Imperial College London

ProfessorBernadetteByrne

Faculty of Natural SciencesDepartment of Life Sciences

Professor of Molecular Membrane Biology
 
 
 
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Contact

 

+44 (0)20 7594 3004b.byrne Website

 
 
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Location

 

504Sir Ernst Chain BuildingSouth Kensington Campus

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Summary

 

Publications

Publication Type
Year
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118 results found

Tiernan H, Byrne B, Kazarian SG, 2020, ATR-FTIR spectroscopy and spectroscopic imaging for the analysis of biopharmaceuticals, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, Vol: 241, Pages: 1-11, ISSN: 1386-1425

Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectroscopy is a label-free, non-destructive technique that can be applied to a vast range of biological applications, from imaging cancer tissues and live cells, to determining protein content and protein secondary structure composition. This review summarises the recent advances in applications of ATR-FTIR spectroscopy to biopharmaceuticals, the application of this technique to biosimilars, and the current uses of FTIR spectroscopy in biopharmaceutical production. We discuss the use of ATR-FTIR spectroscopic imaging to investigate biopharmaceuticals, and finally, give an outlook on the possible future developments and applications of ATR-FTIR spectroscopy and spectroscopic imaging to this field. Throughout the review comparisons will be made between FTIR spectroscopy and alternative analytical techniques, and areas will be identified where FTIR spectroscopy could perhaps offer a better alternative in future studies. This review focuses on the most recent advances in the field of using ATR-FTIR spectroscopy and spectroscopic imaging to characterise and evaluate biopharmaceuticals, both in industrial and academic research based environments.

Journal article

Bae HE, Cecchetti C, Du Y, Katsube S, Mortensen JS, Huang W, Rehan S, Lee HJ, Loland CJ, Guan L, Kobilka BK, Byrne B, Chae PSet al., 2020, Pendant-bearing glucose-neopentyl glycol (P-GNG) amphiphiles for membrane protein manipulation: Importance of detergent pendant chain for protein stabilization., Acta Biomaterialia, Vol: 112, Pages: 250-261, ISSN: 1742-7061

Glucoside detergents are successfully used for membrane protein crystallization mainly because of their ability to form small protein-detergent complexes. In a previous study, we introduced glucose neopentyl glycol (GNG) amphiphiles with a branched diglucoside structure that has facilitated high resolution crystallographic structure determination of several membrane proteins. Like other glucoside detergents, however, these GNGs were less successful than DDM in stabilizing membrane proteins, limiting their wide use in protein structural study. As a strategy to improve GNG efficacy for protein stabilization, we introduced two different alkyl chains (i.e., main and pendant chains) into the GNG scaffold while maintaining the branched diglucoside head group. Of these pendant-bearing GNGs (P-GNGs), three detergents (GNG-2,14, GNG-3,13 and GNG-3,14) were not only notably better than both DDM (a gold standard detergent) and the previously described GNGs at stabilizing all six membrane proteins tested here, but were also as efficient as DDM at membrane protein extraction. The results suggest that the C14 main chain of the P-GNGs is highly compatible with the hydrophobic widths of membrane proteins, while the C2/C3 pendant chain is effective at strengthening detergent hydrophobic interactions. Based on the marked effect on protein stability and solubility, these glucoside detergents hold significant potential for membrane protein structural study. Furthermore, the independent roles of the detergent two alkyl chains first introduced in this study have shed light on new amphiphile design for membrane protein study. STATEMENT OF SIGNIFICANCE: Detergent efficacy for protein stabilization tends to be protein-specific, thus it is challenging to find a detergent that is effective at stabilizing multiple membrane proteins. By incorporating a pendant chain into our previous GNG scaffold, we prepared pendant chain-bearing GNGs (P-GNGs) and identified three P-GNGs that were highly effec

Journal article

Ehsan M, Katsube S, Cecchetti C, Du Y, Mortensen JS, Wang H, Nygaard A, Ghani L, Loland CJ, Kobilka BK, Byrne B, Guan L, Chae PSet al., 2020, New Malonate-Derived Tetraglucoside Detergents for Membrane Protein Stability, ACS CHEMICAL BIOLOGY, Vol: 15, Pages: 1697-1707, ISSN: 1554-8929

Journal article

Tiernan H, Byrne B, Kazarian SG, 2020, Insight into Heterogeneous Distribution of Protein Aggregates at the Surface Layer Using Attenuated Total Reflection-Fourier Transform Infrared Spectroscopic Imaging, ANALYTICAL CHEMISTRY, Vol: 92, Pages: 4760-4764, ISSN: 0003-2700

Journal article

Saouros S, Cecchetti C, Jones A, Cameron AD, Byrne Bet al., 2020, Strategies for successful isolation of a eukaryotic transporter, Protein Expression and Purification, Vol: 166, Pages: 1-8, ISSN: 1046-5928

