Scientists pioneer new method for watching proteins fold

A protein's function depends on both the chains of molecules it is made of and the way those chains are folded. And while figuring out the former is relatively easy, the latter represents a huge challenge with serious implications because many diseases are the result of misfolded proteins. Now, a team of chemists at the University of Pennsylvania has devised a way to watch proteins fold in "real-time," which could lead to a better understanding of protein folding and misfolding in general. 

The research was conducted by Feng Gai, professor in the Department of Chemistry in the School of Arts and Sciences, along with graduate students Arnaldo Serrano, also of Chemistry, and Robert Culik of the Department of Biochemistry and Molecular Biophysics at Penn's Perelman School of Medicine. They collaborated with Michelle R. Bunagan of the College of New Jersey's Department of Chemistry. 

Their research was published in the international edition of the journal Angewandte Chemie, where it was featured on the cover and bestowed VIP (very important paper) status. 

"One of the reasons that figuring out what happens when proteins fold is difficult is that we don't have the equivalent of a high-speed camera that can capture the process, " Gai said. "If the process were slow, we could take multiple 'pictures' over time and see the mechanism at work. Unfortunately, no one has this capability; the folding occurs faster than the blink of an eye." 

Gai's team uses infrared spectroscopy — a technique that measures how much light different parts of a molecule absorbs — to analyze proteins' structure and how this changes. In this case, the researchers looked at a model protein known as Trp-cage with an infrared laser setup. 

In this experiment, Gai's team used two lasers to study structural changes as a function of time. The first laser acts as the starting gun; by heating the molecule, it causes its structure to change. The second laser acts as the camera, following the motions of the protein's constituent amino acids. 

"The protein is made of different groups of atoms, and the different groups can be thought of as springs," Gai said. "Each spring has a different frequency with which it moves back and forth, which is based on the mass of the atom on either end. If the mass is bigger, the spring oscillates slower. Our 'camera' can detect the speed of that motion and we can relate it to the atoms it is made of and how that segment of the protein chain moves." 

Even in a simple protein like Trp-cage, however, there are many identical bonds, and the researchers need to be able to distinguish one from another in order to see which of them are moving while the protein folds. One strategy they used to get around this problem was to employ the molecular equivalent of a tracking device. 

"We use an amino acid with a carbon isotope marker," Culik said. "If it's incorporated into the protein correctly, we'll know where it is." 

With a single carbon atom of the Trp-cage slightly heavier than the others, the research team can use its signature to infer the position of the other atoms as they fold. The researchers could then "tune" the frequency of their laser to match different parts of the protein, allowing them to isolate them in their analyses. 

Similar isotopes could be inserted in more complicated molecules, allowing their folds to also be viewed with infrared spectroscopy. 

"This technique enhances our structural resolution. It allows us to see which part is moving," Gai said. "That would allow us to see exactly how a protein is misfolding in a disease, for example." 

Source: University of Pennsylvania [December 21, 2011]

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Researcher determines how Legionnaires’ bacteria proliferate, cause disease

A University of Louisville scientist has determined for the first time how the bacterium that causes Legionnaires’ disease manipulates our cells to generate the amino acids it needs to grow and cause infection and inflammation in the lungs. The results are published online today (Nov. 17) in Science. 

Yousef Abu Kwaik, Ph.D., the Bumgardner Endowed Professor in Molecular Pathogenesis of Microbial Infections at UofL, and his team believe their work could help lead to development of new antibiotics and vaccines. 

“It is possible that the process we have identified presents a great target for new research in antibiotic and vaccine candidates, not only for Legionnaires’ disease but in other bacteria that cause illness,” he said. 

According to the Centers for Disease Control and Prevention, Legionnaires’ disease is a lung infection caused by the bacterium called Legionella. The bacterium got its name in 1976, when many people who went to a Philadelphia convention of the American Legion suffered from an outbreak of pneumonia of unknown causes that was later determined to be caused by the bacterium. Each year, between 8,000 and 18,000 people are hospitalized with Legionnaires’ disease in the U.S. There is no vaccine currently available for it. 

For two years, the researchers examined Legionella which is an intercellular bacterium that exists in amoebae in the water systems; it is transmitted to humans through inhalation of water droplets. Cooling towers and whirlpools are the major sources of transmission. The bacterium uses the amoeba’s cellular process to “tag” proteins, causing them to degrade into their basic elements of amino acids. These amino acids are used by the bacteria as the main source of energy to grow and cause disease. 

