Montana State University scientists in the Department of Chemistry and Biochemistry published new research this week that could one day affect the lives of millions around the world who suffer from blood iron disorders.
Martin Lawrence (left) and Anoop Sendamarai (MSU photo by Kelly Gorham)
The paper, which will appear in the Proceedings of the National Academy of Sciences, details the work of Associate Professor Martin Lawrence and doctoral candidate Anoop Sendamarai. The pair have spent the past two years studying Steap3, a protein involved in regulating the body’s absorption of iron.

The results of their studies - the first three-dimensional maps of the atoms that make up Steap3 - could allow pharmaceutical companies to someday design drugs to regulate iron levels in the blood.

“Iron is essential,” Lawrence said. “You can’t live without it, but it’s a double-edged sword. Too much of a good thing can kill you.”

Iron serves several important functions in the bloodstream. It carries oxygen, transports electrons within cells and plays an important role in enzyme systems.

Iron irregularities are some of the most common blood disorders in the world. According to the World Health Organization, iron deficiency, which can lead to anemia, affects more than a billion people around the world and can cause developmental and immune system problems.

Conversely, having too much iron, a condition called hemochromatosis, can also hurt the body by releasing destructive free radicals, Lawrence said. Hemochromatosis affects about one in every 300 people and is most common in people of northern European ancestry. Left untreated, it can lead to early death, often by age 50.

“We’re struck by how many people have too much or too little iron,” Lawrence said.

To understand Steap3’s role in transporting and maintaining balanced levels of iron, Lawrence and Sendamarai first had find and purify samples of the protein and then turn those samples into crystals.

Lawrence said the result of the crystallization process, if done correctly, is analogous to the rigid structure of a brick wall. If done incorrectly, it more closely resembles a pile of bricks.

“It’s kind of a black art really more than a science,” Lawrence said. “You can’t always predict the kind of witch’s brew that needs to be around to get it to crystallize.”

He said only a handful of labs in the country are crystallizing iron transport proteins like Steap3, a fact that places MSU on the same shelf as places like Harvard Medical School.

Once crystallized, the samples are shot with a powerful X-ray beam. Electrons in the sample diffract the X-rays, creating patterns on a digital sensor. The technique, called X-ray crystallography, has been used since the 1950s to de-termine the structure of different substances.

In their basement lab in the campus’s New Chemistry Building, Lawrence and Sendamarai then examined the diffraction patterns created by Steap3.

“It’s kind of like a contour map,” Sendamarai said. “Whenever we see the peaks, we know there are atoms.”

Working backward, they can mathematically determine the position of atoms in the protein and display them in three dimensions.

The computer-drawn result, a three-dimensional image that resembles tangled ribbons and strings, is an picture of what the atoms of Steap3 look like.

Sendamarai said having that picture, which depicts all the nooks and crannies on the protein’s surface, could allow drug companies to design drugs to fit those spots like puzzle pieces.

If a future drug fits those nooks just right, it could help treat hemochromatosis. From there, Sendamarai said it would be conceivable to work backward and possibly treat iron deficiencies or anemia.

Lawrence said that Steap3 is only one in a family of proteins that affect iron transport. This summer, in addition to continuing to study Steap3, Lawrence and Sendamarai hope to learn whether the lab will receive a grant from the National Institutes of Health to work on other iron transport proteins.

“It’s a critical step towards toward learning to modulate iron levels in patients with too much or too little iron,” Sendamarai said. “But, there are a lot of question marks left in iron transport. It’s a big field.”

Many scientists have tried for decades to understand the mechanism that allows these channels to open. Using cryo-electron microscopy, in which samples frozen at extremely low temperatures are examined under an electron microscope, some researchers obtained images of the closed ion channel. More recently, others used X-ray crystallography to image the closed-channel conformation. This technique involves crystallizing the protein, creating a lattice that reveals many details of its three-dimensional structure.

But until the Illinois team developed a new method for probing the interior of the open channel, no studies had been able to infer the structure of the open channel conformation in a living cell. The Illinois team was able to do this by exploiting electrical properties of these membrane proteins.

