Just A Little Squeeze Lets Proteins Assess DNA

[Source: ScienceDaily] - To find its target, all a protein needs to do is give quick squeezes as it moves along the DNA strand, suggests new research from The University of Arizona in Tucson.

Scientists had thought DNA-binding proteins primarily used full-body hugs for accurate readings of the information coded in the DNA's sequence.

Even a protein known to use the hug method, called direct readout, can effectively pinpoint sites on DNA using indirect readout, found researcher Nancy C. Horton and her colleagues.
"It was a total surprise," said Horton, a UA associate professor of biochemistry and molecular biophysics. "No one had ever seen it before."

Doing the quick squeezes that scientists call indirect readout probably works faster than requiring full-body contact with all the DNA, the researchers suggest. Quick and accurate identification of key sites on DNA is important for the health of all kinds of cells, from bacteria to humans.

To detect the protein-DNA connection in such detail, Horton and her co-authors Elizabeth J. Little and Andrea C. Babic studied a DNA-binding protein that bacteria use to protect themselves from viral infections.

The finding has implications for the development of designer drugs.

"People have and are developing DNA-binding proteins to turn genes on and off," Horton said. Such designer proteins can be used to cut out the bad copy of a gene and help replace it with good copy.

"We found that indirect readout is important for finding the right sequence, and we now think indirect readout is also important for finding it quickly," she said.

The team published their paper, "Early Interrogation and Recognition of DNA Sequence by Indirect Readout," in the December issue of the journal Structure. First author Little and co-author Babic were postdoctoral research associates in Horton's laboratory when they did the research. The two are now senior scientists at Ventana Medical Systems, Inc. in Tucson, Ariz.
The National Institutes of Health funded the research.

Horton studies proteins that bind to DNA.

Seven years ago, she figured out the structure of a protein called HincII that snips up DNA. The protein is a type of enzyme called a restriction endonuclease and comes from Haemophilus influenzae bacteria.

Since that time, Horton has been trying to learn how HincII interrogates the DNA to find the right place to cut.

The protein protects bacteria by cutting up DNA from invading viruses. Without the protective protein, viral DNA would commandeer the bacterium's cellular machinery to produce viruses and ultimately kill the bacterial cell.

The HincII protein distinguishes between bacterial DNA and viral DNA by recognizing certain sequences on DNA. Such a defense requires speed to prevent the marauding virus from killing the cell and also accuracy so the protein doesn't accidentally hack up the bacterium's own DNA.
Horton knew from her previous work that the HincII protein used the direct readout method to find the particular sequence of DNA that corresponded to enemy DNA. The protein seemed to distort the DNA to read it.

Removing the direct readout contact between the protein and the DNA might show whether the DNA distortion or the contact itself was important, Horton said.

Therefore Little and Babic created a mutant protein that couldn't hug DNA closely and therefore couldn't use the direct readout method. Little described the mutant protein as missing the fingers the normal protein used to probe the DNA.

"If the finger was doing all the recognition, then the mutant should cut any DNA sequence," Horton said.

To see how the mutant interacted with DNA, the researchers crystallized the mutant protein-DNA complex in action.

Initially, Horton thought the assay had gone wrong and almost threw the results in the trash, she wrote in an e-mail.

The mutant protein had chosen the proper site on the DNA with 100 percent specificity, which was opposite from what she expected. In addition, the DNA was distorted, even though the mutant couldn't make the strong contact a normal protein would.

"I did a double-take. I was just taking a picture to have a record that it was non-specific," she said.

Understanding how endonucleases and other DNA binding-proteins recognize a particular DNA sequence provides insight into key cellular processes including the replication, transcription and repair of DNA.

Little said, "In every single one of your cells are proteins looking for the proper sequences in DNA in order to make the proteins you need to stay alive."

Horton added, "Understanding how these processes work helps in the understanding of diseases so that we could potentially cure the disease."

Medipacs named best of show at Invest Southwest

[Source: Phoenix Business Journal,Patrick O'Grady ] - Medipacs, a maker of infusion pumps for the medical industry, was named the top presenter at the annual Invest Southwest conference Thursday in Scottsdale.


The Tucson company received a $200,000 prize from SCF Arizona for the presentation and idea judged to be the best of the year’s crop — just a drop in the bucket toward Medipacs’ quest to land $8 million to $10 million.


