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Article: Assessing Membrane Protein Structural Dynamics within Lipid Nanodiscs.

Reading, Eamonn

2019 Volume 44, Issue 11, Page(s) 989–990

MeSH term(s)	Deuterium/chemistry ; Isotope Labeling ; Kinetics ; Lipid Bilayers/chemistry ; Mass Spectrometry/methods ; Membrane Proteins/chemistry ; Nanostructures/chemistry ; Protein Binding
Chemical Substances	Lipid Bilayers ; Membrane Proteins ; Deuterium (AR09D82C7G)
Language	English
Publishing date	2019-09-11
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Technical Report
ZDB-ID	194216-5
ISSN	1362-4326 ; 0968-0004 ; 0376-5067
ISSN (online)	1362-4326
ISSN	0968-0004 ; 0376-5067
DOI	10.1016/j.tibs.2019.08.003
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Article ; Online: Structural Mass Spectrometry of Membrane Proteins within Their Native Lipid Environments.

Reading, Eamonn

Chemistry (Weinheim an der Bergstrasse, Germany)

2018 Volume 24, Issue 51, Page(s) 13391–13398

Abstract: Mass spectrometry has emerged as an important structural biology tool for understanding membrane protein structure, function, and dynamics. Generally, structural mass spectrometry of membrane proteins has been performed on purified or reconstituted ... ...

Abstract	Mass spectrometry has emerged as an important structural biology tool for understanding membrane protein structure, function, and dynamics. Generally, structural mass spectrometry of membrane proteins has been performed on purified or reconstituted systems which lack the native lipid membrane and cellular environments. However, there has been progress in the use and adaptations of these methods for probing membrane proteins within increasingly more native contexts. In this Concept article the use and utility of structural mass spectrometry techniques for studying membrane proteins within native environments are highlighted.
MeSH term(s)	Cell Line ; Cell Membrane/physiology ; Cellular Microenvironment/physiology ; Lipids/chemistry ; Mass Spectrometry ; Membrane Proteins/chemistry ; Membrane Proteins/metabolism ; Peptides/chemistry ; Peptides/metabolism ; Protein Binding ; Protein Conformation ; Signal Transduction ; Thermodynamics
Chemical Substances	Lipids ; Membrane Proteins ; Peptides
Language	English
Publishing date	2018-07-04
Publishing country	Germany
Document type	Journal Article ; Review
ZDB-ID	1478547-x
ISSN	1521-3765 ; 0947-6539
ISSN (online)	1521-3765
ISSN	0947-6539
DOI	10.1002/chem.201801556
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Article ; Online: Cell-Free Synthesis Strategies to Probe Co-translational Folding of Proteins Within Lipid Membranes.

Harris, Nicola J / Reading, Eamonn / Booth, Paula J

Methods in molecular biology (Clifton, N.J.)

2022 Volume 2433, Page(s) 273–292

Abstract: In order to comprehend the molecular basis of transmembrane protein biogenesis, methods are required that are capable of investigating the co-translational folding of these hydrophobic proteins. Equally, in artificial cell studies, controllable methods ... ...

Abstract	In order to comprehend the molecular basis of transmembrane protein biogenesis, methods are required that are capable of investigating the co-translational folding of these hydrophobic proteins. Equally, in artificial cell studies, controllable methods are desirable for in situ synthesis of membrane proteins that then direct reactions in the synthetic cell membrane. Here we describe a method that exploits cell-free expression systems and tunable membrane mimetics to facilitate co-translational studies. Alteration of the lipid bilayer composition improves the efficiency of the folding system. The approach also enables membrane transport proteins to be made and inserted into artificial cell platforms such as droplet interface bilayers. Importantly, this gives a new facet to the droplet networks by enabling specific transport of molecules across the synthetic bilayer against a concentration gradient. This method also includes a protocol to pause and restart translation of membrane proteins at specified positions during their co-translational folding. This stop-start strategy provides an avenue to investigate whether the proteins fold in sequence order, or if the correct fold of N-terminal regions is reliant on the synthesis of downstream residues.
MeSH term(s)	Cell-Free System/metabolism ; Lipid Bilayers/chemistry ; Membrane Proteins/metabolism ; Membrane Transport Proteins/metabolism ; Protein Folding
Chemical Substances	Lipid Bilayers ; Membrane Proteins ; Membrane Transport Proteins
Language	English
Publishing date	2022-01-05
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-0716-1998-8_17
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Article ; Online: Structural mass spectrometry approaches to understand multidrug efflux systems.

