Young Faculty Member Tackles Tough Structures

 

By Jason S. Bardi

One Monday morning at 8:00 AM, Assistant Professor Geoffrey Chang, Ph.D., is busy in his laboratory tending to a fermenter full of churning cells. In the corner of his office lies a crumpled sleeping bag, recently used. Chang admits that, unlike a few years ago, he needs to nap sometimes when tending to his longer experiments. "I’m not that young anymore," he says.

One of The Scripps Research Institute’s (TSRI) young faculty, Chang, 29, arrived at the Department of Molecular Biology just over a year ago to pursue membrane protein crystallography. He is particularly interested in the structures of several types of membrane transporters. One of these, the drug efflux pumps, may be an important system for deterring the threat of antibiotic-resistant bacteria and addressing the related problem of the sometimes low efficacy of chemotherapy.

Efflux pumps are broad-based defense mechanisms that bacterial and cancerous cells use to resist pharmaceuticals, transporting the drugs out of the cell. "We actually have very good drugs to fight cancer and to kill bacteria," says Chang, "[but] they can’t always get in the cells to work."

One of the eventual goals is the development of a new class of drugs that patients would take in conjunction with antibiotic or chemotherapeutic agents to keep those drugs in the cells and increase their efficacy. Such "pump inhibitors" could potentially enhance many other drugs on the market as well. There are, already, some efflux pump inhibitors commercially available. "It would be very interesting to know the structure of a pump with one of these inhibitors, and use that as the basis for making the inhibitor more potent," says Chang. "That’s why we’re actively trying to get the structures done. Nobody knows what the structure of any of these pumps are."

Department of Molecular Biology chairman Peter Wright attributes the unfamiliarity to the adolescence of this area of research, and he looks forward to the growth of the field at TSRI under Chang. "He has a new methodology that will dramatically increase the chances of success," says Wright, "and we’re excited to have him."

Out of Our Antibacterial Paradise

At the dawn of the 20th century, bacterial infections accounted for several of the leading causes of death in the United States. By the middle of the century, many had begun to believe that the threat had waned. Bacterial infections that in 1900 topped the list as leading causes of death in the United States were no longer even among the top ten. After Alexander Fleming discovered penicillin in 1928, it—along with all the other "wonder drugs" that followed—toppled tuberculosis (TB) and typhoid fever, controlled cholera and gonorrhea, reduced staphylococcal dysentery infections, and lowered the incidence of many other pandemic bacterial infections. At least partly because of these antibiotics, the average life expectancy in the United States has risen from 47.3 years in 1900 to almost 80 years today.

But the tide is turning again. Several bacteria have developed the ability to resist antibiotic treatment, including M. tuberculosis, E. coli, N. gonorrheae, S. dysenteriae, various types of pneumococci, cholera and typhoid bacilli, and Salmonella enteritis. Bacteria once contained by drugs are now outstripping the ability of drugs to contain them. "What’s happening today," says Chang, "is that a lot of these diseases are coming back."

These diseases are coming back resistant to the antibiotics that have been used to treat them for years, and people who are infected with these resistant strains have to be treated with alternative antibiotics, as they have over the last decade.

Now multiple drug-resistant bacteria are emerging as an even greater threat. Multiple drug-resistant TB is no longer susceptible to broad categories of antibiotics, such as the RNA synthesis inhibitor rifampicin, the cell wall synthesis inhibitor isoniazid, and streptomycin, which inhibits the 30 S subunit of the bacterial ribosome. Certain strains of S. dysenteriae, a common hospital pathogen, have even become resistant to all but one single drug—the quinolone ciprofloxacin—and may soon become completely untreatable.

Treating multiple drug-resistant bacterial infections can be a hundred times more expensive than treating normal infections, and the World Health Organization estimates the total cost of treating all hospital-borne antibiotic resistant bacterial infections is around $10 billion a year. Worse, with rapid transit, open borders, and world travel being what they are, multiple drug-resistant bacteria could potentially spread beyond the isolated confines of a hospital and into populations. The greatest fear of all is the grim prospect of a multiple drug-resistant bacterial epidemic.

Uncharted Waters

Bacteria use a host of methods to foil antibiotics. The bacterium S. aureus produces an enzyme, ß-lactamase, which specifically degrades penicillin and its analogues through hydrolytic cleavage of the penicillin ß-lactam ring. This "target modification" mechanism is also used by S. aureus and other bacteria against different classes of antibiotics, such erythromycin and chloramphenicol. There are a host of other mechanisms bacteria employ to evade antibiotics, such as encoding enzymes that sequester antibiotics by binding to them, undergoing small point mutations in the molecular targets that lower a drug’s affinity, overproducing a drug’s substrate in the cell, and efflux pumps.

