136 results found
Ghani L, Zhang X, Munk CF, et al., 2023, Tris(hydroxymethyl)aminomethane Linker-Bearing Triazine-Based Triglucosides for Solubilization and Stabilization of Membrane Proteins., Bioconjug Chem, Vol: 34, Pages: 739-747
High-resolution membrane protein structures are essential for a fundamental understanding of the molecular basis of diverse cellular processes and for drug discovery. Detergents are widely used to extract membrane-spanning proteins from membranes and maintain them in a functional state for downstream characterization. Due to limited long-term stability of membrane proteins encapsulated in conventional detergents, development of novel agents is required to facilitate membrane protein structural study. In the current study, we designed and synthesized tris(hydroxymethyl)aminomethane linker-bearing triazine-based triglucosides (TTGs) for solubilization and stabilization of membrane proteins. When these glucoside detergents were evaluated for four membrane proteins including two G protein-coupled receptors, a few TTGs including TTG-C10 and TTG-C11 displayed markedly enhanced behaviors toward membrane protein stability relative to two maltoside detergents [DDM (n-dodecyl-β-d-maltoside) and LMNG (lauryl maltose neopentyl glycol)]. This is a notable feature of the TTGs as glucoside detergents tend to be inferior to maltoside detergents at stabilizing membrane proteins. The favorable behavior of the TTGs for membrane protein stability is likely due to the high hydrophobicity of the lipophilic groups, an optimal range of hydrophilic-lipophilic balance, and the absence of cis-trans isomerism.
Lee HS, Das M, Mahler F, et al., 2022, 3,4-Bis(hydroxymethyl)hexane-1,6-diol-based Maltosides (HDMs) for membrane-protein study: importance of detergent rigidity-flexibility balance in protein stability, Chemistry: An Asian Journal, Vol: 17, ISSN: 1861-471X
Detergents have been major contributors to membrane-protein structural study for decades. However, membrane proteins solubilized in conventional detergents tend to aggregate or denature over time. Stability of large eukaryotic membrane proteins with complex structures tends to be particularly poor, necessitating development of novel detergents with improved properties. Here, we prepared a novel class of detergents, designated 3,4-bis(hydroxymethyl)hexane-1,6-diol-based maltosides (HDMs). When tested on three membrane proteins, including two G-protein-coupled receptors (GPCRs), the new detergents displayed significantly better behaviors compared with DDM. Moreover, the HDMs were superior or comparable to LMNG, an amphiphile widely used for GPCR structural study. An optimal balance of detergent rigidity vs. flexibility of the HDMs is likely responsible for their favorable behaviors toward membrane-protein stability. Thus, the current study not only introduces the HDMs, with significant potential for membrane-protein structural study, but also suggests a useful guideline for designing novel detergents for membrane-protein research.
Beattie JW, Istrate A, Lu A, et al., 2022, Causes of industrial protein a column degradation, explored using Raman spectroscopy., Analytical Chemistry, Vol: 94, Pages: 15703-15710, ISSN: 0003-2700
Monoclonal antibodies (mAbs) are used extensively as biotherapeutics for chronic and acute conditions. Production of mAbs is lengthy and expensive, with protein A affinity capture the most costly step, due both to the nature of the resin and its marked reduction in binding capacity with repeated use. Our previous studies using in situ ATR-FTIR spectroscopy indicated that loss in protein A binding capacity is not the result of leaching or degradation of protein A ligand, suggesting fouling is the principal cause. Here we explore binding behavior and resin capacity loss using Raman spectroscopy. Our data reveal a distinct Raman spectral fingerprint for mAb bound to the protein A ligand of MabSelect SuRe. The results show that the drop in static binding capacity (SBC) previously observed for used protein A resin is discernible by Raman spectroscopy in combination with partial least-squares regression. The SBC is lowest (35.76 mg mL-1) for used inlet resin compared to used outlet (40.17 mg mL-1) and unused resin samples (70.35 mg mL-1). Depth profiling by Raman spectroscopy indicates that at below saturating concentrations (∼18 mg mL-1), binding of mAb is not homogeneous through used resin beads with protein binding preferentially to the outer regions of the bead, in contrast to fully homogeneous distribution through unused control MabSelect SuRe resin beads. Analysis of the Raman spectra indicates that one foulant is irreversibly bound mAb. The presence of irreversibly bound mAb and host cell proteins was confirmed by mass spectrometric analysis of used resin beads.
