Calixar C2B

Increase your protein stability to grow better diffracting crystals
Calixar™'s new C2B additive kit provides a range of novel, patented surfactants that can aid membrane protein purification. This exciting new kit provides two varieties of stabilizing surfactants: Calixarenes1,2 and fluorinated poly(tris)3-6 and bis-glucose7-10 surfactants.

Introduction

Membrane protein crystallization is notoriously difficult being constrained by the requirement for lipids to structure the membrane domain and the imperfect folding of proteins outside this specific environment. Amphiphilic molecules such as detergents tend to replace lipids for structural bliology as they solubilize the protein in protein/detergent complexes via interactions between protein transmembrane domains and detergent hydrophobic tails while simultaneously exposing hydrophilic detergent head groups and protein soluble domains to aqueous media. Unfortunately, detergents display poor structuring properties – they mask the hydrophobic region of the membrane protein with bulky micelles which prevent the hydrophilic regions from creating protein/protein contacts necessary for the formation and stabilization of crystals and complexes.

In contrast, the molecules in the C2B Kit, developed and exclusively manufactured by CALIXAR™, have been proven to increase both stability in solution and crystallogenesis of membrane proteins. Calixar™'s C2B kit is exclusively available to buy from Molecular Dimensions.


Calixarenes

Calixarene structureCalixarenes facilitate crystallogenesis by micellar-like supramolecular clusters that facilitate protein/protein interactions. Three calixarenes are included in C2B, all are ionic and formed from the grafting of polar groups onto the upper and lower rim of calix-[4]-rene (right). CALX116ACE and CALX163ACE have 3 hydrophilic sodium carboxylate ions on the upper rim and a hydrophobic tail of 1 (CALX116ACE) or 6 carbons (CALX163ACE) on the lower rim. They behave as surfactants forming 2-5 nm micelles with a pH-sensitive critical micellar concentration between 7.5 and 12.5 mM. CALX004SFO has four sulfonate groups on the upper rim.

These molecules are designed to structure the membrane domains of proteins through hydrophobic interactions and a network of salt bridges with the basic residues found at the cytosol-membrane interface of membrane proteins1,2. Calixarenes act in two ways:


  • Their multiple functional groups are able to promote the formation of a salt bridge network through the formation of supramolecular clusters of calixarene and protein. These clusters can strengthen interprotein and interdomain interactions to stabilise your protein or protein complex. As shown on the right: Calixarene cages form networks with their functional groups exposed and available to interlink proteins and thus stabilize a complex or lattice.

  • They can forming a cage around surface lysines, creating a charge shield (see left, Cytochrome C with calixarenes caging two surface lysines). Charge shielding changes the apparent electrostatic surface properties of a protein and can therefore improve crystal packing and sample distribution on carbon-coated cryoEM grids.

Calixarenes have been shown to promote membrane protein crystallisation through improved crystal contacts. they have been used in the crystallization of a number of proteins (pdbids: 5LYC, 3TYI, 4YE1, 4N0J, 4PRU, 4N0K and 4PRQ). Their protein complex stabilising and charge shielding functions can also aid with single particle cryoEM structure analysis.

Fluorinated Surfactants

C2B also contains two varieties of LC molecule surfactants: fluorinated poly(tris) surfactants (LC001 and LC002)3-6 and hydrogenated and fluorinated bis-Glucose compounds (LC021, LC036, LC037, LC039 and LC039)7-10. The chemistry of LC molecules have been developed by Calixar SAS in partnership with the joint laboratory Chem2staB (C2B) at Avignon University, France, and their presence in the new kit will offer new avenues of success for membrane protein crystallisation.

Fluorinated surfactants act in a very different manner to their hydrogenated equivalents due to the increased size of fluorine relative to hydrogen and its highly hydrophobic nature. Fluorinated surfactants are unable to extract proteins from membrane, but can be useful in subsequent purification steps as they do not strip natural lipids and other co-factors from the proteins. In addition, the bulky fluorinated tails can not penetrate into the interior and disrupt structure. Fluorinated surfactants often decrease non-specific aggregation and are thought to result in improved distribution on cryoEM grids and better vitrification for cryo-EM data collection. They are also reported to increase crystallizability.

The bis-glucose compounds have a tetradhedral chiral carbon with two polar headgroups (the glucose moieties) and a long hydrophobic fluorinated tail. In this way they are somewhat reminiscent of the neo-pentyl glycol detergents, such as LMNG that have seen some recent success in structural biology.

Patents
Gordeliy, Vet al. EP 2832741(A1) (2015).
Falson, Pet al. WO 2010116055 (2010).
References
Calixarenes
  1. Matar-Merheb, R et al. PLoS One31: e18036 (2011).
  2. McGovern, RE et al. Nat Chem 4: 527 (2012).
  3. McGovern, RE et al. Chem Commun 50: 10412 (2014).
  4. McGovern, RE et al. Chem Sci 6 442 (2015).
  5. Rennie, ML et al. Angew Chem Int Ed Engl 56: 5517 (2017).
Fluorinated surfactants with poly-tris headgroup
  1. Damian, M et al. FEBS Lett58: 1944 (2007).
  2. Nehmé, R et al. Biochim Biophys Acta 1798: 1100 (2010).
  3. Kyrychenko, A et al. J Mol Biol416: 328 (2012).
  4. Simonyan, L et al. Biochem Biophys Acta 1859: 1144 (2017).
Hydrogenated and Fluorinated surfactants with bis-glucose headgroup
  1. Abla, M et al. J Colloid Interface Sci445: 127 (2015).
  2. Abla, M et al. J Org Chem 73: 8142 (2008).
  3. Breyton, C et al. J Biol Chem288: 30763 (2013).
  4. Abla, M et al. J Fluorine Chem134: 63 (2012).
This product is manufactured and distributed under an exclusive license using CALIXAR™ patented technology and is supplied for research use only. It may not be used for the provision of commercial services for which a separate license must be obtained.
The CALIXAR™ C2B Additive Kit 10 x 50 μL MD1-109 is exclusively licensed to Molecular Dimensions for the manufacture and distribution to final end users only. The use of this kit for commercial service purposes requires a separate license from Calixar SAS.

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