The isolation of integral membrane proteins for structural analysis remains challenging and this is particularly the case for eukaryotic membrane proteins. Here we describe our efforts to isolate OsBOR3, a boron transporter from Oryza sativa. OsBOR3 was expressed as both full length and a C-terminally truncated form lacking residues 643–672 (OsBOR3Δ1-642). While both express well as C-terminal GFP fusion proteins in Saccharomyces cerevisiae, the full length protein isolates poorly in the detergent dodecyl-β-d-maltoside (DDM). The OsBOR3Δ1-642 isolated in DDM in large quantities but was contaminated with GFP tagged protein, indicated incomplete protease removal of the tag. Addition of the reducing agent dithiothreitol (DTT) had no effect on isolation. Detergent screening indicated that the neopentyl glycol detergents, LMNG, UDMNG and DMNG conferred greater stability on the OsBOR3Δ1-642 than DDM. Isolation of OsBOR3Δ1-642 in LMNG both in the presence and absence of DTT produced large quantities of protein but contaminated with GFP tagged protein. Isolation of OsBOR3Δ1-642 in DMNG + DTT resulted in protein sample that does not contain any detectable GFP but elutes at a higher retention volume than that seen for protein isolated in either DDM or LMNG. Mass spectrometry confirmed that the LMNG and DMNG purified protein is OsBOR3Δ1-642 indicating that the DMNG isolated protein is monomer compared to the dimer isolated using LMNG. This was further supported by single particle electron microscopic analysis revealing that the DMNG protein particles are roughly half the size of the LMNG protein particles.

Journal article

Ghani L, Munk CF, Zhang X, Katsube S, Du Y, Cecchetti C, Huang W, Bae HE, Saouros S, Ehsan M, Guan L, Liu X, Loland CJ, Kobilka BK, Byrne B, Chae PSet al., 2019, 1,3,5-Triazine-cored maltoside amphiphiles for membrane protein extraction and stabilization, Journal of the American Chemical Society, Vol: 141, Pages: 19677-19687, ISSN: 0002-7863

Despite their major biological and pharmacological significance, the structural and functional study of membrane proteins remains a significant challenge. A main issue is the isolation of these proteins in a stable and functional state from native lipid membranes. Detergents are amphiphilic compounds widely used to extract membrane proteins from the native membranes and maintain them in a stable form during downstream analysis. However, due to limitations of conventional detergents, it is essential to develop novel amphiphiles with optimal properties for protein stability in order to advance membrane protein research. Here we designed and synthesized 1,3,5-triazine-cored dimaltoside amphiphiles derived from cyanuric chloride. By introducing variations in the alkyl chain linkage (ether/thioether) and an amine-functionalized diol linker (serinol/diethanolamine), we prepared two sets of 1,3,5-triazine-based detergents. When tested with several model membrane proteins, these agents showed remarkable efficacy in stabilizing three transporters and two G protein-coupled receptors. Detergent behavior substantially varied depending on the detergent structural variation, allowing us to explore detergent structure–property–efficacy relationships. The 1,3,5-triazine-based detergents introduced here have significant potential for membrane protein study as a consequence of their structural diversity and universal stabilization efficacy for several membrane proteins.

Journal article

Pyle E, Guo C, Hofmann T, Schmidt C, Ribiero O, Politis A, Byrne Bet al., 2019, Protein-lipid interactions stabilize the oligomeric state of BOR1p from saccharomyces cerevisiae, Analytical Chemistry, Vol: 91, Pages: 13071-13079, ISSN: 0003-2700

The BOR proteins are integral membrane transporters which mediate efflux of boron. Structures of two BOR family members from Arabidopsis thaliana and Saccharomyces mikitiae indicate that the proteins exist as dimers. However, it remains unclear whether dimer formation is dependent on protein-lipid interactions or whether the dimer is the functional form of the protein. Here, we used the BOR1p protein from Saccharomyces cerevisiae (ScBOR1p), recombinantly expressed in its native host, to explore these aspects of BOR transporter structure and function. Native mass spectrometry (MS) revealed that ScBOR1p isolates as a monomer in a range of detergents. Lipidomics analysis showed that ScBOR1p co-isolates with phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI). Delipidation of ScBOR1p followed by addition of PS or PE causes formation of ScBOR1p dimers. Using a homology model of ScBOR1p, we identified a possible lipid binding site at the dimer interface comprising residues Arg265, Arg267, Arg480, and Arg481. A quadruple 4R/A mutant was expressed and isolated and also shown to be monomeric by native MS, and addition of PS or PE to this mutant did not reform the dimer. Functional complementation analysis revealed that the 4R/A mutant had boron efflux activity, suggesting that the ScBOR1p monomer is responsible for transport function. Taken together, these data strongly indicate that the physiological form of the ScBOR1p is the dimer and that dimer formation is dependent on association with membrane lipids.

