The ocean depths offer unique opportunities for uncovering proteins that have evolved under exotic conditions, according to Joseph Ng, Ph.D., associate professor, University of Alabama, Huntsville. Dr. Ng and his coworkers confront the challenges of developing three-dimensional descriptions of protein molecules through the use of x-ray crystallography.
Current technology takes advantage of structural genomics, in which a sequenced genome is screened for coding regions that can be analyzed through a bioinformatics program. Candidate sequences of interest are then optimized with the goal of expressing proteins and building crystals that can be subjected to analysis.
In a natural state it would be selectively disadvantageous for an organism’s protein to crystallize spontaneously. For this reason, most proteins are resistant to crystallization under normal physiological conditions. Yet, knowledge of a molecule’s 3-D structure is essential for a rigorous understanding of its structure and function, and for the design of drugs that would influence its behavior in a therapeutic situation.
Dr. Ng’s program involves identification, cloning, expression, purification, crystallization, and crystallography of promising molecules. “Success depends on the solubility and stability of the protein and determining the optimal nucleation and crystal growth parameters. Problems may arise if the protein solution is heterogeneous, if the molecules lack a compact arrangement and move extensively in solution, or if the molecules undergo nonspecific aggregation. We have a number of tricks in our bag for overcoming these roadblocks.”
The group has access to a number of hyperthermophilic bacterial proteins, obtained from deep-sea explorations, and these are yielding useful information on the adaptations of proteins to harsh environments. The Huntsville team has found that many of these proteins have a high propensity to crystallize.
Not all heat-resilient proteins are good candidates for crystallization, but for those that are, their structures reveal common molecular features. They possess unique architectural and molecular “hot spots” that endow them with stability and the ability to form crystal lattices. Using high-throughput gene-synthesis and protein-engineering methods developed in Dr. Ng’s lab, proteins that do not crystallize are coupled to hyperthermophilic protein fragments as crystallization vehicles.
Protein crystals suitable for x-ray crystallography can now be obtained by this method where they cannot be acquired otherwise. The subsequent processing of the molecules exploits capillary tubes into which the protein solutions are introduced. The tubes are sealed with nail polish, and counter diffusion takes place within the capillary. The crystals are then subjected to analysis in situ. Many proteins have been analyzed including lysozyme, Factor XIII, Con A, insulin, and glucose isomerase.
“Over 50 percent of targeted proteins in structural genomics programs cannot be crystallized using conventional methods,” Dr. Ng pointed out. “So this approach opens the door to a whole realm that was previously inaccessible.”