Membrane-protein stability in a phospholipid-based crystallization medium.
Protein stability is a crucial factor to consider when attempting to crystallize integral membrane proteins. Cubic phase, or in meso, lipid-bilayer crystallization media are thought to provide native-like environments that should facilitate membrane protein crystallization by helping to stabilize the native protein conformation for the duration of the crystallization process. While excellent crystals of bacteriorhodopsin (bR) and other Halobacterial rhodopsins have been obtained in lipid-bilayer gels formed with monoglycerides, success remains elusive in the general application of such media to other membrane proteins. Additionally, we have noted that some mutants of bR are highly unstable in gels formed with monoolein. Phosphatidylethanolamines (PE) and derivatives of PE represent another class of lipids that can form connected-bilayer gels. When wildtype bR and a labile bR mutant were reconstituted into this phospholipid gel, spectroscopy showed that the protein is both more stable and has improved conformational homogeneity as compared to gels formed using monoolein. In addition, we demonstrate that well-diffracting crystals of bR can be grown from a PE-based crystallization medium. Since most proteins lack a stability-indicating chromophore and other structure-based analytical techniques are poorly compatible with the lipid gel, we developed a generally-applicable spectroscopic technique based on the intrinsic fluorescence of tryptophan residues. This fluorescence assay makes possible the rapid evaluation of lipid gels as media for the crystallization of membrane proteins.
Lunde
Christopher S
CS
QB3 Institute, University of California, Berkeley, CA 94720, USA. cslunde@lbl.gov
Rouhani
Shahab
S
Facciotti
Marc T
MT
Glaeser
Robert M
RM
D001426:Bacterial Proteins / Q000737:chemistry; D001436:Bacteriorhodopsins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D003460:Crystallization; D018360:Crystallography, X-Ray; D019605:Euryarchaeota / Q000378:metabolism; D008055:Lipids / Q000737:chemistry; D008956:Models, Chemical; D010714:Phosphatidylethanolamines / Q000378:metabolism; D011487:Protein Conformation; D012243:Rhodopsin / Q000378:metabolism; D013053:Spectrophotometry / Q000295:instrumentation* / Q000379:methods; D014364:Tryptophan / Q000737:chemistry; D014961:X-Ray Diffraction
D001426:Bacterial Proteins; D008055:Lipids; D010714:Phosphatidylethanolamines; D001436:Bacteriorhodopsins; D014364:Tryptophan; D012243:Rhodopsin