Youn T, Yoon S, Byrne B, et al., 2022, Foldable Detergents for Membrane Protein Stability, ChemBioChem: a European journal of chemical biology, Vol: 23, Pages: 1-5, ISSN: 1439-4227
Detergents are widely used for membrane protein structural study. Many recently developed detergents contain multiple tail and head groups, which are typically connected via a small and branched linker. Due to their inherent compact structures, with small inter-alkyl chain distances, these detergents form micelles with high alkyl chain density in the interiors, a feature favorably associated with membrane-protein stability. A recent study on tandem triazine maltosides (TZMs) revealed a distinct trend; despite possession of an apparently large inter-alkyl chain distance, the TZM−Es were highly effective at stabilizing membrane proteins. Thanks to the incorporation of a flexible spacer between the two triazine rings in the linker region, these detergents are prone to folding into a compact architecture in micellar environments instead of adopting an extended conformation. Detergent foldability represents a new concept of novel detergent design with significant potential for future detergent development.
Lee HJ, Ehsan M, Zhang X, et al., 2022, Development of 1,3-acetonedicarboxylate-derived glucoside amphiphiles (ACAs) for membrane protein study, Chemical Science, Vol: 13, Pages: 5750-5759, ISSN: 2041-6520
Detergents are extensively used for membrane protein manipulation. Membrane proteins solubilized in conventional detergents are prone to denaturation and aggregation, rendering downstream characterization of these bio-macromolecules difficult. Although many amphiphiles have been developed to overcome the limited efficacy of conventional detergents for protein stabilization, only a handful of novel detergents have so far proved useful for membrane protein structural studies. Here, we introduce 1,3-acetonedicarboxylate-derived amphiphiles (ACAs) containing three glucose units and two alkyl chains as head and tail groups, respectively. The ACAs incorporate two different patterns of alkyl chain attachment to the core detergent unit, generating two sets of amphiphiles: ACA-As (asymmetrically alkylated) and ACA-Ss (symmetrically alkylated). The difference in the attachment pattern of the detergent alkyl chains resulted in minor variation in detergent properties such as micelle size, critical micelle concentration, and detergent behaviors toward membrane protein extraction and stabilization. In contrast, the impact of the detergent alkyl chain length on protein stability was marked. The two C11 variants (ACA-AC11 and ACA-SC11) were most effective at stabilizing the tested membrane proteins. The current study not only introduces new glucosides as tools for membrane protein study, but also provides detergent structure–property relationships important for future design of novel amphiphiles.
Lee HJ, Lee HS, Youn T, et al., 2022, Impact of novel detergents on membrane protein studies, Chem, Vol: 8, Pages: 980-1013, ISSN: 2451-9294
Detergents are widely used for membrane protein solubilization, purification, and structural analysis. Over the past two decades, much effort has been made to design novel detergents with improved properties. Here, we provide the details of 23 different classes of novel detergents with particular focus on OGNG, LMNG, and GDN, which have been widely applied to the successful determination of a wide range of membrane protein structures. This review provides insights into the biophysical basis of the distinct behaviors of these three detergents by analysis of the stability of protein-detergent complexes (PDCs) in terms of detergent-detergent and detergent-protein interactions. The review also discusses recently developed variants of OGNG, LMNG, and GDN as well as several other classes of amphiphiles in the context of these interactions. Finally, a detailed perspective of a new detergent design is provided as a basis for expanding the applicability of small detergents for membrane protein structural study.