Journal article

Kourkoulou A, Grevias P, Lambrinidis G, Pyle E, Dionysopoulou M, Politis A, Mikros E, Byrne B, Diallinas Get al., 2019, Specific residues in a purine transporter are critical for dimerization, ER-Exit and function., Genetics, ISSN: 1943-2631

Transporters are transmembrane proteins that mediate the selective translocation of solutes across biological membranes. Recently, we have shown that specific interactions with plasma membrane phospholipids are essential for formation and/or stability of functional dimers of the purine transporter, UapA, a prototypic eukaryotic member of the ubiquitous NAT family. Here, we provide strong evidence that distinct interactions of UapA with membrane lipids are essential for ab initio formation of functional dimers in the ER or ER-exit and further subcellular trafficking. Through genetic screens we identify mutations that restore defects in dimer formation and/or trafficking. Suppressors of defective dimerization restore ab initio formation of UapA dimers in the ER. Most of these suppressors are located in the movable core domain, but also in the core-dimerization interface and in residues of the dimerization domain exposed to lipids. Molecular Dynamics suggest the majority of suppressors stabilize interhelical interactions in the core domain and thus assist the formation of functional UapA dimers. Among suppressors restoring dimerization, a specific mutation, T401P, was also isolated independently as a suppressor restoring trafficking, suggesting that stabilization of the core domain restores function by sustaining structural defects caused by abolishment of essential interactions with specific lipids. Importantly, introduction of mutations topologically equivalent to T401P into a rat homologue of UapA, namely rSNBT1, permitted the functional expression of a mammalian NAT in A. nidulans Thus, our results provide a potential route for the functional expression and manipulation of mammalian transporters in the model Aspergillus system.

Journal article

Sarkar K, Joedicke L, Westwood M, Burnley R, Wright M, McMillan D, Byrne Bet al., 2019, Modulation of PTH1R signaling by an ECD binding antibody results in inhibition of beta-arrestin 2 coupling, Scientific Reports, Vol: 9, ISSN: 2045-2322

Parathyroid hormone receptor 1 (PTH1R) belongs to the secretin class of G protein coupled receptors (GPCRs) and natively binds parathyroid hormone (PTH) and parathyroid hormone related peptide (PTHrP). Ligand binding to PTH1R involves binding to the large extracellular domain (ECD) and the orthosteric pocket, inducing conformational changes in the transmembrane domain and receptor activation. PTH1R regulates bone metabolism, signaling mainly through Gs and Gq/11 G-proteins. Here, we used phage display to generate PTH1R ECD-specific antibodies with the aim of modulating receptor functionality. We identified ECD-scFvhFc, which exhibited high affinity binding to both the isolated ECD and to the full-length receptor in styrene-maleic acid (SMA) lipid particles. Epitope mapping using hydrogen-deuterium exchange mass spectrometry (HDX-MS) indicates that the α1 helix of the ECD is ECD-scFvhFc’s epitope which may partially overlap with the known PTH (1–34) binding site. However, PTH (1–34)-mediated Gs activation is Undisturbed by ECD-scFvhFc binding. In contrast, ECD-scFvhFc potently inhibits β-arrestin-2 recruitment after PTH (1–34)-driven receptor activation and thus represents the first monoclonal antibody to selectively inhibit distinct PTH1R signaling pathways. Given the complexity of PTH1R signaling and the emerging importance of biased GPCR activation in drug development, ECD-scFvhFc could be a valuable tool to study PTH1R signaling bias.

Journal article

Kourkoulou A, Grevias P, Lambrinidis G, Pyle E, Dionysopoulou M, Politis A, Mikros E, Byrne B, Diallinas Get al., 2019, Distinct specific interactions of the UapA transporter with membrane lipids are critical for dimerization, ER-exit and function, Genetics

<jats:title>Abstract</jats:title><jats:p>Transporters are transmembrane proteins that mediate the selective translocation of solutes across biological membranes. Recently, we have shown that specific interactions with plasma membrane phospholipids are essential for formation and/or stability of functional dimers of the purine transporter, UapA, a prototypic eukaryotic member of the ubiquitous NAT family. Here, we show that distinct interactions of UapA with specific or annular lipids are essential for <jats:italic>ab initio</jats:italic> formation of functional dimers in the ER or ER-exit and further subcellular trafficking. Through genetic screens we identify mutations that restore defects in dimer formation and/or trafficking. Suppressors of defective dimerization restore <jats:italic>ab initio</jats:italic> formation of UapA dimers in the ER. Most of these suppressors are located in the movable core domain, but also in the core-dimerization interface and in residues of the dimerization domain exposed to lipids. Molecular Dynamics suggest the majority of suppressors stabilize interhelical interactions in the core domain and thus assist the formation of functional UapA dimers. Among suppressors restoring dimerization, a specific mutation, T401P, was also isolated independently as a suppressor restoring trafficking, suggesting that stabilization of the core domain restores function by sustaining structural defects caused by abolishment of essential interactions with specific or annular lipids. Importantly, introduction of mutations topologically equivalent to T401P into a rat homologue of UapA, namely rSNBT1, permitted the functional expression of a mammalian NAT in <jats:italic>A. nidulans</jats:italic>. Thus, our results provide a potential route for the functional expression and manipulation of mammalian transporters in the model Aspergillus system.</jats:p><jats:sec><jats:title>Author Summar

