Laterally mobile, functionalized self-assembled monolayers at the fluorous-aqueous interface in a plug-based microfluidic system: characterization and testing with membrane protein crystallization.
This paper describes a method to generate functionalizable, mobile self-assembled monolayers (SAMs) in plug-based microfluidics. Control of interfaces is advancing studies of biological interfaces, heterogeneous reactions, and nanotechnology. SAMs have been useful for such studies, but they are not laterally mobile. Lipid-based methods, though mobile, are not easily amenable to setting up the hundreds of experiments necessary for crystallization screening. Here we demonstrate a method, complementary to current SAM and lipid methods, for rapidly generating mobile, functionalized SAMs. This method relies on plugs, droplets surrounded by a fluorous carrier fluid, to rapidly explore chemical space. Specifically, we implemented his-tag binding chemistry to design a new fluorinated amphiphile, RfNTA, using an improved one-step synthesis of RfOEG under Mitsunobu conditions. RfNTA introduces specific binding of protein at the fluorous-aqueous interface, which concentrates and orients proteins at the interface, even in the presence of other surfactants. We then applied this approach to the crystallization of a his-tagged membrane protein, Reaction Center from Rhodobacter sphaeroides, performed 2400 crystallization trials, and showed that this approach can increase the range of crystal-producing conditions, the success rate at a given condition, the rate of nucleation, and the quality of the crystal formed.
Department of Chemistry, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA.
Jason E
JE
Kreutz
Liang
L
Li
L Spencer
LS
Roach
Takuji
T
Hatakeyama
Rustem F
RF
Ismagilov
D003460:Crystallization; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006845:Hydrocarbons, Fluorinated / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry*; D008567:Membranes, Artificial; D044085:Microfluidics / Q000379:methods*; D013499:Surface Properties; D014867:Water / Q000737:chemistry
D006845:Hydrocarbons, Fluorinated; D008565:Membrane Proteins; D008567:Membranes, Artificial; D014867:Water; D049452:Green Fluorescent Proteins