Nanotechnology milestone: general method for designing stable proteins

ORIGINAL: Foresight Institute

Comparison of computational models with experimentally determined structure: design model (left) and NMR structure (right). Credit: Nobuyasu Koga et al./Nature)

Yet another milestone along the protein design molecular engineering path to advanced nanotechnology has been reached, thanks to the efforts of the laboratory of David Baker, one of the 2004 winners of the Foresight Feynman Prize in Nanotechnology for Theoretical work. From KurzweilAI “How to design proteins from scratch“:

… By following a set of rules, they designed five proteins from scratch that fold reliably into predicted conformations. In a blind test, the team showed that the synthesized proteins closely match the predicted structures.

“What you have now is a flexible set of building blocks for nanoscale assembly,” says Jeremy England, a molecular biophysicist at the Massachusetts Institute of Technology in Cambridge, who was not involved in the work. …

More detail is given in a commentary in Nature, the journal in which the research was published “Proteins made to order: Researchers design proteins from scratch with predictable structures“:.

… Baker’s proteins are in a sense “platonic ideals”, he says: simple backbone constructs with every amino acid optimized to fold into the prescribed, stable structure. In this way they differ from natural proteins, whose folded structures represent a compromise between the competing requirements of optimum folding and biological function, leading to “frustrated” parts of the sequence that may be essential for function but are destabilizing to the fold. As evidence of their stability, the designed proteins melt at about 100 °C, Koga says, compared to 40–50 °C for a natural protein. …

The best summary of the significance of this result is given by the authors in the conclusion of their paper (reference numbers omitted from quotation):

The design principles and methodology we have described should allow the ready design of a wide range of robust and stable protein building blocks for the next generation of engineered functional proteins. Almost all protein design and engineering efforts so far have repurposed naturally occurring proteins that evolved for some other, often unrelated, function. It should now become possible to custom-design protein scaffolds ideal for the desired function, and to build larger assemblies and materials from robust ideal building blocks.

From the standpoint of advanced nanotechnology/molecular manufacturing, the “build larger assemblies” part is especially interesting. The abstract of the research is available on the journal web site, and the authors have made a full text PDF available on the Baker lab web site.

—James Lewis, PhD

This entry was posted on Wednesday, November 21st, 2012 at 1:13 PM and is filed under Atomically Precise Manufacturing (APM), Bionanotechnology, Computational nanotechnology, Molecular Nanotechnology, Molecular manufacturing, Nano, Nanobiotechnology, Nanotech, Nanotechnology, Productive Nanosystems, Research. 

New online game to design RNA molecules: advancing nanotechnology?

ORIGINAL: www.foresight.org

(Credit: EteRNA)

As we pointed out a few months ago, the greater complexity of folding rules for RNA compared to its chemical cousin DNA gives RNA a greater variety of compact, three-dimensional shapes and a different set of potential functions than is the case with DNA, and this gives RNA nanotechnology a different set of advantages compared to DNA nanotechnology as a road towards atomically precise manufacturing. Proteins have even more complex folding rules and an even greater variety of structures and functions. We also noted here that online gamers playing Foldit topped scientists in redesigning a protein to achieve a novel enzymatic activity that might be especially useful in developing molecular building blocks for molecular manufacturing. Now KurzweilAI.net brings news of an online game that allows players to design RNA molecules “New videogame lets amateur researchers mess with RNA“.

EteRNA, an online game with more than 38,000 registered users, allows players to design molecules of ribonucleic acid — RNA — that have the power to build proteins or regulate genes.

EteRNA players manipulate nucleotides, the fundamental building blocks of RNA, to coax molecules into shapes specified by the game.

Those shapes represent how RNA appears in nature while it goes about its work as one of life’s most essential ingredients.

EteRNA was developed by scientists at Stanford and Carnegie Mellon universities, who use the designs created by players to decipher how real RNA works. The game is a direct descendant of Foldit — another science crowdsourcing tool disguised as entertainment — which gets players to help figure out the folding structures of proteins.

The game’s elite players compete for a unique and wondrous prize: the chance to have RNA designs of their own making brought to life. Every two weeks, four to 16 player-designed molecules are picked to be synthesized in an RNA lab at Stanford.

The chance to win this reward has proven highly motivating for EteRNA‘s players. They carefully study the data that the lab provides on how the synthesized molecules behave when ushered into existence, then use their observations to refine their next designs. In doing so, they — like their Foldit-playing peers — have helped scientists take advantage of the human brain’s unparalleled talent for recognizing patterns and solving puzzles.

But EteRNA players have also done something much more profound: By scrutinizing their creations, learning from their triumphs and mistakes, and using their accumulated wisdom to develop new hypotheses, they aren’t just building better RNA molecules; they’re discovering fundamental aspects of biochemistry that no one — not even the world’s top RNA researchers — knew before. And in doing so, they are blurring the line that separates gamer from scientist …

The article goes on to discuss a growing movement among EteRNA players to synthesize their RNA molecules themselves, linking online scientific game-playing and crowd-sourced molecular design to Open Science and DIY Biotechnology. The EteRNA web site provides tutorials to get new users started and instant feedback. Could games like Foldit and EteRNA represent new crowd-sourced paths to the more rapid development of atomically precise manufacturing?

—James Lewis, PhD

An expanded genetic alphabet could lead to more easily designed proteins

ORIGINAL: Foresight Institute

Floyd E. Romesberg, associate professor at Scripps Research Institute (Credit: The Scripps Research Institute)

Back in 1992, in chapter 15 of Nanosystems, Eric Drexler suggested that it would be easier to design proteins that fold predictably, an important step on the road to advanced nanotechnology (or molecular manufacturing, or atomically precise manufacturing) if additional amino acids beyond the 20 that are genetically coded could be incorporated into proteins. Chemical synthesis of peptides has provided a way to accomplish this for small amounts of short proteins, but to obtain large amounts of long proteins, it would be very convenient to expand the genetic alphabet to encode additional amino acids. This long-standing effort has taken a major step forward with the discovery of how artificial DNA base pairs can be replicated. A hat tip to ScienceDaily for reprinting this Scripps Research Institute news release “Scripps Research Institute study suggests expanding the genetic alphabet may be easier than previously thought“:

A new study led by scientists at The Scripps Research Institute suggests that the replication process for DNA—the genetic instructions for living organisms that is composed of four bases (C, G, A and T)—is more open to unnatural letters than had previously been thought. An expanded “DNA alphabet” could carry more information than natural DNA, potentially coding for a much wider range of molecules and enabling a variety of powerful applications, from precise molecular probes and nanomachines to useful new life forms.

The new study, which appears in the June 3, 2012 issue of Nature Chemical Biology [abstract], solves the mystery of how a previously identified pair of artificial DNA bases can go through the DNA replication process almost as efficiently as the four natural bases.

“We now know that the efficient replication of our unnatural base pair isn’t a fluke, and also that the replication process is more flexible than had been assumed,” said Floyd E. Romesberg, associate professor at Scripps Research, principal developer of the new DNA bases, and a senior author of the new study. The Romesberg laboratory collaborated on the new study with the laboratory of co-senior author Andreas Marx at the University of Konstanz in Germany, and the laboratory of Tammy J. Dwyer at the University of San Diego.

Adding to the DNA Alphabet

Romesberg and his lab have been trying to find a way to extend the DNA alphabet since the late 1990s. In 2008, they developed the efficiently replicating bases NaM and 5SICS, which come together as a complementary base pair within the DNA helix, much as, in normal DNA, the base adenine (A) pairs with thymine (T), and cytosine (C) pairs with guanine (G).

The following year, Romesberg and colleagues showed that NaM and 5SICS could be efficiently transcribed into RNA in the lab dish. But these bases’ success in mimicking the functionality of natural bases was a bit mysterious. They had been found simply by screening thousands of synthetic nucleotide-like molecules for the ones that were replicated most efficiently. And it had been clear immediately that their chemical structures lack the ability to form the hydrogen bonds that join natural base pairs in DNA. Such bonds had been thought to be an absolute requirement for successful DNA replication-—a process in which a large enzyme, DNA polymerase, moves along a single, unwrapped DNA strand and stitches together the opposing strand, one complementary base at a time.

An early structural study of a very similar base pair in double-helix DNA added to Romesberg’s concerns. The data strongly suggested that NaM and 5SICS do not even approximate the edge-to-edge geometry of natural base pairs—termed the Watson-Crick geometry, after the co-discoverers of the DNA double-helix. Instead, they join in a looser, overlapping, “intercalated” fashion. “Their pairing resembles a ‘mispair,’ such as two identical bases together, which normally wouldn’t be recognized as a valid base pair by the DNA polymerase,” said Denis Malyshev, a graduate student in Romesberg’s lab who was lead author along with Karin Betz of Marx’s lab.

Yet in test after test, the NaM-5SICS pair was efficiently replicable. “We wondered whether we were somehow tricking the DNA polymerase into recognizing it,” said Romesberg. “I didn’t want to pursue the development of applications until we had a clearer picture of what was going on during replication.”

Edge to Edge

To get that clearer picture, Romesberg and his lab turned to Dwyer’s and Marx’s laboratories, which have expertise in finding the atomic structures of DNA in complex with DNA polymerase. Their structural data showed plainly that the NaM-5SICS pair maintain an abnormal, intercalated structure within double-helix DNA—but remarkably adopt the normal, edge-to-edge, “Watson-Crick” positioning when gripped by the polymerase during the crucial moments of DNA replication.

“The DNA polymerase apparently induces this unnatural base pair to form a structure that’s virtually indistinguishable from that of a natural base pair,” said Malyshev.

