Krios I-Titan Krios I at Diamond
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Sophie E.
Ayscough
,
Luke A.
Clifton
,
Jörgen
Ådén
,
Sebastian
Köhler
,
Nicolò
Paracini
,
James
Doutch
,
Eilis C.
Bragginton
,
Anna E.
Leung
,
Oliver
Bogojevic
,
Jia-Fei
Poon
,
Tamás Milán
Nagy
,
Hanna P.
Wacklin-Knecht
,
Gerhard
Gröbner
Open Access
Abstract: The Bcl-2 family of proteins governs mitochondrial outer membrane (MOM) permeabilization, a critical step in apoptosis that is dysfunctional in many cancers. Although cellular studies have long implicated direct interactions between the pore-forming apoptotic Bax protein and its opponent, the antiapoptotic Bcl-2 protein in apoptosis regulation, the underlying basic principles behind this control remained unresolved. To provide in-depth insight, we carried out a systematic biophysical study in which we utilized neutron reflectometry (NR) and ATR-FTIR to elucidate the molecular communication between those proteins in and around the mitochondrial membrane environment. The spatial and temporal changes across model MOM surfaces were resolved during the interaction of Bax with Bcl-2. The NR-derived membrane surface Bax distributions suggested that Bcl-2 mediated Bax sequestration through both Bcl-2/Bax heterodimerization and Bax/Bax oligomerization. Kinetic analysis revealed a two-step process: rapid formation of Bcl-2/Bax heterodimers, followed by slower Bax oligomerization on these complexes. Importantly, this sequestration mechanism was also observed in the presence of cardiolipin, a lipid known to promote the formation of an apoptotic pore by Bax in the absence of Bcl-2. These findings suggest a fundamental mechanism by which cancer cells may evade apoptosis by exploiting Bcl-2’s ability to neutralize Bax through structural entrapment, even if excess Bax is present, either in response to treatment or natural death signals.
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Feb 2026
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I07-Surface & interface diffraction
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Alexandra L.
Martin
,
Philip N.
Jemmett
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Thomas
Howitt
,
Mary H.
Wood
,
Liam R.
Cox
,
Timothy R.
Dafforn
,
Mario
Campana
,
Rebecca J. L.
Welbourn
,
Maximilian W. A.
Skoda
,
Luke A.
Clifton
,
Hadeel
Hussain
,
Jonathan L.
Rawle
,
Francesco
Carla
,
Christopher L.
Nicklin
,
Thomas
Arnold
,
Sarah L.
Horswell
Diamond Proposal Number(s):
[22078, 21088]
Open Access
Abstract: The reasons for the wide diversity of lipids found in natural cell membranes are still not fully understood but could potentially be exploited in treating disease and infection. This study aims to establish whether charge alone or specific chemical structure of an anionic lipid headgroup determines the structure and properties of model bacterial cell membranes. We compare different compositions of a zwitterionic lipid di-myristoyl phosphatidylethanolamine (DMPE) and two anionic lipids, di-myristoyl phosphatidylglycerol (DMPG) and tetra-myristoyl cardiolipin (TMCL). TMCL has a distinct condensing effect, increasing packing and decreasing the pressures of the phase transitions. Although relatively well solvated itself, TMCL does not substantially alter the solvation of mixed monolayers or bilayers. DMPE:TMCL mixtures have very similar electrochemical behaviour to mixtures of DMPE with di-myristoyl phosphatidylserine (DMPS) but DMPE:DMPG bilayers have greater surface charges. A ternary mixture representing an Escherichia coli membrane has similar electrochemical response to but is more tightly packed than DMPE:DMPG. These results establish the importance of the anionic lipid in modelling different types of cell membranes: DMPG will be required in model bacterial membranes and should not be replaced with DMPS. Even very small amounts of CL will have a measurable effect on structure, so its inclusion is important. Our results also highlight the importance of diverse techniques in understanding membrane behaviour: reflectivity measurements of monolayers over a range of surface pressure provide excellent insight into the electrochemical responses of lipid bilayers, while surface diffraction and infrared spectroscopy are much more sensitive to differences in packing between lipids.
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Aug 2025
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[33797]
Open Access
Abstract: Model membranes allow for structural and biophysical studies on membrane biochemistry at the molecular level, albeit on systems of reduced complexity which can limit biological accuracy. Floating supported bilayers offer a means of producing planar lipid membrane models not adhered to a surface, which allows for improved accuracy compared to other model membranes. Here we communicate the incorporation of an integral membrane protein complex, the multidomain β-barrel assembly machinery (Bam), into our recently developed in situ self-assembled floating supported bilayers. Using neutron reflectometry and quartz crystal microbalance measurements we show this sample system can be fabricated using a two-step self-assembly process. We then demonstrate the complexity of the model membrane and tuneability of the membrane-to-surface distance using changes in the salt concentration of the bulk solution. Results demonstrate an easily fabricated, biologically accurate and tuneable membrane assay system which can be utilized for studies on integral membrane proteins within their native lipid matrix.
