Recent Publications

May 15, 2012
Untangling mysteries of spider silk

Using high-energy X-rays provided by Argonne's Advanced Photon Source (APS), scientists peered into the structure of orb spiders’ dragline silk. This is the chief thread that allows them to dangle precipitously off branches and window frames.

"Spider silk has a unique combination of mechanical strength and elasticity that make it one of the toughest materials we know," said Professor Jeffery Yarger of Arizona State University, one of the lead researchers of the study.

Paging Peter Parker: Scientists have taken another step closer to producing viable artificial spider silk by zooming-in on the nanoscopic structure of the natural, spider-made stuff, using the brightest X-ray beams in the Western Hemisphere.

And as it turns out, despite the intricate and deliberate patterns woven by common orb spiders, the strongest part of their silk — the threads called dragline silk, which are used to create the scaffolding of the entire web — are mostly made up of extremely random and disordered atoms on the nanoscale, according to the results of a new study by scientists in Arizona and Illinois.

In fact, between 85 and 90 percent of orb spiders’ dragline silk fibers are “amorphous regions,” comprised of random, disorganized atoms, which researchers believe are responsible for providing the silk with its extreme elasticity.

The remaining 15 to 10 percent of the silk is a highly-orderly crystalline lattice structure of atoms which gives the silk its amazing strength — as strong as steel.

See the difference between the orderly regions (multicolored) and random amorphous (blue) in the following image of X-ray data provided by Argonne National Laboratory in Illinois, where the study took place.

“These techniques we develop are getting us closer and closer to know the exact molecular structures within natural spider silk,” said Jeff Yarger, a biochemistry professor at Arizona State University and one of the lead researchers of the study, in an email to TPM.

“This knowledge is required in order to develop synthetic spider silk,” Yarger added.

Scientists have already managed to bioengineer spider silk out of silk worms and goats’ milk, but as Yarger pointed out, they aren’t quite as good as the spider-made silk.

“The problem is that these synthetic silks do not form the correct secondary and tertiary structures that natural spider silk fibers form,” Yarger told TPM.

The thought is that by getting a better view at what makes up real spider silk, scientists will be able to better replicate it on their own.

“Previously scientists had concentrated on the more ordered crystalline regions even though they form a minority of the fiber,” said Chris Benmore, an X-ray scientist on the project, in an email to TPM.

Benmore works specifically at the Advanced Photon Source (APS), the high-powered x-ray facility used to get the new close-up of the spider silk. The APS is a synchrotron facility — a giant particle accelerator measuring over a half-mile around, located at the Argonne National Laboratory in Argonne, Illinois, near Chicago and funded by the Department of Energy.

Scientists from around the country and the globe are allowed to use the x-rays for their own research projects. As Argonne explains, the APS is “open to everyone who has a need for extremely brilliant x-ray photon beams.”

In this case, the APS high-energy X-rays allowed Yarger and his colleagues to achieve an unprecedented level of detail when it came to imaging the amorphous regions of spider silk, zooming in to 10 million times magnification.

“We have recently developed a very powerful high energy x-ray probe at the APS which does not destroy the sample (which is a problem with lower energy x-rays) and provides a detailed insight into both the amorphous and crystalline regions of the spider silk,” Benmore explained.

The results of the X-ray study were published in a paper in the journal Physical Review Letters in late April.

"Total X-Ray Scattering of Spider Dragline Silk", C. J. Benmore, T. Izdebski, and J. L. Yarger Phys. Rev. Lett. 108, 178102 (2012) – Published April 24, 2012

April 18, 2012
Hayes & Herckes explore novel fingerprinting technique

Bioaerosols are diverse and complex dispersed particles that are either living or of biological origin. These include viruses, pollen, fungal spores, bacteria, and debris from vertebrates, including humans, and other biota (plants, insects, etc).

These particles range from ~10 nm to 100 μm, and their existence has been recognized for well over a century. Currently, the central topics of bioaerosol studies are health hazards, effects on the atmosphere, terrorism detection, and global climate.

Over the past decade, several studies focused on molecular and isotopic markers that can be used to track bioaerosols, especially for tracing particles released from soils and a variety of agricultural environments. Molecular marker studies have mainly focused on organic marker compounds, for example saccharides, alkanes, and steroids for tracing soil dust and plant bioaerosols.