Russell Lewis, Benjamin / Lawrence, Ryan / Hammerschmid, Dietmar / Reading, Eamonn

Essays in biochemistry

2022 Volume 67, Issue 2, Page(s) 255–267

Abstract: Multidrug efflux pumps are ubiquitous across both eukaryotes and prokaryotes, and have major implications in antimicrobial and multidrug resistance. They reside within cellular membranes and have proven difficult to study owing to their hydrophobic ... ...

Abstract	Multidrug efflux pumps are ubiquitous across both eukaryotes and prokaryotes, and have major implications in antimicrobial and multidrug resistance. They reside within cellular membranes and have proven difficult to study owing to their hydrophobic character and relationship with their compositionally complex lipid environment. Advances in structural mass spectrometry (MS) techniques have made it possible to study these systems to elucidate critical information on their structure-function relationships. For example, MS techniques can report on protein structural dynamics, stoichiometry, connectivity, solvent accessibility, and binding interactions with ligands, lipids, and other proteins. This information proving powerful when used in conjunction with complementary structural biology methods and molecular dynamics (MD) simulations. In the present review, aimed at those not experts in MS techniques, we report on the current uses of MS in studying multidrug efflux systems, practical considerations to consider, and the future direction of the field. In the first section, we highlight the importance of studying multidrug efflux proteins, and introduce a range of different MS techniques and explain what information they yield. In the second section, we review recent studies that have utilised MS techniques to study and characterise a range of different multidrug efflux systems.
MeSH term(s)	Anti-Bacterial Agents/chemistry ; Anti-Bacterial Agents/metabolism ; Anti-Bacterial Agents/pharmacology ; Drug Resistance, Multiple, Bacterial ; Biological Transport ; Mass Spectrometry
Chemical Substances	Anti-Bacterial Agents
Language	English
Publishing date	2022-12-20
Publishing country	England
Document type	Review ; Journal Article ; Research Support, Non-U.S. Gov't
ISSN	1744-1358 ; 0071-1365
ISSN (online)	1744-1358
ISSN	0071-1365
DOI	10.1042/EBC20220190
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Article ; Online: Chromatographic Phospholipid Trapping for Automated H/D Exchange Mass Spectrometry of Membrane Protein-Lipid Assemblies.

Hammerschmid, Dietmar / Calvaresi, Valeria / Bailey, Chloe / Russell Lewis, Benjamin / Politis, Argyris / Morris, Michael / Denbigh, Laetitia / Anderson, Malcolm / Reading, Eamonn

Analytical chemistry

2023 Volume 95, Issue 5, Page(s) 3002–3011

Abstract: Lipid interactions modulate the function, folding, structure, and organization of membrane proteins. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) has emerged as a useful tool to understand the structural dynamics of these proteins within lipid ... ...

Abstract	Lipid interactions modulate the function, folding, structure, and organization of membrane proteins. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) has emerged as a useful tool to understand the structural dynamics of these proteins within lipid environments. Lipids, however, have proven problematic for HDX-MS analysis of membrane-embedded proteins due to their presence of impairing proteolytic digestion, causing liquid chromatography column fouling, ion suppression, and/or mass spectral overlap. Herein, we describe the integration of a chromatographic phospholipid trap column into the HDX-MS apparatus to enable online sample delipidation prior to protease digestion of deuterium-labeled protein-lipid assemblies. We demonstrate the utility of this method on membrane scaffold protein-lipid nanodisc─both empty and loaded with the ∼115 kDa transmembrane protein AcrB─proving efficient and automated phospholipid capture with minimal D-to-H back-exchange, peptide carry-over, and protein loss. Our results provide insights into the efficiency of phospholipid capture by ZrO
MeSH term(s)	Phospholipids ; Deuterium ; Membrane Lipids ; Mass Spectrometry/methods ; Deuterium Exchange Measurement/methods ; Membrane Proteins ; Peptide Hydrolases
Chemical Substances	Phospholipids ; Deuterium (AR09D82C7G) ; Membrane Lipids ; Membrane Proteins ; Peptide Hydrolases (EC 3.4.-)
Language	English
Publishing date	2023-01-27
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1508-8
ISSN	1520-6882 ; 0003-2700
ISSN (online)	1520-6882
ISSN	0003-2700
DOI	10.1021/acs.analchem.2c04876
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Article ; Online: Chromatographic Phospholipid Trapping for Automated H/D Exchange Mass Spectrometry of Membrane Protein–Lipid Assemblies

