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Tandem neopentyl glycol maltosides (TNMs) for membrane protein stabilisation
Bae, Hyoung Eun,Mortensen, Jonas S.,Ribeiro, Orquidea,Du, Yang,Ehsan, Muhammad,Kobilka, Brian K.,Loland, Claus J.,Byrne, Bernadette,Chae, Pil Seok The Royal Society of Chemistry 2016 Chemical communications Vol.52 No.81
<P>A novel class of detergents, designated tandem neopentyl glycol maltosides (TNMs), were evaluated with four target membrane proteins. The best detergent varied depending on the target, but TNM-C12L and TNM-C11S were notable for their ability to confer increased membrane protein stability compared to DDM. These agents have potential for use in membrane protein research.</P>
A comparative study of branched and linear mannitol-based amphiphiles on membrane protein stability
Hussain, Hazrat,Helton, Tyler,Du, Yang,Mortensen, Jonas S.,Hariharan, Parameswaran,Ehsan, Muhammad,Byrne, Bernadette,Loland, Claus J.,Kobilka, Brian K.,Guan, Lan,Chae, Pil Seok The Royal Society of Chemistry 2018 The Analyst Vol.143 No.23
<P>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 <I>n</I>-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.</P>
Das, M.,Du, Y.,Mortensen, J.,Ribeiro, O.,Hariharan, P.,Guan, L.,Loland, C.,Kobilka, B.,Byrne, B.,Chae, P. THE ROYAL SOCIETY OF CHEMISTRY 2017 Chemical Science Vol.8 No.2
<P>Amphiphile selection is a crucial step in membrane protein structural and functional study. As conventional detergents have limited scope and utility, novel agents with enhanced efficacy need to be developed. Although a large number of novel agents have been reported, so far there has been no systematically designed comparative study of the protein stabilization efficacy of stereo-isomeric amphiphiles. Here we designed and prepared a novel class of stereo-isomeric amphiphiles, designated butane-1,2,3,4-tetraolbased maltosides (BTMs). These stereoisomers showed markedly different behaviour for most of the targeted membrane proteins depending on the chirality of the linker region. These findings indicate an important role for detergent stereochemistry in membrane protein stabilization. In addition, we generally observed enhanced detergent efficacy with increasing alkyl chain length, reinforcing the importance of the balance between hydrophobicity and hydrophilicity in detergent design. The stereo-isomeric difference in detergent efficacy observed provides an important design principle for the development of novel amphiphiles for membrane protein manipulation.</P>
Duc, Nguyen Minh,Du, Yang,Thorsen, Thor S.,Lee, Su Youn,Zhang, Cheng,Kato, Hideaki,Kobilka, Brian K.,Chung, Ka Young Springer New York LLC 2015 Journal of the American Society for Mass Spectrome Vol.26 No.5
<P>G protein-coupled receptors (GPCRs) have important roles in physiology and pathology, and 40% of drugs currently on the market target GPCRs for the treatment of various diseases. Because of their therapeutic importance, the structural mechanism of GPCR signaling is of great interest in the field of drug discovery. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a useful tool for analyzing ligand binding sites, the protein-protein interaction interface, and conformational changes of proteins. However, its application to GPCRs has been limited for various reasons, including the hydrophobic nature of GPCRs and the use of detergents in their preparation. In the present study, we tested the application of bicelles as a means of solubilizing GPCRs for HDX-MS studies. GPCRs (e.g., β<SUB>2</SUB>-adrenergic receptor [β<SUB>2</SUB>AR], μ-opioid receptor, and protease-activated receptor 1) solubilized in bicelles produced better sequence coverage (greater than 90%) than GPCRs solubilized in n-dodecyl-β-D-maltopyranoside (DDM), suggesting that bicelles are a more effective method of solubilization for HDX-MS studies. The HDX-MS profile of β<SUB>2</SUB>AR in bicelles showed that transmembrane domains (TMs) undergo lower deuterium uptake than intracellular or extracellular regions, which is consistent with the fact that the TMs are highly ordered and embedded in bicelles. The overall HDX-MS profiles of β<SUB>2</SUB>AR solubilized in bicelles and in DDM were similar except for intracellular loop 3. Interestingly, we detected EX1 kinetics, an important phenomenon in protein dynamics, at the C-terminus of TM6 in β<SUB>2</SUB>AR. In conclusion, we suggest the application of bicelles as a useful method for solubilizing GPCRs for conformational analysis by HDX-MS.</P> [FIG OMISSION]</BR>