Much like the flow of electrons through an electrical wire, the flow of ions through a cell membrane is a current. In the 1970s, two German researchers developed a technique for measuring the current through a single ion channel, an innovation (known as the patch-clamp technique) that won them a Nobel Prize in 1991. Claudio Grosman, a professor of molecular and integrative physiology at Illinois, and Gisela D. Cymes, a postdoctoral associate in his lab, adopted this technique, and predicted that they could use it as a tool for what they call “in vivo, time-resolved structural biology.”

In a study published in 2005, the Grosman lab showed that ionizable amino acids (that is, those that may alternately be charged or neutral) can be engineered into the inner lining of the channel pore. These changes to the amino acid sequence alter the current, revealing the structure of the open-channel conformation in unprecedented detail.

The neurotransmitter acetylcholine is an essential chemical communicator, carrying impulses from neurons to skeletal muscle cells and many parts of the nervous system. Now researchers at the University of Illinois have painstakingly mapped the interior of a key component of the relay system that allows acetylcholine to get its message across. Their findings, which appear in the current issue of Nature Structure & Molecular Biology, reveal how the muscle nicotinic acetylcholine receptor responds to a burst of acetylcholine on the surface of a cell.

The muscle nicotinic receptor is a neurotransmitter-gated ion channel. This “gate” regulates the flow of information, in the form of charged particles, or ions, across the cell membrane. Although normally closed, when the ion channel encounters acetylcholine – or nicotine – on the surface of the cell the interaction causes the gate to open, allowing positively charged ions (called cations) to flow into the cell.

“As the ionizable amino acids bind and release protons from the watery environment, the pore gains or loses a positive charge that interferes with the normal flow of cations through the channel,” Grosman said.
After analyzing the data, Grosman’s team demonstrated that the discrete changes in current reflect the position of each mutated amino acid in the channel and the extent to which water molecules penetrate the membrane protein.

This approach allowed Grosman’s team to map the relative position of every amino acid that formed the ion channel.

The new study extends this work to more distant portions of the protein.

After comparing these findings to direct studies of the structure of the closed channel, Grosman concluded that the conformational changes that allow the channel to open are quite subtle. The five subunits that make up the protein channel do not rotate or pivot dramatically when opening the gate.

“There are many good reasons why I think a subtle conformational change is advantageous from an evolutionary point of view,” Grosman said.

Muscle nicotinic receptors must respond to acetylcholine with staggering speed, opening within microseconds of their exposure to the neurotransmitter.

“These ion channels are meant to be quick,” he said. “If they are too slow, we have disease.”

Grosman said that the approach developed in his lab is the first to allow scientists to infer the structure of an ion channel in its open conformation as it functions in a living cell.

“I know when the protein is open, because in single-molecule experiments the distinction between open and closed conformations is simple; the channel either passes a current or not,” he said.

In a living cell the protein responds, in measurable ways, to changes in its structure and environment, he said. “It’s not frozen at super low temperatures. It’s not in a crystalline lattice. The cells are alive at the beginning of the experiment and when we finish the experiment, the cells keep living.”

Determining the structure of unknown natural compounds is a slow and expensive part of drug screening and development — but this may now change thanks to a new combination of experimental and computational protocols developed at the University of California, San Diego and presented at RECOMB 2008 (Research in Computational Molecular Biology) on March 31 in Singapore. This image is figure 1 from the paper.

The determination of the structure of unknown natural compounds is a slow and expensive part of drug screening and development – but this may now change thanks to a new combination of experimental and computational protocols developed at the University of California, San Diego and presented at RECOMB 2008 (Research in Computational Molecular Biology) on March 31 in Singapore.

UC San Diego researchers have devised a way to cut the time it takes to determine the structure of peptides derived from natural compounds from six months or a year to as little as one day. This advance may assist drug discovery researchers – who need to know as much as possible as quickly as possible about the natural products with antibiotic, antiviral and other pharmacologically interesting properties that they are probing.

According to the researchers, it is currently difficult, time consuming and costly to determine the molecular structure of a class of natural compounds called nonribosomal peptides (NRPs) that are intensely studied for their drug potential.

To address this issue, UCSD researchers developed a quick, automated and inexpensive way to determine the structure of NRPs through an innovative collaboration between mass spectrometry experts at the UCSD Skaggs School of Pharmacy and Pharmaceutical Sciences and bioinformatics experts and computer scientists from UCSD’s Jacobs School of Engineering.