Hammerschmid, Dietmar / Calvaresi, Valeria / Bailey, Chloe / Russell Lewis, Benjamin / Politis, Argyris / Morris, Michael / Denbigh, Laetitia / Anderson, Malcolm / Reading, Eamonn

Analytical Chemistry. 2023 Jan. 27, v. 95, no. 5 p.3002-3011

2023

Abstract	Lipid interactions modulate the function, folding, structure, and organization of membrane proteins. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) has emerged as a useful tool to understand the structural dynamics of these proteins within lipid environments. Lipids, however, have proven problematic for HDX-MS analysis of membrane-embedded proteins due to their presence of impairing proteolytic digestion, causing liquid chromatography column fouling, ion suppression, and/or mass spectral overlap. Herein, we describe the integration of a chromatographic phospholipid trap column into the HDX-MS apparatus to enable online sample delipidation prior to protease digestion of deuterium-labeled protein–lipid assemblies. We demonstrate the utility of this method on membrane scaffold protein–lipid nanodisc—both empty and loaded with the ∼115 kDa transmembrane protein AcrB—proving efficient and automated phospholipid capture with minimal D-to-H back-exchange, peptide carry-over, and protein loss. Our results provide insights into the efficiency of phospholipid capture by ZrO₂-coated and TiO₂ beads and describe how solution conditions can be optimized to maximize not only the performance of our online but also the existing offline, delipidation workflows for HDX-MS. We envision that this HDX-MS method will significantly ease membrane protein analysis, allowing to better interrogate their dynamics in artificial lipid bilayers or even native cell membranes.
Keywords	analytical chemistry ; automation ; deuterium ; digestion ; lipid bilayers ; liquid chromatography ; mass spectrometry ; peptides ; phospholipids ; protein depletion ; proteinases ; proteolysis ; transmembrane proteins
Language	English
Dates of publication	2023-0127
Size	p. 3002-3011.
Publishing place	American Chemical Society
Document type	Article ; Online
ZDB-ID	1508-8
ISSN	1520-6882 ; 0003-2700
ISSN (online)	1520-6882
ISSN	0003-2700
DOI	10.1021/acs.analchem.2c04876
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Article: Peptide-Based Approach to Inhibition of the Multidrug Resistance Efflux Pump AcrB

Jesin, Joshua A / Stone, Tracy A / Mitchell, Chloe J / Reading, Eamonn / Deber, Charles M

Biochemistry. 2020 Oct. 07, v. 59, no. 41

2020

Abstract: Clinically relevant multidrug-resistant bacteria often arise due to overproduction of membrane-embedded efflux proteins that are capable of pumping antibiotics out of the bacterial cell before the drugs can exert their intended toxic effect. The ... ...