Beyond these studies, viewing animal and human bioaerosols as an information-rich marker of its source has not been seriously considered. Reasons for this absence are lack of sufficient (bio) analytical capabilities and poor understanding of the biochemical fingerprints likely to be present in this type of debris.

Considering the body of evidence that does exist indicating abundant cellular material and proteins in the atmosphere, this is somewhat surprising—limited analytical capabilities notwithstanding. Even though there is imperfect knowledge of the “dead” and fragmented biological fraction of particles in the atmosphere, the mere existence of this type of debris creates an opportunity for its use in many potential applications.

Living organisms, including humans, constantly release a surprisingly large amount of dead skin cells and fragments into the environment. As analytical capabilities are improved and are focused on the characterization of this fraction (and the living fraction), detailed biochemical information will be found within the aerosol. This has the potential to significantly affect fields ranging from biochemical forensics and biodiversity studies to medical profiling and environmental studies.

"Exploring the feasibility of bioaerosol analysis as a novel fingerprinting technique". Josemar A. Castillo, Sarah J. R. Staton, Thomas J. Taylor, Pierre Herckes and Mark A. Hayes, Anal Bioanal Chem, 2012, 403(1), 15-26, DOI: 10.1007/s00216-012-5725-0

One of the outstanding questions of early Earth biology is how ancient organisms made the evolutionary transition from anoxygenic (no oxygen produced) to oxygenic (oxygen-producing) photosynthesis. A team of scientists from ASU has moved us significantly closer to understanding this problem. Photo by: stock.xchng

Photosynthesis is one of the fundamental processes of life on Earth. The evolutionary transition from anoxygenic (no oxygen produced) to oxygenic (oxygen-producing) photosynthesis resulted in the critical development of atmospheric oxygen in amounts large enough to allow the evolution of organisms that use oxygen, including plants and mammals.

One of the outstanding questions of the early Earth is how ancient organisms made this transition. A team of scientists from Arizona State University has moved us closer to understanding how this occurred, in a paper recently published in the Proceedings of the National Academy of Sciences. The paper, titled "Light-driven oxygen production from superoxide by manganese-binding bacterial reaction centers," is authored by James Allen, JoAnn Williams, Tien Le Olson, Aaron Tufts, Paul Oyala and Wei-Jen Lee, all from the Department of Chemistry and Biochemistry in ASU's College of Liberal Arts and Sciences.

Plants and algae, as well as cyanobacteria, use photosynthesis to produce oxygen and “fuels,” the latter being oxidizable substances like carbohydrates and hydrogen. There are two pigment-protein complexes that orchestrate the primary reactions of light in oxygenic photosynthesis: photosystem I and photosystem II.

“In photosynthesis, the oxygen is produced at a special metal site containing four manganese and one calcium atom connected together as a metal cluster,” explains professor James Allen. “ This cluster is bound to the protein called photosystem II that provides a carefully controlled environment for the cluster.”

On illumination, two water molecules bound at the cluster are split into molecular oxygen and four protons. Since water molecules are very stable, this process requires that the metal cluster be capable of efficiently performing very energetic reactions.

Allen, Williams and coworkers are trying to understand how a primitive anoxygenic organism that was capable of performing only simple low energy reactions could have evolved into oxygen-producing photosynthesis.

They have been manipulating the reaction center of the purple bacterium Rhodobacter sphaeroides encouraging it to acquire the functions of photosystem II. In the recent publication, they describe how a mononuclear manganese bound to the reaction center has gained some of the functional features of the metal cluster of photosystem II.

Although the mononuclear manganese cannot split water, it can react with reactive oxygen species to produce molecular oxygen. These results suggest that the evolution of photosynthesis might well have proceeded through intermediates that were capable of oxygen production and served until a protein with a bound manganese-calcium cluster evolved.

Jenny Green, jenny.green@asu.edu
480-965-1430
Department of Chemistry and Biochemistry

"Light-driven oxygen production from superoxide by Mn-binding bacterial reaction centers", James P. Allen, Tien L. Olson, Paul Oyala, Wei-Jen Lee, Aaron A. Tufts, and JoAnn C. Williams, Proceedings of the National Academy of Sciences, (2012) Volume 109, pages 2314-2318

read online

March 12, 2012
What ruled the world before DNA and RNA?

All living organisms use DNA as the carrier of genetic material and RNA as the messenger molecule directing the expression of genes and creation of proteins. This arrangement has lasted 3.5 billion years. But what came before these life-giving molecules?

