RESUMEN
Hierarchical organization of integral membrane proteins (IMP) and lipids at the membrane is essential for regulating myriad downstream signaling. A quantitative understanding of these processes requires both detections of oligomeric organization of IMPs and lipids directly from intact membranes and determination of key membrane components and properties that regulate them. Addressing this, we have developed a platform that enables native mass spectrometry (nMS) analysis of IMP-lipid complexes directly from intact and customizable lipid membranes. Both the lipid composition and membrane properties (such as curvature, tension, and fluidity) of these bilayers can be precisely customized to a target membrane. Subsequent direct nMS analysis of these intact proteolipid vesicles can yield the oligomeric states of the embedded IMPs, identify bound lipids, and determine the membrane properties that can regulate the observed IMP-lipid organization. Applying this method, we show how lipid binding regulates neurotransmitter release and how membrane composition regulates the functional oligomeric state of a transporter.
Asunto(s)
Lípidos , Proteínas de la Membrana , Espectrometría de Masas/métodos , Transporte Biológico , Lípidos/química , Proteínas de la Membrana/química , Membrana Dobles de Lípidos/químicaRESUMEN
Salmonella enterica serovar Typhimurium melibiose permease (MelBSt) is a prototype of the major facilitator superfamily (MFS) transporters, which play important roles in human health and diseases. MelBSt catalyzed the symport of galactosides with Na+, Li+, or H+ but prefers the coupling with Na+. Previously, we determined the structures of the inward- and outward-facing conformation of MelBSt and the molecular recognition for galactoside and Na+. However, the molecular mechanisms for H+- and Na+-coupled symport remain poorly understood. In this study, we solved two x-ray crystal structures of MelBSt, the cation-binding site mutants D59C at an unliganded apo-state and D55C at a ligand-bound state, and both structures display the outward-facing conformations virtually identical as published. We determined the energetic contributions of three major Na+-binding residues for the selection of Na+ and H+ by free energy simulations. Transport assays showed that the D55C mutant converted MelBSt to a solely H+-coupled symporter, and together with the free-energy perturbation calculation, Asp59 is affirmed to be the sole protonation site of MelBSt. Unexpectedly, the H+-coupled melibiose transport exhibited poor activities at greater bulky ΔpH and better activities at reversal ΔpH, supporting the novel theory of transmembrane-electrostatically localized protons and the associated membrane potential as the primary driving force for the H+-coupled symport mediated by MelBSt. This integrated study of crystal structure, bioenergetics, and free energy simulations, demonstrated the distinct roles of the major binding residues in the cation-binding pocket of MelBSt.
Asunto(s)
Sodio , Simportadores , Simportadores/química , Simportadores/metabolismo , Simportadores/genética , Sitios de Unión , Cristalografía por Rayos X , Sodio/metabolismo , Sodio/química , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Salmonella typhimurium/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/química , Melibiosa/metabolismo , Melibiosa/química , Cationes/metabolismo , Cationes/química , Conformación ProteicaRESUMEN
Salmonella enterica serovar Typhimurium melibiose permease (MelBSt) is a prototype of the Na+-coupled major facilitator superfamily transporters, which are important for the cellular uptake of molecules including sugars and small drugs. Although the symport mechanisms have been well-studied, mechanisms of substrate binding and translocation remain enigmatic. We have previously determined the sugar-binding site of outward-facing MelBSt by crystallography. To obtain other key kinetic states, here we raised camelid single-domain nanobodies (Nbs) and carried out a screening against the WT MelBSt under 4 ligand conditions. We applied an in vivo cAMP-dependent two-hybrid assay to detect interactions of Nbs with MelBSt and melibiose transport assays to determine the effects on MelBSt functions. We found that all selected Nbs showed partial to complete inhibitions of MelBSt transport activities, confirming their intracellular interactions. A group of Nbs (714, 725, and 733) was purified, and isothermal titration calorimetry measurements showed that their binding affinities were significantly inhibited by the substrate melibiose. When titrating melibiose to the MelBSt/Nb complexes, Nb also inhibited the sugar-binding. However, the Nb733/MelBSt complex retained binding to the coupling cation Na+ and also to the regulatory enzyme EIIAGlc of the glucose-specific phosphoenolpyruvate/sugar phosphotransferase system. Further, EIIAGlc/MelBSt complex also retained binding to Nb733 and formed a stable supercomplex. All data indicated that MelBSt trapped by Nbs retained its physiological functions and the trapped conformation is similar to that bound by the physiological regulator EIIAGlc. Therefore, these conformational Nbs can be useful tools for further structural, functional, and conformational analyses.
