Story URL: http://news.medill.northwestern.edu/chicago/news.aspx?id=124533
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UIC research on understanding proteins may lead to more effective treatment of infections

by Michelle Minkoff
March 18, 2009


PROTEIN1

Michelle Minkoff/MEDILL

Cultures of bacterial cells, from which protein may be extracted and then examined, are grown on this vibrating table, called a shaker.

PROTEIN2

Courtesy of Connie Jeffery, University of Illinois at Chicago

Crystals, developed from a supersaturated protein solution, are hit with an intense X-ray beam, resulting in scattering light particles that help scientists analyze the structure of proteins at a specific moment in time.

When we are attacked by bacteria, doctors prescribe medications. But those medications can’t do any good if they are ejected before they can fully penetrate the bacterial cells inside our bodies.

That’s why some researchers at the University of Illinois at Chicago are hoping to figure out how to block this ejection.

Antibiotics are often rendered ineffective if they are expelled using an efflux pump sitting in the bacteria cell’s membrane, the first barrier encountered by any material entering the cell.
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Connie Jeffery, an associate professor of biological sciences at UIC, and her team of graduate and undergraduate students, are studying the structure of these membrane proteins, hoping to open the door to better ways to target the rejection of medication.

The research team is looking at proteins called multidrug resistance transmembrane efflux pumps.

“People think about protein as something you eat,” said Jeffery. “Protein is actually the material that makes up most of the little machines in your body. In Chicago, we have steel, and it’s used to make different types of apartment buildings, train stations, trains themselves, and the tracks, and that’s kind of what protein is like. The shape of a protein goes with its function. If you see a boat, you can say that’s a boat and know something about how it works, even if not all boats look the same.”

The drug resistance proteins are attached to the membranes on the bacterial cell, one outer and one inner. A gap, called the periplasmic space, separates the membranes. The efflux pumps are complexes of three proteins that span from the outer membrane through the inner membrane. Scientists know that the proper arrangement of these proteins is needed for the pump to work, but it is still unknown exactly how they are connected. If this pump can be controlled, that may make medications more effective at fighting infections.

And that’s what Jeffery and her team is studying -- these pumps that can expel antibiotics out of the cell. The researchers are focusing on one family of proteins found in Pseudomonas aeruginosa, a common bacterium found in the soil, which can cause serious infections in patients, particularly those with cystic fibrosis, burns, artificial body parts or a suppressed immune system.

“We’ve been looking at how the three proteins assemble into the functional complex, with the idea that if you know how they assemble, maybe you can prevent them from assembling,” Jeffery said.

Jeffery said many of the discoveries in understanding the effect of these proteins on drug resistance have come about in the last decade.

Since the sequences of the genes for the proteins are known, scientists already know the sequence of amino acids within each protein, Jeffery said. Amino acids are building blocks within a protein that help to determine the biological activity of a protein. But to be functional, the protein must fold up in a specific three-dimensional structure, and this gene sequence alone can’t be used to predict what this three-dimensional structure looks like.

"We are functioning in a three-dimensional world," said Andrzej Joachimiak, director of the Structural Biology Center and the Midwest Center for Structural Genomics at Argonne National Laboratory. "When we understand the three-dimensional structure of a protein, we are better positioned to understand what happens in a cell. When we understand these details and basic biological concepts, we can understand how things work,” said Joachimiak, who is not a part of Jeffery's research team.

To conduct their research, team members get a clean sample of the protein and oversaturate it using a salt solution. Jeffery said the process has to be executed with delicacy and precision.

“When conditions are just right, the protein comes out of solution to form a crystal, which looks like a tiny diamond,” she said.

A crystal is a repeating three-dimensional pattern of protein molecules. But a weakness of the crystal is that it only captures an image of the protein at one spot in time, or one step in the efflux process. The shape, or conformation, of a protein undergoes changes as the protein performs its function. That means many crystals need to be made in different conditions to thoroughly understand the full function of the protein complex.

Each crystal is hit with a large, very powerful X-ray beam at the Advanced Photon Source, a synchrotron light source at Argonne National Laboratory.

Joachimiak said crystallography has been used to understand protein structure since around 1961. “Before, work on one part of a structure could take more than 10 years. Now, we can do it much faster,” he said.

The X-rays are scattered as a pattern of spots, and the researchers measure the location and intensity of these spots.

This information is input into a series of computer programs that use a mathematical formula, a Fourier transform, to map out a three-dimensional pattern determined by the arrangement of spots.

Argonne’s X-ray source yields high resolution data that enable researchers to see as precisely as about one angstrom. An angstrom is a minute unit of distance about one-millionth the diameter of a human hair.

“We use a computer graphics program to fit a model of the polypeptide chain into the data as well as we can, then the computer does some calculations, then we make more adjustments, and the cycle continues until the protein structure matches the data,” Jeffery explained.

Once the researchers have a model of the protein structure, the analysis starts, and can proceed in a variety of directions.

“We usually have specific questions, such as ‘What changes in the structure occur during function?’ ‘Where is the channel for these pumps?’ We know the molecule has to go through something,” Jeffery said.

As these questions are answered, researchers in the medical field will gain knowledge that may help them target the pumps to block their actions, or stop them from inhibiting an antibiotic’s action on a cell.