Problems in obtaining diffraction-quality crystals of hetero-oligomeric integral membrane proteins.
The major barrier responsible for the slow pace of structure determination of integral membrane proteins is the difficulty of crystallizing detergent-solubilized hydrophobic proteins, particularly hetero-oligomeric integral membrane proteins. For the latter class of multi-subunit proteins, we have encountered the following problems in addition to the ubiquitous problem of detergent compatibility: (i) instability caused by over-purification that results in delipidation; (ii) protease activity degrading exposed loops and termini of subunits of the complex that could not be inhibited; (iii) poor protein-protein contacts presumably arising from masking by the detergent micelle. Problem (i) could be ameliorated in crystallization of the cytochrome b(6)f complex by augmenting the delipidated complex with synthetic lipid. Problem (ii) has not been solved. Problem (iii) has been solved in other systems by the use of monoclonal antibodies (or other protein ligands) to increase the probability of protein-protein contacts. In the case of the complex formed by the cobalamin and colicin receptor, BtuB, and the receptor binding domain of colicin E3, the latter served as a ligand for protein-protein contacts that facilitated crystallization.
Zhang
Huamin
H
Department of Biological Sciences, Lilly Hall of Life Sciences, Purdue University, West Lafayette, IN 47907-2054, USA. hzhang@bilbo.bio.purdue.edu
Cramer
William A
WA
D003460:Crystallization / Q000379:methods*; D045346:Cytochrome b6f Complex / Q000737:chemistry; D003902:Detergents / Q000737:chemistry; D004492:Edetic Acid; D038983:Galactolipids; D008055:Lipids / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D010713:Phosphatidylcholines; D021122:Protein Subunits / Q000737:chemistry / Q000302:isolation & purification; D040901:Proteomics / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization
D003902:Detergents; D038983:Galactolipids; D008055:Lipids; D008565:Membrane Proteins; D010713:Phosphatidylcholines; D021122:Protein Subunits; C009909:monogalactosyldiacylglycerol; D045346:Cytochrome b6f Complex; D004492:Edetic Acid; C017251:1,2-oleoylphosphatidylcholine