Ehsan M, Wang H, Katsube S, et al., 2022, Glyco-Steroidal Amphiphiles (GSAs) for membrane protein structural study, ChemBioChem: a European journal of chemical biology, Vol: 23, Pages: 1-8, ISSN: 1439-4227
Integral membrane proteins pose considerable challenges to high resolution structural analysis. Maintaining membrane proteins in their native state during protein isolation is essential for structural study of these bio-macromolecules. Detergents are the most commonly used amphiphilic compounds for stabilizing membrane proteins in solution outside a lipid bilayer. We previously introduced a glyco-diosgenin (GDN) detergent that was shown to be highly effective at stabilizing a wide range of membrane proteins. This steroidal detergent has additionally gained attention due to its compatibility with membrane protein structure study via cryo-EM. However, synthetic inconvenience limits widespread use of GDN in membrane protein study. To improve its synthetic accessibility and to further enhance detergent efficacy for protein stabilization, we designed a new class of glyco-steroid-based detergents using three steroid units: cholestanol, cholesterol and diosgenin. These new detergents were efficiently prepared and showed marked efficacy for protein stabilization in evaluation with a few model membrane proteins including two G protein-coupled receptors. Some new agents were not only superior to a gold standard detergent, DDM (n-dodecyl-β-d-maltoside), but were also more effective than the original GDN at preserving protein integrity long term. These agents represent valuable alternatives to GDN, and are likely to facilitate structural determination of challenging membrane proteins.
Ghani L, Kim S, Wang H, et al., 2022, Foldable detergents for membrane protein study: importance of setergent core flexibility in protein stabilization., Chemistry: A European Journal, ISSN: 0947-6539
Membrane proteins are of biological and pharmaceutical significance. However, their structural study is extremely challenging mainly due to the fact that only a small number of chemical tools are suitable for stabilizing membrane proteins in solution. Detergents are widely used in membrane protein study, but conventional detergents are generally poor at stabilizing challenging membrane proteins such as G protein-coupled receptors and protein complexes. In the current study, we prepared tandem triazine-based maltosides (TZMs) with two amphiphilic triazine units connected by different diamine linkers, hydrazine (TZM-Hs) and 1,2-ethylenediamine (TZM-Es). These TZMs were consistently superior to a gold standard detergent (DDM) in terms of stabilizing a few membrane proteins. In addition, the TZM-Es containing a long linker showed more general protein stabilization efficacy with multiple membrane proteins than the TZM-Hs containing a short linker. This result indicates that introduction of the flexible1,2-ethylenediamine linker between two rigid triazine rings enables the TZM-Es to fold into favourable conformations in order to promote membrane protein stability. The novel concept of detergent foldability introduced in the current study has potential in rational detergent design and membrane protein applications.
Sarkar K, Joedicke L, Westwood M, et al., 2022, Modulation of PTH1R signaling by an extracellular binding antibody, PARATHYROID HORMONE, Vol: 120, Pages: 109-132, ISSN: 0083-6729
Ehsan M, Wang H, Cecchetti C, et al., 2021, Maltose-bis(hydroxymethyl)phenol (MBPs) and Maltosetris(hydroxymethyl)phenol (MTPs) Amphiphiles for Membrane Protein Stability, ACS Chemical Biology, Vol: 16, Pages: 1779-1790, ISSN: 1554-8929
Membrane protein structures provide a fundamental understanding of their molecular actions and are of importance for drug development. Detergents are widely used to solubilize, stabilize, and crystallize membrane proteins, but membrane proteins solubilized in conventional detergents are prone to denaturation and aggregation. Thus, developing novel detergents with enhanced efficacy for protein stabilization remains important. We report herein the design and synthesis of a class of phenol-derived maltoside detergents. Using two different linkers, we prepared two sets of new detergents, designated maltose-bis(hydroxymethyl)phenol (MBPs) and maltose-tris(hydroxymethyl)phenol (MTPs). The evaluation of these detergents with three transporters and two G-protein coupled receptors allowed us to identify a couple of new detergents (MBP-C9 and MTP-C12) that consistently conferred enhanced stability to all tested proteins compared to a gold standard detergent (DDM). Furthermore, the data analysis based on the detergent structures provides key detergent features responsible for membrane protein stabilization that together will facilitate the future design of novel detergents.
