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Lysozyme Cubic Phases Crystallization Introduction


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June 2004 J.I. Yeh

Lysozyme Cubic Phases Crystallization
Introduction:

Lipic cubic phases are complex materials composed of immiscible, hydrophilic, and hydrophobic compartments. These highly viscous and transparent materials can be closed micellar and bicontinuous, depending of the lipid. The main difference is the supramolecular assembly: discrete, discontinuous versus curved, continuous bilayers. In either array, intercommunicating water channels pervade the entire phase. Bicontinuous phases allow crystallization of membrane proteins by virtue of their lateral diffusion in curved bilayer, thought to mimic native membrane bilayers; in discrete micelles, this type of crystal growth is predicted not to occur. Although particularly apt for membrane proteins, water-soluble proteins can also be crystallized in these phases.


Nucleation of protein followed by growth of membrane proteins may be due to the ability of biocontinuous phases to allow for lateral diffusion in the curved bilayer. In discrete micelles, lateral diffusion is not thought to occur.
Crystallization of small soluble molecules and hydrophilic proteins are likely to occur in aqueous compartments, into which these solutes partition, independent of the type of cubic phase and packing of the lipidic compartments.
Cubic phases allow incorporation of additives that are used routinely in the crystallization of proteins. Different cubic phases can be doped with wide concentrations of additives. Proteins, contained within the aqueous compartments of the cubic phases, can crystallize irrespective of the presence of cubic phases. In membrane proteins, they are thought to diffuse through the bicontinuous layer, through the matrix to form packing interactions.


From Landau and Rosenbusch (1996).

PNAS U.S.A. 93:14532-14535.

Experimental: final volumes 40 L and final concentrations:

I. MO Cubic Phases:

63% MO

50 mM Na-acetate pH 4.5



0.8 M NaCl

[lysozyme]=40 mg/mL


II. MP Cubic Phases:

60% MP


50 mM Na-acetate, pH 4.5

0.8 M NaCl

[lysozyme]=34 mg/mL

III. PLPC Closed Micellar Cubic Phases:

40% PLPC

50 mM Na-acetate pH 4.5

0.8 M NaCl

[lysozyme]=34 mg/mL


IV. Aqueous (control):

40 mM Na-acetate, pH 4.5

1.3 M NaCl

[lysozyme]= 50 mg/mL


Stock Solutions:

  1. 4M NaCl

  2. 50 mM Sodium acetate buffer, pH 4.6

  3. 400 mg/mL lysozyme

  4. MO / MP: 25 mg each per setup


Procedure:

  1. Mix together in an eppendorf: 8 L NaCl and 28 L NaAc buffer; add 4 L of lysozyme and mix gently.

  2. Cubic phases are prepared by mixing the crystallization solution, from Step 1, with the appropriate lipid in a polypropylene eppendorf*, which is clear and allows visualization of the crystals more easily. Upon mixing of the lipid with the solution, a very viscous, semi-solid material forms so thorough mixing is required.

  3. Centrifuge above mixture at 10,000xg for 150 minutes in a desktop centrifuge at 20 oC.

  4. Stability can be assessed under a light microscope, where the cubic phases are gel-like, transparent, and nonbirefringent material with crystals embedded in the material. Lysozyme crystals grow at the interface of the cubic and a small amount of extruded liquid phase, just above (or below) the semi-solid lipid phase.

  5. Time for crystallization is dependent on concentrations of protein, additive, and phase used for crystallization; MO will yield lysozyme crystals within a day while MP may take more time.



Crystal Mounting#:

Crystals of soluble protein can be extracted from the LCP fairly readily as they form at the matrix/water interface and may be free-floating. For membrane proteins grown embedded in the cubic phases, lipase can be used to hydrolyze the mono-olein (or MP), which will form liquefied droplets of oleic acid dispersed in glycerol/water solution. After lipase addition, centrifugation results in crystals at the water/oil interface. These crystals can be subjected to a second lipase treatment or rinsed in aml to remove adhering oleic acid droplets and lipase and mounted using standard methods.


Abbreviations:

MO: 1-monooleoyl-rac-glycerol (Sigma)

MP: 1-monopalmitoleoyl-rac-glycerol (Sigma)

PLPC: palmitoyl lysophosphatidylcholine (Fluka)



* Landau, Rummel, Cowan-Jacob, and Rosenbusch (1997). J. Phys. Chem. B. 101 (11) 1935-1937(uses glass vials [id=2mm, od=4mm, length=20mm] to Vf= 10-20 L].

# Nollert and Landau (1998). Biochem. Soc. Trans. 26: 709-713.


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