“The bacteria live on an ‘Atkins diet’ of low carbs and high protein, and they trick the host cell to provide that specialized diet,” Abu Kwaik said. 

The same process occurs in a host – animal or human – who inhales the bacterium and is diagnosed with Legionnaires’ disease. However, the bacteria do not tag the proteins, but rather trick the host into tagging the proteins for degradation to generate the amino acids. 

In the laboratory, Abu Kwaik and his team saw that by inactivating the bacterial virulence factor responsible for tricking the cell into tagging proteins for degradation in mice models, the pulmonary disease was totally prevented. This was totally due to disabling the bacteria from generating amino acids, he said. 

The process was then reversed, and the disease became evident when the mice, infected by the disabled bacteria, were injected with amino acids to compensate for the inability of the altered bacteria. 

“Bacteria need to live on high protein and amino acids as sources of nutrition and energy in order to replicate in a host. This is what causes pulmonary disease,” Abu Kwaik said. “No one has known how they generate sufficient sources of nutrients from the host to proliferate. Our work is the first to identify this process for any bacteria that cause disease.” 

He added that the type of host infected does not appear to affect the process. “Whether in a single-cell amoeba or a multi-cellular mammal, Legionella seems to know what to do; the process is the same, and is highly conserved through evolution.  By interfering with the bacterium’s sources of nutrients, we can stop it from thriving and causing disease.” 

Examining nutrient sources for organisms with the goal of stopping them from acquiring nutrients is a relatively new arena of basic research that deserves further study, he said. “We went after the basics – the food and energy source – which are prerequisite for the bacteria to grow and cause disease. It is not a process that is well understood yet, but by first discovering how an organism gets nutrients by tricking the host into degrading proteins, and then interfering with that process, we can, in effect, starve it to death and prevent or treat the disease.” 

Source: University of Louisville [November 17, 2011]

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Report answers questions about E. coli: The good, the bad and the deadly

It has been the cause of infamous international foodborne disease outbreaks and yet it is the most studied bacterium in science, an essential part of the human digestive tract, and a backbone of the biotech industry. To enhance public understanding of the bacterium Escherichia coli, the American Academy of Microbiology brought together the nation's leading experts to consider and answer some of the most frequently asked questions about this multifaceted microorganism. 

"The story of E. coli, what we are trying to tell in this report, is really much larger than just its role as a pathogen. It's been such a large component of research for so long – so much of what we know about biology has come from studying E. coli," says Michael Doyle of the University of Georgia Center for Food Safety, a member of the steering committee. 

The report, entitled FAQ – E.coli: Good, Bad and Deadly is based on the deliberations of 13 of the nation's leading experts who met for one day in September 2011 to develop clear answers to frequently asked questions regarding the role of E. coli in scientific research, human health and foodborne disease. 

Some of the questions considered by the report are: 

• What is E. coli anyway? 

• How has E. coli contributed to our understanding of biology? 

• What does naturally occurring E. coli in our GI tract do? 

• What is the difference between "good" E. coli that inhabits our GI tract and the "bad" E. coli that makes us sick? 

• Why does E. coli make some people sick and not others? 

• How does E. coli become pathogenic? 

• How does our food become contaminated with E. coli? 

• What steps are being taken to protect our food from contamination by pathogenic E. coli? 

• What types of food are most commonly associated with E. coli, and why do there seem to be more cases of contamination recently? 

Most answers begin with a simple paragraph summarizing what is known, followed by a more detailed explanation. In addition, spread throughout the report are sidebar boxes discussing issues related to the questions such as a list of Nobel Prizes awarded for work done on E. coli and a discussion of toxins created by the bacterium. 

FAQ – E. coli: Good, Bad and Deadly is the latest offering in a series of reports designed to provide a rapid response to emerging issues. Traditionally Academy reports are based on multi-day colloquia after which the final report can take up to a year to develop. The FAQ series are based on single-day meetings focused on specific questions after which a final report is published in 2-3 months. 

"The Academy FAQ reports explain complex microbiological problems in a timely, balanced format that is easily understandable by the public, the media and policy makers," says Stanley Maloy of San Diego State University, who moderated the colloquium. 

Source: American Society for Microbiology [November 15, 2011]

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