Protein Acceleration Become Easy !

All living things require proteins, members of a vast family of molecules that nature "makes to order" according to the blueprints in DNA. Through the natural process of evolution, DNA mutations generate new or more effective proteins.

Humans have found so many alternative uses for these molecules - as foods, industrial enzymes, anti-cancer drugs - that scientists are eager to better understand how to engineer protein variants designed for specific uses.

Now Stanford engineers have invented a technique to dramatically accelerate protein evolution for this purpose. This technology, described in Nature Chemical Biology, allows researchers to test millions of variants of a given protein, choose the best for some task and determine the DNA sequence that creates this variant.

"Evolution, the survival of the fittest, takes place over a span of thousands of years, but we can now direct proteins to evolve in hours or days," said Jennifer Cochran, a professor of bioengineering who co-authored the paper with Thomas Baer, director of the Stanford Photonics Research Center.

 "This is a practical, versatile system with broad applications that researchers will find easy to use," Baer said. 

By combining Cochran's protein engineering knowhow with Baer's expertise in laser-based instrumentation, the team created a tool that can test millions of protein variants in a matter of hours.

"The demonstrations are impressive and I look forward to seeing this technology more widely adopted," said Frances Arnold, a professor of chemical engineering at Caltech who was not affiliated with the study. Making a million mutants The researchers call their tool µSCALE, or Single Cell Analysis and Laser Extraction.

The "µ" stands for the microcapillary glass slide that holds the protein samples. The slide is roughly the size and thickness of a penny, yet in that space a million capillary tubes are arrayed like straws, open on the top and bottom.

1 / 3 The microcapillary glass slide that holds the protein samples is roughly the size and thickness of a penny, yet in that space a million capillary tubes are arrayed like straws. Credit: Cochran Lab, Stanford The power of µSCALE is how it enables researchers to build upon current biochemical techniques to run a million protein experiments simultaneously, then extract and further analyze the most promising results.

The researchers first employ a process termed "mutagenesis" to create random variations in a specific gene. These mutations are inserted into batches of yeast or bacterial cells, which express the altered gene and produce millions of random protein variants. A µSCALE user mixes millions of tiny opaque glass beads into a sample containing millions of yeast or bacteria and spreads the mixture on a microcapillary slide.

Tiny amounts of fluid trickle into each tube, carrying individual cells. Surface tension traps the liquid and the cell in each capillary. The slide bearing these million yeast or bacteria, and the protein variants they produce, is inserted into the µSCALE device. A software-controlled microscope peers into each capillary and takes images of the biochemical reaction occurring therein. Once a µSCALE user identifies a capillary of interest, the researcher can direct the laser to extract the contents of that tube without disrupting its neighbors, using an ingenious method devised by Baer. "The beads are what enable extraction," Baer said.

"The laser supplies energy to move the beads, which breaks the surface tension and releases the sample from the capillary." Thus µSCALE empties the contents of a single capillary onto a collector plate, where the DNA of the isolated cell can be sequenced and the gene variant responsible for the protein of interest can be identified.

More information: http://phys.org/news/2015-12-protein-evolution.html

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Oil and water: An icy interaction when oil chains are short, steamy when chains are long

Water transforms into a previously unknown structure in between a liquid and a vapor when in contact with alcohol molecules containing long oily chains, according to Purdue University researchers. However, around short oily chains water is more icelike.




Water plays a huge role in biological processes, from protein folding to membrane formation, and it could be that this transformation is useful in a way not yet understood, said Dor Ben-Amotz, the professor of chemistry who led the research.

Ben-Amotz's research team found that as they examined alcohols with increasingly long carbon chains, the transformation occurred at lower and lower temperatures.

When in contact with a chain seven carbon atoms long, the water molecules became much looser and more vaporlike at a temperature of 140 degrees Fahrenheit, which is about halfway between the melting and boiling points.

"For oils with chains longer than four carbons, or about one nanometer in length, we saw the water transform into a completely new structure as the temperature rose," Ben-Amotz said. "If the trend we saw holds true, then this transformation could be happening at body temperature around important physiological molecules like proteins and phospholipids.Water responds very sensitively in its structure to small changes, he said.

"Water's versatility is what makes it so special," he said. "For instance, the surfaces of proteins have both oily and charged regions; and water changes itself to accommodate these very different components and everything in between. We are learning more about exactly how it does this."

The researchers found that water molecules interacting with the oil always formed a more ordered, icelike structure at lower temperatures, while the bulk of the water remained liquid. This ice-like structure would melt away as the temperatures increased and in longer molecules a new structure would appear, he said.

A paper detailing the National Science Foundation-funded work is published in the current issue of Nature and is also highlighted in a news and views article in the same issue. In addition to Ben-Amotz, co-authors include Purdue graduate student Joel Davis and postdoctoral fellows Kamil Gierszal and Ping Wang.