Abstract	Clinically relevant multidrug-resistant bacteria often arise due to overproduction of membrane-embedded efflux proteins that are capable of pumping antibiotics out of the bacterial cell before the drugs can exert their intended toxic effect. The Escherichia coli membrane protein AcrB is the archetypal protein utilized for bacterial efflux study because it can extrude a diverse range of antibiotic substrates and has close homologues in many Gram-negative pathogens. Three AcrB subunits, each of which contains 12 transmembrane (TM) helices, are known to trimerize to form the minimal functional unit, stabilized noncovalently by helix–helix interactions between TM1 and TM8. To inhibit the efflux activity of AcrB, we have rationally designed synthetic peptides aimed at destabilizing the AcrB trimerization interface by outcompeting the subunit interaction sites within the membrane. Here we report that peptides mimicking TM1 or TM8, with flanking N-terminal peptoid tags, and C-terminal lysine tags that aid in directing the peptides to their membrane-embedded target, decrease the AcrB-mediated efflux of the fluorescent substrate Nile red and potentiate the effect of the antimicrobials chloramphenicol and ethidium bromide. To further characterize the motif encompassing the interaction between TM1 and TM8, we used Förster resonance energy transfer to demonstrate dimerization. Using the TM1 and TM8 peptides, in conjunction with several selected mutant peptides, we highlight residues that may increase the potency and specificity of the peptide drug candidates. In targeting membrane-embedded protein–protein interactions, this work represents a novel approach to AcrB inhibition and, more broadly, a potential route to a new category of efflux pump inhibitors.
Keywords	Escherichia coli ; Gram-negative bacteria ; N-substituted glycines ; antibiotics ; chloramphenicol ; dimerization ; energy transfer ; ethidium ; fluorescence ; lysine ; multiple drug resistance ; mutants ; protein-protein interactions ; synthetic peptides ; toxicity ; transporters
Language	English
Dates of publication	2020-1007
Size	p. 3973-3981.
Publishing place	American Chemical Society
Document type	Article
Note	NAL-light
ZDB-ID	1108-3
ISSN	1520-4995 ; 0006-2960
ISSN (online)	1520-4995
ISSN	0006-2960
DOI	10.1021/acs.biochem.0c00417
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Article ; Online: Peptide-Based Approach to Inhibition of the Multidrug Resistance Efflux Pump AcrB.

Jesin, Joshua A / Stone, Tracy A / Mitchell, Chloe J / Reading, Eamonn / Deber, Charles M

Biochemistry

2020 Volume 59, Issue 41, Page(s) 3973–3981

Abstract	Clinically relevant multidrug-resistant bacteria often arise due to overproduction of membrane-embedded efflux proteins that are capable of pumping antibiotics out of the bacterial cell before the drugs can exert their intended toxic effect. The
MeSH term(s)	Binding Sites ; Drug Resistance, Multiple, Bacterial/genetics ; Drug Resistance, Multiple, Bacterial/physiology ; Escherichia coli/chemistry ; Escherichia coli/metabolism ; Escherichia coli Proteins/chemistry ; Escherichia coli Proteins/metabolism ; Fluorescence Resonance Energy Transfer ; Membrane Transport Proteins/chemistry ; Membrane Transport Proteins/metabolism ; Multidrug Resistance-Associated Proteins/chemistry ; Multidrug Resistance-Associated Proteins/metabolism ; Peptides/chemistry ; Peptides/metabolism ; Protein Conformation
Chemical Substances	Escherichia coli Proteins ; Membrane Transport Proteins ; Multidrug Resistance-Associated Proteins ; Peptides
Language	English
Publishing date	2020-10-07
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1108-3
ISSN	1520-4995 ; 0006-2960
ISSN (online)	1520-4995
ISSN	0006-2960
DOI	10.1021/acs.biochem.0c00417
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Article ; Online: Structural dynamics in the evolution of SARS-CoV-2 spike glycoprotein.

Calvaresi, Valeria / Wrobel, Antoni G / Toporowska, Joanna / Hammerschmid, Dietmar / Doores, Katie J / Bradshaw, Richard T / Parsons, Ricardo B / Benton, Donald J / Roustan, Chloë / Reading, Eamonn / Malim, Michael H / Gamblin, Steve J / Politis, Argyris

Nature communications

2023 Volume 14, Issue 1, Page(s) 1421

Abstract: SARS-CoV-2 spike glycoprotein mediates receptor binding and subsequent membrane fusion. It exists in a range of conformations, including a closed state unable to bind the ACE2 receptor, and an open state that does so but displays more exposed antigenic ... ...

