May 5, 2003
Purdue biologists crystallize technique to expand protein research
WEST LAFAYETTE, Ind. Purdue University scientists have managed to crystallize a particularly troublesome type of protein, an accomplishment that could overcome a 20-year hurdle in fighting a wide range of diseases.
William Cramer and three other scientists have tackled a major problem confronting protein researchers how to crystallize fat-soluble proteins in order to study them. Most proteins are water-soluble and form crystals readily, and research on these crystals has revealed a wealth of information that could have wide applications in medicine. However, although about 30 percent of the proteins in nature are fat-soluble, it has only been possible to crystallize a few of these. Any means of crystallizing the fat-soluble variety could thus bring many more diseases within the realm of biomedical understanding.
The team has thus far only crystallized one such protein, but Cramer said they are hopeful their technique can be applied to others.
"By dissolving the protein in a synthetic detergent and adding a bit of synthetic fat as 'glue,' we have produced crystals that we can study with standard methods of X-ray analysis," said Cramer, the Henry Koffler Distinguished Professor of Biological Sciences in Purdue's School of Science. "This method could be generally important to solving the structure of many biomedically important proteins."
The research appeared in the April 29 issue of the Proceedings of the National Academy of Sciences. Cramer co-authored the paper with his Purdue colleagues Huamin Zhang and visiting scholar Genji Kurisu of Osaka University, and with Janet Smith of the Department of Biological Sciences.
A living thing contains upwards of 5,000 proteins, which the cells use for everything from growth to development to adaptation. Much of the work takes place in the cell membrane, the boundary between the cell and its environment. Membrane proteins which numerically make up about 30 percent of the proteins in an organism are crucial for the life of the cell because they regulate the cell's energy level and the transport of vitamins, minerals and other essential materials across the boundary.
"If cells were countries, the membrane proteins would be both the border guards and the border industries," Cramer said. "Most of the cellular commerce is in their hands."
Scientists would like to be able to study these proteins because they play different roles in many diseases. The usual method to study proteins making a crystal of the protein and examining it with high-energy X-rays or electrons has proven difficult with membrane proteins, which dissolve only in fat, not water.
"A critical step in forming a protein crystal is dissolving it in water," Cramer said. "The trouble with membrane proteins is they only mix with fat. If you pull them out of the cell membrane, which is made of fat and protein, the protein congeals like grease on a frying pan after you dip it in cold water."
The protein molecules in such clumps are so disordered that they cannot easily form crystals. As a result, scientists have made very little progress in understanding membrane proteins. So troublesome has the problem been that when a team of German researchers managed to crystallize a membrane protein in the early 1980s, their work won them the 1988 Nobel Prize in Chemistry. Their approach to the problem at that time was to use a detergent to dissolve and crystallize the protein.
"To continue the frying pan analogy, the original method was essentially to put a little dish soap into the pan," Cramer said. "It dissolved the grease clumps so that they could crystallize."
While the approach was innovative, it unfortunately did not prove widely applicable to other fat-soluble proteins. In the nearly two decades since their success, more than 20,000 water-soluble proteins have been solved, compared to only about 50 that are fat-soluble. But Cramer's group has come up with a refinement of the original approach that could greatly expand our knowledge of these proteins' structures.
"We knew that using detergent alone didn't keep most fat-soluble protein molecules orderly enough to crystallize," Cramer said. "That's why so few other proteins have yielded to the Germans' technique. So we needed a little bit of 'glue' to keep a protein organized even in solution."
To solve the problem, Cramer's team ended up reintroducing a small quantity of synthetic fat back into the mixture not a sufficient amount to make the proteins congeal again, but just enough to keep the complex twists and turns in a protein molecule's structure from losing their uniform shape. They called their creation a "protein-lipid-detergent complex."
"After we put a touch of fat back in with the protein and detergent, the crystals formed literally overnight," Cramer said. "That's incredibly fast in this business."
The protein they studied purified from a bacterium, which uses it for photosynthesis formed crystals of a deep reddish-brown color, which the group is now analyzing as they would any water-soluble protein crystal. While the qualities of this particular protein is of major interest, Cramer said that perhaps the most important finding was that the crystallization technique itself worked.
"Our method, which may be the next major step in analysis of membrane protein complexes, could be used to push the door open a lot wider for the study of fat-soluble proteins," he said. "We hope such an opportunity could lead to treatments for a number of diseases, including cystic fibrosis, chronic muscle wasting, and many neurological abnormalities."
Cramer cautioned, however, that he is not yet certain the technique can be applied to other membrane proteins.
"We are currently encouraging our colleagues in other laboratories to attempt our approach with other fat-soluble proteins," he said. "We believe it will prove generally successful, but it will take a lot of trials with other such proteins before we can be sure."
This research has been supported in part by the National Institutes of Health Institute of General Medical Sciences and the Japanese Ministry of Science and Education.
Writer: Chad Boutin, (765) 494-2081, firstname.lastname@example.org
Source: William Cramer, (765) 494-4956, email@example.com
A publication-quality graphic is available at ftp://ftp.purdue.edu/pub/uns/cramer.crystal.jpeg.
A defined proteindetergentlipid complex for crystallization of
The cytochrome b6 f complex of
By Huamin Zhang, Genji Kurisu, Janet L. Smith,
The paucity of integral membrane protein structures creates a major bioinformatics gap, whose origin is the difficulty of crystallizing these detergent-solubilized proteins. The problem is particularly formidable for hetero-oligomeric integral membrane proteins, where crystallization is impeded by the heterogeneity and instability of the protein subunits and the small lateral pressure imposed by the detergent micelle envelope that surrounds the hydrophobic domain. In studies of the hetero (eight subunit)- dimeric 220,000 molecular weight cytochrome b6f complex, derived from the thermophilic cyanobacterium, Mastigocladus laminosus, crystals of the complex in an intact state could not be obtained from highly purified delipidated complex despite exhaustive screening. Crystals of proteolyzed complex could be obtained that grew very slowly and diffracted poorly. Addition to the purified lipid-depleted complex of a small amount of synthetic nonnative lipid, dioleolyl-phosphatidylcholine, resulted in a dramatic improvement in crystallization efficiency. Large crystals of the intact complex grew overnight, whose diffraction parameters are as follows: 94% complete at 3.40 Å spacing; Rmerge 5 8.8% (38.5%), space group, P6122; and unit cell parameters, a5b5156.3 Å, c 5 364.0 Å, a 5 b 5 90o, g 5 120o. It is proposed that the methodology of augmentation of a well-defined lipid-depleted integral membrane protein complex with synthetic nonnative lipid, which can provide conformational stability to the protein complex, may be of general use in the crystallization of integral membrane proteins.