These two massive molecules are wonderfully versatile, capable of assembling and replicating themselves while also folding into all the different shapes needed for proper biological functions. Both DNA and RNA work by stringing together lots of different combinations of molecules called nucleotides, and it's the arrangement of these nucleotides that provides all the larger scale genetic information.

Both DNA and RNA are held together by "backbones" that are made of phosphates and sugar. These chemical bonds provide the structure needed to hold together the long chain of nucleotides in a single giant DNA or RNA molecule. In fact, it's these backbones that give the two forms of genetic material their name - the sugar holding together DNA is deoxyribose, hence deoxyribonucleic acid, while RNA contains ribose.
It's these sugars that have led researchers at Arizona State to consider the possibility of a simpler genetic material that ruled the primordial world more than 3.5 billion years ago. Both ribose and deoxyribose are what's known as five-carbon, or pentose, sugars. This simply means that their chemical structure features five carbon atoms.

The problem with pentose sugars is that they don't emerge and combine easily, especially in an ancient Earth that had not yet been transformed by the presence of RNA and DNA. That's a bit of a biological Catch-22: for pentose sugars to emerge, you need a world that's already full of pentose sugars. The way out of this paradox, according to Arizona State researcher John Chaput, is to presuppose an earlier, less complex form of genetic material that could be a bridge between the lifeless prebiotic world and our current, DNA-dominated planet.
The likeliest candidate, according to Chaput, is a molecule called threose nucleic acid, or TNA. Unlike ribose and deoxyribose, threose is a tetrose sugar, meaning it only has four carbon atoms in its molecular structure. From a chemical standpoint, four is a much easier number than five - threose could easily have formed from the combination of a pair of carbon fragments, each featuring two of the necessary atoms.

That's all well and good, but the theory doesn't mean much if TNA can't actually function as a genetic material for simple organisms. The initial experiments on this point are encouraging, as Chaput reports that TNA "can fold into complex shapes that can bind to a desired target with high affinity and specificity." These functional TNA molecules were created from initial random sequences through a process of Darwinian evolution, the same process by which DNA and RNA first emerged as the carriers of genetic material in the real world.

What's more, TNA is able to bond chemically with both DNA and RNA molecules and still function as a source of genetic information. That's crucial because, if TNA really did once provide genetic data for our planet's very earliest organisms, there must have eventually been a transition point from this early molecule to its immediate successor RNA. In this model, TNA serves as a simpler way for basic life to get started in the unforgiving primordial Earth, which was then able to give way to more complex macromolecules that were able to carry out a wider range of biological functions.
Right now, there's no way to prove that Earth was once a world of TNA - indeed, biologists are still struggling to find hard evidence that RNA once served as the primary source of genetic material before the dominance of DNA. The way forward is to demonstrate that synthetic TNA really can provide all the functions required by early lifeforms. If that holds up, TNA could well then become the likeliest explanation for the first origins of life.

"Darwinian evolution of an alternative genetic system provides support for TNA as an RNA progenitor" Hanyang Yu, Su Zhan and John C.Chaput, Nature Chemistry (2012) doi:10.1038/nchem.1241.
read online

October 10, 2011
New technique unlocks secrets of ancient ocean

Earth’s largest mass extinction event, the end-Permian mass extinction, occurred some 252 million years ago. An estimated 90 percent of Earth’s marine life was eradicated. To better understand the cause of this “mother of all mass extinctions,” researchers from Arizona State University and the University of Cincinnati used a new geochemical technique. The team measured uranium isotopes in ancient carbonate rocks and found that a large, rapid shift in the chemistry of the world’s ancient oceans occurred around the extinction event.

The mechanism of the end-Permian mass extinction has been much debated. One proposed cause for the extinction, the release of toxic hydrogen sulfide gas, is directly related to oceanic anoxia, which is a depletion of dissolved oxygen from the ocean.

Widespread evidence exists for oceanic anoxia before the extinction, but the timing and extent of anoxia remain unknown. Previous hypotheses posited that the deep ocean was depleted of oxygen for millions of years before the end-Permian extinction. The new research using measurements of uranium isotopes in ancient carbonate rocks indicates that the period of ocean-wide anoxia was much shorter.

