LCP-Tm: An Assay to Measure and Understand Stability of Membrane Proteins in a Membrane Environment

Department of Molecular Biology, The Scripps Research Institute, La Jolla, California, USA.
Biophysical Journal (Impact Factor: 3.97). 04/2010; 98(8):1539-48. DOI: 10.1016/j.bpj.2009.12.4296
Source: PubMed


Structural and functional studies of membrane proteins are limited by their poor stability outside the native membrane environment. The development of novel methods to efficiently stabilize membrane proteins immediately after purification is important for biophysical studies, and is likely to be critical for studying the more challenging human targets. Lipidic cubic phase (LCP) provides a suitable stabilizing matrix for studying membrane proteins by spectroscopic and other biophysical techniques, including obtaining highly ordered membrane protein crystals for structural studies. We have developed a robust and accurate assay, LCP-Tm, for measuring the thermal stability of membrane proteins embedded in an LCP matrix. In its two implementations, protein denaturation is followed either by a change in the intrinsic protein fluorescence on ligand release, or by an increase in the fluorescence of a thiol-binding reporter dye that measures exposure of cysteines buried in the native structure. Application of the LCP-Tm assay to an engineered human beta2-adrenergic receptor and bacteriorhodopsin revealed a number of factors that increased protein stability in LCP. This assay has the potential to guide protein engineering efforts and identify stabilizing conditions that may improve the chances of obtaining high-resolution structures of intrinsically unstable membrane proteins.

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    • "Studies on bacteriorhodopsin revealed a marked difference in stability of the protein in phases made from different lipids. The LCP- T m assay describes the use of the CPM dye [85] for direct analysis of the folded state of GPCR which has been incorporated into LCP. There is a somewhat time-consuming heating and centrifugation process required to ensure changes in the LCP do not interfere with the fluorescence measurements . "
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    ABSTRACT: G protein-coupled receptors are integral membrane cell surface receptors with key roles in mediating the cellular responses to a wide range of biologically relevant molecules including hormones, neurotransmitters and importantly the majority of currently available drugs. The first high-resolution, X-ray crystallographic structure of a GPCR, that of rhodopsin, was obtained in 2000. It took a further seven years for the next structure, that of the β2 adrenergic receptor. Remarkably, at the time of writing, there have been an astonishing 18 further independent high-resolution GPCR structures published in the last five years (overall total of 68 structures in different conformations or bound to different ligands). Of particular note is the recent structure of the β2 adrenergic receptor in complex with its cognate heterotrimeric G-protein revealing for the first time molecular details of the interaction between a GPCR and the complete G-protein. Together these structures have provided unprecedented detail into the mechanism of action of these incredibly important proteins. This review describes several key methodological advances that have made such extraordinarily fast progress possible.
    Biochimica et Biophysica Acta 07/2013; 1828(11). DOI:10.1016/j.bbamem.2013.07.013 · 4.66 Impact Factor
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    • "Clearly, membrane proteins embedded in meso phases exhibit an increased half-life compared to the detergent-solubilized state [26]. Nevertheless, a reduction of the required incubation temperature is desirable and may be attempted by doping the mesophase with lipids like cholesterol or by the exchange of the major component monoolein [26]–[28]. "
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    ABSTRACT: We investigated in meso crystallization of membrane proteins to develop a fast screening technology which combines features of the well established classical vapor diffusion experiment with the batch meso phase crystallization, but without premixing of protein and monoolein. It inherits the advantages of both methods, namely (i) the stabilization of membrane proteins in the meso phase, (ii) the control of hydration level and additive concentration by vapor diffusion. The new technology (iii) significantly simplifies in meso crystallization experiments and allows the use of standard liquid handling robots suitable for 96 well formats. CIMP crystallization furthermore allows (iv) direct monitoring of phase transformation and crystallization events. Bacteriorhodopsin (BR) crystals of high quality and diffraction up to 1.3 Å resolution have been obtained in this approach. CIMP and the developed consumables and protocols have been successfully applied to obtain crystals of sensory rhodopsin II (SRII) from Halobacterium salinarum for the first time.
    PLoS ONE 04/2012; 7(4):e35458. DOI:10.1371/journal.pone.0035458 · 3.23 Impact Factor
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    • "Using this method, one can now pre-screen host lipids, precipitant conditions, and identities of ligands with microgram quantities of material to magnify the chances of crystallogenesis and rule out conditions that are not conducive to diffusion, nucleation, and crystal growth83. LCP-Tm, another LCP tool to measure the stability of the receptor in the host's lipids without the use of labels, will also facilitate the screening of better constructs and ligands84. Assisted by different LCP tools, the variables of the multi-dimensional crystallization space are notably reduced, and thus, the bottleneck in obtaining initial crystal leads is substantially overcome. "
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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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