The conference brought together a dozen companies from throughout the Western U.S. — and for the first time, a company from Mexico. Unima Bioseguridad Integra presented its method for using natural methods to prevent food-borne illnesses. The company already has another technology, Custovac, being distributed by German pharmaceutical company Boehringer Ingelheim for use in animal vaccinations.


The conference had more attendees this year than last, a feat praised by organizers given the economic climate. Still, it may be a few weeks or months before this year’s attendees find out whether they will garner funding.


Other presenters at the event:
• AmpliMed Corp of Tucson brought in its phased cancer drug approach. The company has gone through three funding rounds and is working on its fourth as it moves to a trial for the drug.
• CaptiveMotion LLC of Tempe brought its computer face modeling technology. The startup by former video-game developer Adam Kraver allows game and film companies an easier way to convert images of the human face for digital uses.
• CellTrust Corp. displayed its technology to encrypt and store secure text and voice messages. The Scottsdale-based company sees a large market for the technology, particularly in medical fields where new federal laws require patient data be kept confidential.
• Consolidated Energy Systems LLC of Salt Lake City brought its technology for taking petroleum-based fuels that ordinarily could not be used and converting them to run large diesel electrical generating plants. To keep the project carbon-neutral, the plan would be to use the carbon dioxide in underground oil recovery.
• Eco Pool Technologies Inc. of Phoenix has begun production of a solar pool cleaner on a limited basis. The company also is developing a new version that would be able to work 24 hours a day, and other products that could lower electric costs for pool owners.
• Grip Audio LLC of Glendale presented a way to use computer software and local-area networks to replicate the cabling necessary for live music and studio productions. The company is looking at the professional audio market and has begun testing with several businesses and groups.
• Targeting the home video market, iMemories LLC of Scottsdale has developed a way to transfer VHS tapes to digital format and gives users the option of storing them online.
• MedTrust Online LLC, a spinoff of the Translational Genomics Research Institute, is an online resource for oncologists looking for up-to-date information in the fight against cancer. The database will provide physicians with data to help them better and more accurately treat their patients.
• NanoMR of Albuquerque, N.M., offered a patented medical diagnostic tool that could examine blood cultures in minutes, targeting detection of blood-borne bacteria.
• Protein Genomics Inc. of Sedona made its pitch for Tropoelastin, a device that can help in healing and preventing scarring.

UA Researchers Tackling Unsolved Questions about Protein Structures

[Source: Deborah Dawn, BIO5] - A University of Arizona research team is exploring the evolutionary origins of protein structures. Their findings help us better understand how proteins evolved to carry out the instructions encoded in the genes of every living thing.

Protein molecules are made up of chains of amino acids. These chains bend and fold into a dizzying array of three-dimensional shapes and structures, depending on the order of the amino acids in a given chain. Those varied structures are part of what allow the proteins—which are assembled based on instructions coded in DNA—to regulate everything from an organism's growth and metabolism to the ways messages are transmitted from cell to cell. Protein structures are at the heart of how organisms function.

However, the evolution of those structures is still poorly understood, because there are few observed examples of proteins that have clearly evolved from one shape to another. "The origin of the diversity of protein structures is a major unsolved problem," explains BIO5 member Matthew H.J. Cordes, an associate professor of Biochemistry and Molecular Biophysics at the UA.

Cordes' lab is solving that problem. Two graduate students in his lab, Christian M. Roessler and Branwen M. Hall, have located protein molecules in two different viruses that have dramatically different structures: one protein has a helical, or corkscrew shape, while the other is shaped more like a hairpin. Yet these very different proteins have similar amino acid sequences and perform similar functions--binding to DNA to help the viruses replicate and spread--making it likely that they had a common ancestor.

"Somehow, mutations converted the corkscrew structure to the hairpin structure," Cordes says of the finding, which was recently reported in the Proceedings of the National Academy of Sciences.

While this isn't the first example of structural differences among proteins with a common ancestor, it may be the most dramatic natural example of related proteins retaining clear similarity in amino acid sequence while undergoing major reorganization of their structure. "This finding strongly confirms that evolutionary processes produce new protein shapes," Cordes says. "It could become a textbook example of the reality and beauty of evolutionary changes in structure." He adds that some proteins in this family with the hairpin shape bind to DNA more strongly than those with the corkscrew shape, though it is too early to tell if this is always the case. It's not yet known whether such an advantage helped drive the hairpin structure's evolution.

Cordes' graduate students found their protein pair via an unusual method: Roessler and Hall used a stepping-stone technique to make a series of small "jumps" among closely related proteins, following minute structural changes from one protein to another until they "landed" at a protein that was dramatically different from the one they'd started with, yet was still related to it.