Journal article

Ehsan M, Du Y, Mortensen JS, Hariharan P, Qu Q, Ghani L, Das M, Grethen A, Byrne B, Skiniotis G, Keller S, Loland CJ, Guan L, Kobilka BK, Chae PSet al., 2019, Self-assembly behavior and application of terphenyl-cored trimaltosides for membrane-protein studies: impact of detergent hydrophobic group geometry on protein stability, Chemistry - A European Journal, Vol: 25, Pages: 11545-11554, ISSN: 0947-6539

Amphipathic agents are widely used in various fields including biomedical sciences. Micelle-forming detergents are particularly useful for in vitro membrane-protein characterization. As many conventional detergents are limited in their ability to stabilize membrane proteins, it is necessary to develop novel detergents to facilitate membrane-protein research. In the current study, we developed novel trimaltoside detergents with an alkyl pendant-bearing terphenyl unit as a hydrophobic group, designated terphenyl-cored maltosides (TPMs). We found that the geometry of the detergent hydrophobic group substantially impacts detergent self-assembly behavior, as well as detergent efficacy for membrane-protein stabilization. TPM-Vs, with a bent terphenyl group, were superior to the linear counterparts (TPM-Ls) at stabilizing multiple membrane proteins. The favorable protein stabilization efficacy of these bent TPMs is likely associated with a binding mode with membrane proteins distinct from conventional detergents and facial amphiphiles. When compared to n-dodecyl-β-d-maltoside (DDM), most TPMs were superior or comparable to this gold standard detergent at stabilizing membrane proteins. Notably, TPM-L3 was particularly effective at stabilizing the human β2 adrenergic receptor (β2 AR), a G-protein coupled receptor, and its complex with Gs protein. Thus, the current study not only provides novel detergent tools that are useful for membrane-protein study, but also suggests a critical role for detergent hydrophobic group geometry in governing detergent efficacy.

Journal article

Sadaf A, Ramos M, Mortensen JS, Du Y, Bae HE, Munk CF, Hariharan P, Byrne B, Kobilka BK, Loland CJ, Guan L, Chae PSet al., 2019, Conformationally restricted monosaccharide-cored glycoside amphiphiles: the effect of detergent headgroup variation on membrane protein stability., ACS Chemical Biology, Vol: 14, Pages: 1717-1726, ISSN: 1554-8929

Detergents are widely used to isolate membrane proteins from lipid bilayers, but many proteins solubilized in conventional detergents are structurally unstable. Thus, there is major interest in the development of novel amphiphiles to facilitate membrane protein research. In this study, we have designed and synthesized novel amphiphiles with a rigid scyllo-inositol core, designated scyllo-inositol glycosides (SIGs). Varying the headgroup structure allowed the preparation of three sets of SIGs that were evaluated for their effects on membrane protein stability. When tested with a few model membrane proteins, representative SIGs conferred enhanced stability to the membrane proteins compared to a gold standard conventional detergent (DDM). Of the novel amphiphiles, a SIG designated STM-12 was most effective at preserving the stability of the multiple membrane proteins tested here. In addition, a comparative study of the three sets suggests that several factors, including micelle size and alkyl chain length, need to be considered in the development of novel detergents for membrane protein research. Thus, this study not only describes new detergent tools that are potentially useful for membrane protein structural study but also introduces plausible correlations between the chemical properties of detergents and membrane protein stabilization efficacy.

Journal article

Nguyen TTM, Byrne B, Mcmanus JJ, 2019, Understanding Membrane Protein Interactions Using Phase Diagrams, Joint 12th EBSA European Biophysics Congress / 10th IUPAP International Conference on Biological Physics (ICBP), Publisher: SPRINGER, Pages: S230-S230, ISSN: 0175-7571

Conference paper

Ehsan M, Kumar A, Mortensen JS, Du Y, Hariharan P, Kumar KK, Ha B, Byrne B, Guan L, Kobilka BK, Loland CJ, Chae PSet al., 2019, Self-assembly behaviors of a penta-phenylene maltoside and its application for membrane protein study, Chemistry: An Asian Journal, Vol: 14, Pages: 1926-1931, ISSN: 1861-471X

We prepared an amphiphile with a penta-phenylene lipophilic group and a branched trimaltoside head group. This new agent, designated penta-phenylene maltoside (PPM), showed a marked tendency to self-assembly into micelles via strong aromatic-aromatic interactions in aqueous media, as evidenced by 1 H NMR spectroscopy and fluorescence studies. When utilized for membrane protein studies, this new agent was superior to DDM, a gold standard conventional detergent, in stabilizing multiple proteins long term. The ability of this agent to form aromatic-aromatic interactions is likely responsible for enhanced protein stabilization when associated with a target membrane protein.

Journal article

Das M, Du Y, Mortensen JS, Ramos M, Ghani L, Lee HJ, Bae HE, Byrne B, Guan L, Loland CJ, Kobilka BK, Seok Chae Pet al., 2019, Trehalose-cored amphiphiles for membrane protein stabilization: importance of the detergent micelle size in GPCR stability (vol 17, pg 3249, 2019), ORGANIC & BIOMOLECULAR CHEMISTRY, Vol: 17, Pages: 4919-4920, ISSN: 1477-0520

Journal article

Das M, Du Y, Mortensen JS, Ramos M, Ghani L, Lee HJ, Bae HE, Byrne B, Guan L, Loland CJ, Kobilka BK, Chae PSet al., 2019, Trehalose-cored amphiphiles for membrane protein stabilization: importance of the detergent micelle size in GPCR stability, Organic and Biomolecular Chemistry, Vol: 17, Pages: 3249-3257, ISSN: 1477-0520

Despite their importance in biology and medicinal chemistry, structural and functional studies of membrane proteins present major challenges. To study diverse membrane proteins, it is crucial to have the correct detergent to efficiently extract and stabilize the proteins from the native membranes for biochemical/biophysical downstream analyses. But many membrane proteins, particularly eukaryotic ones, are recalcitrant to stabilization and/or crystallization with currently available detergents and thus there are major efforts to develop novel detergents with enhanced properties. Here, a novel class of trehalose-cored amphiphiles are introduced, with multiple alkyl chains and carbohydrates projecting from the trehalose core unit are introduced. A few members displayed enhanced protein stabilization behavior compared to the benchmark conventional detergent, n-dodecyl-β-d-maltoside (DDM), for multiple tested membrane proteins: (i) a bacterial leucine transporter (LeuT), (ii) the R. capsulatus photosynthetic superassembly, and (iii) the human β2 adrenergic receptor (β2AR). Due to synthetic convenience and their favourable behaviors for a range of membrane proteins, these agents have potential for membrane protein research. In addition, the detergent property-efficacy relationship discussed here will guide future design of novel detergents.

Journal article

Cecchetti C, Pyle E, Byrne B, 2019, Transporter oligomerisation: roles in structure and function, Biochemical Society Transactions, Vol: 47, Pages: 433-440, ISSN: 0300-5127

Oligomerisation is a key feature of integral membrane transporters with roles in structure, function and stability. In this review, we cover some very recent advances in our understanding of how oligomerisation affects these key transporter features, with emphasis on a few groups of transporters, including the nucleobase ascorbate transporters, neurotransmitter sodium symporters and major facilitator superfamily members.

Journal article

Hussain H, Helton T, Du Y, Mortensen JS, Hariharan P, Ehsan M, Byrne B, Loland CJ, Kobilka BK, Guan L, Chae PSet al., 2018, A comparative study of branched and linear mannitol-based amphiphiles on membrane protein stability, The Analyst, Vol: 143, Pages: 5702-5710, ISSN: 0003-2654

The study of membrane proteins is extremely challenging, mainly because of the incompatibility of the hydrophobic surfaces of membrane proteins with an aqueous medium. Detergents are essential agents used to maintain membrane protein stability in non-native environments. However, conventional detergents fail to stabilize the native structures of many membrane proteins. Development of new amphipathic agents with enhanced efficacy for membrane protein stabilization is necessary to address this important problem. We have designed and synthesized linear and branched mannitol-based amphiphiles (MNAs), and comparative studies showed that most of the branched MNAs had advantages over the linear agents in terms of membrane protein stability. In addition, a couple of the new MNAs displayed favorable behaviors compared to n-dodecyl-β-D-maltoside and the previously developed MNAs in maintaining the native protein structures, indicating potential utility of these new agents in membrane protein study.

Journal article

Das M, Du Y, Mortensen JS, Hariharan P, Lee HS, Byrne B, Loland CJ, Guan L, Kobilka BK, Chae PSet al., 2018, Rationally engineered tandem facial amphiphiles for improved membrane protein stabilization efficacy, ChemBioChem: a European journal of chemical biology, Vol: 19, Pages: 2225-2232, ISSN: 1439-4227

A new family of tandem facial glucosides/maltosides (TFGs/TFMs) for membrane protein manipulation was prepared. The best detergent varied depending on the hydrophobic thickness of the target protein, but ether‐based TFMs (TFM‐C0E, TFM‐C3E, and TFM‐C5E) were notable for their ability to confer higher membrane protein stability than the previously developed amide‐based TFA‐1 (P. S. Chae, K. Gotfryd, J. Pacyna, L. J. W. Miercke, S. G. F. Rasmussen, R. A. Robbins, R. R. Rana, C. J. Loland, B. Kobilka, R. Stroud, B. Byrne, U. Gether, S. H. Gellman, J. Am. Chem. Soc. 2010, 132, 16750–16752). Thus, this study not only introduces novel agents with the potential to be used in membrane protein research but also highlights the importance of both the hydrophobic length and linker functionality of the detergent in stabilizing membrane proteins.

