LCP-FRAP Assay for Pre-Screening Membrane Proteins for in Meso Crystallization.

Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 USA.
Crystal Growth & Design (Impact Factor: 4.89). 02/2008; 8(12):4307-4315. DOI: 10.1021/cg800778j
Source: PubMed


Fluorescence recovery after photobleaching was used to study the diffusion of two integral membrane proteins, bacteriorhodopsin and beta2-adrenergic receptor, in lipidic cubic phase (LCP). We found that the diffusion properties within the LCP matrix strongly depend on the protein construct and applied screening conditions. Common precipitants often induce restriction on diffusion of proteins in LCP and thereby impede their chances for crystallization. A high protein mobile fraction and a fast diffusion rate correlate very well with known crystallization conditions. Using this knowledge, one can now pre-screen precipitant conditions with microgram quantities of material to rule out conditions that are not conducive to diffusion, nucleation, and crystal growth. The results of this assay will narrow membrane protein crystallization space by identifying suitable protein constructs, stabilizing compounds and precipitant conditions amenable to in meso crystallization. Crystallization pre-screening will significantly increase the chances of obtaining initial crystal hits, expediting efforts in generating high-resolution structures of challenging membrane protein targets.

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Available from: Jeffrey Liu, Apr 30, 2014
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    • "Unlike the theoretical analysis, progress was made by experimental studies of the lipidic cubic phase guiding the crystallization studies. One powerful approach, fluorescence recovery after photobleaching (FRAP), seeks to assess the ability that GPCRs have while in the lipidic bilayer-based meso phase and identify conditions that favor diffusion in two dimensions as freely as possible to find other protein partners with which to build the two-dimensional array82. A major advance is the application of fluorescence to assay the diffusion rates, seeking conditions that maximize the diffusibility. "
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    ABSTRACT: G-protein-coupled receptors (GPCRs) are one of the most challenging targets in structural biology. To successfully solve a high-resolution GPCR structure, several experimental obstacles must be overcome, including expression, extraction, purification, and crystallization. As a result, there are only a handful of unique structures reported from this protein superfamily, which consists of over 800 members. In the past few years, however, there has been an increase in the amount of solved GPCR structures, and a few high-impact structures have been determined: the peptide receptor CXCR4, the agonist bound receptors, and the GPCR-G protein complex. The dramatic progress in GPCR structural studies is not due to the development of any single technique, but a combination of new techniques, new tools and new concepts. Here, we summarize the progress made for GPCR expression, purification, and crystallization, and we highlight the technical advances that will facilitate the future determination of GPCR structures.
    Acta Pharmacologica Sinica 03/2012; 33(3):324-34. DOI:10.1038/aps.2011.187 · 2.91 Impact Factor
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    • ") . In a second approach , since used to determine the majority of nonrhodopsin GPCR structures to date , a T4 lysozyme fusion was introduced within the third cyto - plasmic loop of heterologously expressed ␤ 2 - adrenergic receptor to aid crystallization in a modified lipid cubic phase matrix and resulted in high - order diffracting crys - tals ( Cherezov et al . , 2008 ; Hanson et al . , 2008 ; Roth et al . , 2008 ) . In addition to the high - resolution 2 . 4 - Å cara - zolol - bound structure ( PDB ID 2RH1 ) , a series of adren - ergic receptor structures have been solved that con - tained a variety of antagonist and inverse agonist compounds ( Hanson et al . , 2008 ; Roth et al . , 2008 ; Wacker et"
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    ABSTRACT: Crucial as molecular sensors for many vital physiological processes, seven-transmembrane domain G protein-coupled receptors (GPCRs) comprise the largest family of proteins targeted by drug discovery. Together with structures of the prototypical GPCR rhodopsin, solved structures of other liganded GPCRs promise to provide insights into the structural basis of the superfamily's biochemical functions and assist in the development of new therapeutic modalities and drugs. One of the greatest technical and theoretical challenges to elucidating and exploiting structure-function relationships in these systems is the emerging concept of GPCR conformational flexibility and its cause-effect relationship for receptor-receptor and receptor-effector interactions. Such conformational changes can be subtle and triggered by relatively small binding energy effects, leading to full or partial efficacy in the activation or inactivation of the receptor system at large. Pharmacological dogma generally dictates that these changes manifest themselves through kinetic modulation of the receptor's G protein partners. Atomic resolution information derived from increasingly available receptor structures provides an entrée to the understanding of these events and practically applying it to drug design. Supported by structure-activity relationship information arising from empirical screening, a unified structural model of GPCR activation/inactivation promises to both accelerate drug discovery in this field and improve our fundamental understanding of structure-based drug design in general. This review discusses fundamental problems that persist in drug design and GPCR structural determination.
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    • "Solubilization and purification of the nAChR using LFC-16 produced ~82% monomeric composition and a high mobile fraction of ~85%; FC-16 produced ~74% monomeric composition and an ~87% mobile fraction (Fig. 4a). These mobile fractions are similar to those reported as crystallization conditions of the β 2 -adrenergic receptor–T4L in LCP (Cherezov et al. 2008). These results indicate that the percentage of monomer in the nAChR sample positively affects the receptor mobile fraction in the LCP matrix. "
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