Science through cooperation

Protein crystallography has a key role to play in a project that is making a significant contribution to understanding human diseases. The Structural Genomics Consortium (SGC) has already achieved one landmark, and looks set to continue in a similar vein. Tim Lloyd spoke with Alexey Bochkarev Ph.D., Principal Investigator, Crystallography, at SGC Toronto, to learn more.

Spread across two continents, the SGC operates from the Universities of Toronto, Canada; Karolinska Institutet, Sweden and Oxford, UK. The Consortium, which is a not-for-profit organisation, aims to determine the three dimensional structures of proteins of medical relevance, and make them available in the public domain, free of any restrictions.

From a target list of approximately 2000 proteins the SGC aims to deposit the structures of 384 proteins, between July 2004 and June 2007. This equates to a deposition rate of 200 structures per year and in addition the SGC is aiming to reduce the cost/structure to below USD$125,000.

The target list comprises only proteins that are relevant to human health issues. Diabetes and cancer are obvious candidates, but infectious diseases such as malaria are also part of the objective. This is not surprising when one considers the prevalence of this disease and the attention it receives from healthcare companies and institutions globally.

The consortium’s research is divided into three areas:

1. Structural genomics of soluble proteins
2. Structural genomics of integral membrane proteins
3. Structural chemistry of soluble proteins

Alexey Bochkarev is Principal Investigator for Protein Crystallography at the SGC’s Toronto laboratory and has worked within the Consortium since 2004.

“The aims of SGC Toronto are the same as those of Oxford and Sweden” says Bochkarev, “it is a single project run through conjunction of the three centres. Aled Edwards, Chief Executive of the Consortium, set up the three centres and they collaborate intensively. National funding supports each site.”

In terms of progress, the SGC deposited its 200th structure into the Protein Data Bank in March 2006, at an overall pace of 170 structures a year at a cost of USD$125,000.

The SGC is also involved with a pilot project relating to the structural genomics of integral membrane proteins. The first stages have yielded two new bacterial ion channels and continued pilot-scale work is devoted to membrane proteins from humans.

Structural chemistry is a large part of the Consortium’s progress and the SGC performs regular chemical screens on target proteins to identify compounds that promote protein purification or crystallisation. Approximately 20 per cent of the SGC protein structures were determined in the presence of a compound derived from SGC screening initiatives.

“We are also looking at the mechanism of interaction between proteins and ligands,” says Bochkarev, “by determining additional complexes of SGC targets with natural and synthetic ligands.”

The SGC plans to make the results of its chemical screens freely available through public databases.

The Toronto chapter

“We (SGC Toronto) do not compete with the other centres in the Consortium,” explains Bochkarev, “our research is divided into small biology groups, based on function and protein families.”

The Faculty of Medicine at the University of Toronto is home to the SGC Toronto laboratories, under the direction of Prof. Cheryl Arrowsmith. Scientists are drawn from several departments within the University, including the Banting and Best Department of Medical Research, the Departments of Physiology, Pharmacology, Medical Biophysics, and Medical Genetics and Microbiology. This variety of disciplines reflects the broad range of skills required for the Consortium’s research.

There are four dedicated research teams looking at:

• Insulin Signaling and Diabetes
• Steroid Metabolism
• Adenosine Triphosphatases and Guanosine Triphosphatases
• Malarial Parasites and Other Apicomplexans

The jewel in the crown for Toronto must surely be the protein crystallography group headed by Bochkarev, who understands the challenges to be met: “We are always asking ourselves: How do we transfer crystallography knowledge to a biology group? How do we make it more effective? We are proud to offer the most powerful crystallography facility in Canada – a great benefit for the university students as we are the only institution like this.”

The group’s protein crystallisation process is based on preparing blocks of crystal screens in 96-well deep well plates using a liquid handling robot. Bochkarev uses a TTP Mosquito robot to dispense protein drops from 100 nanoliters to 1.2 microliters and mixed with mother liquors in pre-determined ratios.

One of the most interesting facets of the group is their commitment to automation. “We are well automated, but always seeking ways to arrange our work in an even more automated fashion.” The group collaborates with Canada’s synchrotron remotely and is in the early stages of developing this project. Despite the physical distance between the two facilities, Bochkarev acknowledges “We are not magicians, but we are working to develop a more automated process.”

Experience ideal for the task

Bochkarev began his working life working at the Space Research Institute at the Russian Academy of Science, before moving into the field of Molecular Biology as a graduate student. In 1994 he moved to Canada as a post-doc Fellow with Dr A Edwards at McMaster University in Ontario, from where he progressed to Assistant Professor at the University of Oklahoma Health Sciences Center in 1998. In 2004 Bochkarev returned to Canada to join the SGC.

Alexey’s background with computers, programming, biology and crystallography have armed him with the necessary experience to manage and develop a highly-automated crystallography group: “my experience with computers and programming is important as this forms the basis for all automation.”

Evolve and learn

There are exciting times ahead for Alexey’s group, and the SGC overall. Bochkarev is keen to point out the significance of his group’s work to the development of protein crystallography as a technology. “Crystallography is definitely important and still evolving, because of the way we are trying to do it now. It has become part of a larger project.” Alexey has set views on the path that drug discovery is taking: “chemical screening is interesting, but over-simplified. Many companies have dedicated groups to identify natural plants, but depend on synthesis. We must learn from nature and try to modify.”