Determining membrane protein structures: Still a challenge!

INSERM, U773, Centre de Recherche Biomédicale Bichat Beaujon CRB3, Faculté de Médecine X. Bichat, Université Paris 7, BP 416, F-75018, Paris, France.
Trends in Biochemical Sciences (Impact Factor: 13.52). 07/2007; 32(6):259-70. DOI: 10.1016/j.tibs.2007.04.001
Source: PubMed

ABSTRACT Determination of structures and dynamics events of transmembrane proteins is important for the understanding of their function. Analysis of such events requires high-resolution 3D structures of the different conformations coupled with molecular dynamics analyses describing the conformational pathways. However, the solution of 3D structures of transmembrane proteins at atomic level remains a particular challenge for structural biochemists--the need for purified and functional transmembrane proteins causes a 'bottleneck'. There are various ways to obtain 3D structures: X-ray diffraction, electron microscopy, NMR and modelling; these methods are not used exclusively of each other, and the chosen combination depends on several criteria. Progress in this field will improve knowledge of ligand-induced activation and inhibition of membrane proteins in addition to aiding the design of membrane-protein-targeted drugs.

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Available from: Jean-Jacques Lacapere, Jul 28, 2015
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    • "Structural studies of membrane protein are intricate and several approaches are possible [12]. Among them, electron microscopy can be used to study membrane protein organization within the artificial membranes either by two-dimensional crystallization in solution or by reorganization of adsorbed proteins under a lipid monolayer at the air/water interface [13] [14] [15]. "
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    ABSTRACT: Translocator protein TSPO is a membrane protein highly conserved in evolution which does not belong to any structural known family. TSPO is involved in physiological functions among which transport of molecules such as cholesterol to form steroids and bile salts in mammalian cells. Membrane protein structure determination remains a difficult task and needs concomitant approaches (for instance X-ray- or Electron-crystallography and NMR). Electron microscopy and two-dimensional crystallization under functionalized monolayers have been successfully developed for recombinant tagged proteins. The difficulty comes from the detergent carried by membrane proteins that disrupt the lipid monolayer. We identified the best conditions for injecting the histidine tagged recombinant TSPO in detergent in the subphase and to keep the protein stable. Reconstituted recombinant protein into a lipid bilayer favors its adsorption to functionalized monolayers and limits the disruption of the monolayer by reducing the amount of detergent. Finally, we obtained the first transmission electron microscopy images of recombinant mouse TSPO negatively stained bound to the lipid monolayer after injection into the subphase of pre-reconstituted TSPO in lipids. Image analysis reveals that circular objects could correspond to an association of at least four monomers of mouse TSPO. The different amino acid compositions and the location of the polyhistidine tag between bacterial and mouse TSPO could account for the formation of dimer versus tetramer, respectively. The difference in the loop between the first and second putative transmembrane domain may contribute to distinct monomer interaction, this is supported by differences in ligand binding parameters and biological functions of both proteins.
    Biochimica et Biophysica Acta 07/2012; 1818(11):2791-800. DOI:10.1016/j.bbamem.2012.06.020 · 4.66 Impact Factor
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    • "This is the first report, to our knowledge, of the successful expression of recombinant hENT1 (N-HAT-3ÂFLAG- hENT1) in a bacterial host. This is a significant step towards three-dimensional analysis of hENT1, a clinically important drug transporter, because it represents an important step towards overcoming the problem of low yields of mammalian ENTs and producing sufficient concentrations of protein (up to 10 mg/mL) for future structural studies (Lacapère et al. 2007). This approach can be scaled up and uses a lac promoter to drive expression plus tandem affinity purification to isolate N-HAT-3ÂFLAG-hENT1. "
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    ABSTRACT: Nucleoside transporters (NTs) are integral membrane proteins necessary for the cellular entry of nucleoside analog drugs used in chemotherapeutic treatment of conditions such as cancer and viral or parasitic infections. NTs are also the targets of certain drugs used in the treatment of various cardiovascular conditions. Because of the importance of NTs in drug uptake, determination of the three-dimensional structure of these proteins, particularly hENT1, has the potential to improve these treatments through structure-based design of more specifically targeted and transported drugs. In this paper, we use NMR spectroscopy to investigate the structure of the large intracellular loop between transmembrane domains 6 and 7 and we also describe a method for the successful overexpression of full-length hENT1 in a bacterial system. Recombinant tandem histidine-affinity (HAT) and 3×FLAG tagged hENT1 was overexpressed in E. coli, affinity purified, and functionally characterized by nitrobenzylthioinosine (NBTI) binding. Anti-3×FLAG immunodetection confirmed the expression of N-HAT-3×FLAG-hENT1, while increased NBTI binding (3.2-fold compared with controls) confirmed the conformational integrity of the recombinant hENT1 within the bacterial inner membrane. Yields of recombinant hENT1 using this approach were ~15 µg/L of bacterial culture and this approach provides a basis for large-scale production of protein for a variety of purposes.
    Biochemistry and Cell Biology 04/2011; 89(2):246-55. DOI:10.1139/o10-155 · 2.35 Impact Factor
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    • "This may be obtained with the help of a structural alphabet (Offmann et al. 2007; Joseph et al. 2010) as it has been used for defining DARC structural model (de Brevern et al. 2005; de Brevern 2009; de Brevern et al. 2009). The results herein described are quite important for molecular modelling of transmembrane proteins (de Graaf and Rognan 2009; Mornon et al. 2009), which are major medical drug targets, (Jacoby et al. 2006; Lacapere et al. 2007; Landry and Gies 2008; Arinaminpathy et al. 2009) and to improve protein topology prediction approaches (Harrington and Ben-Tal 2009; Klammer et al. 2009; Nugent and Jones 2009). "
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    ABSTRACT: α-Helical transmembrane proteins (TMPα) are composed of a series of helices embedded in the lipid bilayer. Due to technical difficulties, few 3D structures are available. Therefore, the design of structural models of TMPα is of major interest. We study the secondary structures of TMPα by analyzing the influence of secondary structures assignment methods (SSAMs). For this purpose, a published and updated benchmark databank of TMPα is used and several SSAMs (9) are evaluated. The analysis of the results points to significant differences in SSA depending on the methods used. Pairwise comparisons between SSAMs led to more than 10% of disagreement. Helical regions corresponding to transmembrane zones are often correctly characterized. The study of the sequence-structure relationship shows very limited differences with regard to the structural disagreement. Secondary structure prediction based on Bayes' rule and using only a single sequence give correct prediction rates ranging from 78 to 81%. A structural alphabet approach gives a slightly better prediction, i.e., only 2% less than the best equivalent approach, whereas the prediction rate with a very different assignment bypasses 86%. This last result highlights the importance of the correct assignment choice to evaluate the prediction assessment.
    Amino Acids 03/2010; 39(5):1241-54. DOI:10.1007/s00726-010-0559-6 · 3.65 Impact Factor
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