidrug Exporters

13. Regulation of the Expression of Bacterial Multidrug Exporters by Two-Component Signal Transduction Systems

Kunihiko Nishino

14. Study of the Expression of Bacterial Multidrug Efflux Pumps in Anaerobic Conditions

Jingjing Sun, Ziqing Deng, Danny Ka Chun Fung, and Aixin Yan

15. Identification of a Staphylococcus aureus Efflux Pump Regulator Using a DNA-Protein Affinity Technique

Que Chi Truong-Bolduc and David C. Hooper

16. High Throughput Flow Cytometry Screening of Multidrug Efflux Systems

Mark K. Haynes, Matthew Garcia, Ryan Peters, Anna Waller, Pietro Tedesco, Oleg Ursu, Cristian G. Bologa, Radleigh G. Santos, Clemencia Pinilla, Terry H. Wu, Julie A. Lovchik, Tudor I. Oprea, Larry A. Sklar, and George P. Tegos

Part V: Advanced Technologies Expected for Application to Multi

drug Efflux Transport Studies

17. Single-Molecule Analysis of Membrane Transporter Activity by Means of a Microsystem

Rikiya Watanabe, Naoki Soga, Shin-ya Ohdate, and Hiroyuki Noji

18. Large Scale Femtoliter Droplet Array for Single Cell Efflux Assay of Bacteria

Ryota Iino, Shouichi Sakakihara, Yoshimi Matsumoto, and Kunihiko Nishino

19. Reconstitution and Transport Analysis of Eukaryotic Transporters in the Post-Genomic Era

Hiroshi Omote and Yoshinori Moriyama