Byrne B, Renard K, 2021, Insights into the role of membrane lipids in structure, function and regulation of integral membrane proteins., International Journal of Molecular Sciences, Vol: 22, Pages: 1-20, ISSN: 1422-0067
Membrane proteins exist within the highly hydrophobic membranes surrounding cells and organelles, playing key roles in cellular function. It is becoming increasingly clear that the membrane does not just act as an appropriate environment for these proteins, but that the lipids that make up these membranes are essential for membrane protein structure and function. Recent technological advances in cryogenic Electron Microscopy and in advanced Mass Spectrometry methods, as well as the development of alternative membrane mimetic systems, have allowed experimental study of membrane protein-lipid complexes. These have been complemented by computational approaches, exploiting the ability of Molecular Dynamics simulations to allow exploration of membrane protein conformational changes in membranes of defined lipid content. These studies have revealed the importance of lipids in stabilising the oligomeric forms of membrane proteins, mediating protein-protein interactions, maintaining a specific conformational state of a membrane protein and activity. Here we review some of the key recent advances in the field of membrane protein-lipid studies, with major emphasis on respiratory complexes, transporters, channels and G-protein coupled receptors.
Byrne B, 2021, It takes two to transport via an elevator, Cell Research, Vol: 27, Pages: 965-966, ISSN: 1001-0602
Membrane transporter proteins are critical for cellular uptake and export of molecules, and are reported to function by a number of different molecular mechanisms. The new occluded state structure of the uracil transporter, UraA, from Escherichia coli, reveals that both coordinated movement of the two domains of a single protomer together with dimer formation are important for transport activity.
Beattie J, Rowland-Jones R, Farys M, et al., 2021, Insight into purification of monoclonal antibodies in industrial columns via studies of Protein A binding capacity by in situ ATR-FTIR spectroscopy, The Analyst, Vol: 146, Pages: 5177-5185, ISSN: 0003-2654
Therapeutic monoclonal antibodies (mAbs) are effective treatments for a range of cancers and other serious diseases, however mAb treatments cost on average ∼$100 000 per year per patient, limiting their use. Currently, industry favours Protein A affinity chromatography (PrAc) as the key step in downstream processing of mAbs. This step, although highly efficient, represents a significant mAb production cost. Fouling of the Protein A column and Protein A ligand leaching contribute to the cost of mAb production by shortening the life span of the resin. In this study, we assessed the performance of used PrAc resin recovered from the middle inlet, center and outlet as well as the side inlet of a pilot-scale industrial column. We used a combination of static binding capacity (SBC) analysis and Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) spectroscopy to explore the used resin samples. SBC analysis demonstrated that resin from the inlet of the column had lower binding capacity than resin from the column outlet. ATR-FTIR spectroscopy with PLS (partial least square) analysis confirmed the results obtained from SBC analysis. Importantly, in situ ATR-FTIR spectroscopy also allowed both measurement of the concentration and assessment of the conformational state of the bound Protein A. Our results reveal that PrAc resin degradation after use is dependent on column location and that neither Protein A ligand leaching nor denaturation are responsible for binding capacity loss.
Byrne B, Cecchetti C, Strauss J, et al., 2021, A novel high-throughput screen for identifying lipids that stabilise membrane proteins in detergent based solution, PLoS One, Vol: 16, Pages: 1-20, ISSN: 1932-6203
Membrane proteins have a range of crucial biological functions and are the target of about 60% of all prescribed drugs. For most studies, they need to be extracted out of the lipid-bilayer, e.g. by detergent solubilisation, leading to the loss of native lipids, which may disturb important protein-lipid/bilayer interactions and thus functional and structural integrity. Relipidation of membrane proteins has proven extremely successful for studying challenging targets, but the identification of suitable lipids can be expensive and laborious. Therefore, we developed a screen to aid the high-throughput identification of beneficial lipids. The screen covers a large lipid space and was designed to be suitable for a range of stability assessment methods. Here, we demonstrate its use as a tool for identifying stabilising lipids for three membrane proteins: a bacterial pyrophosphatase (Tm-PPase), a fungal purine transporter (UapA) and a human GPCR (A2AR). A2AR is stabilised by cholesteryl hemisuccinate, a lipid well known to stabilise GPCRs, validating the approach. Additionally, our screen also identified a range of new lipids which stabilised our test proteins, providing a starting point for further investigation and demonstrating its value as a novel tool for membrane protein research. The pre-dispensed screen will be made commercially available to the scientific community in future and has a number of potential applications in the field.
