Monday, March 19, 2012

Semi-automated 'pathwalking' to build a protein model

In a report that appears online in the journal Structure, the BCM team describes the development of the semi-automated protocol that enables researchers to "rapidly generate an ensemble of initial models for individual proteins, which can later be optimized to produce full atomic models."

Taking the 3-D images generated through the process of electron cryo-microscopy and X-ray crystallography, the team developed this computational approach to produce these first-generation models of the proteins' structure or fold without prior knowledge of the protein's sequence or other information.

"This is important in working with big complexes made up of 10 to 30 proteins," said Dr. Matthew Baker, instructor in biochemistry and molecular biology at BCM and the paper's corresponding author. "You might know the structure of one or two proteins, but you want to know how all of those proteins interact with each other. As long as you can separate one from another, you can use this technique to make a model of each of the proteins in the complex."

"We borrowed from a classic computer science problem called the 'traveling salesman problem,'" said Dr. Mariah Baker, the paper's first author and a postdoctoral fellow at BCM. "It is in effect a connect-the-dots puzzle without the numbers."

In the traveling salesman problem, computer programmers are asked to figure the best route for a salesman who wants to visits all the cities where he sells just once while minimizing the distance traveled. Pathwalking solves a similar problem for proteins by looking for the optimal path through a 3-D image that connects C-alpha atoms, rather than cities, to form the protein's structure.

The tool is the answer to the dilemma presented by the near-atomic structures that are in the "middle" – not of the highest resolution or the lowest resolution, said Matthew Baker.

As many as 25 percent of all structures imaged by electron cryo-microscopy and one-third of large protein complexes solved by X-ray crystallography are in the 3 to 10 angstroms range, said Matthew Baker.

Until now, the methodology used to annotate or trace the structure of protein from these density maps was usually tailored to specific cases, said Mariah Baker.

"They involved a lot of user intervention and the possibility to include bias," she said. That sparked a determination to automate the process with better routines that required less specific information.

"The question we asked was, can we trace a protein fold in a density map without a priori knowledge," she said. "The answer is that we can."

Provided by Baylor College of Medicine (news : web)

Crystal structure of archael chromatin clarified in new study

Three distinct evolutionary branches of organisms make up all natural forms of life on the planet: bacteria, archaea and eukaryotes. Among these three, the domain known as archaea includes a variety of organisms that live in similar to those of an , thus offering arguably the greatest glimpse of what life may have looked like 4 billion years ago.

One area of great interest is the process by which DNA bind to proteins to compact and regulate the availability of , a process which is essential in all cellular organisms. In eukaryotes, proteins known as "histones" package and order DNA into a compact protein-DNA structure called chromatin. Archaea, in contrast, have no such universal chromatin proteins, instead using two or more DNA-binding proteins to package DNA. Alba is the most widespread and abundant such archaeal chromatin protein, present in the of every archaeal species that lives in high-temperature environments (thermophilic or hyperthermophilic).

While researchers know about the existence of Alba in archaea, the question of how these proteins bind to and compact DNA has remained a mystery. To answer this question, the researchers analyzed the of the Alba2-DNA complex from the archaea A. pernix K1 at atomic-level resolution using synchrotron radiation from the RIKEN SPring-8 facility in Harima, Japan. Their results indicate that unlike the chromatin structure of eukaryotes, Alba2 in archaea forms a hollow pipe with the duplex DNA running through it, with the hairpin structure of Alba2 stabilizing the pipe.

Published in the February 10th issue of the Journal of Biological Chemistry, this newly-discovered mechanism for compacting DNA marks a major step forward in our understanding of the evolution of chromatin structure. The results promise to clarify how abnormalities in chromatin structure can contribute to cancers and gene disorders, while also providing inspiration for the development of new types of biodevices.

Provided by RIKEN (news : web)

Image or mirror image? Chiral recognition by femtosecond laser

The trick is to replace the individual high-energy photon with three laser photons that excite the molecule through intermediate levels until it releases an electron (this method is known as REMPI, Resonance-Enhanced Multi-Photon Ionization). “It is thus possible to eject with less energetic but more intense light,” explains Thomas Baumert of the University of Kassel.

For the measurements, the light must be circularly polarized. What does this mean? “Ordinary” light consists of waves that oscillate in all spatial directions perpendicular to their direction of travel. If light is linearly polarized, the light waves oscillate exclusively in one plane. When light is circularly polarized, the light wave oscillates in a helical form, because its amplitude describes a circle around the axis of travel – either to the right or the left.

Molecules in the are randomly oriented and thus encounter the laser light from all possible angles; the ejected electrons also fly off in every possible direction as they leave the molecule. By using both a special configuration for measurement and special calculation processes, the team is able to determine the distribution of the angles of the electrons’ flight paths. In the case of linearly polarized light, the distribution is symmetrical. “However, when the electrons are ejected by circularly polarized light, we find a distinct asymmetry to the angles at which the free electrons are found in relation to the laser beam,” reports Baumert. “This asymmetry is inverted if left circularly polarized light is used instead of right, an effect known as photoelectron circular dichroism. We observe the same effect when we keep the circular polarization the same but change from the “right handed” to the “left handed” structure of the chiral molecule being observed.” The researchers were able to demonstrate this with the chiral compounds camphor and fenchone.

