Membrane protein crystallization in micelles conjugated by nucleoside base-pairing: A different concept.

Abstract

The dearth of high quality, three dimensional crystals of membrane proteins, suitable for X-ray diffraction analysis, constitutes a serious barrier to progress in structural biology. To address this challenge, we have developed a new crystallization medium that relies on the conjugation of surfactant micelles via base-pairing of complementary hydrophobic nucleosides. Base-pairs formed at the interface between micelles bring them into proximity with each other; and when the conjugated micelles contain a membrane protein, crystal nucleation centers can be stabilized, thereby promoting crystal growth. Accordingly, two hydrophobic nucleoside derivatives - deoxyguanosine (G) and deoxycytidine (C), each covalently bonded to a 10 carbon chain were synthesized and added to an aqueous solution containing octyl β-d-thioglucopyranoside micelles. These hydrophobic nucleosides induced the formation of oil-rich globules after 2days incubation at 19°C or after a few hours in the presence of ammonium sulfate; however, phase separation was inhibited by 100mM GMP. The presence of the membrane protein bacteriorhodopsin in the conjugated - micellar dispersion resulted in the growth within the colorless globules of a variety of purple crystals, the color indicating a functional protein. On this basis, we suggest that conjugation of micelles via base-pair complementarity may provide significant assistance to the structural determination of integral membrane proteins.