Journal article

Pyle E, Kalli AC, Amillis S, Hall Z, Lau AM, Hanyaloglu AC, Diallinas G, Byrne B, Politis Aet al., 2018, Structural lipids enable the formation of Ffnctional oligomers of the eukaryotic purine symporter UapA, Cell Chemical Biology, Vol: 25, Pages: 840-848.e4, ISSN: 2451-9456

The role of membrane lipids in modulating eukaryotic transporter assembly and function remains unclear. We investigated the effect of membrane lipids in the structure and transport activity of the purine transporter UapA from Aspergillus nidulans. We found that UapA exists mainly as a dimer and that two lipid molecules bind per UapA dimer. We identified three phospholipid classes that co-purified with UapA: phosphatidylcholine, phosphatidylethanolamine (PE), and phosphatidylinositol (PI). UapA delipidation caused dissociation of the dimer into monomers. Subsequent addition of PI or PE rescued the UapA dimer and allowed recovery of bound lipids, suggesting a central role of these lipids in stabilizing the dimer. Molecular dynamics simulations predicted a lipid binding site near the UapA dimer interface. Mutational analyses established that lipid binding at this site is essential for formation of functional UapA dimers. We propose that structural lipids have a central role in the formation of functional, dimeric UapA.

Journal article

Das M, Du Y, Mortensen JS, Bae HE, Byrne B, Loland CJ, Kobilka BK, Chae PSet al., 2018, An engineered lithocholate-based facial amphiphile stabilizes membrane proteins: assessing the impact of detergent customizability on protein stability, Chemistry: A European Journal, Vol: 24, Pages: 9860-9868, ISSN: 0947-6539

Amphiphiles are critical tools for the structural and functional study of membrane proteins. Membrane proteins encapsulated by conventional head‐to‐tail detergents tend to undergo structural degradation, necessitating the development of structurally novel agents with improved efficacy. In recent years, facial amphiphiles have yielded encouraging results in terms of membrane protein stability. Herein, we report a new facial detergent (i.e., LFA‐C4) that confers greater stability to tested membrane proteins than the bola form analogue. Owing to the increased facial property and the adaptability of the detergent micelles in complex with different membrane proteins, LFA‐C4 yields increased stability compared to n‐dodecyl‐β‐d‐maltoside (DDM). Thus, this study not only describes a novel maltoside detergent with enhanced protein‐stabilizing properties, but also shows that the customizable nature of a detergent plays an important role in the stabilization of membrane proteins. Owing to both synthetic convenience and enhanced stabilization efficacy for a range of membrane proteins, the new agent has major potential in membrane protein research.

Journal article

Ehsan M, Das M, Stern V, Du Y, Mortensen JS, Hariharan P, Byrne B, Loland CJ, Kobilka BK, Guan L, Chae PSet al., 2018, Steroid-based amphiphiles for membrane protein study: the importance of alkyl spacers for protein stability, ChemBioChem: a European journal of chemical biology, Vol: 19, Pages: 1433-1443, ISSN: 1439-4227

Membrane proteins allow effective communication between cells and organelles and their external environments. Maintaining membrane protein stability in a non‐native environment is the major bottleneck to their structural study. Detergents are widely used to extract membrane proteins from the membrane and to keep the extracted protein in a stable state for downstream characterisation. In this study, three sets of steroid‐based amphiphiles—glyco‐diosgenin analogues (GDNs) and steroid‐based pentasaccharides either lacking a linker (SPSs) or containing a linker (SPS‐Ls)—have been developed as new chemical tools for membrane protein research. These detergents were tested with three membrane proteins in order to characterise their ability to extract membrane proteins from the membrane and to stabilise membrane proteins long‐term. Some of the detergents, particularly the SPS‐Ls, displayed favourable behaviour with the tested membrane proteins. This result indicates the potential utility of these detergents as chemical tools for membrane protein structural study and a critical role of the simple alkyl spacer in determining detergent efficacy.

Journal article

Ehsan M, Du Y, Molist I, Seven AB, Hariharan P, Mortensen JS, Ghani L, Loland CJ, Skiniotis G, Guan L, Byrne B, Kobilka BK, Chae PSet al., 2018, Vitamin E-based glycoside amphiphiles for membrane protein structural studies, ORGANIC & BIOMOLECULAR CHEMISTRY, Vol: 16, Pages: 2489-2498, ISSN: 1477-0520