Asunto(s)
Anticuerpos de Dominio Único , Simportadores , Anticuerpos de Dominio Único/metabolismo , Melibiosa/metabolismo , Simportadores/metabolismo , Transporte Iónico , Sodio/metabolismoRESUMEN
Membrane protein structures are essential for the molecular understanding of diverse cellular processes and drug discovery. Detergents are not only widely used to extract membrane proteins from membranes but also utilized to preserve native protein structures in aqueous solution. However, micelles formed by conventional detergents are suboptimal for membrane protein stabilization, necessitating the development of novel amphiphilic molecules with enhanced protein stabilization efficacy. In this study, we prepared two sets of tandem malonate-derived glucoside (TMG) variants, both of which were designed to increase the alkyl chain density in micelle interiors. The alkyl chain density was modulated either by reducing the spacer length (TMG-Ms) or by introducing an additional alkyl chain between the two alkyl chains of the original TMGs (TMG-Ps). When evaluated with a few membrane proteins including a G protein-coupled receptor, TMG-P10,8 was found to be substantially more efficient at extracting membrane proteins and also effective at preserving protein integrity in the long term compared to the previously described TMG-A13. This result reveals that inserting an additional alkyl chain between the two existing alkyl chains is an effective way to optimize detergent properties for membrane protein study. This new biochemical tool and the design principle described have the potential to facilitate membrane protein structure determination.
Asunto(s)
Detergentes , Proteínas de la Membrana , Proteínas de la Membrana/metabolismo , Detergentes/química , MicelasRESUMEN
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-ß-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.
Asunto(s)
Detergentes , Proteínas de la Membrana , Proteínas de la Membrana/química , Detergentes/química , Trometamina , Triazinas , Glucósidos/química , SolubilidadRESUMEN
The melibiose permease of Salmonella typhimurium (MelBSt) catalyzes the stoichiometric symport of galactopyranoside with a cation (H+, Li+, or Na+) and is a prototype for Na+-coupled major facilitator superfamily (MFS) transporters presenting from bacteria to mammals. X-ray crystal structures of MelBSt have revealed the molecular recognition mechanism for sugar binding; however, understanding of the cation site and symport mechanism is still vague. To further investigate the transport mechanism and conformational dynamics of MelBSt, we generated a complete single-Cys library containing 476 unique mutants by placing a Cys at each position on a functional Cys-less background. Surprisingly, 105 mutants (22%) exhibit poor transport activities (<15% of Cys-less transport), although the expression levels of most mutants were comparable to that of the control. The affected positions are distributed throughout the protein. Helices I and X and transmembrane residues Asp and Tyr are most affected by cysteine replacement, while helix IX, the cytoplasmic middle-loop, and C-terminal tail are least affected. Single-Cys replacements at the major sugar-binding positions (K18, D19, D124, W128, R149, and W342) or at positions important for cation binding (D55, N58, D59, and T121) abolished the Na+-coupled active transport, as expected. We mapped 50 loss-of-function mutants outside of these substrate-binding sites that suffered from defects in protein expression/stability or conformational dynamics. This complete Cys-scanning mutagenesis study indicates that MelBSt is highly susceptible to single-Cys mutations, and this library will be a useful tool for further structural and functional studies to gain insights into the cation-coupled symport mechanism for Na+-coupled MFS transporters.
Asunto(s)
Cisteína/metabolismo , Simportadores/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Transporte Biológico Activo , Transporte Iónico , Modelos Moleculares , Mutagénesis/genética , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Sodio/metabolismo , Simportadores/metabolismoRESUMEN
Here, we present the atomic resolution crystallographic structure, the function, and the ion-binding properties of the KcsA mutants, G77A and G77C, that stabilize the 2,4-ion-bound configuration (i.e., water, K+, water, K+-ion-bound configuration) of the K+ channel's selectivity filter. A full functional and thermodynamic characterization of the G77A mutant revealed wild-type-like ion selectivity and apparent K+-binding affinity, in addition to showing a lack of C-type inactivation gating and a marked reduction in its single-channel conductance. These structures validate, from a structural point of view, the notion that 2 isoenergetic ion-bound configurations coexist within a K+ channel's selectivity filter, which fully agrees with the water-K+-ion-coupled transport detected by streaming potential measurements.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Canales de Potasio/química , Canales de Potasio/metabolismo , Permeabilidad de la Membrana Celular , Cristalografía por Rayos X , Activación del Canal Iónico , Iones , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Conformación Proteica , Estabilidad ProteicaRESUMEN
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-ß-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.
Asunto(s)
Ciclopentanos/química , Maltosa/química , Maltosa/farmacología , Proteínas de la Membrana/química , Diseño de Fármacos , Glucósidos/química , Interacciones Hidrofóbicas e Hidrofílicas , Estabilidad Proteica/efectos de los fármacos , Solubilidad/efectos de los fármacos , EstereoisomerismoRESUMEN
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-ß-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 ß2 adrenergic receptor (ß2 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.