Determining the structure of unknown natural compounds is a slow and expensive part of drug screening and development — but this may now change thanks to a new combination of experimental and computational protocols developed at the University of California, San Diego and presented at RECOMB 2008 (Research in Computational Molecular Biology) on March 31 in Singapore. This image is figure 2 from the paper.

If you imagine the structure of an NRP as a cyclic string of beads, then the new algorithms both decipher the mass of each bead based on the mass spectrometry and determine the order of the beads within the ring – crucial pieces of information for uncovering both the structure of the molecule and its pharmacological activities.

In addition to screening for new drugs and studying natural compounds, the authors say this work may aid biosynthetic engineering efforts to reprogram E. coli strains in order to turn them into NRP assembly lines, now that researchers have a rapid method for characterizing the resulting NRPs.

NRPs such as penicillin, and other natural products, have an unparalleled track record in pharmacology: nine out of the top 20 best-selling drugs were either inspired by or derived from natural products, the authors say.

Nonribosomal peptides evolved over millions of years and often serve chemical defense and communication purposes for the organisms that manufacture them, explained first author Nuno Bandiera, a UCSD postdoctoral researcher and successful Ph.D. candidate from the computer science department at UCSD’s Jacobs School of Engineering.

It is notoriously difficult to determine the structure of NRPs because the usual peptide sequencing tools do not work. The cyclic structures of NRPs, the prevalence of non-standard amino acids that thwart database lookups, and the lack of structural information directly inscribed in the genomic DNA due to the nonribosomal nature of the peptides are all major contributors to the roadblock. Researchers have had to rely on slow, manual, expensive and not always reliable approaches to deciphering the structure of NRPs.

“This work removes a particularly troublesome bottleneck in the drug discovery pipeline for this class of therapeutics,” said Pieter Dorrestein, assistant professor in the Skaggs School of Pharmacy and Pharmaceutical Sciences and the Departments of Pharmacology, Chemistry and Biochemistry. “We have shown a way to quickly, structurally characterize nonribosomal peptides. Our next step is to replicate our findings with newly discovered, potentially therapeutic peptides.”

The UCSD researchers have shown that it is possible to break NRP rings apart and then break the resulting peptide strings into smaller and smaller subunits of the original ring using multiple passes with a mass spectrometer. This approach – called multistage mass spectrometry – allowed the UCSD Skaggs School researchers to collect data on the weights of ring fragments as these fragments got progressively shorter and more numerous with each pass of the mass spectrometer.

The UCSD Jacobs School computer scientists designed algorithms that literally pick up the pieces from here. The algorithms glue the overlapping pieces together until they have reassembled a series of possible original ring structures, explains Julio Ng, a graduate student in UCSD’s Interdisciplinary Bioinformatics Ph.D. program and RECOMB 2008 paper co-author.

The algorithms make use of data on the weights of the various NRP ring fragments collected at each stage using mass spectrometry. This work is an extension of an award-winning automated approach Bandeira and colleagues used to reconstruct snake venom peptides.

“Our Recomb 2008 paper represents the first demonstration of de novo sequencing of nonribosomal peptides. Without knowing the structure of the original compound, we can determine it,” explains computer science professor Pavel Pevzner, the last author on the RECOMB 2008 paper and the director of UCSD’s Center for Algorithmic and Systems Biology which is part of the UCSD Division of the California Institute for Telecommunications and Information Technology (Calit2).

In their RECOMB 2008 paper, the researchers document how they used de novo sequencing to determine the structure of two different nonribosomal peptides. In order to be able to verify their results, the researchers chose peptides that had been independently sequenced using a slow, labor intensive, costly and somewhat inconsistent nuclear magnetic resonance (NMR) approach. NMR provides information on the position of specific atoms within a molecule by using the magnetic properties of nuclei. The team is now working on more than ten additional compounds and has filed a provisional patent for the technique.

This project arose after Roger Linington from UC Santa Cruz, a co-author on the RECOMB 2008 paper, approached Dorrestein with the hope that Dorrestein’s group would be able to use mass spectrometry to obtain the molecular structure of a natural compound that is very effective against malaria. When Dorrestein found that the data being collected from a strictly mass spectrometer approach was getting extremely complicated – in large part due to the cyclic structure of the compound, he contacted Pevzner. What followed was a fruitful back and forth between the mass spectrometry team and the computer scientists that eventually led to this novel and creative solution.

