Nitrogen Generator Helps NMR Uncover New Methods of Targeting Anthrax
A nitrogen generator is playing a key role by improving the resolution of nuclear magnetic resonance (NMR) spectrometers in studies aimed at finding biological targets with which to attack anthrax bacteria. One of the things that makes anthrax so dangerous is its ability to form spores that are so tough you can put them in boiling water without killing the bacteria. John Cavanagh, Professor of Biochemistry at North Carolina State University, Raleigh, North Carolina, is studying signal transduction pathways that allow bacteria such as anthrax to respond to environmental stress with the goal of eventually developing new therapeutic targets. A critical part of this work involves the use of NMR to determine the three-dimensional structures and interactions of biological molecules in solution. Cavanagh has long used nitrogen to maintain the physiological temperature of the sample undergoing NMR spectroscopy. In the past, his laboratory spent about $400 per month purchasing liquid nitrogen dewars and nitrogen gas cylinders. Recently he invested in a gaseous nitrogen generator that can continually produce nitrogen direct from his building’s compressed air at minimal cost and requires practically no maintenance. “The NMR machine we are using cost exactly one million dollars but we were able to substantially improve its performance with a simple device that cost under $11,000,” Cavanagh said.
Bacteria are highly adaptable organisms occupying an inexhaustible variety of ecological niches. They possess exceptional protective and responsive capabilities by encoding, at the correct times, a repertoire of genes normally unexpressed unless called upon. The key to bacterial adaptability lies in their capacity to invoke the necessary signal transduction pathways needed for protection and maximal growth in the specific situation in which they find themselves. They accomplish this by sensing signals emanating from their environment, recognizing its composition and subsequently initiating the correct response to ensure survival. Bacteria are extraordinarily accomplished at all these tasks and rapidly establish complex communication networks to cope with a myriad of stressful circumstances. The commitment for self-protection requires an enormous investment of energy, and there would be little advantage in such an undertaking if the environmental hostility was fleeting. Consequently the cell first enters a transition-state. During this time the cell decides which particular protective strategy is the most appropriate in light of the stress it faces. The transition-state is under the control of so-called transition-state regulators. These communication modules are very important targets for antimicrobial therapeutic agents.
Transition-state regulators
Transition-state regulators provide the cell with some well-needed breathing room as it contemplates its future. Not surprisingly they play essential roles in inducing synthesis of virulence factors in many pathogens in response to nutrient and metabolite deprivation. Studies to this point have centered on broad genetic characterization of transition-state regulators and little is known about their detailed mechanism of action. Cavanagh’s laboratory was responsible for the first detailed structural characterization of any transition state regulatory protein, AbrB (antibiotic resistance protein B) from Bacillus subtilis bacteria. AbrB from B. subtilis shares 100% identity in the DNA-binding domain and 85% identity overall with AbrB from anthrax, lending his studies extra significance. The AbrB protein from anthrax is known to be a negative regulator of the three anthrax toxins, lethal factor, edema factor and protective antigen. Anthrax secretes the three subunits of its toxin into the bloodstream of its host. Seven protective-antigen molecules then assemble into a pre-pore. The pre-spore undergoes a change in shape, forming a mature spore that allows lethal factor and edema factor to enter cells. Once inside, these toxin subunits destroy the cell, paving the way to disease.
NMR spectroscopy is the most powerful analytical technique available for determining the three-dimensional solution structure of proteins that form the molecular basis for these and many other biological processes. NMR is a phenomenon that occurs when the nuclei of certain atoms are immersed in a static magnetic field and exposed to a second oscillating magnetic field. Some nuclei experience this phenomenon, and others do not, depending upon whether or not they possess a property called spin. Under proper conditions, such nuclei absorb electromagnetic radiation in the radio-frequency region at frequencies governed by their chemical environment. This environment is influenced by chemical bonds, molecular conformations, and dynamic processes, for example. By measuring the frequencies at which these absorptions occur and their strengths, it is usually possible to deduce facts about the structure of the molecule being examined. NMR spectroscopy is routinely used by chemists to study chemical structure using simple one-dimensional techniques. Two, three- and four--dimensional techniques are used by structural biologists to determine the structure of more complicated molecules. Time domain NMR spectroscopic techniques are used to probe molecular dynamics in solutions. These techniques are challenging and complementing x-ray crystallography for the determination of protein structure.
