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

ArticleinBiophysical Journal 98(8):1539-48 · April 2010with26 Reads
DOI: 10.1016/j.bpj.2009.12.4296 · Source: PubMed
Abstract
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.
    • "Therefore a generic assay independent of membrane protein used in reconstitution is required. Cherezov and co-workers have used CPM assay successfully in lipid cubic phase [14]. We hypothesized that CPM assay could be used to study nanodisc reconstituted receptors. "
    [Show abstract] [Hide abstract] ABSTRACT: Membrane proteins are generally unstable in detergents. Therefore, biochemical and biophysical studies of membrane proteins in lipidic environments provides a near native-like environment suitable for membrane proteins. However, manipulation of proteins embedded in lipid bilayer has remained difficult. Methods such as nanodiscs and lipid cubic phase have been developed for easy manipulation of membrane proteins and have yielded significant insights into membrane proteins. Traditionally functional reconstitution of receptors in nanodiscs has been studied with radioligands. We present a simple and faster method for studying the functionality of reconstituted membrane proteins for routine characterization of protein batches after initial optimization of suitable conditions using radioligands. The benefits of the method are • Faster and generic method to assess functional reconstitution of membrane proteins. •Adaptable in high throughput format (≥96 well format). •Stability measurement in near-native lipid environment and lipid dependent melting temperatures.
    Full-text · Article · Mar 2016
    • "By measuring a peak height for aliquots of the same sample incubated at different temperatures, FSEC can also be used to monitor thermostability of a GPCR sample (Hattori et al., 2012). Thermostability of a pure sample can be determined in the differential scanning fluorimetry based on the dye for which fluorescence increases after its covalent binding to the denatured protein in water solution (Alexandrov et al., 2008) or LCP (Liu et al., 2010). Of course, purity of the final GPCR sample should always be examined in polyacrylamide gel electrophoresis and, if possible, the protein molecular weight and presence of posttranslational modifications checked by mass spectrometry (Ho et al., 2008). "
    [Show abstract] [Hide abstract] ABSTRACT: Structural studies of G protein-coupled receptors (GPCRs) gave insights into molecular mechanisms of their action and contributed significantly to molecular pharmacology. This is primarily due to technical advances in protein engineering, production and crystallization of these important receptor targets. On the other hand, NMR spectroscopy of GPCRs, which can provide information about their dynamics, still remains challenging due to difficulties in preparation of isotopically labeled receptors and their low long-term stabilities. In this review, we discuss methods used for expression and purification of GPCRs for crystallographic and NMR studies. We also summarize protein engineering methods that played a crucial role in obtaining GPCR crystal structures.
    Full-text · Article · Mar 2015
    • "The success of LCP crystallization can be attributed to several factors. First, the membrane-like environment of LCP confers higher stability on reconstituted MPs compared with the environment of detergent micelles [19]. Second, LCP crystallization supports type I packing that exhibits extensive interactions between transmembrane segments, leading to higher level ordering and high-resolution diffraction [7]. "
    [Show abstract] [Hide abstract] ABSTRACT: Despite recent technological advances in heterologous expression, stabilization and crystallization of membrane proteins (MPs), their structural studies remain difficult and require new transformative approaches. During the past two years, crystallization in lipidic cubic phase (LCP) has started gaining a widespread acceptance, owing to the spectacular success in high-resolution structure determination of G protein-coupled receptors (GPCRs) and to the introduction of commercial instrumentation, tools and protocols. The recent appearance of X-ray free-electron lasers (XFELs) has enabled structure determination from substantially smaller crystals than previously possible with minimal effects of radiation damage, offering new exciting opportunities in structural biology. The unique properties of LCP material have been exploited to develop special protocols and devices that have established a new method of serial femtosecond crystallography of MPs in LCP (LCP-SFX). In this method, microcrystals are generated in LCP and streamed continuously inside the same media across the intersection with a pulsed XFEL beam at a flow rate that can be adjusted to minimize sample consumption. Pioneering studies that yielded the first room temperature GPCR structures, using a few hundred micrograms of purified protein, validate the LCP-SFX approach and make it attractive for structure determination of difficult-to-crystallize MPs and their complexes with interacting partners. Together with the potential of femtosecond data acquisition to interrogate unstable intermediate functional states of MPs, LCP-SFX holds promise to advance our understanding of this biomedically important class of proteins.
    Article · Jul 2014
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