Sadaf A, Kim S, Bae HE, et al., 2021, Conformationally flexible core-bearing detergents with a hydrophobic or hydrophilic pendant: effect of pendant polarity on detergent conformation and membrane protein stability, Acta Biomaterialia, Vol: 128, Pages: 393-407, ISSN: 1742-7061
Membrane protein structures provide atomic level insight into essential biochemical processes and facilitate protein structure-based drug design. However, the inherent instability of these bio-macromolecules outside lipid bilayers hampers their structural and functional study. Detergent micelles can be used to solubilize and stabilize these membrane-inserted proteins in aqueous solution, thereby enabling their downstream characterizations. Membrane proteins encapsulated in detergent micelles tend to denature and aggregate over time, highlighting the need for development of new amphiphiles effective for protein solubility and stability. In this work, we present newly-designed maltoside detergents containing a pendant chain attached to a glycerol-decorated tris(hydroxymethyl)methane (THM) core, designated GTMs. One set of the GTMs has a hydrophobic pendant (ethyl chain; E-GTMs), and the other set has a hydrophilic pendant (methoxyethoxylmethyl chain; M-GTMs) placed in the hydrophobic-hydrophilic interfaces. The two sets of GTMs displayed profoundly different behaviors in terms of detergent self-assembly and protein stabilization efficacy. These behaviors mainly arise from the polarity difference between two pendants (ethyl and methoxyethoxylmethyl chains) that results in a large variation in detergent conformation between these sets of GTMs in aqueous media. The resulting high hydrophobic density in the detergent micelle interior is likely responsible for enhanced efficacy of the M-GTMs for protein stabilization compared to the E-GTMs and a gold standard detergent DDM. A representative GTM, M-GTM-O12, was more effective for protein stability than some recently developed detergents including LMNG. This is the first case study investigating the effect of pendant polarity on detergent geometry correlated with detergent efficacy for protein stabilization. STATEMENT OF SIGNIFICANCE: This study introduces new amphiphiles for use as biochemical tools in membrane protein stud
Byrne B, Saouros S, Mohan T, et al., 2021, Structural and functional insights into the mechanism of action of plant borate transporters, Scientific Reports, Vol: 11, Pages: 1-12, ISSN: 2045-2322
Boron has essential roles in plant growth and development. BOR proteins are key in the active uptake and distribution of boron, and regulation of intracellular boron concentrations. However, their mechanism of action remains poorly studied. BOR proteins are homologues of the human SLC4 family of transporters, which includes well studied mammalian transporters such as the human Anion Exchanger 1 (hAE1). Here we generated Arabidopsis thaliana BOR1 (AtBOR1) variants based (i) on known disease causing mutations of hAE1 (S466R, A500R) and (ii) a loss of function mutation (D311A) identified in the yeast BOR protein, ScBOR1p. The AtBOR1 variants express in yeast and localise to the plasma membrane, although both S466R and A500R exhibit lower expression than the WT AtBOR1 and D311A. The D311A, S466R and A500R mutations result in a loss of borate efflux activity in a yeast bor1p knockout strain. A. thaliana plants containing these three individual mutations exhibit substantially decreased growth phenotypes in soil under conditions of low boron. These data confirm an important role for D311 in the function of the protein and show that mutations equivalent to disease-causing mutations in hAE1 have major effects in AtBOR1. We also obtained a low resolution cryo-EM structure of a BOR protein from Oryza sativa, OsBOR3, lacking the 30 C-terminal amino acid residues. This structure confirms the gate and core domain organisation previously observed for related proteins, and is strongly suggestive of an inward facing conformation.