“De Novo Sequencing of Nonribosomal Peptides,” presented at RECOMB 2008 by Nuno Bandeira, Julio Ng, Dario Meluzzi, Pieter Dorrestein and Pavel A. Pevzner from University of California, San Diego, USA; Roger G. Linington from University of California, Santa Cruz, USA.

Shown is an image of bacteriophage Epsilon15 studied by Wen Jiang, an assistant professor of biological sciences at Purdue. The bacteriophage is shown at a resolution of 4.5 angstrom — the highest resolution achieved for a living organism of this size. Credit Graphic/Wen Jiang lab

WEST LAFAYETTE, Ind. - A team led by a Purdue University researcher has achieved images of a virus in detail two times greater than had previously been achieved.

Wen Jiang, an assistant professor of biological sciences at Purdue, led a research team that used the emerging technique of single-particle electron cryomicroscopy to capture a three-dimensional image of a virus at a resolution of 4.5 angstroms. Approximately 1 million angstroms would equal the diameter of a human hair.

“This is one of the first projects to refine the technique to the point of near atomic-level resolution,” said Jiang, who also is a member of Purdue’s structural biology group. “This breaks a threshold and allows us to now see a whole new level of detail in the structure. This is the highest resolution ever achieved for a living organism of this size.”

Details of the structure of a virus provide valuable information for development of disease treatments, he said.

“If we understand the system - how the virus particles assemble and how they infect a host cell - it will greatly improve our ability to design a treatment,” Jiang said. “Structural biologists perform the basic science and provide information to help those working on the clinical aspects.”

A paper detailing the work was published in the Feb. 28 issue of Nature.

Roger Hendrix, a professor of biological sciences at the University of Pittsburgh, said what is learned about viruses can be applied to many other biological systems.

“Understanding the proteins that create the structure of a virus gives us insight into the tiny biological machines found throughout our bodies,” he said. “Getting to 4.5 angstrom using this technique is a watershed of sorts because it is the first time we can actually trace the polypeptide chain - the backbone of proteins. Now we can see the tiny gears and levers that allow the proteins to move and interact as they carry out their intricate biological roles.”

The imaging technique, called cryo-EM, has the added benefit of maintaining the sample being studied in a state very similar to its natural environment. Other imaging techniques used regularly, such as X-ray crystallography, require the sample be manipulated.

“This method offers a new approach for modeling the structure of proteins in other macromolecular assemblies, such as DNA, at near-native states,” Jiang said. “The sample is purified in a solution that is very similar to the environment that would be found in a host cell. It is as if the virus is frozen in glass and it is alive and infectious while we examine it.”

In addition to Jiang, Matthew L. Baker, Joanita Jakana and Wah Chiu from Baylor College of Medicine, and Peter R. Weigele and Jonathan King from Massachusetts Institute of Technology worked on the project, which was funded by the National Institutes of Health and the National Science Foundation.

The team obtained a three-dimensional map of the capsid, or protein shell, of the epsilon15 bacteriophage, a virus that infects bacteria and is a member of a family of viruses that are the most abundant life forms on Earth, Jiang said.

Other methods of determining the structure could not be used for this family of virus. None had been successfully crystallized, and the complexity of members of this family had prevented evaluation through the genome sequence alone.

“This demonstration shows that cryo-EM is doable and is a major step in reaching the full potential of this technique,” he said. “The goal is to have it reach a 3 to 4 angstrom resolution, which would allow us to clearly see the amino acids that make up a protein.”

In electron microscopy, a beam of electrons takes the place of the light beam used in a conventional microscope. The use of electrons instead of light allows the microscope to “see” in much greater detail.

Cryo-EM cools specimens to temperatures well below the freezing point of water. This decreases damage from the electron beam and allows the specimens to be examined for a longer period of time. Longer exposure time allows for sharper, more detailed images.

Researchers using cryo-EM had obtained images at a resolution of 6-9 angstroms but could not differentiate between smaller elements of the structure spaced only 4.5 angstroms apart.

“There are different elements that make up the protein building blocks of the virus,” Jiang said. “It is like examining a striped blanket. From a distance, the stripes blur together and the blanket appears to be one solid color. As you get closer you can see the different stripes, and if you use a magnifying glass you can see the strands of string that make up the material. The resolution needs to be smaller than the distance between the strands of thread in order to see two separate strands.

