A high-throughput method for membrane protein solubility screening: the ultracentrifugation dispersity sedimentation assay

Abstract

One key to successful crystallization of membrane proteins is the identification of detergents that maintain the protein in a soluble, monodispersed state. Because of their hydrophobic nature, membrane proteins are particularly prone to forming insoluble aggregates over time. This nonspecific aggregation of the molecules reduces the likelihood of the regular association of the protein molecules essential for crystal lattice formation. Critical buffer components affecting the aggregation of membrane proteins include detergent choice, salt concentration, and presence of glycerol. The optimization of these parameters is often a time- and protein-consuming process. Here we describe a novel ultracentrifugation dispersity sedimentation (UDS) assay in which ultracentrifugation of very small (5 microL) volumes of purified, soluble membrane protein is combined with SDS-PAGE analysis to rapidly assess the degree of protein aggregation. The results from the UDS method correlate very well with established methods like size-exclusion chromatography (SEC), while consuming considerably less protein. In addition, the UDS method allows rapid screening of detergents for membrane protein crystallization in a fraction of the time required by SEC. Here we use the UDS method in the identification of suitable detergents and buffer compositions for the crystallization of three recombinant prokaryotic membrane proteins. The implications of our results for membrane protein crystallization prescreening are discussed.

Figure 1.

Schematic workflow of UDS is shown for a membrane protein where no previous information on behavior in different detergents is available.

Figure 2.

(A) SECs for MP-A maintained in OG (broken line) and DDM (solid line). (B) UDS of MP-A in a range of different detergents in the presence of 10% glycerol and 100 mM NaCl. (C) UDS of MP-A in C₁₂E₈, FC-12, and TriDM in 0% glycerol and decreasing concentrations of NaCl. (D) SEC of MP-A in buffer containing 0.05% C₁₂E₈ and 100 mM NaCl. (E) Crystals of MP-A obtained in 0.1% FC-12. Bar, 50 μm.

Figure 3.

(A) Crystals of MP-B grown in the presence of DDM. Bar, 200 μm. (B) MP-B diffracted X-rays to up to 4.0 Å. (C) Crystals of MP-C grown in buffer containing DDM; the crystals were photographed under a polarizer for clarity. Bar, 200 μm. (D) MP-C crystals diffracted anisotropically with some reflections to 6.5 Å.

Figure 4.

UDS of MP-B (A) and MP-C (C) in a range of different detergents. Arrows indicate protein bands. SEC analysis of MP-B in DDM (B) and MP-C (D) in DM.