Cordes' lab is now working out the details of the specific mutations that might have caused their two proteins to diverge from one another. They also plan to use their stepping-stone technique to shed light on the evolutionary links among other proteins.

"This is like space exploration," Cordes says. "We're journeying through the protein universe, step by step."

Integrated Biosystems Research 2007 Symposium

"Advances in Proteomics Research," sponsored by the Arizona Proteomics Alliance, will be held Friday, November 16, 2007:

• 8 am ~ Registration (& Coffee), Poster set up
• 8:45 am – 5 pm ~ Lectures, Lunch, Posters Session, Panel Discussion
• 5 – 6 pm ~ Reception

Location: Lectures at The Great Hall, Armstrong Hall (LAW); Lunch, Poster Session & Reception at The Biodesign Institute, Arizona State University, Tempe, AZ ; Visitor Parking entrance off Terrace Road in Rural Road Parking Structure.

Purpose:

• To foster a collaborative research network in Arizona advancing systems biology and its translation to personalized medicine.
• To advance the proteomics capabilities in the state through improved awareness of the most cutting-edge methodologies and their novel applications.

Invited speakers will address topics of: Protein Biomarkers – Discovery and Validation; Avances in Protein Analysis; Protein Networks, Pathways, and Clinical Applications. The symposium will also feature panel discussions with invited guests:

• Dr. Leland Hartwell, Fred Hutchinson Cancer Research Center
• Dr. Brian Balgley, Calibrant Biosystems, Inc.
• Dr. James Kerwin, House Ear Institute
• Dr. Amanda Paulovich, Fred Hutchison Cancer Research Center
• Dr. Tom Grogan, Ventana Medical Systems
• Dr. Russ Finley, Wayne State University
• Dr. Jeffrey Trent, Translational Genomics Research Institute
• Dr. George Poste, The Biodesign Institute

For more information, visit http://www.integrativeproteomics.org/. To register before the November 9 deadline, contact Pam Young at 480 727-0378 or Pamela.Young@asu.edu

NIH awards UA nearly $1M for mass spectrometer

[Source: Deborah Daun, BIO5] - The National Institutes of Health awarded a $924,995 High-End Instrumentation (HEI) grant to The University of Arizona for a mass spectrometer that will assist a wide range of UA scientists, from those working on the prevention and cures for diseases such as colorectal cancer and Valley Fever to those researching the atmospheric chemistry on Titan, the largest moon on Saturn.

“This new Fourier Transform Ion Cyclotron Resonance Mass Spectrometer gives the highest mass accuracy available and thus adds tremendous capacity to our mass spectrometry resources at the UA,” says Vicki Wysocki, PhD, BIO5 member and professor, UA Chemistry Department. Dr. Wysocki is the lead investigator on the HEI grant. The new instrument will be located in the UA Department of Chemistry and will be available to researchers through UA’s Chemistry Mass Spectrometry Facility and UA’s Arizona Proteomics Consortium, which is located in the new BIO5 building on the UA campus.

A mass spectrometer measures the masses of individual molecules that have been converted to ions (i.e. have been electrically charged). Mass spectrometry is a powerful analytical technique used to identify unknown compounds, quantify known materials, and elucidate the structure and chemical properties of molecules.

The UA is one of only 14 national recipients of HEI grants for cutting-edge equipment required to advance biomedical research. The grants come from The National Center for Research Resources (NCRR), a part of the NIH. Awarded to research institutions around the country, these one-time HEI grants support the purchase of sophisticated instruments costing more than $750,000.

“These high-performance advanced technologies enable both basic discoveries that shed light on the underlying causes of disease and the development of novel therapies to treat them,” said Dr. Barbara Alving, director, NCRR. “The value of this investment in advanced equipment is greatly leveraged because each of these rare tools is used by a number of investigators, advancing a broad range of research projects.”

Alliance Aims to Boost Biomedicine

Arizona’s bioscience efforts continue to grow through an extensive, statewide collaborative network of initiatives. Now, research capabilities are being allied around the state in a new effort to improve medical diagnostics and human health.