“By being able to zoom in, researchers were able to see components that blurred together at the earlier achieved resolution.”

Cryo-EM requires high-end electron microscopes and powerful computing resources. The research team used the Baylor College of Medicine’s cryoelectron microscope. It is expected that Purdue will install a state-of-the-art cryoelectron microscope in 2009.

In 2006 Purdue received a $2 million grant from the National Institute of Health to purchase the microscope. It will be installed in Hockmeyer Hall of Structural Biology, expected to open in 2009.

Computer programs are used to extract the signal from the microscope and to combine thousands of two-dimensional images into an accurate three-dimensional image that maps the structure of the virus. This requires use of a large data set and could not have been done without the resources of Purdue’s Office of Information Technology, or ItaP, Jiang said.

Jiang used Purdue’s Condor program - which links computers including desktop machines and large, powerful research computers - to create the largest distributed computing network at a university.

“ITaP provided us with computational power at the supercomputer scale that was necessary for this work,” he said. “Purdue’s Condor program allowed us to take advantage of the power of 7,000 computers. This was a critical element to our success.”

Jiang plans to continue to refine every step of the process to improve the capabilities of the technique and to examine more medically relevant virus species.

Purdue’s structural biology group studies a diverse group of problems, including cellular signaling pathways, RNA catalysis, bioremediation, molecular evolution, viral entry, viral replication and viral pathogenesis. Researchers use a combination of X-ray crystallography, electron cryomicroscopy, NMR spectroscopy, and advanced computational and modeling tools to study these problems.

Illinois postdoctoral researcher Feng-Jie Sun (left) and crop sciences professor Gustavo Caetano-Anollés began with the idea that understanding the structural properties of tRNA would shed light on how organisms and viruses evolved. Photo by L. Brian Stauffer, U. of I. News Bureau.

Transfer RNA (also called tRNA) is an ancient molecule, central to every task a cell performs and thus essential to all life. A new study from the University of Illinois indicates that it is also a great historian, preserving some of the earliest and most profound events of the evolutionary past in its structure.

The study, co-written by Gustavo Caetano-Anollés, a professor of crop sciences, and postdoctoral researcher Feng-Jie Sun, appears March 7 in PLoS Computational Biology. Caetano-Anollés is an affiliate of the U. of I. Institute for Genomic Biology.

Of the thousands of RNAs so far identified, transfer RNA (tRNA) is the most direct intermediary between genes and proteins. Like many other RNAs (ribonucleic acids), tRNA aids in translating genes into the chains of amino acids that make up proteins. With the help of a highly targeted enzyme, each tRNA molecule recognizes and latches onto a specific amino acid, which it carries into the protein-building machinery. In order to successfully add its amino acid to the end of a growing protein, tRNA must also accurately read a coded segment of messenger RNA, which gives instructions for the exact sequence of amino acids in the protein.

The fact that tRNA is so central to the task of building proteins probably means that it has been around for a long time, Caetano-Anollés said. His inquiry began with a hunch that understanding the structural properties of tRNA would shed light on how organisms and viruses evolved.

“Perhaps in evolution there are things that are so fundamental that they are kept, held onto, for millions or even billions of years,” Caetano-Anollés said. “Those are the fossils, the molecular fossils, that tell us about the past. Therefore, studying these molecules can address fundamental questions in biology and evolution.”

All tRNAs assemble themselves into a shape that, if flattened, resembles a cloverleaf. Patterns in these structures give clues to early evolutionary history. The red areas of the molecule pictured above are the most ancient. Image courtesy of Gustavo Caetano-Anollés.

All tRNAs assemble themselves into a shape that, if flattened, resembles a cloverleaf. The team began by looking for patterns in this cloverleaf structure, using detailed data from hundreds of molecules representing viruses and each of the three superkingdoms of life: archaea, bacteria and eukarya.

The researchers converted all distinguishing features of the individual tRNA cloverleaf structures into coded characters, a process that allowed a computerized search for the most “parsimonious” (that is, the simplest, most probable) tRNA family tree. They conducted the same analysis on the tRNAs of each of the superkingdoms, to see how far these groupings diverged from the overall tree. This comparison allowed them to determine the order in which viruses and each of the superkingdoms diverged.