Membrane proteins play critical roles in a variety of cellular processes. For a detailed molecular level understanding of their biological functions and roles in disease, it is necessary to extract them from the native membranes. While the amphipathic nature of these bio-macromolecules presents technical challenges, amphiphilic assistants such as detergents serve as useful tools for membrane protein structural and functional studies. Conventional detergents are limited in their ability to maintain the structural integrity of membrane proteins and thus it is essential to develop novel agents with enhanced properties. Here, we designed and characterized a novel class of amphiphiles with vitamin E (i.e., α-tocopherol) as the hydrophobic tail group and saccharide units as the hydrophilic head group. Designated vitamin E-based glycosides (VEGs), these agents were evaluated for their ability to solubilize and stabilize a set of membrane proteins. VEG representatives not only conferred markedly enhanced stability to a diverse range of membrane proteins compared to conventional detergents, but VEG-3 also showed notable efficacy toward stabilization and visualization of a membrane protein complex. In addition to hydrophile–lipophile balance (HLB) of detergent molecules, the chain length and molecular geometry of the detergent hydrophobic group seem key factors in determining detergent efficacy for membrane protein (complex) stability.

Journal article

Sadaf A, Du Y, Santillan C, Mortensen JS, Molist I, Seven AB, Hariharan P, Skiniotis G, Loland CJ, Kobilka BK, Guan L, Byrne B, Chae PSet al., 2017, Dendronic trimaltoside amphiphiles (DTMs) for membrane protein study, Chemical Science, Vol: 8, Pages: 8315-8324, ISSN: 2041-6520

The critical contribution of membrane proteins in normal cellular function makes their detailed structure and functional analysis essential. Detergents, amphipathic agents with the ability to maintain membrane proteins in a soluble state in aqueous solution, have key roles in membrane protein manipulation. Structural and functional stability is a prerequisite for biophysical characterization. However, many conventional detergents are limited in their ability to stabilize membrane proteins, making development of novel detergents for membrane protein manipulation an important research area. The architecture of a detergent hydrophobic group, that directly interacts with the hydrophobic segment of membrane proteins, is a key factor in dictating their efficacy for both membrane protein solubilization and stabilization. In the current study, we developed two sets of maltoside-based detergents with four alkyl chains by introducing dendronic hydrophobic groups connected to a trimaltoside head group, designated dendronic trimaltosides (DTMs). Representative DTMs conferred enhanced stabilization to multiple membrane proteins compared to the benchmark conventional detergent, DDM. One DTM (i.e., DTM-A6) clearly outperformed DDM in stabilizing human β2 adrenergic receptor (β2AR) and its complex with Gs protein. A further evaluation of this DTM led to a clear visualization of β2AR-Gs complex via electron microscopic analysis. Thus, the current study not only provides novel detergent tools useful for membrane protein study, but also suggests that the dendronic architecture has a role in governing detergent efficacy for membrane protein stabilization.

Journal article

Ehsan M, Ghani L, Du Y, Hariharan P, Mortensen JS, Ribeiro O, Hu H, Skiniotis G, Loland CJ, Guan L, Kobilka BK, Byrne B, Chae PSet al., 2017, New penta-saccharide-bearing tripod amphiphiles for membrane protein structure studies, The Analyst, Vol: 142, Pages: 3889-3898, ISSN: 0003-2654

Integral membrane proteins either alone or as complexes carry out a range of key cellular functions. Detergents are indispensable tools in the isolation of membrane proteins from biological membranes for downstream studies. Although a large number of techniques and tools, including a wide variety of detergents, are available, purification and structural characterization of many membrane proteins remain challenging. In the current study, a new class of tripod amphiphiles bearing two different penta-saccharide head groups, designated TPSs, were developed and evaluated for their ability to extract and stabilize a range of diverse membrane proteins. Variations in the structures of the detergent head and tail groups allowed us to prepare three sets of the novel agents with distinctive structures. Some TPSs (TPS-A8 and TPS-E7) were efficient at extracting two proteins in a functional state while others (TPS-E8 and TPS-E10L) conferred marked stability to all membrane proteins (and membrane protein complexes) tested here compared to a conventional detergent. Use of TPS-E10L led to clear visualization of a receptor-Gs complex using electron microscopy, indicating profound potential in membrane protein research.

Journal article

Pyle E, Kalli AC, Amillis S, Hall Z, Hanyaloglu AC, Diallinas G, Byrne B, Politis Aet al., 2017, Structural lipids enable the formation of functional oligomers of the eukaryotic purine symporter UapA, Publisher: Cold Spring Harbor Laboratory

<jats:title>Abstract</jats:title><jats:p>The role of membrane lipids in modulating eukaryotic transporter structure and function remains poorly understood. We used native mass spectrometry in combination with molecular dynamics simulations and <jats:italic>in vivo</jats:italic> analyses to investigate the roles of membrane lipids in the structure and transport activity of the purine transporter, UapA, from <jats:italic>Aspergillus nidulans</jats:italic>. We revealed that UapA exists mainly as a dimer and that two lipid molecules bind per UapA dimer. We identified three classes of phospholipids: phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) which co-purified with UapA. Delipidation of UapA caused dissociation of the dimer into individual protomers. Subsequent addition of PI or PE rescued the UapA dimer and allowed recovery of bound lipids, suggesting a central role of these lipids in stabilising the dimer. We predicted a putative lipid-binding site near the UapA dimer interface. Mutational analyses established that lipid binding at this site is essential for formation of functional UapA dimers. Our findings reveal unprecedented level of detail into the nature of UapA-lipid interactions and provide a framework for studying similar eukaryotic systems.</jats:p>