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Jun 2024
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I04-Macromolecular Crystallography
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Benjamin F.
Cooper
,
Giedrė
Ratkevičiūtė
,
Luke A.
Clifton
,
Hannah
Johnston
,
Rachel
Holyfield
,
David J.
Hardy
,
Simon G.
Caulton
,
William
Chatterton
,
Pooja
Sridhar
,
Peter
Wotherspoon
,
Gareth W.
Hughes
,
Stephen C. L.
Hall
,
Andrew L.
Lovering
,
Timothy J.
Knowles
Diamond Proposal Number(s):
[26803]
Open Access
Abstract: The E. coli Paraquat Inducible (Pqi) Pathway is a putative Gram-negative phospholipid transport system. The pathway comprises three components: an integral inner membrane protein (PqiA), a periplasmic spanning MCE family protein (PqiB) and an outer membrane lipoprotein (PqiC). Interactions between all complex components, including stoichiometry, remain uncharacterised; nevertheless, once assembled into their quaternary complex, the trio of Pqi proteins are anticipated to provide a continuous channel between the inner and outer membranes of diderms. Here, we present X-ray structures of both the native and a truncated, soluble construct of the PqiC lipoprotein, providing insight into its biological assembly, and utilise neutron reflectometry to characterise the nature of the PqiB-PqiC-membrane interaction. Finally, we employ phenotypic complementation assays to probe specific PqiC residues, which imply the interaction between PqiB and PqiC is less intimate than previously anticipated.
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Jan 2024
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I07-Surface & interface diffraction
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Diamond Proposal Number(s):
[22995]
Open Access
Abstract: Hypothesis: The attractive interaction between a cationic surfactant monolayer at the air–water interface and vesicles, incorporating anionic lipids, is sufficient to drive the adsorption and deformation of the vesicles. Osmotic rupture of the vesicles produces a continuous lipid bilayer beneath the monolayer. Experimental: Specular neutron reflectivity has been measured from the surface of a purpose-built laminar flow trough, which allows for rapid adsorption of vesicles, the changes in salt concentration required for osmotic rupture of the adsorbed vesicles into a bilayer, and for neutron contrast variation of the sub-phase without disturbing the monolayer. Findings: The neutron reflectivity profiles measured after vesicle addition are consistent with the adsorption and flattening of the vesicles beneath the monolayer. An increase in the buffer salt concentration results in further flattening and fusion of the adsorbed vesicles, which are ruptured by a subsequent decrease in the salt concentration. This process results in a continuous, high coverage, bilayer suspended 11 Å beneath the monolayer. As the bilayer is not constrained by a solid substrate, this new mimetic is well-suited to studying the structure of lipid bilayers that include transmembrane proteins.
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Mar 2023
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Stephen C. I.
Hall
,
Luke
Clifton
,
Pooja
Sridhar
,
David J.
Hardy
,
Peter
Wotherspoon
,
Jack
Wright
,
James
Whitehouse
,
Nadisha
Gamage
,
Claire S.
Laxton
,
Caitlin
Hatton
,
Gareth W.
Hughes
,
Mark
Jeeves
,
Timothy J.
Knowles
Abstract: The outer membrane of Gram-negative bacteria presents a robust physicochemical barrier protecting the cell from both the natural environment and acting as the first line of defense against antimicrobial materials. The proteins situated within the outer membrane are responsible for a range of biological functions including controlling influx and efflux. These outer membrane proteins (OMPs) are ultimately inserted and folded within the membrane by the β-barrel assembly machine (Bam) complex. The precise mechanism by which the Bam complex folds and inserts OMPs remains unclear. Here, we have developed a platform for investigating Bam-mediated OMP insertion. By derivatizing a gold surface with a copper-chelating self-assembled monolayer, we were able to assemble a planar system containing the complete Bam complex reconstituted within a phospholipid bilayer. Structural characterization of this interfacial protein-tethered bilayer by polarized neutron reflectometry (PNR) revealed distinct regions consistent with known high-resolution models of the Bam complex. Additionally, by monitoring changes of mass associated with OMP insertion by quartz crystal microbalance with dissipation monitoring (QCM-D), we were able to demonstrate the functionality of this system by inserting two diverse OMPs within the membrane, pertactin and OmpT. This platform has promising application in investigating the mechanism of Bam-mediated OMP insertion, in addition to OMP function and activity within a phospholipid bilayer environment.
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Oct 2021
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Open Access
Abstract: Over recent years, there has been a rapid development of membrane-mimetic systems to encapsulate and stabilize planar segments of phospholipid bilayers in solution. One such system has been the use of amphipathic copolymers to solubilize lipid bilayers into nanodiscs. The attractiveness of this system, in part, stems from the capability of these polymers to solubilize membrane proteins directly from the host cell membrane. The assumption has been that the native lipid annulus remains intact, with nanodiscs providing a snapshot of the lipid environment. Recent studies have provided evidence that phospholipids can exchange from the nanodiscs with either lipids at interfaces, or with other nanodiscs in bulk solution. Here we investigate kinetics of lipid exchange between three recently studied polymer-stabilized nanodiscs and supported lipid bilayers at the silicon-water interface. We show that lipid and polymer exchange occurs in all nanodiscs tested, although the rate and extent differs between different nanodisc types. Furthermore, we observe adsorption of nanodiscs to the supported lipid bilayer for one nanodisc system which used a polymer made using reversible addition-fragmentation chain transfer polymerization. These results have important implications in applications of polymer-stabilized nanodiscs, such as in the fabrication of solid-supported films containing membrane proteins.
