NEW YORK (GenomeWeb News) – Researchers have developed a high-throughput method for determining protein structure based on small angle X-ray scattering, or SAXS, that they said is much quicker that current approaches based on X-ray crystallography or nuclear magnetic resonance.
The effort to speed up the determination of protein structure stemmed from a realization that "growing metagenomics, proteomics, and bioinformatics contributions are outpacing classical structural biology approaches, creating an increasing structural knowledge gap," the researchers said in a paper describing the approach that was published in an advanced online paper in Nature Methods.
The research was supported through the US Department of Energy's "Molecular Assemblies: Genes and Genomes Integrated Efficiently," or MAGGIE, program.
While the team explained that crystallography and nuclear magnetic resonance spectroscopy provide a great deal of information about protein structure, they noted that the speed and ease of such approaches is limited. For instance, some proteins are easier to crystallize than others and NMR tends to be time-consuming and limited to proteins that fall within a specific size range.
In an effort to come up with a protein structure determination method that is rapid and reliable, the researchers explored SAXS, an approach based on the way proteins in solution scatter X-rays.
Although SAXS has been slower to catch on in structural biology research than crystallography, the study's authors explained, it is becoming more commonly used with the advent of better synchrotron X-ray sources and detectors that offer improved speed and reduced sample requirements.
Because it allows analysis of proteins in solution, co-lead author Gregory Hura, a scientist with Lawrence Berkeley National Laboratory's Advanced Light Source, told GenomeWeb Daily News, SAXS shifts the bottleneck in determining protein structure away from crystallization and onto the protein purification step.
Hura and his co-workers team developed a SAXS pipeline for doing partially automated structural analyses of proteins from very small (microliter volumes) of protein in solution. The approach relied on a pipetting robot to transfer samples from a 96-well plate and LBL's Advanced Light Source.
The team tested their high-throughput SAXS method by looking for structural information for 50 proteins, including 34 from an archeal species called Pyrococcus furiosus and 16 samples from collaborators at the Joint Center for Structural Genomics.
The researchers obtained structural information for 82 percent of the proteins tested, including 31 P. furiosus proteins and ten proteins from other microbes. In contrast, Hura said, the expected success rate using an approach such as crystallography is just 12 to 15 percent.
"We can now obtain structural information in solution on most samples, rather than the 15 percent obtained by the best of the current Structural Genomics Initiative efforts employing nuclear magnetic resonance and crystallography," senior author John Tainer, a researcher affiliated with the Scripps Research Institute and LBL's Life Sciences Division, said in a statement.
The SAXS approach does have its drawbacks. For instance, the structural information it provides is still lower resolution than that available from crystallography. Hura noted that it also takes some experience to understand when a signal is worth analyzing, since everything in a solution will show a scatter pattern in SAXS. And because it looks at proteins in solution, the SAXS method is better suited for soluble rather than membrane proteins.
In the future, Hura said, he and his colleagues intend to address the latter issue by exploring ways to obtain structural information for more membrane proteins. They also plan to continue refining their SAXS method in an effort to come up with a fully automated approach
Eventually, the team hopes to apply SAXS for looking at protein complexes within entire metabolic networks. They are particularly interested in learning more about P. furiosus' hydrogen-producing pathway, Hura explained, since it may have potential applications for bioenergy research. That will involve studying various P. furiosus protein combinations under different conditions.
"This is where our system will have a big impact," Hura said in a statement. "We can do this type of structural analysis in a matter of weeks, as opposed to years with crystallography."