The new analysis supports an earlier study that suggested that the archaea were the first to arise as an evolutionarily distinguishable group. Archaea are microbes that can survive in boiling acid, near sulfurous ocean vents or in other extreme environments. The earlier study, also led by Caetano-Anollés, analyzed the vast catalog of protein folds – those precisely configured regions in proteins that give them their functionality – as a guidebook to evolutionary history.

“The transfer RNA data matches our earlier data,” Caetano-Anollés said. “This is important because two lines of independent evidence are supporting each other.”

The new analysis also indicates that viruses emerged not long after the archaea, with the superkingdoms eukarya and bacteria following much later – and in that order. This finding may influence the ongoing debate over whether viruses existed prior to, or after, the emergence of living cells, Caetano-Anollés said.

“This supports the idea that viruses arose from the cellular domain,” he said.

UPTON, NY - Biologists at the U.S. Department of Energy’s Brookhaven National Laboratory, Stony Brook University, and the University of Wurzburg, Germany, have deciphered the structure of a large protein complex responsible for adding sugar molecules to newly formed proteins - a process essential to many proteins’ functions. The structure offers insight into the molecular “sugar-coating” mechanism, and may help scientists better understand a variety of diseases that result when the process goes awry. The research will appear in the March 12, 2008, issue of the journal Structure.”Proteins perform their functions by interacting at their surfaces with other molecules. So you can imagine that adding or removing sugar molecules will change the protein’s surface structure, and therefore its function,” said Huilin Li, a biologist at Brookhaven Lab who holds a joint appointment at Stony Brook and is co-corresponding author on the Structure paper. “Messing up this process can lead to the production of malformed proteins that are unable to do their jobs,” he added.

The results can be devastating. Failure of glycosylation, as the “sugar-coating” process is known, can lead to a variety of genetic disorders characterized by neurological problems including seizures and stroke-like episodes, feeding disorders, and possibly even some forms of muscular dystrophy.

“We studied one enzyme involved in glycosylation, the one that recognizes the protein sequence and adds the sugar chains to the protein as it is being synthesized by the cell,” said William J. Lennarz of Stony Brook University, a coauthor on the paper. “The challenge is that the enzyme, known as oligosaccharide transferase (OT), is large by protein standards, has eight intricately linked components, and sits embedded in a membrane within the cell’s protein-manufacturing machinery.”

“Membrane proteins, particularly large ones, are very difficult to study structurally,” added Li.

So the scientists turned to a technique called cryo-electron microscopy (cryo-EM), which shows great promise in deciphering large membrane protein structures.

“We imaged the purified OT complex by cryo-EM and obtained a first snapshot of the complex by computer reconstruction of the micrographs,” said Li, a cryo-EM expert.

In cryo-EM, he explained, samples are frozen in vitreous ice and maintained at cryogenic temperatures (-274° Fahrenheit) using liquid nitrogen while the samples are photographed in the high vacuum of an electron microscope. The sophisticated cryo-EM machine resides in Brookhaven Lab’s biology department. Li and his collaborators also measured the mass of the OT complex at Brookhaven’s Scanning Transmission Electron Microscope (STEM) facility.

The structure deciphered by the group helps to explain many biochemical phenomena observed about the enzyme complex over the past two decades. It also offers hints as to how the enzyme performs its various jobs, from recognizing the sugar molecules to be added to the protein, scanning the protein as it is formed to identify the sites where sugars should be attached, and transferring the sugar molecules to the protein at the right positions.

“OT physically associates with the protein translocation channel which moves a protein across a membrane and the cell’s protein synthesis machinery, forming an efficient three-machine assembly line for protein translation, translocation, and glycosylation,” Li said.

The researchers say further research is needed to illuminate the molecular mechanisms of disorders of glycosylation involving oligosaccharide transferase. For example, they would like to do structural studies of the enzyme at higher resolution in complex with substrates or in association with the cell’s protein translocation and protein synthesis machinery. A new facility Brookhaven Lab hopes to begin construction on next year, known as the National Synchrotron Light Source II, would greatly increase the precision of this work.

This research was supported by the National Institutes of Health and by Brookhaven National Laboratory’s Laboratory-Directed Research and Development funds.