Dendronic trimaltoside amphiphiles (DTMs) for membrane protein study
Sadaf, Aiman,Du, Yang,Santillan, Claudia,Mortensen, Jonas S.,Molist, Iago,Seven, Alpay B.,Hariharan, Parameswaran,Skiniotis, Georgios,Loland, Claus J.,Kobilka, Brian K.,Guan, Lan,Byrne, Bernadette,Cha Royal Society of Chemistry 2017 Chemical Science Vol.8 No.12
<▼1><P>A novel amphiphile with a dendronic hydrophobic group (DTM-A6) was markedly effective at stabilizing and visualizing a GPCR-G<SUB>s</SUB> complex.</P></▼1><▼2><P>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>i.e.</I>, DTM-A6) clearly outperformed DDM in stabilizing human β<SUB>2</SUB> adrenergic receptor (β<SUB>2</SUB>AR) and its complex with G<SUB>s</SUB> protein. A further evaluation of this DTM led to a clear visualization of β<SUB>2</SUB>AR-G<SUB>s</SUB> complex <I>via</I> 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.</P></▼2>
Glucose-Neopentyl Glycol (GNG) amphiphiles for membrane protein study
Chae, Pil Seok,Rana, Rohini R.,Gotfryd, Kamil,Rasmussen, Søren G. F.,Kruse, Andrew C.,Cho, Kyung Ho,Capaldi, Stefano,Carlsson, Emil,Kobilka, Brian,Loland, Claus J.,Gether, Ulrik,Banerjee, Surajit,Byrn The Royal Society of Chemistry 2013 Chemical communications Vol.49 No.23
<P>The development of a new class of surfactants for membrane protein manipulation, “GNG amphiphiles”, is reported. These amphiphiles display promising behavior for membrane proteins, as demonstrated recently by the high resolution structure of a sodium-pumping pyrophosphatase reported by Kellosalo <I>et al.</I> (<I>Science</I>, 2012, 337, 473).</P> <P>Graphic Abstract</P><P>A new class of surfactants, the GNGs, tends to form small protein–detergent complexes, which may be favorable for crystallization. <IMG SRC='http://pubs.rsc.org/services/images/RSCpubs.ePlatform.Service.FreeContent.ImageService.svc/ImageService/image/GA?id=c2cc36844g'> </P>
New penta-saccharide-bearing tripod amphiphiles for membrane protein structure studies
Ehsan, Muhammad,Ghani, Lubna,Du, Yang,Hariharan, Parameswaran,Mortensen, Jonas S.,Ribeiro, Orquidea,Hu, Hongli,Skiniotis, Georgios,Loland, Claus J.,Guan, Lan,Kobilka, Brian K.,Byrne, Bernadette,Chae, Royal Society of Chemistry 2017 The Analyst Vol.142 No.20
<P>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.</P>
Das, Manabendra,Du, Yang,Ribeiro, Orquidea,Hariharan, Parameswaran,Mortensen, Jonas S.,Patra, Dhabaleswar,Skiniotis, Georgios,Loland, Claus J.,Guan, Lan,Kobilka, Brian K.,Byrne, Bernadette,Chae, Pil S American Chemical Society 2017 JOURNAL OF THE AMERICAN CHEMICAL SOCIETY - Vol.139 No.8
<P>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-beta-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.</P>
Bae, Hyoung Eun,Du, Yang,Hariharan, Parameswaran,Mortensen, Jonas S.,Kumar, Kaavya K.,Ha, Betty,Das, Manabendra,Lee, Hyun Sung,Loland, Claus J.,Guan, Lan,Kobilka, Brian K.,Chae, Pil Seok Royal Society of Chemistry 2019 Chemical Science Vol.10 No.4
<▼1><P>An asymmetric MNG, MNG-8,12, provided enhanced stability to human G protein-coupled receptors (GPCRs) compared to the symmetric MNG, MNG-3.</P></▼1><▼2><P>Maintaining protein stability in an aqueous solution is a prerequisite for protein structural and functional studies, but conventional detergents have increasingly showed limited ability to maintain protein integrity. A representative novel agent, maltose neopentyl glycol-3 (MNG-3), has recently substantially contributed to membrane protein structural studies. Motivated by the popular use of this novel agent, we prepared asymmetric versions of MNG-3 and evaluated these agents with several membrane proteins including two G protein-coupled receptors in this study. We found that some new MNGs were significantly more effective than MNG-3 at preserving protein integrity in the long term, suggesting that these asymmetric MNGs will find a wide use in membrane protein studies. In addition, this is the first study addressing the favorable effect of detergent asymmetric nature on membrane protein stability.</P></▼2>
1,3,5-Triazine-Cored Maltoside Amphiphiles for Membrane Protein Extraction and Stabilization
Ghani, Lubna,Munk, Chastine F.,Zhang, Xiang,Katsube, Satoshi,Du, Yang,Cecchetti, Cristina,Huang, Weijiao,Bae, Hyoung Eun,Saouros, Savvas,Ehsan, Muhammad,Guan, Lan,Liu, Xiangyu,Loland, Claus J.,Kobilka American Chemical Society 2019 JOURNAL OF THE AMERICAN CHEMICAL SOCIETY - Vol.141 No.50
<P>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.</P> [FIG OMISSION]</BR>