Abstract	SARS-CoV-2 spike glycoprotein mediates receptor binding and subsequent membrane fusion. It exists in a range of conformations, including a closed state unable to bind the ACE2 receptor, and an open state that does so but displays more exposed antigenic surface. Spikes of variants of concern (VOCs) acquired amino acid changes linked to increased virulence and immune evasion. Here, using HDX-MS, we identified changes in spike dynamics that we associate with the transition from closed to open conformations, to ACE2 binding, and to specific mutations in VOCs. We show that the RBD-associated subdomain plays a role in spike opening, whereas the NTD acts as a hotspot of conformational divergence of VOC spikes driving immune evasion. Alpha, beta and delta spikes assume predominantly open conformations and ACE2 binding increases the dynamics of their core helices, priming spikes for fusion. Conversely, substitutions in omicron spike lead to predominantly closed conformations, presumably enabling it to escape antibodies. At the same time, its core helices show characteristics of being pre-primed for fusion even in the absence of ACE2. These data inform on SARS-CoV-2 evolution and omicron variant emergence.
MeSH term(s)	Humans ; Spike Glycoprotein, Coronavirus/genetics ; Angiotensin-Converting Enzyme 2 ; COVID-19 ; SARS-CoV-2/genetics ; Mutation
Chemical Substances	spike protein, SARS-CoV-2 ; Spike Glycoprotein, Coronavirus ; Angiotensin-Converting Enzyme 2 (EC 3.4.17.23)
Language	English
Publishing date	2023-03-14
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2553671-0
ISSN	2041-1723 ; 2041-1723
ISSN (online)	2041-1723
ISSN	2041-1723
DOI	10.1038/s41467-023-36745-0
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Article ; Online: Conformational restriction shapes the inhibition of a multidrug efflux adaptor protein.

Russell Lewis, Benjamin / Uddin, Muhammad R / Moniruzzaman, Mohammad / Kuo, Katie M / Higgins, Anna J / Shah, Laila M N / Sobott, Frank / Parks, Jerry M / Hammerschmid, Dietmar / Gumbart, James C / Zgurskaya, Helen I / Reading, Eamonn

Nature communications

2023 Volume 14, Issue 1, Page(s) 3900

Abstract: Membrane efflux pumps play a major role in bacterial multidrug resistance. The tripartite multidrug efflux pump system from Escherichia coli, AcrAB-TolC, is a target for inhibition to lessen resistance development and restore antibiotic efficacy, with ... ...

Abstract	Membrane efflux pumps play a major role in bacterial multidrug resistance. The tripartite multidrug efflux pump system from Escherichia coli, AcrAB-TolC, is a target for inhibition to lessen resistance development and restore antibiotic efficacy, with homologs in other ESKAPE pathogens. Here, we rationalize a mechanism of inhibition against the periplasmic adaptor protein, AcrA, using a combination of hydrogen/deuterium exchange mass spectrometry, cellular efflux assays, and molecular dynamics simulations. We define the structural dynamics of AcrA and find that an inhibitor can inflict long-range stabilisation across all four of its domains, whereas an interacting efflux substrate has minimal effect. Our results support a model where an inhibitor forms a molecular wedge within a cleft between the lipoyl and αβ barrel domains of AcrA, diminishing its conformational transmission of drug-evoked signals from AcrB to TolC. This work provides molecular insights into multidrug adaptor protein function which could be valuable for developing antimicrobial therapeutics.
MeSH term(s)	Membrane Transport Proteins/metabolism ; Escherichia coli Proteins/metabolism ; Multidrug Resistance-Associated Proteins/metabolism ; Biological Transport ; Escherichia coli/metabolism ; Anti-Bacterial Agents/pharmacology ; Anti-Bacterial Agents/metabolism ; Bacterial Outer Membrane Proteins/metabolism
Chemical Substances	Membrane Transport Proteins ; Escherichia coli Proteins ; Multidrug Resistance-Associated Proteins ; Anti-Bacterial Agents ; Bacterial Outer Membrane Proteins ; AcrB protein, E coli
Language	English
Publishing date	2023-07-18
Publishing country	England
Document type	Journal Article ; Research Support, U.S. Gov't, Non-P.H.S. ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2553671-0
ISSN	2041-1723 ; 2041-1723
ISSN (online)	2041-1723
ISSN	2041-1723
DOI	10.1038/s41467-023-39615-x
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