The Arizona Proteomics Alliance (AZPA), a statewide consortium, has been formed to advance the emergent science of proteomics, a science whose broad vision is to understand the biological role of the complete set of proteins in the human body, or proteome. The initiative combines the expertise of nine leading Arizona institutions, including ASU, Banner Health, Barrow Neurological Institute, Carl T. Hayden Veterans Affairs Medical Center, Intrinsic Bioprobes Inc., Mayo Clinic, Sun Health Research Institute, Translational Genomics Research Institute (TGen) and the University of Arizona (U of A). “This alliance places Arizona in the forefront of proteomics research capabilities and will no doubt have broad impact on biomedical research and personalized medicine,” said Jeff Trent, president and scientific director of TGen.
AZPA promotes a team approach to solving problems of significant biomedical interest, according to Michael Mobley, administrative director of AZPA and associate director of the Biodesign Institute at ASU. The alliance will advance the role of proteomics research in the understanding of human health and disease by creating a network of scientists who share resources and expertise.

“AZPA facilitates access to technical and intellectual resources that are rarely surpassed within the United States, and provides the state with a distinct competitive advantage in this field,” says Serrine Lau, scientific director of AZPA, director of Southwest Environmental Health Sciences Center at the U of A’s College of Pharmacy, and a member of U of A’s BIO5 Institute. “We are pleased that the resources of the Arizona Proteomics Consortium at U of A will be part of this state-wide alliance,” Lau says.

The Mayo Clinic is also expecting to be able to expand its efforts and bring new diagnostic and treatment options to its patients as part of the proteomics initiative, according to Laurence Miller, director of research and the cancer center at the Arizona Mayo Clinic. “The alliance will put Arizona in a stronger position to compete for federal and private research funding,” Mobley says.

The effort builds on the vast knowledge generated by the Human Genome Project, which has mapped roughly 25,000 genes. Each gene codes for the production of protein, but with all the modifications possible to both the gene transcript and protein, the human body could have well over a million different proteins, according to the Human Proteome Initiative. This entourage of proteins, the proteome, is ultimately responsible for everything, good or bad, related to human health and disease. Developing tests to rapidly obtain protein profiles as a predictive marker for disease or identifying therapeutic targets benefit health care and medicine.

Leslie Tolbert, vice president for research, graduate studies and economic development at U of A, said that the pooling of technologies statewide would allow researchers to maximize capabilities and minimize costs in a field that requires the development of new research methods. For example, this collaborative effort is expected to foster an expansion of the cancer research initiative at Banner Health, the state’s largest hospital system, says Susan Edwards, president of the Arizona region for Banner Health. A unifying research approach within the alliance is “integrative proteomics,” which involves advancing the processes, methods and technologies that elucidate the role of proteins within an integrated understanding of human biology, according to Mark Hayes, director of Arizona Applied NanoSensors in the College of Liberal Arts and Sciences at ASU. “This means developing proteomics research capabilities so that they are complementary to other advancing fields like genomics, bioinformatics and clinical research,” says Hayes, who was an early champion for the expansion of regional proteomics and the formation of this alliance.

The alliance has just launched a Web site (http://www.integrativeproteomics.org/) where researchers can exchange information about resources and the important problems they are working together to solve. “Collaboration is one of the things that sets Arizona science apart from other states, where competition is more often the rule,” says Joseph Rogers, president and senior scientist of the Sun Health Research Institute. The important partnerships formed in proteomics are expected to not only expand research efforts and improve chances for greater funding, but also to promote the future recruitment and training of talented individuals for the field of health care in Arizona.

According to ASU, there's a new wrinkle in evolution -- man-made proteins

Nature, through the trial and error of evolution, has discovered a vast diversity of life from what can only presumed to have been a primordial pool of building blocks. Inspired by this success, a new Biodesign Institute research team, led by John Chaput, is now trying to mimic the process of Darwinian evolution in the laboratory by evolving new proteins from scratch. Using new tricks of molecular biology, Chaput and co-workers have evolved several new proteins in a fraction of the 3 billion years it took nature. Their most recent results, published in the May 23rd edition of the journal PLoS ONE, have led to some surprisingly new lessons on how to optimize proteins which have never existed in nature before, in a process they call ‘synthetic evolution.’

"The goal of our research is to understand certain fundamental questions regarding the origin and evolution of proteins," said Chaput, a researcher in the institute’s Center for BioOptical Nanotechnology and assistant professor in Arizona State University’s department of chemistry and biochemistry. "Would proteins that we evolve in the lab look like proteins we see today in nature or do they look totally different from the set of proteins nature ultimately chose" By gaining a better understanding of these questions, we hope to one day create new tailor-made catalysts that can be used as therapeutics in molecular medicine or biocatalysts in biotechnology."

The building blocks of proteins are 20 different amino acids that are strung together and folded to make the unique globular shape, stability and function of every protein. The mixing and matching of the amino acid chain like numbers in the lottery are what favor the odds in nature of finding just the right combinations to help generate biological diversity. Yet no one can predict how the string of amino acids sequence folds to make the 3-D functional structure of a protein.

John Chaput, a researcher at the Biodesign Institute at Arizona State University, has used molecular biology tricks to rapidly evolve proteins that have improved stability when compared to naturally occuring... To select the raw ingredients to create the proteins, Chaput’s group (which includes Harvard collaborator Jack Szostak, and ASU colleagues Jim Allen, Meitian Wang, Matthew Rosenow and Matthew Smith) began their quest by further evolving a protein that had been previously selected from a pool of random sequences.

Jack Szostak and Anthony Keefe first made the parental protein in 2001. To achieve their feat, they stacked the odds of finding just one or two new proteins and generated a library of random amino acid sequences so vast — 400 trillion — that it dwarfs the number of items in the entire Library of Congress (134 million). They started with a small protein stretch 80 amino acids long. This basic protein segment acts as a protein scaffold that can be selected for the ability to strongly clutch its target molecule, ATP. There was only one problem, the parental protein could bind ATP, but it wasn’t very stable without it. "It turns out that protein stability is a major problem in biology," said Chaput. "As many as half of the 30,000 genes discovered from the human genome project contain proteins that we really don’t know what their structure is or whether or not they would be stable. So for our goal, we wanted to learn more about the evolution of protein folding and stability."

Chaput’s group decided to speed up protein evolution once again by randomly mutating the parental sequence with a selection specically designed to improve protein stability. The team upped the ante and added increasing amounts of a salt, guanidine hydrochloride, making it harder for the protein fragment to bind its target (only the top 10 percent of strongest ATP binders remained). After subjecting the protein fragments to several rounds of this selective environmental pressure, only the ‘survival of the fittest’ ATP binding protein fragments remained. The remaining fragments were identified and amino acid sequences compared with one another. Surprisingly, Chaput had bested nature’s designs, as the test tube derived protein was not only stable, but could bind ATP twice as tight as anything nature had come up with before. To understand how this information is encoded in a protein sequence, Chaput and colleagues solved the 3-D crystal structures for their evolutionary optimized protein, termed DX, and the parent sequence.

In a surprising result, just two amino acids changes in the protein sequence were found to enhance the binding, solubility and heat stability. "We were shocked, because when we compared the crystal structures of the parent sequence to the DX sequence, we didn’t see any significant changes," said Chaput. "Yet no one could have predicted that these two amino acids changes would improve the function of the DX protein compared to the parent. The results have helped provide a new understanding of how subtle amino acid changes contribute to the protein folding and stability. Chaput’s team has developed the technology potential to take any of nature’s proteins and further improve its stability and function. "We have the distinct advantage over nature of being able to freeze the evolution of our lab-evolved proteins at different time points to begin to tease apart this random process and relate it to the final protein function," said Chaput. Next, Chaput plans on further expanding his efforts to evolve proteins with new therapeutic features or catalytic functions.

For more information, contact Joe Caspermeyer, joseph.caspermeyer@asu.edu, 480-727-0369

Biodesign Institute scientists offer new view of photosynthesis

A research team led by Neal Woodbury, at ASU's Biodesign Institute, has come up with a new insight into the mechanism of photosynthesis, which involves the orchestrated movement of proteins on the timescale of a millionth of a millionth of a second. Their findings are described in in the May 4 issue of Science, "Protein Dynamics Control the Kinetics of Initial Electron Transfer in Photosynthesis."

During photosynthesis, plants are capable of scavenging nearly every photon of available light energy to produce food. In order to examine the processes involved in photosynthesis, Dr Woodbury’s team used the earliest photosynthetic bacteria to evolve called Rhodobacter sphaeroides and created mutants that allowed them to uncover more of the physical mechanism driving photosynthesis and theoretically tweak the electron transfer relationships between molecules in the reaction center. The reaction center is where light energy is funneled into specialized chlorophyll binding proteins that form a scaffold, holding chlorophyll molecules at a highly optimized distances and orientations so that electrons can hop from one chlorophyll to another.

The research team includes lead author Haiyu Wang, Biodesign Institute; Su Lin, Biodesign Institute; James Allen, ASU Department of Chemistry and Biochemistry; JoAnn Williams, ASU Department of Chemistry and Biochemistry; Sean Blankert and Christa Laser, Biodesign Institute.

For More Information