“Our study shows that the ocean was anoxic for at most tens of thousands of years before the extinction event. That’s much shorter than prior estimates,” says Gregory Brennecka, the lead author of the study and a graduate student in ASU’s School of Earth and Space Exploration in the College of Liberal Arts and Sciences.

Brennecka, working in Professor Ariel Anbar’s research group, conducted the analysis of the samples. Anbar is a professor in ASU’s School of Earth and Space Exploration and the Department of Chemistry and Biochemistry. Achim Herrmann, a senior lecturer at Barrett, the Honors College at ASU, and Thomas Algeo of the University of Cincinnati, who collected the samples in China, helped guide the selection of samples and interpretation of data.

The team studied samples of carbonate rock from Dawen in southern China for uranium isotope ratios (²³⁸U/²³⁵U) and thorium to uranium ratios (Th/U). The study presumes that carbonate rocks capture ²³⁸U/²³⁵U and Th/U of the seawater in which they were deposited. If so, they can be used to study changes in the chemistry of ancient oceans. In separate, related work, the team is testing the limits of this assumption.

In a section of rock spanning the time of the extinction, the team found a marked shift in ²³⁸U/²³⁵U in the carbonate rocks immediately prior to the mass extinction, which signals an increase in oceanic anoxia. The team also found higher Th/U ratios in the same interval, which indicate a decrease in the uranium content of seawater. Lower concentrations of uranium in seawater also serve as signals of oceanic anoxia.

These decreases in ²³⁸U/²³⁵U and increases in Th/U only occur at the section of rock that contains the end-Permian extinction horizon. This shows that a period of oceanic anoxia existed only briefly prior to the mass extinction, rather than the previously hypothesized much longer timeframe.

The team’s findings represent an increase in knowledge about the ocean’s chemistry at a critical period of the Earth’s history. “This technique gives us a better understanding of how ocean chemistry can change over time, and how sensitive it is to certain environmental factors,” says Brennecka.

The implications of the new geochemical tool the researchers developed are just as important as the study’s findings.

Uranium isotope ratios have been utilized to study the ocean’s chemistry before, but only in black shale, a different and less common type of rock. This study represents the first time uranium isotope ratios have been studied in carbonates for paleo-redox purposes, which is a promising new geochemical tool for future research.

“One of the important outcomes of this study is that we were able to quantify the relative change in the amount of oceanic anoxia across the extinction event in the global ocean. Previous studies were only able to show whether anoxic conditions existed or not. We can now compare this event to other events in Earth history and develop a better understanding of how the amount of oxygen in the Earth’s ocean has changed through time and how this might have affected marine diversity,” says Herrmann.

Carbonates are much more widespread than black shales on Earth through space and time. “By focusing on carbonates we can study ancient anoxic events in many more places and times,” says Anbar. “This was our major motivation in developing the uranium isotope technique.”

It is only recently that researchers have developed the ability to precisely measure slight variations in uranium ratios, largely due to research completed at ASU. Most of the team’s research in this study was conducted at ASU. The study samples were analyzed at ASU’s W. M. Keck Foundation Laboratory for Environmental Biogeochemistry.

“Over the past decade, my research group has worked with many collaborators to develop new techniques to study changes in oxygen in the Earth’s ocean through time,” says Anbar. “We are especially interested in the connections between ocean oxygenation and biological evolution. The uranium isotope technique is the newest method. We expect it will be very useful. This study shows that it is yielding insights pretty quickly.”

“It is exciting to be here, because most of the development work to measure uranium isotopes was done at ASU over the past five years. It is exciting to be at the forefront of these advancements,” says Brennecka.

The team’s results will be published in the Proceedings of National Academy of Sciences Oct. 10 in a paper titled, “Rapid expansion of oceanic anoxia immediately before the end-Permian mass extinction.”

"Rapid expansion of oceanic anoxia immediately before the end-Permian mass extinction", Gregory A. Brenneckaa, Achim D. Herrmanna,Thomas J. Algeoc, and Ariel D. Anbar, Published online before print October 10, 2011, doi: 10.1073/pnas.1106039108 PNAS October 10, 2011

read online

Using the energy of visible light to fuel chemical conversions in a similar manner to photosynthesis is extremely attractive. Multiple chromophore systems, attached to dendrimers or protein assemblies, have been constructed that are able to capture light and transport the excitation energy in one direction through donor-acceptor energy-transfer processes, but organizing a large number of multiple chromophores with precision remains challenging.

