Tunable Phase Separation of Detergents for the Crystallization of Membrane Proteins and Block Copolymers

Membrane proteins and the biomimetic membranes they produce are promising materials for membrane separations. The high protein packing in self-assembled 2D sheet structures is ideal for desirable characteristics such as high selectivity and permeability. One potential method for synthesizing these 2D sheets involves heating a mixture of proteins, amphiphilic block copolymers, and detergents. In this method, a critical component of 2D sheet formation is the phase separation of detergent at elevated temperatures, resulting in the aggregation of detergent micelles containing block copolymers and membrane proteins. However, some membrane proteins may be unstable and unfit for 2D sheet formation at extreme temperatures. To address this issue, we investigated using additives in detergent mixtures to reduce the temperature required for phase separation. The phase separation temperatures were determined using cloud point measurements of n-octyl-oligo-oxyethylene (Octyl-POE) detergent solutions containing various amounts of glucose. In these solutions, the phase separation temperature was lowered from 56°C to 29°C by increasing the glucose concentration from 0 to 2.2 M. [add data on lowest concentration where phase separation was observed and how it does not change much when Octyl-POE is present]. Adding 1.4 M of glucose to a solution of Outer Membrane Protein F (OmpF), poly(butadiene-b-ethylene oxide) (PB-PEO) and Octyl-POE enabled the formation of 2D sheets containing OmpF and PB-PEO at 45 °C. This temperature was far less than the necessary 60 °C required in the absence of glucose. These results showed the promising use of additives to decrease the required temperature for phase separation and the subsequent 2D sheet formation, enabling this method to be more broadly applied to other membrane protein-derived nanosheets.