Tripod Amphiphiles for Membrane Protein Manipulation

Department of Chemistry, University of Wisconsin-Madison, 1101 University Avenue, Madison, WI 53706, USA. .
Molecular BioSystems (Impact Factor: 3.21). 12/2009; 6(1):89-94. DOI: 10.1039/b915162c
Source: PubMed


Integral membrane proteins (IMPs) are crucial biological components, mediating the transfer of material and information between cells and their environment. Many IMPs have proven to be difficult to isolate and study. High-resolution structural information on this class of proteins is limited, largely because of difficulties in generating soluble forms of such proteins that retain native folding and activity, and difficulties in generating high-quality crystals from such preparations. Isolated IMPs typically do not dissolve in aqueous solution, a property that arises from the large patches of hydrophobic surface necessary for favorable interactions with the core of a lipid bilayer. Detergents are generally required for IMP solubilization: hydrophobic segments of detergent molecules cluster around and shield from water the hydrophobic protein surfaces. The critical role played by detergents in membrane protein manipulation, and the fact that many IMPs are recalcitrant to solubilization and/or crystallization with currently available detergents, suggest that it should be valuable to explore new types of amphiphiles for these purposes. This review constitutes a progress report on our long-term effort to develop a new class of organic molecules, collectively designated "tripod amphiphiles," that are intended as alternatives to conventional detergents for membrane protein manipulation. One long-range goal of this research is to identify new types of amphiphiles that facilitate IMP crystallization. This review should help introduce an important biochemical need to organic chemists, and perhaps inspire new approaches to the problem.

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    • "This carbon in the lipophilic region limits the conformational freedom of this class of molecules, thereby making them rigid relative to conventional detergents. This rigidification likely enabled us to solve the crystal structures of several N-oxide TPAs themselves [37] and has the potential to facilitate the crystallization of a wide array of membrane proteins; for example, TPA-solubilized bR and potassium channel from Streptomyces lividans have been crystallized, although their structures have not yet been solved [34] [36]. Recent TPA advances had led to a series of molecules with bifurcated glucose headgroups with favorable solubilization and stabilization efficacy [35]. "
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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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    ABSTRACT: The advantages of using hemifluorinated surfactants as an efficient alternative to detergents for manipulating membrane proteins in aqueous solution have been demonstrated in recent reports. However, the large-scale synthesis of these surfactants is still considered as a major matter and has limited their use for biochemical purposes. We report herein the synthesis of a novel series of perfluorohexane-based surfactants endowed with a short propyl hydrocarbon tip and whose polar head size is modulated by the presence of two or three glucose moieties. The synthetic route is based on the radical addition of two alkenes onto the 1,6-diiodoperfluorohexane using AIBN as a radical initiator, affording the surfactants in satisfactory overall yields. The self-assembling properties of these hemifluorinated surfactants were studied by surface tension measurements, dynamic light scattering, as well as their behavior upon reversed-phase chromatography and were compared with those of their perfluorinated analogues. Our findings strongly suggest the predominant influence of the propyl tip on both adsorption and micellization phenomena as well as on the hydrophobic character of the surfactants, whereas as previously observed, the shorter ethyl tip does not greatly affect these properties when compared to the perfluorinated analogues. Moreover, all the surfactants reported here self-assemble into small and monodisperse aggregates, a feature of crucial importance for biochemistry applications.
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    ABSTRACT: IntroductionPreparation of Expression ConstructsTransfer of Plasmid DNA to Rhodobacter via Conjugal MatingSmall-Scale Screening for Expression and Localization of Target Protein in RhodobacterLarge-Scale CultureDetergent Solubilization and Chromatographic Purification of Expressed Membrane ProteinsProtein Identification and Assessment of PurityPreparations of Specialized Rhodobacter MembranesAppendix: Media and Buffer FormulationsAbbreviationsReferences
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