Yan Liu and co-workers from Arizona State University have now used a DNA scaffold to prepare rapid and efficient light-harvesting antennas. A seven-helix DNA bundle, in which six helices surround a protruding one, holds cyclic arrays of three chromophores attached to the DNA fairly rigidly - either through a base or directly incorporated into the backbone - at well-controlled inter-chromophore distances. They are arranged to favour a stepwise energy transfer from the primary donor to the intermediate donor, then to the acceptor at the protruding site.

The researchers prepared a series of antennas in which the relative ratios between the three chromophores are varied, and studied their energy-transfer processes and light-harvesting abilities. In all cases, the only energy transfer observed was a stepwise, unidirectional transfer cascade from the primary donor to the secondary, and then to the acceptor, demonstrating the efficacy of the DNA scaffold. The light-harvesting abilities of the antennas were most efficient when a large number of donor chromophores were present.

"Holding on to DNA", Anne Pichon Nature Chemistry 3, 654 (2011) doi:10.1038/nchem.1134, Published online 23 August 2011  

Structural overlay of the activase C-domain(yellow) onto the hexameric assembly of FtsH, a remote homolog (blue)

September 14, 2011
How do higher plants regulate CO ₂ assimilation under heat-stress conditions?

In recent years, interest in understanding the structure and function of Rubisco activase, a motor protein in plants, has intensified, propelled in part by its purported role in modulating the thermotolerance of photosynthesis.

The first X-ray structural information on Rubisco activase has recently been provided by post-doctoral research associate Nathan Henderson and associate professor Rebekka Wachter who work in Arizona State University’s Department of Chemistry and Biochemisty. The work was published online in the Journal of Biological Chemistry in a rapid report format (http://www.jbc.org/content/early/2011/08/31/jbc.C111.289595), and will appear in print later this year.

Rubisco activase is a motor protein thought to play a central role in coordinating the rate of CO 2 fixation with the light reactions of photosynthesis. In spite of intense efforts by a number of researchers, structural information on activase has remained elusive for well over 20 years.

Rubisco is an extremely abundant enzyme responsible for net atmospheric carbon fixation in all photosynthetic organisms. Its catalytic competence plays an important role in heat-related limitations on biomass production. Activase regulates Rubisco activity by sensing the ATP/ADP ratio and redox poise of the chloroplast stroma, and has been linked to the inhibition of net photosynthesis under heat stress conditions.

The activase domain elucidated in the Wachter lab is essential in the specificity of Rubisco recognition, and will therefore serve as a structural framework to test models for the physical interaction between activase and Rubisco.

This long-awaited piece of a highly complex puzzle will accelerate research efforts aimed at understanding the down-regulation of carbon fixation at elevated temperatures, and will allow for structure-guided genetic engineering of activases to identify more thermostable variants.

"Atomic resolution X-ray structure of the substrate recognition domain of higher plant Rubisco activase", J. Nathan Henderson, Agnieszka M. Kuriata, Raimund Fromme, Michael E. Salvucci and Rebekka M. Wachter, JBC, 2011, doi: 10.1074/jbc.C111.289595

August 31, 2011
Molecules as mini-computers

Photochromes are photoswitchable, bistable chromophores which, like transistors, can implement binary logic operations. When several photochromes are combined in one molecule, interactions between them such as energy and electron transfer allow design of simple Boolean logic gates and more complex logic devices with all-photonic inputs and outputs. Selective isomerization of individual photochromes can be achieved using light of different wavelengths, and logic outputs can employ absorption and emission properties at different wavelengths, thus allowing a single molecular species to perform several different functions, even simultaneously. This manuscript reports a molecule consisting of three linked photochromes that can be configured as AND, XOR, INH, half-adder, half-subtractor, multiplexer, demultiplexer, encoder, decoder, keypad lock, and logically reversible transfer gate logic devices, all with a common initial state. The system demonstrates the advantages of light-responsive molecules as multifunctional, reconfigurable nanoscale logic devices that represent an approach to true molecular information processing units.

The beguiling idea that molecules can manipulate and process information using logic, as do electronic computers and human brains, has spawned numerous studies of molecule-based systems that perform not only as simple logic gates but also as more complex devices such as adders and subtractors, multi- plexers/demultiplexers, encoders/decoders, keypad locks, and multivalued logic devices. In recent years, such decision-making molecular systems have been explored in clever applications taking advantage of binary and multivalued information processing for multiparameter chemosensing, pro-drug activation, medical diagnostics, object labeling, data storage, and smart materials and surfaces. Molecular computing, which requires more than a single logic function/device, has been critically discussed in recent years. One of the foremost problems is the physical integration of many logic gates in arrays in order to achieve functional complexity beyond basic logic gates such as AND, OR, NOR, NAND, XOR, INH, etc. This would require efficient wiring (concatenation) of simple logic switches. Only a few proof-of-principle examples of such concatenation are known in the literature. One approach to circumvent this problem is functional integration. This means that a unimolecular system can mimic a complex logic circuit, composed of various logic gates, without the necessity of representing each logic gate by a different structural feature. The complex logic devices mentioned above are examples of this approach. For example, a molecular half-adder is a combination of AND and XOR logic gate functions in the same molecule.

Many of the molecular logic systems that have been described use chemicals as inputs and optical as well as chemical signals as outputs. However, the use of photons for both inputs and outputs offers several advantages. First, due to the physical equality of input and output, i.e., an all-photonic operation, one barrier for the concatenation of logic devices is removed. Also, light does not require physical access, allowing monolithic structures containing many individual logic elements. This is a potential advantage for transferring molecular logic devices from solution operation to the solid phase in the future. Light does not lead to the buildup of waste products, thus in principle allowing nearly infinite cycling of the system (although this has not been achieved yet in practice). Optical input signals can be delivered by remote control and as pulses, even on the subpicosecond time scale, permitting rapid cycling. Importantly, different chromo- phores in the same molecule interact differently with light of different wavelengths. Moreover, a single substance can perform several logic operations with the same set of inputs, depending upon the readout selected, and may be reconfigured for different operations simply by changing the input wavelengths, output wavelengths, or initial state.

This paper reports a photochromic triad acting as a unimolecular, multifunctional, and reconfigurable logic system that performs the functions of the following devices: AND, XOR (exclusive OR), INH (inhibit), half-adder, half-subtractor, multiplexer, demultiplexer, encoder, decoder, keypad lock, and logically reversible transfer gate. Uniquely, all operations use a common initial state, and the majority (including the half-adder and half- subtractor) share the same two inputs, differing only in the choice of the optical output signal. This not only allows parallel logic operations with the same set of inputs but also makes switching (reconfiguration) among the various logic operations extremely rapid and convenient. The thereby achieved level of functional integration through implementation of logic gate arrays without the need of physical gate-to-gate connections is unprecedented. Additionally, the triad can be reset by a universal optical signal, independent of the preceding input combination/logic operation.

"All-Photonic Multifunctional Molecular Logic Device",  oakim Andrasson, Uwe Pischel, Stephen D. Straight Thomas A. Moore, Ana L. Moore, and Devens Gust, JACS, August 3, 201, Volume 133, Issue 30 Pages 11641-11648.

read online

June 6, 2011
DNA PACKAGING

While efforts to unlock the subtleties of DNA have produced remarkable insights into the code of life, researchers still grapple with fundamental questions. For example, the underlying mechanisms by which human genes are turned on and off—generating essential proteins, determining our physical traits, and sometimes causing disease—remain poorly understood. Marcia Levitus, faculty member in the department of chemistry and biochemistry and Kaushik Gurunathan at the Biodesign Institute at Arizona State University along with their colleagues Hannah S. Tims, and Jonathan Widom of Northwestern University in Evanston, Illinois have been preoccupied with tiny, spool-like entities known as nucleosomes. Their latest insights into how these structures wrap and unwrap, permitting regulatory proteins to access, bind with and act on regions of DNA, recently appeared in the Journal of Molecular Biology. ;Nucleosomes, Levitus explains, are essential components of the genome, acting to regulate access to DNA and protect it from harm. Nucleosome structure permits the entire strand of human DNA, roughly 6 feet in length, to be densely packed into the nucleus of every cell—an area just 10 microns in diameter. This occurs after nucleosomes assemble and fold into higher order structures, culminating in the formation of chromosomes. Each nucleosome (there are roughly 30 million per cell) consists of a 147 base pair segment of DNA. This length of DNA thread is wound 1.67 times around the spool-like protein units, known as histones. The histone complex, together with its windings of DNA, forms the nucleosome core particle. A multitude of proteins must act on regions of the DNA strand, by binding with appropriate target sites. Essential functions rely on these operations, including gene expression, replication and repair of damaged regions of the DNA molecule. But in eukaryotic cells like those of humans, some 75-80 percent of the DNA strand is curled up and hidden in the nucleosomes—inaccessible to protein binding interactions. In earlier work, the group was able to show that nucleosomes are dynamic structures, quite different from the static pictures produced by X-ray crystallography.  Lengths of DNA make themselves available for protein interaction by unwrapping and rewrapping around the histone core. When nucleosomes unwrap, proteins present in sufficient concentration can find their DNA targets and bind with them. In order to observe and characterize the dynamic behavior of nucleosomes, the team relied on a versatile imaging method known as Fluorescence Resonance Energy Transfer or FRET. The technique allows researchers to look at a pair of fluorescent molecules or fluorophores, one of which is attached to the end of the exposed DNA strand, the other, to one of the histones around which the DNA is coiled.