Cecchetti C, Scull NJ, Mohan TC, et al., 2021, Transfer of stabilising mutations between different secondary active transporter families, FEBS Open Bio, Vol: 11, Pages: 1685-1694, ISSN: 2211-5463
Integral membrane transporters play essential roles in the movement of substrates across biological membranes. One approach to produce transporters suitable for structural studies is to introduce mutations that reduce conformational flexibility and increase stability. However, it can be difficult to predict which mutations will result in a more stable protein. Previously, we stabilised the uric acid-xanthine transporter, UapA, a member of the SLC23 family, through introduction of a single-point mutation, G411V, trapping the protein in the inward-facing conformation. Here, we attempted to stabilise the structurally related BOR1 transporter from Arabidopsis thaliana, a member of the SLC4 family, by introducing the equivalent substitution. We identified possible residues, P362 and M363, in AtBOR1, likely to be equivalent to the G411 of UapA, and generated four mutants, P362V or L and M363F or Y. Stability analysis using heated Fluorescent Size Exclusion Chromatography indicated that the M363F/Y mutants were more stable than the WT AtBOR1 and P362V/L mutants. Furthermore, functional complementation analysis revealed that the M363F/Y mutants exhibited reduced transport activity compared to the P362V/L and WT proteins. Purification and crystallisation of the M363F/Y proteins yielded crystals that diffracted better than WT (5.5 vs 7 Å). We hypothesise that the increased bulk of the F and Y substitutions limits the ability of the protein to undergo the conformational rearrangements associated with transport. These proteins represent a basis for future studies on AtBOR1.
Tiernan H, Byrne B, Kazarian SG, 2021, ATR-FTIR spectroscopy and spectroscopic imaging to investigate the behaviour of proteins subjected to freeze-thaw cycles in droplets, wells, and under flow., The Analyst, Vol: 146, Pages: 2902-2909, ISSN: 0003-2654
Biopharmaceuticals are used to treat a range of diseases from arthritis to cancer, however, since the advent of these highly specific, effective drugs, there have been challenges involved in their production. The most common biopharmaceuticals, monoclonal antibodies (mAbs), are vulnerable to aggregation and precipitation during processing. Freeze thaw cycles (FTCs), which can be required for storage and transportation, can lead to a substantial loss of product, and contributes to the high cost of antibody production. It is therefore necessary to monitor aggregation levels at susceptible points in the production pathway, such as during purification and transportation, thus contributing to a fuller understanding of mAb aggregation and providing a basis for rational optimisation of the production process. This paper uses attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy and spectroscopic imaging to investigate the effect of these potentially detrimental FTCs on protein secondary structure in both static wells and under flowing conditions, using lysozyme as a model protein. The results revealed that the amount of protein close to the surface of the ATR crystal, and hence level of aggregates, increased with increasing FTCs. This was observed both within wells and under flow conditions, using conventional ATR-FTIR spectroscopy and ATR-FTIR spectroscopic imaging. Interestingly, we also observed changes in the Amide I band shape indicating an increase in β-sheet contribution, and therefore an increase in aggregates, with increasing number of FTCs. These results show for the first time how ATR-FTIR spectroscopy can be successfully applied to study the effect of FTC cycles on protein samples. This could have numerous broader applications, such as in biopharmaceutical production and rapid diagnostic testing.
Das M, Mahler F, Hariharan P, et al., 2020, Diastereomeric Cyclopentane-Based Maltosides (CPMs) as tools for membrane protein study, Journal of the American Chemical Society, Vol: 142, Pages: 21382-21392, ISSN: 0002-7863
Amphiphilic agents, called detergents, are invaluable tools for studying membrane proteins. However, membrane proteins encapsulated by conventional head-to-tail detergents tend to denature or aggregate, necessitating the development of structurally distinct molecules with improved efficacy. Here, a novel class of diastereomeric detergents with a cyclopentane core unit, designated cyclopentane-based maltosides (CPMs), were prepared and evaluated for their ability to solubilize and stabilize several model membrane proteins. A couple of CPMs displayed enhanced behavior compared with the benchmark conventional detergent, n-dodecyl-β-d-maltoside (DDM), for all the tested membrane proteins including two G-protein-coupled receptors (GPCRs). Furthermore, CPM-C12 was notable for its ability to confer enhanced membrane protein stability compared with the previously developed conformationally rigid NBMs [J. Am. Chem. Soc.2017, 139, 3072] and LMNG. The effect of the individual CPMs on protein stability varied depending on both the detergent configuration (cis/trans) and alkyl chain length, allowing us draw conclusions on the detergent structure–property–efficacy relationship. Thus, this study not only provides novel detergent tools useful for membrane protein research but also reports on structural features of the detergents critical for detergent efficacy in stabilizing membrane proteins.
