Avantis Nickel-Chelating Lipids

Two-Dimensional CrysTallizaTion of HisTiDine-TaggeD PePTiDes anD ProTeins

Proteins containing a short sequence of adjacent histidine residues (~6; His-tag) can be immobilized on a two dimensional lipid film containing a nickel-chelating group, N,Nbis[carboxymethyl]-L-lysine (nitriloacetic acid) (NTA), covalently attached to the lipid molecules. NTA-lipids provide a general approach to two-dimensional crystallization of His-tagged proteins for structure determination using electron microscopy and image processing. The technique is rapid, with crystals forming within minutes to hours from dilute protein solutions, and requires only small quantities of material. The use of NTA-lipids has the potential advantage of binding proteins in a unique orientation and allowing crystal formation under a wide range of solution conditions, including physiological conditions.

DOGS-NTA-Ni Avanti Number 790404

DOGS-NTA Avanti Number 790528

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references overleaf

Avantis Nickel-Chelating Lipids

References Kubalek, E., S.F.J. LeGrice, and P Brown. (1994). Two-dimensional crystallization of histidine-tagged HIV-1 . reverse transcriptase promoted by a novel nickel-chelating lipid. J Struct Biol 113,117-123. Schmitt, L., L. Dietrich, and R.J. Tamp. (1994). Synthesis and characterization of chelator-lipids for reversible immobilization of engineered proteins at self-assembled lipid interfaces. Am Chem Soc 166,8485-8491. Dietrich, C., O. Boscheinen, K-D. Scharf, L. Schmitt, and R. Tamp. (1996). Functional immobilization of a DNA-binding protein at a membrane interface via histidine tag and synthetic chelator lipids. Biochemistry 35,1100-1105. Barklis, E., J. McDermott, S. Wilkens, E. Schabtach, M.F. Schmid, S. Fuller, S. Karanjia, Z. Love, R. Jones, Y. Rui, X. Zhao, and D. Thompson. (1997). Structural analysis of membrane-bound retrovirus capsid proteins. EMBO J 16,1199-1213. Vnien-Bryan, C., F. Balavoine, B. Toussaaint, C. Miskowski, E.A. Hewat, B. Helme, and P .M. Vignais. (1997). Structural study of the response regulator HupR from Rodobacter capsulatus. Electron microscopy of two-dimensional crystals on a nickel-chelating lipid. J Mol Biol 274,687-692. Bischler, N., F. Balavoine, P Milkereit, H. Tschochner, C. Mioskowski, and P Schultz. (1998). Specific . . interaction and two-dimensional crystallization of histidine tagged yeast RNA polymerase I on nickelchelating lipids. Biophys J 74,1522-1532. Barklis, E., J. McDermott, S. Wilkens, S. Fuller, and D. Thompson. (1998). Organization of HIV-1 capsid proteins on a lipid monolayer. J Biol Chem 273,7177-7180 Vnien-Bryan, C., P-F. Lenne, C. Zakri, A. Renault, A. Brisson, J-F. Legrand, and B. Berge. (1998). Characterization of the growth of 2D protein crystals on a lipid monolayer by ellipsometry and rigidity measurements coupled to electron microscopy. Biophysical J 74,2649-2657. Wilson-Kubalek, E.M., R.E. Brown, H. Celia, and R.A. Milligan. (1998). Lipid nanotubes as substrates for helical crystallization of macromolecules. Proc Natl Acad Sci USA 95,8040-8045.

2D Protein Crystallization

His6-Protein solution is placed in a Teflon well. The surface of the solution is coated with NTA-lipids and various helper lipids. The device is incubated for 3-48 hours at room temperature in a closed humid chamber. After negative staining the crystals are examined in an electron microscope.

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