Challenge of distinguishing spectral lines
“For the most part the results of NMR spectroscopy – the resonances - are displayed as a two-dimensional map with the axes representing two different, identifying nuclear frequencies in the protein.” Cavanagh said. “As the size of the molecule being studied increases, the resonance lines generated by its various atomic components broaden and can begin to overlap which makes it difficult or impossible to determine its structure. Keeping resonances as narrow as possible to alleviate any overlap is a huge part of performing high-resolution NMR on complex biomolecules. At the same time, the protein sample we study must typically be maintained around room temperature and this is generally accomplished by blowing air over it. The problem with air is that it partly consists of oxygen atoms with one unpaired electron. That single electron often couples to the nuclei which greatly broadens the lines on the spectrum, increasing their tendency to overlap and making them that much more difficult to interpret. We overcome this problem by delivering temperature-controlled nitrogen instead because it doesn’t interfere with the sample. We have found that nitrogen can dramatically improve the quality of the results that we are able to obtain with NMR.”
When Cavanagh’s laboratory first began using nitrogen, he purchased nitrogen tanks from a local gas supplier. The tanks cost in the neighborhood of $100 each and lasted about one week. This represented a significant expense and also meant that he and other laboratory personnel had to pay close attention to the amount of gas left and, when it was nearly empty, take the time to change the tank and order replacements. Moving the tanks was a particularly delicate operation because the magnets in the NMR spectrometer are so powerful that if you drop a flashlight near them it will never hit the floor. The time spent dealing with nitrogen supply subtracted from the amount time that was available to set up analysis runs and manage the facility.
Nitrogen generator provides continuous supply
In an effort to eliminate these problems, Cavanagh investigated the new breed of gas generators that produce nitrogen to a high level of purity by separating it from air. The basic advantage of this approach is that the laboratory eliminates the need to purchase and handle gas tanks. Cavanagh selected a Balston® N2-2010 nitrogen generator from Parker Hannifin Corporation, Filtration and Separation Division, Haverhill, Massachusetts that produces up to 124 standard liters per minute at a purity level ranging from 95% to 99.5% without using any electricity. The generator requires virtually no attention because it uses simple electromechanical components such as pressure vessels, and valves with a history of reliability in laboratory applications. Since the nitrogen generator simply separates air into its constituent parts, it has no adverse environmental effects. Gas generators are also much safer than high-pressure tanks as the generator typically operates at a low pressure in the neighborhood of 100 psig and stores small volumes of compressed gas.
The N2-2010 produces nitrogen by utilizing a combination of filtration and membrane separation technologies. A high efficiency pre-filtration system pre-treats the compressed air to remove all contaminants down to 0.01 micron. Hollow fibers separate the clean air into concentrated nitrogen output stream and oxygen enriched permeate stream that is vented form the system. The combination of technologies produces a continuous on demand supply of pure nitrogen. Routine maintenance is limited to periodic replacement of filter cartridges, requires no factory servicing and can easily be performed by the user.
“This machine paid for itself very quickly by eliminating the need to purchase gas dewars and cylinders,” Cavanagh concluded. “It is very robust. We couldn’t break it even if we tried. While I am continually working with a million-dollar NMR machine, I have to admit that I get an enormous kick out of this one, which cost only a few thousand dollars. It’s a very simple but elegant device. If we didn’t have it our lives would be much more complicated.”
NCSU1) The N2-2010 nitrogen generator installed at NCSU.
NCSU2) A shot of the Varian Inova 600MHz NMR spectrometer with the N2 generator in the background.
NCSU3) A shot of John Cavanagh at the the generator.
NCSU4) A shot of John Cavanagh up on the NMR magnet looking at the generator.
NCSU5) A shot of John Cavanagh with the generator