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Apr 2020
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[21035]
Abstract: Staufen is a dsRNA binding protein that plays an essential role in many aspects of RNA regulation, such as mRNA transport, Staufen-mediated mRNA decay and the regulation of mRNA translation. Staufen is a modular protein characterized by the presence of conserved consensus amino acid sequences that fold into double-stranded RNA binding domains (RBDs) as well as degenerated RBDs that maintain the α-β-β-β-α fold but are unable to bind RNA and are instead involved in protein-protein interactions. The variety of biological processes in which Staufen participates in the cell suggests that this protein associates with many diverse RNA targets, some of which have been identified experimentally. Staufen binding mediates the recruitment of effectors via protein-protein and protein-RNA interactions. The structural determinants of a number of these interactions, as well as the structure of full-length Staufen, remain unknown. Here, we present the first solution structure models for full-length human Staufen155, showing that its domains are arranged as beads-on-a-string in the absence of RNA.
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Dec 2019
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I07-Surface & interface diffraction
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Abstract: Clupeine, a cationic antimicrobial peptide found in fish, is of interest as a food additive but non-specific binding of the peptide to anionic molecules reduces its antimicrobial activity. The overall positive charge of clupeine can be reduced by blocking 10% of its arginine residues with 1,2-cyclohexanedione (CHD). The modified peptide retains antimicrobial activity but it is not known if its effect on the structure of Gram-negative model membranes is the same as the native peptide. In the presented paper, neutron reflectometry (NR) and X-ray reflectometry were used to investigate the effect of native and modified clupeine on the structure of model monolayer membranes composed of Phosphatidylethanolamine (PE), Phosphatidylglycerol (PG), and Cardiolipin (CL). The effect of the peptides on the structure of 1,2-dipalmitoyl (d62)-sn-glycero-3-phosphocholine (DPPC)/PE:PG:CL bilayers were also examined by NR. In both model systems, modified clupeine demonstrated a greater effect on the lipid structure. Charge reduction in the modified sample also resulted in improved hydrophobicity, and the formation of thicker peptide layers in the membrane models. Some lipid translocation was observed in the inner tail region (∼69 ± 0.24% DPPC and ∼24 ± 0.02% PE:PG:CL); and in the outer tail region (∼24 ± 0.02% DPPC and ∼56 ± 0.01% PE:PG:CL). Improved hydrophobicity and electrostatic interactions with lipid head groups, strongly suggests that the modified clupeine may use the carpet mechanisms to exert its effect on model membranes. These findings suggest that changing the charge on the native peptide changes the way in which the modified peptide disrupts Gram-negative model membranes.
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Oct 2019
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I04-Macromolecular Crystallography
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Gareth W.
Hughes
,
Stephen C. L.
Hall
,
Claire S.
Laxton
,
Pooja
Sridhar
,
Amirul H.
Mahadi
,
Caitlin
Hatton
,
Thomas J.
Piggot
,
Peter J.
Wotherspoon
,
Aneika C.
Leney
,
Douglas G.
Ward
,
Mohammed
Jamshad
,
Vaclav
Spana
,
Ian T.
Cadby
,
Christopher
Harding
,
Georgia L.
Isom
,
Jack A.
Bryant
,
Rebecca J.
Parr
,
Yasin
Yakub
,
Mark
Jeeves
,
Damon
Huber
,
Ian R.
Henderson
,
Luke A.
Clifton
,
Andrew L.
Lovering
,
Timothy J.
Knowles
Diamond Proposal Number(s):
[14692]
Abstract: The Mla pathway is believed to be involved in maintaining the asymmetrical Gram-negative outer membrane via retrograde phospholipid transport. The pathway is composed of three components: the outer membrane MlaA–OmpC/F complex, a soluble periplasmic protein, MlaC, and the inner membrane ATPase, MlaFEDB complex. Here, we solve the crystal structure of MlaC in its phospholipid-free closed apo conformation, revealing a pivoting β-sheet mechanism that functions to open and close the phospholipid-binding pocket. Using the apo form of MlaC, we provide evidence that the inner-membrane MlaFEDB machinery exports phospholipids to MlaC in the periplasm. Furthermore, we confirm that the phospholipid export process occurs through the MlaD component of the MlaFEDB complex and that this process is independent of ATP. Our data provide evidence of an apparatus for lipid export away from the inner membrane and suggest that the Mla pathway may have a role in anterograde phospholipid transport.
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Jun 2019
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