Crystallization of transmembrane proteins in cubo: mechanisms of crystal growth and defect formation.
Crystallization of membrane proteins is a major stumbling block en route to elucidating their structure and understanding their function. The novel concept of membrane protein crystallization from lipidic cubic phases, "in cubo", has yielded well-ordered crystals and high-resolution structures of several membrane proteins, yet progress has been slow due to the lack of understanding of the molecular mechanisms of protein transport, crystal nucleation, growth, and defect formation in cubo. Here, we examine at molecular and mesoscopic resolution with atomic force microscopy the morphology of in cubo grown bacteriorhodopsin crystals in inert buffers and during etching by detergent. The results reveal that crystal nucleation occurs following local rearrangement of the highly curved lipidic cubic phase into a lamellar structure, which is akin to that of the native membrane. Crystals grow within the bulk cubic phase surrounded by such lamellar structures, whereby transport towards a growing crystalline layer is constrained to within an individual lamella. This mechanism leads to lack of dislocations, generation of new crystalline layers at numerous locations, and to voids and block boundaries. The characteristic macroscopic lengthscale of these defects suggests that the crystals grow by attachment of single molecules to the nuclei. These insights into the mechanisms of nucleation, growth and transport in cubo provide guidance en route to a rational design of membrane protein crystallization, and promise to further advance the field.
Qutub
Yasser
Y
Department of Chemical Engineering, University of Houston, Houston, TX 77204, USA.
Reviakine
Ilya
I
Maxwell
Carrie
C
Navarro
Javier
J
Landau
Ehud M
EM
Vekilov
Peter G
PG
D001436:Bacteriorhodopsins / Q000737:chemistry / Q000302:isolation & purification*; D003460:Crystallization; D005989:Glycerides; D019617:Halobacterium salinarum / Q000737:chemistry; D018625:Microscopy, Atomic Force
D005989:Glycerides; D001436:Bacteriorhodopsins; C005953:monoolein