Preparation and functional characterisation

A robust protocol for refolding TolC and other outer membrane components of tripartite efflux pumps from inclusion bodies.

Daufel A, Cordova A, Budiardjo SJ, Slusky JSG. 

Methods Enzymol. 2025;724:3-20. 

doi: 10.1016/bs.mie.2025.09.010. Epub 2025 Oct 11. 

PMID: 41309176.

A refined protocol for refolding TolC and its homologs (e.g., VceC and CmeC) from inclusion bodies, significantly improving protein yield. 

=> importance of detergent choice and protein concentration as key factors for successful refolding. 

=> common pitfalls + solutions for optimizing protein stability.

Functional analysis and NMR studies of multi-drug efflux pumps.

Ralph D, Yeh V, Goode A, Blair JMA, Williams P, Bonev BB. 

Methods Enzymol. 2025;724:21-37. 

doi: 10.1016/bs.mie.2025.09.005. Epub 2025 Sep 29. 

PMID: 41309172.

Workflow for characterizing IM efflux pumps, combining in vivo functional assays with in vitro structural analysis using ssNMR. 

Fluorescence-based assay => efflux activity in whole cells : real-time assessment of transport dynamics. 

NMR techniques => protein-substrate interactions and conformational changes.

Using hydrogen/deuterium exchange mass spectrometry to understand bacterial membrane efflux proteins.

Russell Lewis B, Hammerschmid D, Sys J, Reading E. 

Methods Enzymol. 2025;724:39-82. 

doi: 10.1016/bs.mie.2025.08.026. Epub 2025 Sep 18. 

PMID: 41309178.

HDX-MS = powerful tool for studying the dynamics of MPs. 

Here : HDX-MS can reveal conformational changes in efflux pumps, such as AcrB, when interacting with substrates or inhibitors. 

AcrB in NDs => HDX-MS identifies inhibitor-induced perturbations in protein backbone motions. 

Doxorubicin transport measurements for a bacterial multidrug transport protein.

Hsieh PY, van Veen HW. 

Methods Enzymol. 2025;724:83-97. 

doi: 10.1016/bs.mie.2025.09.018. Epub 2025 Oct 14. 

PMID: 41309185.

NorM-VC = MATE family multidrug transporter from Vibrio cholerae, confers resistance by extruding antibiotics like doxorubicin. 

Here: detailed protocols for expressing, purifying, and reconstituting NorM-VC into proteoliposomes, followed by transport activity measurements in Lactococcus lactis and proteoliposomes (adaptable for HT screening).

Molecular investigation of drug efflux pumps by deep mutational scanning.

Thavarasah S, Meier G, Partas A, Seeger MA. 

Methods Enzymol. 2025;724:99-131. 

doi: 10.1016/bs.mie.2025.09.007. Epub 2025 Oct 1. 

PMID: 41309186.

DMS = HT method for systematically assessing the functional impact of mutations in drug efflux pumps. 

Here :  DMS to the ABC exporter EfrCD from Enterococcus faecalis, linking variant activity to bacterial growth phenotypes in the presence of transport substrates. 

Site-saturation mutagenesis + NGS => comprehensive mutational landscapes. 

Reconstitution of tripartite efflux pumps MexAB-OprM and MacAB-TolC in biomimetic systems.

Lodé A, Novelli M, Madigou C, Picard M. 

Methods Enzymol. 2025;724:133-158. 

doi: 10.1016/bs.mie.2025.09.019. Epub 2025 Oct 23. 

PMID: 41309169.

Detailed protocols for reconstituting ABC and RND tripartite efflux pumps into proteoliposomes, nanodiscs, or amphipols, for functional and structural studies. 

Preparation and activity characterization of a type IV ABC transporter efflux pump in peptidiscs.

Kerboeuf J, Galisson F, Gonzalez C, Jault JM, Kaplan E. 

Methods Enzymol. 2025;724:159-179. 

doi: 10.1016/bs.mie.2025.09.008. Epub 2025 Sep 29. 

PMID: 41309170.

PatAB from Streptococcus pneumoniae = key ABC in fluoroquinolone resistance, but their structural and functional analysis is challenging. 

Here : reconstitution of PatAB into peptidiscs. 

Mass photometry and SEC-MALS => transporter mass and ATPase activity in different environments. 

Reconstitution and functional characterization of MmpL3, an essential transporter from Mycobacterium tuberculosis.

Babii S, Choukate K, Zgurskaya HI. 

Methods Enzymol. 2025;724:181-210. 

doi: 10.1016/bs.mie.2025.08.025. Epub 2025 Sep 19. 

PMID: 41309171.

MmpL3 = essential transporter in Mycobacterium tuberculosis, involved in OM assembly and drug efflux. 

Here : protocols for reconstituting MmpL3 and characterizing its proton-coupled lipid transport and TM proton transfer activities. 

Challenges in assaying MmpL3 function, including substrate extraction from membranes and inhibitor binding. 

Discovery and functional characterization of archaeal efflux transporters.

Thompson TP, Fakhoury AA, Rahman KM, Gilmore BF. 

Methods Enzymol. 2025;724:211-274. 

doi: 10.1016/bs.mie.2025.09.014. Epub 2025 Oct 28. 

PMID: 41309173.

Archaeal efflux transporters are emerging as important contributors to antimicrobial resistance, yet their functional characterization remains limited. 

Here : methodologies for identifying and studying efflux pumps in Halorubrum amylolyticum as a model. 

=> genomic identification, functional assays with fluorescent substrates, and heterologous expression in E. coli for activity testing + structural approaches (homology modeling and molecular docking).

Structural and computational studies

Crystallographic studies of efflux transporters with transport substrates and inhibitors.

Murakami S. 

Methods Enzymol. 2025;724:277-297. 

doi: 10.1016/bs.mie.2025.09.006. Epub 2025 Oct 6. 

PMID: 41309174.

X-ray crystallography methods for studying multidrug efflux transporters. 

Strategies for obtaining high-resolution structures of transporter-ligand complexes.

Importance of conformational dynamics in substrate recognition.

Computational approaches for modelling multidrug efflux pumps of the resistance nodulation-cell division superfamily.

Athar M, Gervasoni S, Malloci G, Ruggerone P, Vargiu AV. 

Methods Enzymol. 2025;724:299-361. 

doi: 10.1016/bs.mie.2025.10.001. Epub 2025 Nov 8. 

PMID: 41309175.

Computational modeling => atomistic insights into substrate recognition, transport, and inhibitor binding. 

Here : protocols for homology modeling, molecular docking, and MD simulations, along with guidelines for estimating binding free energy. 

Best practices for ensuring reproducibility and avoiding common pitfalls in computational studies.

MexB/inhibitor complexes preparation for crystallography and cryo-EM, comparison and possible pitfalls.

Wehbi H, Cece Q, Phan G, Broutin I. 

Methods Enzymol. 2025;724:365-396. 

doi: 10.1016/bs.mie.2025.08.028. Epub 2025 Sep 18. 

PMID: 41309177.

Structural studies of MexB + inhibitors = careful optimization to ensure high-quality data. 

Here : comparison of protocols for crystallography and cryo-EM => key parameters for successful structure determination. 

Common pitfalls, such as sample heterogeneity and ligand binding stability, and provide strategies to overcome them.

Stabilization of multidrug efflux transporter MdfA and antibody fragments complex by cross-linking for Cryo-EM single-particle analysis.

Inaba-Inoue S, Moriya T, Nomura N, Tanabe M. 

Methods Enzymol. 2025;724:397-412. 

doi: 10.1016/bs.mie.2025.09.011. Epub 2025 Oct 6. 

PMID: 41309179.

Cryo-EM analysis of small MPs like MdfA can be challenging due to sample instability during grid preparation. 

Here : cross-linking protocol to stabilize MdfA-antibody fragment complexes, improving particle alignment and resolution. 

Step-by-step guide for sample preparation and data collection.

Reconstitution of RND tripartite multidrug complexes for single-particle electron microscopy.

Boyer E, Daury L, Giraud MF, Gerbod-Giannone MC, Poussard S, Broutin I, Taveau JC, Lambert O. 

Methods Enzymol. 2025;724:413-437. 

doi: 10.1016/bs.mie.2025.08.024. Epub 2025 Sep 13. 

PMID: 41309180.

Protocols for reconstituting efflux pumps into nanodiscs and analyzing their structure using single-particle EM. 

=> pump assembly with cognate and non-cognate partners : insights into molecular recognition and functional coordination.

Cryo-electron microscopy (Cryo-EM) structural determination of the MmpL family of transporters.

Kundracik E, Gregor WD, Maharjan R, Zhang Z, Klenotic PA, Yu EW. 

Methods Enzymol. 2025;724:439-467. 

doi: 10.1016/bs.mie.2025.10.002. Epub 2025 Oct 28. 

PMID: 41309181.

Protocols for obtaining high-resolution structures of MmpL proteins, even from heterogeneous samples. 

In situ structural analysis of tripartite efflux assemblies by cryoET.

Zhou X, Wang Z. 

Methods Enzymol. 2025;724:469-485. 

doi: 10.1016/bs.mie.2025.09.009. Epub 2025 Oct 8. 

PMID: 41309182.

cryoET => in situ visualization of tripartite efflux pumps like AcrAB-TolC and MacAB-TolC within their native cellular context. 

Here : detailed protocol for cryoET-based structural characterization, allowing direct observation of pump assembly and function. 

Adaptable to other bacterial strains; powerful tool for studying antibiotic resistance mechanisms.

Functional interplay and single cell heterogeneity

Methods for investigating functional interplay between efflux pumps in Escherichia coli.

Wright M, Caswell MR, Cox G. 

Methods Enzymol. 2025;724:489-502. 

doi: 10.1016/bs.mie.2025.10.003. Epub 2025 Oct 29. 

PMID: 41309183.

Efflux pumps in E. coli exhibit functional interplay, contributing to complex resistance phenotypes. 

Here : genetic and phenotypic methods for studying these interactions, using efflux-deficient mutants to overcome redundancies. 

Protocols for constructing strains and assessing the impact of dual efflux pump combinations on resistance. 

Quantitative single-cell imaging of efflux pump heterogeneity and antibiotic response dynamics.

Li R, Bakshi S. 

Methods Enzymol. 2025;724:503-535. 

doi: 10.1016/bs.mie.2025.08.029. Epub 2025 Oct 25. 

PMID: 41309184.

Understanding bacterial survival under antibiotic stress requires tools to resolve single-cell heterogeneity in efflux pump expression. 

Here : microfluidic imaging platform for quantifying AcrAB-TolC dynamics in E. coli during antibiotic exposure. 

=> combination of lineage tracking with fluorescence imaging to dissect non-genetic variability in efflux activity.