Two-Dimensional Crystallization of Membrane Proteins by Reconstitution Through Dialysis

Abstract

Studies of membrane proteins by two-dimensional (2D) crystallization and electron crystallography have provided crucial information on the structure and function of a rapidly growing number of these intricate proteins within a close-to-native lipid bilayer. Here we provide protocols for planning and executing 2D crystallization trials by detergent removal through dialysis, including the preparation of phospholipids and the dialysis setup. General factors to be considered, such as the protein preparation, solubilizing detergent, lipid for reconstitution, and buffer conditions are discussed. Several 2D crystallization conditions are highlighted that have shown great promise to grow 2D crystals within a surprisingly short amount of time. Finally, conditions for optimizing order and size of 2D crystals are outlined.

Detergent-free Ultrafast Reconstitution of Membrane Proteins into Lipid Bilayers Using Fusogenic Complementary-charged Proteoliposomes.

“…The latter approach is much faster (~30 min 1 ) and is therefore preferable for reconstitution of fragile and sensitive membrane proteins, while the first two approaches are limited by detergent removal speed, which takes many hours and may cause a substantial loss of activity and loss of structural integrity of the proteins. Functionalization of larger vesicles (large unilamellar vesicles, LUV, up to 1 µm diameter) by this approach is more challenging, as vesicle size gets reduced after detergent removal, and it is not possible for giant unilamellar vesicles (GUV, >1 µm), as they are destabilized by detergents (but see Johnson et al 2 for slow 2D-crystallization of membrane proteins in large bilayers). Alternative approaches for GUV membrane functionalization 3 4 5 exist but are laborious, time consuming, and still require some detergent at concentrations below CMC.…”

Detergent-free Ultrafast Reconstitution of Membrane Proteins into Lipid Bilayers Using Fusogenic Complementary-charged Proteoliposomes.

“…The latter approach is much faster (~30 min 1 ) and is therefore preferable for reconstitution of fragile and sensitive membrane proteins, while the first two approaches are limited by detergent removal speed, which takes many hours and may cause a substantial loss of activity and loss of structural integrity of the proteins. Functionalization of larger vesicles (large unilamellar vesicles, LUV, up to 1 µm diameter) by this approach is more challenging, as vesicle size gets reduced after detergent removal, and it is not possible for giant unilamellar vesicles (GUV, >1 µm), as they are destabilized by detergents (but see Johnson et al 2 for slow 2Dcrystallization of membrane proteins in large bilayers). Alternative approaches for GUV membrane functionalization 3,4,5 exist but are laborious, time consuming, and still require some detergent at concentrations below CMC.…”