Controlled 2D crystallization of membrane proteins using methyl-beta-cyclodextrin.
High-resolution structural data of membrane proteins can be obtained by studying 2D crystals by electron crystallography. Finding the right conditions to produce these crystals is one of the major bottlenecks encountered in 2D crystallography. Many reviews address 2D crystallization techniques in attempts to provide guidelines for crystallographers. Several techniques including new approaches to remove detergent like the biobeads technique and the development of dedicated devices have been described (dialysis and dilution machines). In addition, 2D crystallization at interfaces has been studied, the most prominent method being the 2D crystallization at the lipid monolayer. A new approach based on detergent complexation by cyclodextrins is presented in this paper. To prove the ability of cyclodextrins to remove detergent from ternary mixtures (lipid, detergent and protein) in order to get 2D crystals, this method has been tested with OmpF, a typical beta-barrel protein, and with SoPIP2;1, a typical alpha-helical protein. Experiments over different time ranges were performed to analyze the kinetic effects of detergent removal with cyclodextrins on the formation of 2D crystals. The quality of the produced crystals was assessed with negative stain electron microscopy, cryo-electron microscopy and diffraction. Both proteins yielded crystals comparable in quality to previous crystallization reports.
Signorell
Gian A
GA
M. E. Müller Institute for Microscopy at the Biozentrum, University of Basel, Basel, Switzerland.
Kaufmann
Thomas C
TC
Kukulski
Wanda
W
Engel
Andreas
A
Rémigy
Hervé-W
HW
D020346:Aquaporins / Q000737:chemistry; D003460:Crystallization / Q000379:methods*; D003902:Detergents / Q000032:analysis; D004926:Escherichia coli; D005656:Fungal Proteins / Q000737:chemistry; D010843:Pichia; D018272:Porins / Q000737:chemistry; D017186:Titrimetry; D047392:beta-Cyclodextrins / Q000494:pharmacology*
D020346:Aquaporins; D003902:Detergents; D005656:Fungal Proteins; C109237:OmpF protein; D018272:Porins; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin