Robust, High-Throughput Analysis of Protein Structures

Scientists have developed a fast and efficient way to determine the structure of proteins, shortening a process that often takes years into a matter of days. The Structurally Integrated BiologY for Life Sciences (SIBYLS) beamline at the ALS has implemented the world's highest-throughput biological-solution x-ray scattering beamline enabling genomic-scale protein-structure characterization. Coupling brilliant x rays from one of the superconducting bend magnets at the ALS to liquid-handling robotics has enabled the collection of 96 samples in 4 hours. Importantly, the sample format and the amount of material required are practical for most biological problems. The beamline's high-throughput capability is set to have a large impact on many fields that require genomic-scale information, such as Berkeley Lab's bioenergy efforts and cancer biology studies.

Artist's depiction SAXS

Artist's abstract depiction of high-throughput SAXS combining high-brightness x rays, robotic handling, and computation as applied to all the gene products (i.e., proteins) of a microorganism, resulting in the shape and assembly of each macromolecule.

Sometimes Less is More
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Tremendous progress has been made in our ability to extract exquisitely detailed information about protein structures using crystallography. Yet, the process is still frustratingly slow and painstaking, given the sheer volume of proteins coded into the genomes (i.e., the full genetic blueprint) of even the simplest of organisms. Hura et al. have developed a high-throughput protein pipeline that could expedite protein analysis by trading atomic-level detail for speed and simplicity. In many cases, given the critical mass of structural data we have already accumulated, knowledge of a protein's molecular "envelope" is sufficient to address key biological questions.

For example, future synthetic biology efforts may involve taking a useful protein, or a network of proteins, from one microbe and importing it into another. To do this, scientists must learn how much of the network needs to be imported in order for it to do its job. This requires analyzing individual proteins in hundreds of different conditions. Older techniques that are more expensive and time consuming are unlikely to yield more useful information than the technique described here, which the research team believes will help usher in the next phase of genomics research.

The high flux, brilliance, and focus of the x rays from ALS Beamline 12.3.1 are ideal for high-throughput, time-resolved experiments that cannot be replicated using in-house light sources. The SIBYLS team used a technique called small-angle x-ray scattering (SAXS) to image proteins in their natural state, such as in a solution, and at a resolution of about 10 angstroms, enough to determine three-dimensional shape. Although x-ray crystallography yields higher-resolution images, SAXS makes up for what it lacks in precision by providing fast, accurate information on the shape, assembly, and conformational changes of proteins. Also, by studying protein crystals in solution, smaller protein crystals of any shape can be analyzed and radiation damage is significantly reduced, making the technique practical for almost any biomolecule.

Because of the high throughput rate, the bottlenecks in the process occur during sample transfer, washing, and data analysis rather than data collection. To maximize speed, a robot automatically pipettes protein samples into position and supercomputer clusters analyze the resulting data. In this work, the researchers reported the results of 50 purified proteins analyzed over a period of 2 weeks. The 50 proteins were mostly from a single organism that has a total of 2000 genes. With the demonstrated throughput, 2000 proteins would be completed in a year and a half. The shape and assembly were reported for over 80% of the samples analyzed.

Aside from the record-breaking throughput, several additional important results were obtained from this study. Twelve proteins with no previous structural information were solved to 1.5-nm resolution. Over 50% of the proteins studied were multimeric (formed complexes of dimers and trimers, etc.). This finding underscores the importance of multimerization, as several multimeric states had been characterized incorrectly by other techniques. Finally, while genomics enables the quantification of the number of proteins an organism contains, identifying what each protein actually does is very challenging. A significant fraction of known proteins remain mysterious in terms of function. By comparing a protein's measured SAXS profile like a fingerprint to those calculated from existing available structures with known function, the researchers could obtain important clues to the purpose of the unknown protein.

SAXS shape and assembly solution

(a) For proteins with structural homologs or existing structures, experimental scattering data (colors) are compared with the scattering curve calculated for the matching structure (black). (b) The envelope determinations (colored as in a) were overlaid with the existing structures (ribbons). (c) For proteins with no available structural information, envelope predictions from two independent programs, DAMMIN (gray mesh) and GASBOR (blue solid), are compared and generally agree.

The SIBYLS beamline's success relies on applying SAXS to focused biological problems. Current directions include the analysis of DNA repair pathways, which, if malfunctioning, are a leading cause of cancer. An equally important focus is on bioenergy production through the understanding of metabolic pathways in organisms capable of living in extreme industrial environments such as high temperature, salt,or pH. These organisms contain novel proteins that, for example, create hydrogen, a potential alternative fuel, as a by-product. In general, SAXS can quickly provide information at resolutions often sufficient for functional insights into how proteins work. With the number of genes being identified growing at a high rate, high-throughput SAXS helps us keep pace and is an enabling technology that may change the way that structural genomics research is done.

SAXS at Beamline 12.3.1

Configuration of the SAXS endstation at SIBYLS Beamline 12.3.1.

Research conducted by G.L. Hura, M. Hammel, R.P. Rambo, S.E. Tsutakawa, S. Classen, and K.A. Frankel (Berkeley Lab); A.L. Menon, F.L. Poole II, F.E. Jenney Jr., R.C. Hopkins, S.-J. Yang, J.W. Scott, B.D. Dillard, and M.W.W. Adams (University of Georgia); and J.A. Tainer (The Scripps Research Institute and Berkeley Lab).

Research Funding: National Institutes of Health and U.S. Department of Energy (DOE), Offices of Biological and Environmental Research, Advanced Scientific Computing, and Basic Energy Sciences (BES). Operation of the ALS is supported by DOE BES.

Publication about this research: G.L. Hura, A.L. Menon, M. Hammel, R.P. Rambo, F.L. Poole II, S.E. Tsutakawa, F.E. Jenney Jr, S. Classen, K.A. Frankel, R.C. Hopkins, S.-J. Yang, J.W. Scott, B.D. Dillard, M.W.W. Adams, and J.A. Tainer, "Robust, high-throughput solution structural analyses by small angle x-ray scattering (SAXS)," Nat. Methods 6, 606 (2009).

ALSNews Vol. 303, October 28, 2009

Robust, High-Throughput Analysis of Protein Structures

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