“This circular dichroism effect has previously only been observed with synchrotron radiation. In contrast, our procedure uses a compact , so that this method is not limited to basic laboratory research but, because of the magnitude of the observed effects, may also find its way into analysis,” according to Baumert.

More information: Thomas Baumert, Circular Dichroism in the Photoelectron Angular Distributions of Camphor and Fenchone from Multiphoton Ionization with Femtosecond Laser Pulses, Angewandte Chemie International Edition, … ie.201109035

Provided by Wiley (news : web)

That caffeine in your drink -- is it really 'natural?'

Maik A. Jochmann, Ph.D., and colleagues point to the growing consumer preference for foods and beverages that contain only natural ingredients. Coffee, , colas, energy drinks and other caffeine-containing drinks are the most popular beverages in the world. Food regulatory agencies require that be listed on package labels, but do not require an indication of whether the caffeine is from natural or sources. The scientists set out to develop a faster, simpler method for categorizing caffeine's origins.

In the study, they describe use of a technique called stable-isotope analysis to differentiate between natural and synthetic caffeine. The test makes use of differences in the kinds of carbon isotopes – slight variations of the same element – found in caffeine made by plants and caffeine made in labs with petroleum-derived molecular building blocks. Their analysis, which takes as little as 15 minutes, found four products that contained synthetic caffeine, despite a "" label.

More information: Caffeine in Your Drink: Natural or Synthetic? Anal. Chem., Article ASAP. DOI: 10.1021/ac203197d

Owing to possible adulteration and health concerns, it is important to discriminate between natural and synthetic food ingredients. A new method for compound-specific isotope analysis (CSIA) by coupling high-temperature reversed-phase liquid chromatography to isotope ratio mass spectrometry (HT-RPLC/IRMS) was developed for discrimination of natural and synthetic caffeine contained in all types of drinks. The analytical parameters such as stationary phase, column inner diameter, and column temperature were optimized for the separation of caffeine directly from drinks (without extraction). On the basis of the carbon isotope analysis of 42 natural caffeine samples including coffee beans, tea leaves, guaraná powder, and maté leaves, and 20 synthetic caffeine samples from different sources by high-temperature reversed-phase liquid chromatography coupled to isotope ratio mass spectrometry, it is concluded that there are two distinguishable groups of caffeine ?13C-values: one between -25 and -32‰ for natural caffeine, and the other between -33 and -38‰ for synthetic caffeine. Isotope analysis by HT-RPLC/IRMS has been applied to identify the caffeine source in 38 drinks. Four mislabeled products were detected due to added but nonlabeled synthetic caffeine with ?13C-values lower than -33‰. This work is the first application of HT-RPLC/IRMS to real-world food samples, which showed several advantages: simple sample preparation (only dilution), high throughput, long-term column stability, and high precision of ?13C-value. Thus, HT-RPLC/IRMS can be a very promising tool in stable isotope analysis of nonvolatile compounds.

Provided by American Chemical Society (news : web)

Finding cancer cells in blood: Chip-based method for the rapid, sensitive isolation of rare cells in blood

The detection of is a difficult challenge because it requires the detection of quantities as low as one to ten per milliliter of blood—in the presence of large numbers of red blood cells and other cells. Conventional methods cannot manage this, but scientists led by Daniel T. Chiu have now developed a microfluidic system that allows for the analysis of 1 mL of blood within 20 minutes. The secret of their success is to virtually divide the sample into aliquots (portions) and to search these for the presence or absence of the desired cell types.

The blood is initially marked with fluorescent markers that specifically bind to the desired tumor cells. The sample is then passed through a system of microchannels, where it passes through a zone that is irradiated by a laser. The size of this zone determines the volume of the virtual aliquot; 2 nanoliters was found to work well. The laser causes the marker to fluoresce if marked cells are present. It can thus be determined whether or not an aliquot contains one (or more) of the desired cells. If the aliquot fluoresces, it is automatically pumped into a different channel than the fractions that do not fluoresce. The positive aliquots enter a filtration chamber. Red blood cells and the majority of blood cells pass through the filter; tumor cells are larger and are trapped. They can be counted on the filter, examined by microscope, or removed by micropipette for further examination. By using a second marker, certain subpopulations, such as tumor stem cells, can be identified.

Experiments with blood containing a known number of breast yielded a recovery rate of 93 % and a false positive rate of zero. Real blood samples from patients were also examined and the results compared with those from a clinically established system. The new microfluidic system proved to be significantly more sensitive. This new procedure has potential for monitoring treatment, aftercare, and the early detection of cancer.

More information: Daniel T. Chiu, Sensitive and High-Throughput Isolation of Rare Cells from Peripheral Blood with Ensemble-Decision Aliquot Ranking, Angewandte Chemie International Edition, … ie.201108695

Provided by Wiley (news : web)