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New Techniques in Collaborative X-ray Crystallography

By Andrea Moon
August 2008

Lars Pedersen
Collaborative Crystallography Group Leader Lars Pedersen (Photo courtesy of Steve McCaw)

On the afternoon of July 10, NIEHS Staff Scientist Lars Pedersen, Ph.D., presented his most recent research results as part of the Laboratory of Structural Biology seminar series. Pedersen heads the NIEHS Structure & Function Group, also known as the Collaborative Crystallography Group, which exists to aid intramural scientists in structural determination for proteins of current research interest.

In a talk titled "You Don't Always Have to Punt on 4th and Long: Crystal Structures of RACK1A from Arabidopsis thaliana and a Heparan Sulfate (HS) Sulfotransferase from Chicken," Pedersen shared some of the challenges he has encountered collaborating with scientists in other groups to express, purify and solve the structure of a protein.

The metaphor in Pedersen's title refers to those desperate moments in American football - fourth down and much more than ten yards to go - when getting that elusive first down seems impossible. In these kinds of situations, Pedersen's group has implemented a new "trick play" to obtain that far off first down. Two recent accomplishments of this kind include co-authorship on studies pertaining to signal transduction with members of the Laboratory of Neurobiology and heparan sulfate biosynthesis (http://www.ncbi.nlm.nih.gov/pubmed/18223645?ordinalpos=7&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum) Exit NIEHS Website with investigators at the University of North Carolina at Chapel Hill (UNC-CH).

Structural biologists have long used x-ray crystallography as a tool for studying protein structure. The technique allows scientists to obtain atomic resolution three-dimensional "pictures" of the protein structure. According to Pedersen, since structure dictates function throughout nature, determining the structure of a protein opens the door to a better understanding of how it works.

To illustrate the importance of employing new ideas and techniques in X-ray crystallography to overcome problematic projects, Pederson described a successful collaboration with Jian Liu, Ph.D., at the UNC-CH Eshelman School of Pharmacy. After unsuccessfully attempting to express and crystallize a heparan sulfate (HS) sulfotransferase from hamster, Pedersen and his colleagues decided to switch species and use chicken instead. Because chickens have a higher body temperature than humans, their proteins are thought to be more thermostable than human proteins. When the chicken protein still refused to crystallize, the team used a fusion protein system that employed the maltose binding protein (MBP). The fusion protein was much more stable and yielded crystals which diffracted to 2.65Å, a measure that is close to the 1.5 Å distance between two covalently bonded atoms.

Another "stubborn" project that Pedersen had to contend with involved the protein RACK1A, a signal transduction protein that has a similar structure to that of the Gβ protein, but lacks a similar function. The project was done in collaboration with David Armstrong, Ph.D., head of the Membrane Signaling Group at NIEHS. After attempting to crystallize RACK1A in its native form from the plant A. thaliana, Pedersen made an MBP-fusion construct. In this case, merely using the fusion system was not enough. Pedersen and team members also needed to alter the ability of the fusion protein to crystallize by making mutations of surface residues. The resulting crystals diffracted to 2.4Å and produced solid data.

Obtaining the crystal structure of RACK1A and the HS sulfotransferase proteins has allowed for a greater understanding of their function.

(Andrea Moon is a biologist in the NIEHS Collaborative X-ray Crystallography Group, a part of the Laboratory of Structural Biology)



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