Researchers View Protein's Structural Changes In Real Time; X-ray Crystallography Records Changes In One 10-billionth Of A Second

The ability to watch a protein functioning on the inside may lead to a better understanding of how it works, enabling scientists to engineer improved proteins for blood substitutes and improved treatments for genetic diseases.

Researchers at the National Institutes of Health (NIH), Rice University and the European Synchrotron Radiation Facility (ESRF) in France collaborated on the study, which is published in the June 20 issue of the journal Science.

Philip Anfinrud, senior biomedical research scientist in the Laboratory of Chemical Physics at the NIH's National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), headed the study of the protein myoglobin, which provides oxygen to muscles when they contract.

The scientists aimed a laser pulse at the protein for a trillionth of a second to break the chemical bond that links carbon monoxide to the protein. Noting the time when this reaction began, the scientists then used X-ray crystallography to take snapshots of the protein's structure, starting at one 10-billionth of a second. By stitching together the series of still images, the researchers created a moving picture of the changes taking place within the protein.

"The movie shows quite clearly the structural changes that contribute to the efficient expulsion of the toxic carbon monoxide from the protein," Anfinrud said.

For the study, the researchers used a mutant of myoglobin developed in the lab of John Olson at Rice, because in this variant of the protein, the rate at which carbon monoxide is swept away from the binding site is sped up by 1,000 times. Carbon monoxide, a toxic gas molecule, was used instead of oxygen because the protein-carbon monoxide complex is more stable and can endure the thousands of repeated measurements required for this study.

Olson, a professor of biochemistry and cell biology, said the moving pictures reveal the route by which carbon monoxide wanders through the protein to find its way out. He is engineering molecules that change the pathway that carbon monoxide uses to exit the protein, which could benefit the development of artificial blood substitutes by enhancing the rate at which oxygen is released while slowing down its reaction with other gas molecules.

"The real-time pictures of carbon monoxide migrating through protein allow us to refine our engineering strategies," Olson said.

Anfinrud noted that the research builds upon the pioneering work by Keith Moffat at the University of Chicago, who published the first nanosecond time-resolved study of myoglobin in 1996. The time resolution of the current work is 50 times better, and the improved spatial resolution shows even subtle changes in the protein structure, thanks, in part, to improvements in the X-ray source developed by Michael Wulff at the ESRF in Grenoble, France.

In addition to Anfinrud, Olson and Wulff, co-authors of the paper are Friedrich Schotte and Aleksandr Smirnov at the NIDDK, Jayashree Soman at Rice University, Manho Lim now at Pusan National University, Timothy Jackson now at Harvard Medical School, and George Phillips Jr. now at the University of Wisconsin-Madison.

The research was supported by grants from the Robert A. Welch Foundation, the National Institutes of Health, the W.M. Keck Center for Computational Biology and the Korea Science and Engineering Foundation.