 As Levitus explains, spontaneous unwrapping and rewrapping of DNA changes the distance between fluorophores, signaling that the process has occurred and allowing the group to quantify the frequency and rate of DNA exposure and concealment. “Although FRET has been used for decades to measure molecular distances in biological systems, dynamic biomolecules such as nucleosomes present particular challenges,” notes Levitus. Traditionally, FRET experiments are performed with protein solutions containing  many billions of particles. In the case of nucleosomes however, the dynamic behavior of each particle is crucial and bulk measurements using FRET are not effective. “In simple terms, if one wanted to understand how humans clap, it would be useless to listen to the whole planet clapping at once. Instead, one would listen to a few individuals, and that is exactly what we did with nucleosomes,” Levitus says. The results of initial studies were revealing. For base pair sequences along the nucleosomes’ outer rind, spontaneous DNA unwrapping occurs at a rapid rate— about 4 times per second. This corresponds to a period of only 250 milliseconds during which this region of DNA remains fully wrapped and occluded by the histone complex. Once unwrapped, the DNA remains exposed for 10-50 milliseconds. These findings present a plausible mechanism to allow protein binding with unwrapped DNA in vivo, so long as the binding sites occur near the ends of wrapped nucleosomal DNA. The new study also examines, for the first time, the condition of DNA sequences occurring further along the wound length of nucleosomal DNA, that is, closer to the nucleosome’s center. Here, rates of DNA unwrapping decreased by orders of magnitude.

Written by: Richard Harth

"Dynamics of Nucleosome Invasion by DNA Binding Proteins" Hannah S. Tims , Kaushik Gurunathan, Marcia Levitus, Jonathan Widom, J. Mol. Biol. (2011) 411, 430–448.

Read online

August 23, 2011
Peptide-Modified Surfaces for Enzyme Immobilization
Jinglin Fu, Jeremy Reinhold and Neal W. Woodbury

Chemistry and particularly enzymology at surfaces is a topic of rapidly growing interest, both in terms of its role in biological systems and its application in biocatalysis. Existing protein immobilization approaches, including noncovalent or covalent attachments to solid supports, have difficulties in controlling protein orientation, reducing nonspecific absorption and preventing protein denaturation. New strategies for enzyme immobilization are needed that allow the precise control over orientation and position and thereby provide optimized activity.

In this study Fu, Reinhold and Woodbury present a method for utilizing peptide ligands to immobilize enzymes on surfaces with improved enzyme activity and stability. The appropriate peptide ligands have been rapidly selected from high-density arrays and when desirable, the peptide sequences were further optimized by single-point variant screening to enhance both the affinity and activity of the bound enzyme. For proof of concept, the peptides that bound to b-galactosidase and optimized its activity were covalently attached to surfaces for the purpose of capturing target enzymes. Compared to conventional methods, enzymes immobilized on peptide-modified surfaces exhibited higher specific activity and stability, as well as controlled protein orientation.

This approach will be applicable to the immobilization of a wide variety of enzymes on surfaces with optimized orientation, location and performance, and provides a potential mechanism for the patterned self-assembly of multiple enzymes on surfaces.

"Peptide-Modified Surfaces for Enzyme Immobilization.", Fu J, Reinhold J, Woodbury NW, PLoS ONE 6(4):e18692 (2011). doi: 10.1371 / journal.pone. 0018692