The team's observations add to a more than 70-year debate over the interaction of oil and water, with some studies suggesting that water forms little icebergs around the oil molecules, while others point to a more disordered, vaporlike water structure.

 

"This question was really up for grabs until we introduced an experimental method that could see these subtle changes in water structure," Ben-Amotz said. "Surprisingly, we found that both sides are right, and it depends on the size of the oil."

The challenge of the experiment was that the team needed to see the very small number of water molecules that are in contact with the oil chains in the presence of a very large number of other water molecules.

The team combined Raman scattering and multivariate curve resolution to create an analysis method capable of managing an unprecedented signal-to-noise ratio of 10,000-to-1.

"Most people never take a spectrum with a signal-to-noise ratio greater than 100-to-1, but if we performed this experiment that way we wouldn't see anything," Ben-Amotz said. "We needed to have a higher signal-to-noise ratio because we were looking for a needle in a mountain-sized haystack."

Raman scattering involves shooting a beam of light containing photons into a sample. As the photons hit molecules within the sample, they lose or gain energy. Such measurements create a spectrum of peaks that reveal the vibrational motions of the molecules present in the sample. Shifts in the peaks' shapes can show changes in the strength of bonds between water molecules and whether the molecules are becoming more or less ordered.

"With Raman scattering the bulk of the water creates a mountainous peak in the spectrum that buries everything else," Ben-Amotz said. "Multivariate curve resolution lets us see small changes in water structure under that mountain. As is often the case in science, the key was combining two already established techniques in a new way."

Davis said the team next plans to explore the effects of changes in pH and ionic charges on this transformation with the goal of making the experiments more relevant to proteins and biological systems.

"We are trying to better understand the driving forces of the behavior of proteins and cell membranes that are critical to our health," he said. "The role of water is an important piece of the puzzle."

Note: This story has been adapted from a news release issued by the Purdue University
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Protein coat prepares catalyst for cascades

By protecting a transition metal catalyst with a protein coat, scientists have managed to couple up biocatalysts and chemical catalysts to perform a cascade reaction.

When students and post-docs moved from Nick Turner’s lab at the University of Manchester, UK, to Tom Ward’s at the University of Basel, Switzerland, it prompted an intriguing collaboration between a group with experience with naturally occurring enzymes and Ward’s group, which creates artificial metalloenzymes by combining transition metal catalysts with proteins. The result was a way of combining traditional enzyme biocatalysts with chemical catalysts to perform reaction cascades.

In our cells, enzymes happily react with a substrate and then pass the product on to another enzyme to perform the next bit of chemistry. Unfortunately for chemists, just introducing a transition metal catalyst into one of these cascades doesn’t work, the catalyst and the enzyme tend to inactivate each other. Instead, to introduce new chemistry, naturally occurring enzymes have to be modified. Or at least that used to be the only option.

Ward’s group has been working on making artificial metalloenzymes by sitting traditional transition metal catalysts inside proteins, creating catalysts that can perform chemistry traditionally unavailable to enzymes. He says it was the transfer of staff from Turner’s lab that led to the next step of combining the artificial metalloenzymes with naturally occurring enzymes.

To test the hypothesis, the group decided to try and use an enzyme Turner was familiar with, monoamine oxidase (MAO), to synthesis enantiopure amines. MAO is a highly selective oxidase which, in collaboration with a reducing agent or catalyst, converts racemic amines just one enantiomer. Usually the catalyst used is heterogeneous, it’s solid and used after the MAO. An artificial metalloenzyme would be homogeneous and allow the reaction to proceed without any intervention between the two steps.

A streptavidin coat protects the metal catalyst and enzyme from each other, so both can function in tandem © NPG

The team created their artificial transfer hydrogenase (ATH) by shielding an iridium containing reduction catalyst with a streptavidin protein. Using just the MAO and the iridium catalyst resulted in nothing, but when the ATH was used instead, the team saw the starts of a cascade. Modifying and improving the enzymes eventually led to conversion efficiencies for some reactions of 99%, with similar enantiomeric excesses.

Corinna Hess from Durham University, UK, says that this work ‘nicely illustrates that artificial enzymes aren’t merely a biochemical novelty, and demonstrates their great promise for catalysis.’ Before adding that ‘the results are highly encouraging for the use of artificial enzymes in industrial biotransformations, and many inorganic motifs still remain to be explored.’

However, for Ward and Turner, improving industrial chemistry is not the ultimate goal. ‘There are better ways of doing that chemistry,’ explains Ward. Instead, the team see their work as a starting point towards introducing inorganic chemistry into cells. 
RSC

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