Tandem Facial Amphiphiles for Membrane Protein Stabilization

Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706, United States.
Journal of the American Chemical Society (Impact Factor: 12.11). 11/2010; 132(47):16750-2. DOI: 10.1021/ja1072959
Source: PubMed


We describe a new type of synthetic amphiphile that is intended to support biochemical characterization of intrinsic membrane proteins. Members of this new family displayed favorable behavior with four of five membrane proteins tested, and these amphiphiles formed relatively small micelles.

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    • "The most intriguing aspect of the discrepancy between the two binding site models from Javitch and colleagues and the data from the laboratory of Gouaux is that it has not been possible to reproduce the two-leucine stoichiometry pr. LeuT in Gouaux' laboratory even when they attempted to reproduce the preparation of protein according to the methods of Javitch and colleagues or in the recently developed lauryl maltose neopentyl glycol (MNG-3) detergent [91] which has been shown to increase stability and functionality of purified LeuT [10] [11] [12]. Neither has it been possible for them to reduce [ 3 H]leucine binding by 50% by introducing the L400S mutant or by the addition of clomipramine which should bind exclusively to the S2 site [91]. "
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    ABSTRACT: Background: The mammalian neurotransmitter transporters are complex proteins playing a central role in synaptic transmission between neurons by rapid reuptake of neurotransmitters. The proteins which transport dopamine, noradrenaline and serotonin belong to the Neurotransmitter:Sodium Symporters (NSS). Due to their important role, dysfunctions are associated with several psychiatric and neurological diseases and they also serve as targets for a wide range of therapeutic and illicit drugs. Despite the central physiological and pharmacological importance, direct evidence on structure-function relationships on mammalian NSS proteins has so far been unsuccessful. The crystal structure of the bacterial NSS protein, LeuT, has been a turning point in structural investigations. Scope of review: To provide an update on what is known about the binding sites for substrates and inhibitors in the LeuT. The different binding modes and binding sites will be discussed with special emphasis on the possible existence of a second substrate binding site. It is the goal to give an insight into how investigations on ligand binding in LeuT have provided basic knowledge about transporter conformations and translocation mechanism which can pave the road for a deeper understanding of drug binding and function of the mammalian transporters. Major conclusions: The LeuT is a suitable model for the structural investigation of NSS proteins including the possible location of drug binding sites. It is still debated whether the LeuT is a suitable model for the molecular mechanisms behind substrate translocation. General significance: Structure and functional aspects of NSS proteins are central for understanding synaptic transmission. With the purification and crystallization of LeuT as well as the dopamine transporter from Drosophila melanogaster, the application of biophysical methods such as fluorescence spectroscopy, neutron- or x-ray scattering and NMR for understanding its function becomes increasingly available. This article is part of a Special Issue entitled Structural biochemistry and biophysics of membrane proteins.
    Biochimica et Biophysica Acta 04/2014; 1850(3). DOI:10.1016/j.bbagen.2014.04.011 · 4.66 Impact Factor
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    • "Amphiphilic polymers (amphipols) [17] [18], nanodiscs (NDs) [18] [19] [20] [21], and lipodisqs [22] are innovative approaches to overcome the limitation of current tools for membrane protein stability. Tandem facial amphiphiles (TFAs) [23] and hemifluorinated surfactants (HFSs) [24] [25] [26] are other examples of recent inventions that have shown to be excellent in retaining the native structures of delicate membrane proteins. Amphipathic peptides such as lipopeptide detergents (LPDs) [27] and short designer peptides [28] proved to be effective for several classes of membrane protein systems as well. "
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    ABSTRACT: Membrane proteins operate in unique cellular environments. Once removed from their native context for the purification that is required for most types of structural or functional analyses, they are prone to denature if not properly stabilized by membrane mimetics. Detergent micelles have prominently been used to stabilize membrane proteins in aqueous environments as their amphipathic nature allows for shielding of the hydrophobic surfaces of these bio-macromolecules while supporting solubility and monodispersity in water. This study expands the utility of branched diglucoside-bearing tripod agents, designated ganglio-tripod amphiphiles, with introduction of key variations in their hydrophobic sections and shows how these latter elements can be fine-tuned to maximize membrane protein solubilization while preserving characteristics of these molecules that afford stabilization of rather fragile assemblies. Their efficacy rivals benchmark detergents heavily used today, such as n-dodecyl-β-D-maltoside.
    Biochimica et Biophysica Acta 09/2013; 1838(1). DOI:10.1016/j.bbamem.2013.09.011 · 4.66 Impact Factor
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    • "It is possible that this amphiphile may be more suitable for vapor diffusion crystallization of GPCRs than the MNGs because of its tendency to form small protein– detergent complexes. Further advances in the area of alternative detergents have the potential to facilitate structural studies of GPCRs as well as other integral membrane proteins [77] [78]. "
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    ABSTRACT: G protein-coupled receptors are integral membrane cell surface receptors with key roles in mediating the cellular responses to a wide range of biologically relevant molecules including hormones, neurotransmitters and importantly the majority of currently available drugs. The first high-resolution, X-ray crystallographic structure of a GPCR, that of rhodopsin, was obtained in 2000. It took a further seven years for the next structure, that of the β2 adrenergic receptor. Remarkably, at the time of writing, there have been an astonishing 18 further independent high-resolution GPCR structures published in the last five years (overall total of 68 structures in different conformations or bound to different ligands). Of particular note is the recent structure of the β2 adrenergic receptor in complex with its cognate heterotrimeric G-protein revealing for the first time molecular details of the interaction between a GPCR and the complete G-protein. Together these structures have provided unprecedented detail into the mechanism of action of these incredibly important proteins. This review describes several key methodological advances that have made such extraordinarily fast progress possible.
    Biochimica et Biophysica Acta 07/2013; 1828(11). DOI:10.1016/j.bbamem.2013.07.013 · 4.66 Impact Factor
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