Working paper

Byrne B, 2017, It takes two to transport via an elevator, CELL RESEARCH, Vol: 27, Pages: 965-966, ISSN: 1001-0602

Journal article

Hussain H, Mortensen JS, Du Y, Santillan C, Ribeiro O, Go J, Hariharan P, Loland CJ, Guan L, Kobilka BK, Byrne B, Chae PSet al., 2017, Tandem malonate-based glucosides (TMGs) for membrane protein structural studies, Scientific Reports, Vol: 7, ISSN: 2045-2322

High-resolution membrane protein structures are essential for understanding the molecular basis of diverse biological events and important in drug development. Detergents are usually used to extract these bio-macromolecules from the membranes and maintain them in a soluble and stable state in aqueous solutions for downstream characterization. However, many eukaryotic membrane proteins solubilized in conventional detergents tend to undergo structural degradation, necessitating the development of new amphiphilic agents with enhanced properties. In this study, we designed and synthesized a novel class of glucoside amphiphiles, designated tandem malonate-based glucosides (TMGs). A few TMG agents proved effective at both stabilizing a range of membrane proteins and extracting proteins from the membrane environment. These favourable characteristics, along with synthetic convenience, indicate that these agents have potential in membrane protein research.

Journal article

Hussain H, Du Y, Tikhonova E, Mortensen JS, Ribeiro O, Santillan C, Das M, Ehsan M, Loland CJ, Guan L, Kobilka BK, Byrne B, Chae PSet al., 2017, Resorcinarene-Based Facial Glycosides: Implication of Detergent Flexibility on Membrane-Protein Stability, CHEMISTRY-A EUROPEAN JOURNAL, Vol: 23, Pages: 6724-6729, ISSN: 0947-6539

As a membrane-mimetic system, detergent micelles are popularly used to extract membrane proteins from lipid environments and to maintain their solubility and stability in an aqueous medium. However, many membrane proteins encapsulated in conventional detergents tend to undergo structural degradation during extraction and purification, thus necessitating the development of new agents with enhanced properties. In the current study, two classes of new amphiphiles are introduced, resorcinarene-based glucoside and maltoside amphiphiles (designated RGAs and RMAs, respectively), for which the alkyl chains are facially segregated from the carbohydrate head groups. Of these facial amphiphiles, two RGAs (RGA-C11 and RGA-C13) conferred markedly enhanced stability to four tested membrane proteins compared to a gold-standard conventional detergent. The relatively high water solubility and micellar stability of the RGAs compared to the RMAs, along with their generally favourable behaviours for membrane protein stabilisation described here, are likely to be, at least in part, a result of the high conformational flexibility of these glucosides. This study suggests that flexibility could be an important factor in determining the suitability of new detergents for membrane protein studies.

Journal article

Das M, Du Y, Ribeiro O, Hariharan P, Mortensen JS, Patra D, Skiniotis G, Loland CJ, Guan L, Kobilka BK, Byrne B, Chae PSet al., 2017, Conformationally Preorganized Diastereomeric Norbornane-Based Maltosides for Membrane Protein Study: Implications of Detergent Kink for Micellar Properties, Journal of the American Chemical Society, Vol: 139, Pages: 3072-3081, ISSN: 1520-5126

Detergents are essential tools for functional and structural studies of membrane proteins. However, conventional detergents are limited in their scope and utility, particularly for eukaryotic membrane proteins. Thus, there are major efforts to develop new amphipathic agents with enhanced properties. Here, a novel class of diastereomeric agents with a preorganized conformation, designated norbornane-based maltosides (NBMs), were prepared and evaluated for their ability to solubilize and stabilize membrane proteins. Representative NBMs displayed enhanced behaviors compared to n-dodecyl-β-d-maltoside (DDM) for all membrane proteins tested. Efficacy of the individual NBMs varied depending on the overall detergent shape and alkyl chain length. Specifically, NBMs with no kink in the lipophilic region conferred greater stability to the proteins than NBMs with a kink. In addition, long alkyl chain NBMs were generally better at stabilizing membrane proteins than short alkyl chain agents. Furthermore, use of one well-behaving NBM enabled us to attain a marked stabilization and clear visualization of a challenging membrane protein complex using electron microscopy. Thus, this study not only describes novel maltoside detergents with enhanced protein-stabilizing properties but also suggests that overall detergent geometry has an important role in determining membrane protein stability. Notably, this is the first systematic study on the effect of detergent kinking on micellar properties and associated membrane protein stability.

Journal article

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