LCP-FRAP Assay for Pre-Screening Membrane Proteins for In Meso Crystallization

Cherezov, Vadim; Liu, Jeffrey; Griffith, Mark T.; Hanson, Michael A.; Stevens, Raymond C.

doi:10.1021/cg800778j

Cited by 65 publications

(81 citation statements)

References 39 publications

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“…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 array [82] . A major advance is the application of fluorescence to assay the diffusion rates, seeking conditions that maximize the diffusibility.…”

Section: Bicelle Methodsmentioning

confidence: 99%

Ice breaking in GPCR structural biology

Zhao

2012

Acta Pharmacol Sin

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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 highimpact 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.

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Section: Bicelle Methodsmentioning

confidence: 99%

Ice breaking in GPCR structural biology

Zhao

2012

Acta Pharmacol Sin

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“…Because of the high curvature and small aqueous channel size of the first lipid used, monoolein, which is the preferred lipid, it seemed that this technique might be only suited to relatively small membrane proteins like GPCRs. This does not appear to be the case, and crystallisation in LCP has become one of the standard membrane protein techniques, and especially for membrane proteins with small soluble domains [81,82].…”

Section: B Lipidic Cubic Phasementioning

confidence: 99%

“…Nevertheless, the high viscosity of the phase making it difficult to extract and difficulties visualising the crystals represent significant hurdles. Recent new technologies, like using commercial robots and pre-crystallization assays like LCP-FRAP (Fluorescence recovery after photobleaching) [82], have made this technology more accessible and improved the success rate.…”

Section: B Lipidic Cubic Phasementioning

confidence: 99%

Artificial membranes for membrane protein purification, functionality and structure studies

Parmar

Lousa

Muench

et al. 2016

Biochemical Society Transactions

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Membrane proteins represent one of the most important targets for pharmaceutical companies. Unfortunately, technical limitations have long been a major hindrance in our understanding of the function and structure of such proteins. Recent years have seen the refinement of classical approaches and the emergence of new technologies that have resulted in a significant step forward in the field of membrane protein research. This review summarises some of the current techniques used for studying membrane proteins, with overall advantages and drawbacks for each method. Micelles and classical detergent techniquesMembrane proteins account for~30% of both prokaryotic and eukaryotic proteins [1]. Integral proteins such as transporters or ion channels, as well as peripheral membrane proteins such as G-proteins, all perform essential tasks in signal transduction, cell metabolism and transport of small molecules [2][3][4]. Integral and peripheral membrane proteins are respectively embedded in or closely associated with the phospholipid bilayer of cell membranes. Therefore, their function often relies on their precise lipid environment [5,6]. For instance, cardiolipin, which constitutes about 20% of the inner mitochondrial membrane, is essential to the function of many mitochondrial transporters such as the ADP/ATP carriers, the enzymes of the respiratory chain, and bacterial proteins [7][8][9]. This is because cardiolipin offers polar and electrostatic interactions that increase protein stability[10]; similar interactions have been observed for other lipids [11,12]. Unfortunately, classical techniques to study membrane proteins involve the use of detergents that solubilise the protein but also destabilise its interaction with membrane lipids. In many cases, membrane proteins may be stable in only a few detergents, limiting the range of conditions that can be used for crystallization trials [13]. Consequently, much time is spent testing various detergents in different ratios and concentrations in the hope of finding conditions that will mimic the essential interactions of the protein with its natural lipidic environment -and this way stabilise the protein and preserve its functional state. These problems have hindered membrane protein research for many years: both biophysical characterisation and structure solution have suffered due to difficulties of extracting proteins from membranes and keeping them stable away from their native environment. The past few years have seen the emergence of new techniques aimed at providing a membrane-like natural environment. These novel techniques include liposomes, bicelles, discs, polymer and lipids based strategies.

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“…h Serial femtosecond crystallography in LCP (LCP-SFX; Liu et al 2013;Weierstall et al 2014). i MP diffusion in LCP, lipidic cubic phase fluorescence recovery after photobleaching (LCP-FRAP; Cherezov et al 2008;Xu et al 2011). j MP stability in LCP (LCP-Tm; Liu et al 2010).…”

Section: Syringe Mixermentioning

confidence: 99%