The effect of increasing concentrations of precipitating salts used to crystallize proteins on the structure of the lipidic Q224 cubic phase

Vargas, Rodolfo; Mateu, L.; Romero, Antonio

doi:10.1016/j.chemphyslip.2003.09.006

Cited by 15 publications

(28 citation statements)

References 26 publications

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“…The further decrease of the lattice parameter in the re-swelled system is characteristic of the system tending towards the inverse hexagonal phase 42 . The balance between the sources of electrostatic forces in the system are indicative that salt solution concentration in such systems has a subtle effect, and that single-concentration crystallisation screening experiments may not fully consider experimental optimisation 18 , 43 .…”

Section: Resultsmentioning

confidence: 99%

Effects of Cations on the Behaviour of Lipid Cubic Phases

Brasnett

Longstaff

Compton

et al. 2017

Sci Rep

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Inverse bicontinuous cubic structures formed by lipids have been demonstrated in a wide variety of applications, from a host matrix for proteins for crystallisation, to templates for nanoscale structures. Recent work has focused on tuning their properties to realize such applications, often by manipulating the structure by introducing other lipids with different properties such as charge or packing. However, they are often prepared in the presence of solutions containing salt, counteracting the effects, for example, charged lipids, and fundamentally changing the structures obtained. Here, we demonstrate the delicate interplay between electrostatic swelling in bicontinuous structures formed by monoolein (MO) doped with both negatively charged dioleyl phosphatidylglycerol (DOPG), and zwitterionic dioleyl phosphatidylethanolamine (DOPE), with the addition of mono- and divalent salts. The effect of adding salt to the charged phase changes the structure from the primitive cubic () to the double diamond phase () whilst still allowing for modest increases in lattice parameter of up to a nanometer. Contrasting this, the addition of salts to the non-charged phase, has minimal effect on the lattice parameter but now the transition from the () to the inverse hexagonal phase (HII) is observed occurring at higher mole fractions of DOPE than in pure water.

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Section: Resultsmentioning

confidence: 99%

Effects of Cations on the Behaviour of Lipid Cubic Phases

Brasnett

Longstaff

Compton

et al. 2017

Sci Rep

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“…Effects of soluble and lipid-like components on LCP are described in refs. 22, 30, 31. Other parameters that we found to be critical for optimization of crystal growth in meso are the size and the shape of LCP bolus and the protein concentration.…”

Section: Methodsmentioning

confidence: 90%

Recent Progress in the Structure Determination of GPCRs, a Membrane Protein Family with High Potential as Pharmaceutical Targets

Cherezov

Abola

Stevens

2010

Methods in Molecular Biology

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SummaryG protein-coupled receptors (GPCRs) constitute a highly diverse and ubiquitous family of integral membrane proteins, transmitting signals inside the cells in response to an assortment of disparate extracellular stimuli. Their strategic location on the cell surface and their involvement in crucial cellular and physiological processes turn these receptors into highly important pharmaceutical targets. Recent technological developments aimed at stabilization and crystallization of these receptors have led to significant breakthroughs in GPCR structure determination efforts. One of the successful approaches involved receptor stabilization with the help of a fusion partner combined with crystallization in lipidic cubic phase (LCP). The success of using LCP matrix for crystallization is generally attributed to the creation of a more native, membrane-like stabilizing environment for GPCRs just prior to nucleation and to the formation of type I crystal lattices, thus, generating highly ordered and strongly diffracting crystals. Here we describe protocols for reconstituting purified GPCRs in LCP, performing pre-crystallization assays, setting up crystallization trials in manual mode, detecting crystallization hits, optimizing crystallization conditions, harvesting, and collecting crystallographic data The protocols provide a sensible framework for approaching crystallization of stabilized GPCRs in LCP, however, as in any crystallization experiment extensive screening and optimization of crystallization conditions as well as optimization of protein construct and purification steps are required. The process remains risky and these protocols do not necessarily guarantee success.

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“…One has to be careful with the interpretation of LCP-T m data. Similar to the temperature effect, most salts and other kosmotropes, as well as some nonlamellar phase forming lipids, can decrease the lattice parameter of LCP, increase membrane curvature and change the membrane thickness (32,38). In contrast, the addition of chaotropes, certain polymers such as PEG 400, detergents, or lamellar phase forming lipids can induce swelling of the cubic phase and decrease the membrane curvature (32,37,44).…”

Section: Discussionmentioning

confidence: 98%

“…Transient formation of an inverse hexagonal H II phase was induced by mixing LCP with 2 M sodium sulfate at RT (37,38). Formation of a birefringent phase was confirmed by polarized light microscopy.…”

Section: Effect Of Mesophasementioning

confidence: 88%

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

Liu

Hanson

Stevens

et al. 2010

Biophysical Journal

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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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The effect of increasing concentrations of precipitating salts used to crystallize proteins on the structure of the lipidic Q224 cubic phase

Cited by 15 publications

References 26 publications

Effects of Cations on the Behaviour of Lipid Cubic Phases

Effects of Cations on the Behaviour of Lipid Cubic Phases

Recent Progress in the Structure Determination of GPCRs, a Membrane Protein Family with High Potential as Pharmaceutical Targets

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

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