Efflux pumps and transporters are perhaps two of the most difficult structures to study, though, because—like all other membrane proteins—they are notoriously hard to solve. Less than one half of one percent of the structures contained in the Brookhaven National Laboratory Protein Data Bank are of integral membrane proteins, despite the fact that over a third of all proteins in the body are in the membrane.

The difficulty with solving membrane proteins begins with obtaining them. Producing enough protein to work with can be insurmountable. A crystallographer might need several milligrams of protein to start with, but since most channels and transporters constitute such a small percentage of the cellular composition, getting enough material to work with becomes a challenge. "You just can’t grow that many cells," says Chang, who adds that all the membrane structures solved before 1998 are from naturally abundant sources.

Even assuming success in producing sufficient quantities, the proteins must be purified in their native state, which entails purifying them in the presence of detergents so that their hydrophobic membrane-spanning region can be surrounded by the hydrophobic ends of detergent molecules in micelle-like formations. The conditions are highly sensitive to such variables as detergent type, concentration, pH, and ionic strength. Worse, these conditions are almost always unique, demanding a lengthy trial and error search of an unknown biochemical landscape for that exact novel solution in which the protein-detergent complexes will be soluble and stable. "You have to draw on your own experience," says Chang, "You have to have quite a lot of it, actually."

Then a whole separate set of novel conditions must be worked out for growing the crystals, described by Chang as, "crystallizing out of soap." Several tricks can be employed, such as using antibody fragments, cubic lipid phases, or non-detergent systems. But, says Chang, these techniques are highly system specific and may only work with one or two proteins. Crystallizing presents another large problem in that the costs of the detergents can easily add up to hundreds of thousands of dollars.

Finally, any crystals that are grown can be difficult to work with because of their solvent content. Most water-soluble protein crystals have a protein-solvent ratio as high as 3:4—almost as hard or as dense as salt crystals. Membrane protein crystals, however, have a much higher solvent content because of the added bulk of the detergent micelles. As much as 88 percent of the membrane protein crystal may be a mixture of solvent and detergent, which makes the crystals unusually fragile under an x-ray beam. Chang calls it "shooting through Jell-O."

Every part of the process must come together for the science to work. No protein, no crystals; no crystals, no structure. Chang’s solution follows Thomas Edison’s—try as many different targets and conditions as necessary, build on these experiences, and go with those that work. "It’s really still a new field," says Chang. "There’s almost no literature on how to crystallize membrane proteins."

Once he has crystals, Chang will then make data collection trips to synchrotrons at the Stanford Synchrotron Radiation Laboratory and the Advanced Light Source at UC Berkeley. Synchrotrons are radiation sources that use x-ray radiation produced by electrons moving close to the speed of light in particle accelerator storage rings. These x-rays are intense, collimated, and tunable to various wavelengths, which make synchrotrons particularly useful for examining protein crystals. Chang, for example, will bombard his frozen membrane protein crystals with synchrotron x-rays and collect diffraction patterns for a variety of crystals. He will spend months analyzing and refining this data, and eventually—hopefully—solve a novel structure of a membrane complex involved in antibiotic and chemotherapy drug resistance.

Chang Honored at White House

Chang came to TSRI after spending three years as a postdoctoral fellow in Douglas Rees’s laboratory at the California Institute of Technology. In October, just over a year after he arrived, Chang was named one of the recipients of the Fifth Annual Presidential Early Career Awards for Scientists and Engineers, the highest honor bestowed by the United States government on young professionals at the outset of their careers. He spent a day in Washington, D.C., touring the National Institutes of Health and standing for a presentation ceremony at the White House.

Chang takes his award out of a file in his desk drawer and shows it to me—a simple design with nice lettering and William Jefferson Clinton’s bold signature across the bottom. It is still in the presidential blue folder it came in, and after a few moments, he flips the cover closed and slips it back in the file drawer. "I haven’t had time to get it framed," he explains.

 

 

 

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One of the youngest of TSRI's faculty, Geoffrey Chang, pursues membrane protein crystallography.

 

 

 

 

 



Protein crystals, such as those of insulin pictured here, are notoriously difficult to make. "You have to draw on your own experience," says Chang. "You have to have quite a lot of it, actually." (Photo courtesy of NASA).