Crystallizing transmembrane peptides in lipidic mesophases.

Membrane Structural and Functional Biology Group, School of Biochemistry and Immunology, and School of Medicine, Trinity College, Dublin, Ireland.
Biophysical Journal (Impact Factor: 3.67). 08/2010; 99(3):L23-5. DOI: 10.1016/j.bpj.2010.05.011
Source: PubMed

ABSTRACT Structure determination of membrane proteins by crystallographic means has been facilitated by crystallization in lipidic mesophases. It has been suggested, however, that this so-called in meso method, as originally implemented, would not apply to small protein targets having </=4 transmembrane crossings. In our study, the hypothesis that the inherent flexibility of the mesophase would enable crystallogenesis of small proteins was tested using a transmembrane pentadecapeptide, linear gramicidin, which produced structure-grade crystals. This result suggests that the in meso method should be considered as a viable means for high-resolution structure determination of integral membrane peptides, many of which are predicted to be coded for in the human genome.

1 Bookmark
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The lipidic cubic phase (LCP) has repeatedly proven to serve as a successful membrane-mimetic matrix for a variety of difficult-to-crystallize membrane proteins. While monoolein has been the predominant lipid of choice, there is a growing need for the characterization and use of other LCP host lipids, allowing exploration of a range of structural parameters such as bilayer thickness and curvature for optimal insertion, stability and crystallogenesis of membrane proteins. Here, we describe the development of a high-throughput (HT) pipeline to employ small angle X-ray scattering (SAXS) - the most direct technique to identify lipid mesophases and measure their structural parameters - to interrogate rapidly a large number of lipid samples under a variety of conditions, similar to those encountered during crystallization. Leveraging the identical setup format for LCP crystallization trials, this method allows the quickly assessment of lipid matrices for their utility in membrane protein crystallization, and could inform the tailoring of lipid and precipitant conditions to overcome specific crystallization challenges. As proof of concept, we present HT LCP-SAXS analysis of lipid samples made of monoolein with and without cholesterol, and of monovaccenin, equilibrated with solutions used for crystallization trials and LCP fluorescence recovery after photobleaching (FRAP) experiments.
    Methods 08/2011; 55(4):342-9. · 3.64 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: NMR spectroscopy of helical membrane proteins has been very challenging on multiple fronts. The expression and purification of these proteins while maintaining functionality has consumed countless graduate student hours. Sample preparations have depended on whether solution or solid-state NMR spectroscopy was to be performed – neither have been easy. In recent years it has become increasingly apparent that membrane mimic environments influence the structural result. Indeed, in these recent years we have rediscovered that Nobel laureate, Christian Anfinsen, did not say that protein structure was exclusively dictated by the amino acid sequence, but rather by the sequence in a given environment (C.B. Anfinsen, 1973, Science 181:223-230). The environment matters, molecular interactions with the membrane environment are significant and many examples of distorted, non-native membrane protein structures have recently been documented in the literature. However, solid-state NMR structures of helical membrane proteins in proteoliposomes and bilayers are proving to be native structures that permit a high resolution characterization of their functional states. Indeed, solid-state NMR is uniquely able to characterize helical membrane protein structures in lipid environments without detergents. Recent progress in expression, purification, reconstitution, sample preparation and in the solid-state NMR spectroscopy of both oriented samples and magic angle spinning samples has demonstrated that helical membrane protein structures can be achieved in a timely fashion. Indeed, this is a spectacular opportunity for the NMR community to have a major impact on biomedical research through the solid-state NMR spectroscopy of these proteins.
    Journal of Magnetic Resonance 01/2013; · 2.30 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The crystal structure of the β(2)-adrenergic receptor in complex with an agonist and its cognate G protein has just recently been determined. It is now possible to explore in molecular detail the means by which this paradigmatic transmembrane receptor binds agonist, communicates the impulse or signaling event across the membrane, and sets in motion a series of G protein-directed intracellular responses. The structure was determined using crystals of the ternary complex grown in a rationally designed lipidic mesophase by the so-called in meso method. The method is proving to be particularly useful in the G protein-coupled receptor field where the structures of 13 distinct receptor types have been determined in the past 5 years. In addition to receptors, the method has proven to be useful with a wide variety of integral membrane protein classes that include bacterial and eukaryotic rhodopsins, light-harvesting complex II (LHII), photosynthetic reaction centers, cytochrome oxidases, β-barrels, an exchanger, and an integral membrane peptide. This attests to the versatility and range of the method and supports the view that the in meso method should be included in the arsenal of the serious membrane structural biologist. For this to happen, however, the reluctance to adopt it attributable, in part, to the anticipated difficulties associated with handling the sticky, viscous cubic mesophase in which crystals grow must be overcome. Harvesting and collecting diffraction data with the mesophase-grown crystals are also viewed with some trepidation. It is acknowledged that there are challenges associated with the method. Over the years, we have endeavored to establish how the method works at a molecular level and to make it user-friendly. To these ends, tools for handling the mesophase in the pico- to nanoliter volume range have been developed for highly efficient crystallization screening in manual and robotic modes. Methods have been implemented for evaluating the functional activity of membrane proteins reconstituted into the bilayer of the cubic phase as a prelude to crystallogenesis. Glass crystallization plates that provide unparalleled optical quality and sensitivity to nascent crystals have been built. Lipid and precipitant screens have been designed for a more rational approach to crystallogenesis such that the method can now be applied to an even wider variety of membrane protein types. In this work, these assorted advances are outlined along with a summary of the membrane proteins that have yielded to the method. The prospects for and the challenges that must be overcome to further develop the method are described.
    Biochemistry 07/2012; 51(32):6266-88. · 3.38 Impact Factor


Available from