Guests, and hosts, and microfluidics
Rustem Ismagilov group (University of Chicago)
The Ismagilov group from the University of Chicago recently
proposed to use simple guest host chemistry to modulate crystallization of
membrane proteins by capturing detergents with methyl-β-cyclodextrin
(MBCD) in membrane protein samples. MBCD forms a complex with detergent,
breaking up free detergent micelles and extracting loosely bound detergent in
the protein-detergent complex (PDC). It was shown that with detergent
capture, the membrane protein sample had improved homogeneity. Using a
plug-based microfluidic system, which is compatible with crystallization of
membrane proteins, they screened for the optimal concentration of MBCD as
additive and crystallized reaction center (RC) from Blastochloris viridis in
a different space group than had been previously observed. Using the
crystals grown directly in their 10-nL plugs, they solved the structure to
3.2 Å, with an X-ray diffraction experiment performed at GM/CA CAT's
23ID-D. The RC crystals in the new space group show protein contacts closer
to the membrane plane. This observation supported their hypothesis that MBCD
extracted detergent loosely bound to protein, thus making more residues
accessible to protein-protein contacts. The Ismagilov group also proposed to
add MBCD during the process of concentrating membrane proteins, in order to
reduce the detergent concentration in the concentrated sample and prevent the
formation of free micelles.
Figure: MBCD captures free and
loosely-bound detergent and changes the morphology and packing of RC
crystals. (A) Trigonal crystal grown with 4 mM MBCD and 4.6 mM LDAO.
(B) Trigonal crystal in a microfluidic plug. (C) Tetragonal crystal
grown at 44 mM LDAO (~ 40 CMC). (D) Tetragonal crystal in a microfluidic
plug. (E) Tetragonal crystal grown with 8 mM γ-CD and 4.6 mM LDAO.
(F) Tetragonal crystals in a plug containing 8 mM γ-CD. (G) Packing of
RC proteins in trigonal crystals P3121, a = b = 241.2 Å, c =
113.4 Å. (H) Packing of RC proteins in tetragonal crystals
P43212 a = b = 220.4 Å, c = 113.0 Å. (G)
and (H) are viewed along the c axis (31 axis for G and
43 axis for H). The a and b axial directions are shown on the top
right of each figure. Green: subunit C; cyan: subunit H; yellow: subunit M;
and pink: subunit L.
Li, L, Mustafi, D, Fu, Q, Tereshko, V, Chen, DL, Tice, JD, Ismagilov, RF.
Nanoliter microfluidic hybrid method for simultaneous screening and
optimization validated with crystallization of membrane proteins, Proc. Natl.
Acad. Sci. USA 103 (51), 19243-19248 (2006). DOI: 10.1073/pnas.0607502103