Asunto(s)
Detergentes/química , Maltosa/química , Proteínas de la Membrana/química , Materiales Biomiméticos/química , Interacciones Hidrofóbicas e Hidrofílicas , Micelas , Conformación Molecular , Estabilidad Proteica , Solubilidad , Compuestos de Terfenilo/químicaRESUMEN
BACKGROUND: Membrane lipids play critical roles in the structure and function of membrane-embedded transporters. Salmonella typhimurium MelB (MelBSt) is a symporter coupling melibiose translocation with a cation (Na+, Li+, or H+). We present an extensive study on the effects of specific phospholipids on the structure of MelBSt and the melibiose transport catalyzed by this protein. RESULTS: Lipidomic analysis and thin-layer chromatography (TLC) experiments reveal that at least one phosphatidylethanolamine (PE) and one phosphatidylglycerol (PG) molecule associate with MelBSt at high affinities. Solid-state nuclear magnetic resonance (ssNMR) spectroscopy experiments confirmed the presence of lipid tails and glycerol backbones that co-purified with MelBSt; headgroups of PG were also observed. Studies with lipid-engineered strains, including PE-deficient, cardiolipin (CL)- and PG-deficient, or CL-deficient strains, show that lack of PE or PG, however not CL, largely inhibits both H+- and Na+-coupled melibiose active transport to different extents. Interestingly, neither the co-substrate binding (melibiose or Na+) nor MelBSt folding and stability are affected by changing lipid compositions. Remarkably, the delipidated MelBSt with only 2-3 bound lipids, regardless of the headgroup species, also exhibits unchanged melting temperature values as shown by circular dichroism spectroscopy. CONCLUSIONS: (1) Lipid tails and glycerol backbones of interacting PE and PG may contribute to the stability of the structure of MelBSt. (2) The headgroups of PE and PG, but not of CL, play important roles in melibiose transport; however, lipid headgroups do not modulate the folding and stability of MelBSt.
Asunto(s)
Proteínas Bacterianas/genética , Melibiosa/metabolismo , Salmonella typhimurium/genética , Simportadores/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Cardiolipinas/química , Cardiolipinas/metabolismo , Melibiosa/química , Fosfatidiletanolaminas/química , Fosfatidilgliceroles/química , Salmonella typhimurium/metabolismo , Simportadores/química , Simportadores/metabolismoRESUMEN
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.â S. Chae, K. Gotfryd, J. Pacyna, L.â J.â W. Miercke, S.â G.â F. Rasmussen, R.â A. Robbins, R.â R. Rana, C.â J. Loland, B. Kobilka, R. Stroud, B. Byrne, U. Gether, S.â 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.
Asunto(s)
Sistemas de Transporte de Aminoácidos/química , Proteínas Bacterianas/química , Detergentes/química , Proteínas de la Membrana/química , Receptores Adrenérgicos beta 2/química , Simportadores/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Micelas , Estabilidad Proteica , Salmonella typhimurium/metabolismo , SolubilidadRESUMEN
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.
Asunto(s)
Sistemas de Transporte de Aminoácidos/química , Colestanos/química , Detergentes/química , Oligosacáridos/química , Receptores Adrenérgicos beta 2/química , Simportadores/química , Proteínas Bacterianas/química , Colestanos/síntesis química , Detergentes/síntesis química , Humanos , Micelas , Oligosacáridos/síntesis química , Estabilidad Proteica , Salmonella typhimurium/química , EstereoisomerismoRESUMEN
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-ß-d-maltoside and the previously developed MNAs in maintaining the native protein structures, indicating potential utility of these new agents in membrane protein study.
Asunto(s)
Proteínas Bacterianas/química , Detergentes/química , Manitol/análogos & derivados , Proteínas de la Membrana/química , Detergentes/síntesis química , Interacciones Hidrofóbicas e Hidrofílicas , Manitol/síntesis química , Micelas , Estructura Molecular , Estabilidad Proteica , SolubilidadRESUMEN
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.
Asunto(s)
Detergentes/química , Glicósidos/química , Proteínas de la Membrana/química , Vitamina E/análogos & derivados , Aspergillus nidulans/química , Bacterias/química , Proteínas Bacterianas/química , Proteínas Fúngicas/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Micelas , Estructura Molecular , SolubilidadRESUMEN
In a variety of bacteria, the phosphotransferase protein IIA(Glc) plays a key regulatory role in catabolite repression in addition to its role in the vectorial phosphorylation of glucose catalyzed by the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). The lactose permease (LacY) of Escherichia coli catalyzes stoichiometric symport of a galactoside with an H(+), using a mechanism in which sugar- and H(+)-binding sites become alternatively accessible to either side of the membrane. Both the expression (via regulation of cAMP levels) and the activity of LacY are subject to regulation by IIA(Glc) (inducer exclusion). Here we report the thermodynamic features of the IIA(Glc)-LacY interaction as measured by isothermal titration calorimetry (ITC). The studies show that IIA(Glc) binds to LacY with a Kd of about 5 µM and a stoichiometry of unity and that binding is driven by solvation entropy and opposed by enthalpy. Upon IIA(Glc) binding, the conformational entropy of LacY is restrained, which leads to a significant decrease in sugar affinity. By suppressing conformational dynamics, IIA(Glc) blocks inducer entry into cells and favors constitutive glucose uptake and utilization. Furthermore, the studies support the notion that sugar binding involves an induced-fit mechanism that is inhibited by IIA(Glc) binding. The precise mechanism of the inhibition of LacY by IIA(Glc) elucidated by ITC differs from the inhibition of melibiose permease (MelB), supporting the idea that permeases can differ in their thermodynamic response to binding IIA(Glc).
Asunto(s)
Entropía , Escherichia coli/enzimología , Proteínas de Transporte de Membrana/metabolismo , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/metabolismo , Calorimetría , Cristalografía por Rayos X , Galactósidos/metabolismo , Glucosa/metabolismo , Calor , Cinética , Modelos Moleculares , Proteínas Mutantes/metabolismo , Unión ProteicaRESUMEN
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-ß-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.
RESUMEN
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.
RESUMEN
Camelid nanobodies (Nbs) raised against the outward-facing conformer of a double-Trp mutant of the lactose permease of Escherichia coli (LacY) stabilize the permease in outward-facing conformations. Isothermal titration calorimetry is applied herein to dissect the binding thermodynamics of two Nbs, one that markedly improves access to the sugar-binding site and another that dramatically increases the affinity for galactoside. The findings presented here show that both enthalpy and entropy contribute favorably to binding of the Nbs to wild-type (WT) LacY and that binding of Nb to double-Trp mutant G46W/G262W is driven by a greater enthalpy at an entropic penalty. Thermodynamic analyses support the interpretation that WT LacY is stabilized in outward-facing conformations like the double-Trp mutant with closure of the water-filled cytoplasmic cavity through conformational selection. The LacY conformational transition required for ligand binding is reflected by a favorable entropy increase. Molecular dynamics simulations further suggest that the entropy increase likely stems from release of immobilized water molecules primarily from the cytoplasmic cavity upon closure.
Asunto(s)
Proteínas de Transporte de Membrana/metabolismo , Anticuerpos de Dominio Único , Calorimetría , Escherichia coli/enzimología , Simulación de Dinámica Molecular , Especificidad por Sustrato , TermodinámicaRESUMEN
Membrane proteins encapsulated by detergent micelles are widely used for structural study. Because of their amphipathic property, detergents have the ability to maintain protein solubility and stability in an aqueous medium. However, conventional detergents have serious limitations in their scope and utility, particularly for eukaryotic membrane proteins and membrane protein complexes. Thus, a number of new agents have been devised; some have made significant contributions to membrane protein structural studies. However, few detergent design principles are available. In this study, we prepared meta and ortho isomers of the previously reported para-substituted xylene-linked maltoside amphiphiles (XMAs), along with alkyl chain-length variation. The isomeric XMAs were assessed with three membrane proteins, and the meta isomer with a C12 alkyl chain was most effective at maintaining solubility/stability of the membrane proteins. We propose that interplay between the hydrophile-lipophile balance (HLB) and alkyl chain length is of central importance for high detergent efficacy. In addition, differences in inter-alkyl-chain distance between the isomers influence the ability of the detergents to stabilise membrane proteins.
Asunto(s)
Detergentes/química , Proteínas de la Membrana/metabolismo , Micelas , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Isomerismo , Proteínas de la Membrana/química , Estabilidad Proteica , Salmonella typhimurium/enzimología , Solubilidad , Simportadores/química , Simportadores/metabolismo , Temperatura , Xilenos/químicaRESUMEN
Detergents serve as useful tools for membrane protein structural and functional studies. Their amphipathic nature allows detergents to associate with the hydrophobic regions of membrane proteins whilst maintaining the proteins in aqueous solution. However, widely used conventional detergents are limited in their ability to maintain the structural integrity of membrane proteins and thus there are major efforts underway to develop novel agents with improved properties. We prepared mesitylene-cored glucoside amphiphiles (MGAs) with three alkyl chains and compared these agents with previously developed xylene-linked maltoside agents (XMAs) with two alkyl chains and a conventional detergent (DDM). When these agents were evaluated for four membrane proteins including a G protein-coupled receptor (GPCR), some agents such as MGA-C13 and MGA-C14 resulted in markedly enhanced stability of membrane proteins compared to both DDM and the XMAs. This favourable behaviour is due likely to the increased hydrophobic density provided by the extra alkyl chain. Thus, this study not only describes new glucoside agents with potential for membrane protein research, but also introduces a new detergent design principle for future development.