Saouros S, Mohan TC, Cecchetti C, et al., 2020, Structural and functional insights into the mechanism of action of plant boron transporters, Publisher: Cold Spring Harbor Laboratory
Boron has essential roles in plant growth and development. BOR proteins are key in the active uptake and distribution of boron, and regulation of intracellular boron concentrations. However, their mechanism of action remains poorly studied. BOR proteins are members of the SLC4 family of transporters and thus homologues of well studied mammalian transporters including the human Anion Exchanger 1 (hAE1). Here we generated Arabidopsis thaliana BOR1 (AtBOR1) variants based i) on known disease causing mutations of hAE1 (S466R, A500R) and ii) a loss of function mutation (D311A) identified in the yeast BOR protein, ScBOR1p. The AtBOR1 variants express in yeast and localise to the plasma membrane, although both S466R and A500R exhibit lower expression than the WT AtBOR1 and D311A. The D311A, S466R and A500R mutations result in a loss of boron efflux activity in a yeast bor1p knockout strain. A. thaliana plants containing these three individual mutations exhibit substantially decreased growth phenotypes in soil under conditions of low boron. These data confirm an important role for D311 in the function of the protein and show that mutations equivalent to disease causing mutations in hAE1 have major effects in AtBOR1. We also obtained a low resolution cryo-EM structure of a BOR protein from Oryza sativa, OsBOR3 lacking the 30 C-terminal amino acids. This structure confirms the gate and core domain organisation previously observed for related proteins, and is strongly suggestive of an inward facing conformation.
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.
Bae HE, Cecchetti C, Du Y, et 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
Ehsan M, Katsube S, Cecchetti C, et al., 2020, New malonate-derived tetraglucoside detergents for membrane protein stability, ACS Chemical Biology, Vol: 15, Pages: 1697-1707, ISSN: 1554-8929
Membrane proteins are widely studied in detergent micelles, a membrane-mimetic system formed by amphiphilic compounds. However, classical detergents have serious limitations in their utility, particularly for unstable proteins such as eukaryotic membrane proteins and membrane protein complexes, and thus, there is an unmet need for novel amphiphiles with enhanced ability to stabilize membrane proteins. Here, we developed a new class of malonate-derived detergents with four glucosides, designated malonate-derived tetra-glucosides (MTGs), and compared these new detergents with previously reported octyl glucose neopentyl glycol (OGNG) and n-dodecyl-β-d-maltoside (DDM). When tested with two G-protein coupled receptors (GPCRs) and three transporters, a couple of MTGs consistently conferred enhanced stability to all tested proteins compared to DDM and OGNG. As a result of favorable behaviors for a range of membrane proteins, these MTGs have substantial potential for membrane protein research. This study additionally provides a new detergent design principle based on the effect of a polar functional group (i.e., ether) on protein stability depending on its position in the detergent scaffold.
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
Saouros S, Cecchetti C, Jones A, et 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.
Ghani L, Munk CF, Zhang X, et 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.
Kourkoulou A, Grevias P, Lambrinidis G, et al., 2019, Distinct specific interactions of the UapA transporter with membrane lipids are critical for dimerization, ER-exit and function, Genetics: a periodical record of investigations bearing on heredity and variation, Vol: 213, Pages: 1357-1372, ISSN: 0016-6731
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 Summary</jats:title><jats:p>Transporters are proteins
Pyle E, Guo C, Hofmann T, et 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.
Sarkar K, Joedicke L, Westwood M, et 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.
Ehsan M, Du Y, Mortensen JS, et 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.
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