Simple host-guest chemistry to modulate the process of concentration and crystallization of membrane proteins by detergent capture in a microfluidic device.
This paper utilizes cyclodextrin-based host-guest chemistry in a microfluidic device to modulate the crystallization of membrane proteins and the process of concentration of membrane protein samples. Methyl-beta-cyclodextrin (MBCD) can efficiently capture a wide variety of detergents commonly used for the stabilization of membrane proteins by sequestering detergent monomers. Reaction Center (RC) from Blastochloris viridis was used here as a model system. In the process of concentrating membrane protein samples, MBCD was shown to break up free detergent micelles and prevent them from being concentrated. The addition of an optimal amount of MBCD to the RC sample captured loosely bound detergent from the protein-detergent complex and improved sample homogeneity, as characterized by dynamic light scattering. Using plug-based microfluidics, RC crystals were grown in the presence of MBCD, giving a different morphology and space group than crystals grown without MBCD. The crystal structure of RC crystallized in the presence of MBCD was consistent with the changes in packing and crystal contacts hypothesized for removal of loosely bound detergent. The incorporation of MBCD into a plug-based microfluidic crystallization method allows efficient use of limited membrane protein sample by reducing the amount of protein required and combining sparse matrix screening and optimization in one experiment. The use of MBCD for detergent capture can be expanded to develop cyclodextrin-derived molecules for fine-tuned detergent capture and thus modulate membrane protein crystallization in an even more controllable way.
Li
Liang
L
Department of Chemistry and Institute for Biophysical Dynamics, University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA.
Nachtergaele
Sigrid
S
Seddon
Annela M
AM
Tereshko
Valentina
V
Ponomarenko
Nina
N
Ismagilov
Rustem F
RF
D003460:Crystallization; D018360:Crystallography, X-Ray; D003902:Detergents / Q000737:chemistry*; D041921:Hyphomicrobiaceae / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry*; D008823:Micelles; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000379:methods*; D008958:Models, Molecular; D010316:Particle Size; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry*; D013499:Surface Properties; D047392:beta-Cyclodextrins / Q000737:chemistry*
D003902:Detergents; D008565:Membrane Proteins; D008823:Micelles; D045322:Photosynthetic Reaction Center Complex Proteins; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin