Membrane protein reconstitution and crystallization by controlled dilution.
Efficient reconstitution of membrane proteins for functional analyses can be achieved by dilution of a ternary mixture containing proteins, lipids and detergents. Once the dilution reaches the point where the free detergent concentration would become lower than the critical micellar concentration, detergent is recruited from the bound detergent pool, and association of proteins and lipids is initiated. Here we show that dilution is also suitable for the assembly of two-dimensional crystals. A device has been designed that allows controlled dilution of a protein-lipid-detergent mixture to induce formation of densely packed or crystalline proteoliposomes. Turbidity is used to monitor the progress of reconstitution on-line, while dilution is achieved by computer-controlled addition of buffer solution in sub-microliter steps. This system has mainly been tested with porin OmpF, a typical beta-barrel protein, and aquaporin-1, a typical alpha-helical protein. The results demonstrate that large, highly ordered two-dimensional crystals can be produced by the dilution method.
Rémigy
H-W
HW
M.E. Müller Institute for Microscopy, Biozentrum, University of Basel, Klingelbergstr. 70, CH-4056 Basel, Switzerland.
Caujolle-Bert
D
D
Suda
K
K
Schenk
A
A
Chami
M
M
Engel
A
A
D051398:Aquaporin 1; D020346:Aquaporins / Q000302:isolation & purification; D003460:Crystallization; D003902:Detergents; D029968:Escherichia coli Proteins / Q000302:isolation & purification; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000648:ultrastructure; D008823:Micelles; D008854:Microscopy, Electron; D045332:Photosystem II Protein Complex / Q000302:isolation & purification; D018272:Porins / Q000302:isolation & purification; D012995:Solubility
D020346:Aquaporins; D003902:Detergents; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D008823:Micelles; C109237:OmpF protein; D045332:Photosystem II Protein Complex; D018272:Porins; D051398:Aquaporin 1