NaM and 5SICS, lacking hydrogen bonds, are held together in the DNA double-helix by “hydrophobic” forces, which cause certain molecular structures (like those found in oil) to be repelled by water molecules, and thus to cling together in a watery medium. “It’s very possible that these hydrophobic forces have characteristics that enable the flexibility and thus the replicability of the NaM-5SICS base pair,” said Romesberg. “Certainly if their aberrant structure in the double helix were held together by more rigid covalent bonds, they wouldn’t have been able to pop into the correct structure during DNA replication.”

An Arbitrary Choice?

The finding suggests that NaM-5SICS and potentially other, hydrophobically bound base pairs could some day be used to extend the DNA alphabet. It also hints that Evolution’s choice of the existing four-letter DNA alphabet—on this planet—may have been somewhat arbitrary. “It seems that life could have been based on many other genetic systems,” said Romesberg.

He and his laboratory colleagues are now trying to optimize the basic functionality of NaM and 5SICS, and to show that these new bases can work alongside natural bases in the DNA of a living cell.

“If we can get this new base pair to replicate with high efficiency and fidelity in vivo, we’ll have a semi-synthetic organism,” Romesberg said. “The things that one could do with that are pretty mind blowing.”

Now that these scientists have demonstrated that DNA replication is far more flexible than had been thought, it will be fascinating to see what researchers do to expand the genetic alphabet, what additional amino acids they choose to incorporate into what proteins, and what this expansion of the set of amino acids composing proteins means for predicable protein folding and for artificial protein molecular machines.

—James Lewis, PhD

Computational analysis of scattered images brings atomic resolution to electron microscopy

ORIGINAL: The Foresight Institute, Nature

The full field-of-view is shown in the inset image (scale bar, 15 nm); the main image is a blow up of the region indicated by the yellow box, showing 0.236 nm atomic plane fringes (scale bar, 5 nm). The modulus and phase of the reconstructions are combined in these images, with phase represented by colour and modulus by brightness, as indicated on the colour wheel scale. Nature

Courtesy of The University of Sheffield,
project leader Professor John Rodenburg,
of the University of Sheffield´s Department
of Electronic and Electrical Engineering

Although not visible at this magnification, the high resolution image in the open access research publication clearly shows the gold atoms, with a spacing of 0.236 nm between atomic planes.

In his famous visionary 1959 talk in which he described molecular machines building with atomic precision, Feynman suggested that if physicists wanted to help biologists, they should improve the electron microscope by a hundred times to see individual atoms. Many improvements have been made over the years, such as aberration-correcting electron lenses, but a recent contribution from the University of Sheffield has succeeded in showing for the first time that it is possible to recover the complex exit wave from a diffraction image at atomic resolution, over a wide field of view, and using low-energy electrons. A hat tip to ScienceDaily for reprinting this University of Sheffield news release “Scientists revolutionise electron microscope“:

For over 70 years, transmission electron microscopy (TEM), which `looks through´ an object to see atomic features within it, has been constrained by the relatively poor lenses which are used to form the image.

The new method, called electron ptychography, dispenses with the lens and instead forms the image by reconstructing the scattered electron-waves after they have passed through the sample using computers.

Scientists involved in the scheme consider their findings to be a `first step´ in a `completely new epoch of electron imaging´. The process has no fundamental experimental boundaries and it is thought it will transform sub-atomic scale transmission imaging.

Experiments were carried out on an FEI Quanta 600 SEM fitted with a thermally assisted Schottky field emission gun and operating at 30 keV. The probe wavefront was formed using the microscope condenser and objective lenses and was scanned across the specimen using the microscope scanning coils. The specimen was mounted on a compact rig attached to the objective lens pole piece assembly. The door of the microscope was replaced in order to accommodate a flange for a Gatan Orius SC200 CDD camera that was cantilevered into a position below the specimen plane. Nature

Project leader Professor John Rodenburg, of the University of Sheffield´s Department of Electronic and Electrical Engineering, said: “To understand how material behaves, we need to know exactly where the atoms are. This approach will enable us to look at how atoms sit next to one another in a solid object as if we´re holding them in our hands.

“We´ve shown we can improve upon the resolution limit of an electron lens by a factor of five. An extension of the same method should reach the highest resolution transmission image ever obtained; about one tenth of an atomic diameter. No longer does TEM have to be bound by the paradigm of the lens, its Achilles´ heel since its invention in 1933.” …

Professor Rodenburg added: “We measure diffraction patterns rather than images. What we record is equivalent to the strength of the electron, X-ray or light waves which have been scattered by the object – this is called their intensity. However, to make an image, we need to know when the peaks and troughs of the waves arrive at the detector – this is called their phase.

“The key breakthrough has been to develop a way to calculate the phase of the waves from their intensity alone. Once we have this, we can work out backwards what the waves were scattered from: that is, we can form an aberration-free image of the object, which is much better than can be achieved with a normal lens.

“A typical electron or X-ray microscope image is about one hundred times more blurred than the theoretical limit defined by the wavelength. In this project, the eventual aim is to get the best-ever pictures of individual atoms in any structure seen within a three-dimensional object.” …

The research was published in Nature Communications as an open access article “Ptychographic electron microscopy using high-angle dark-field scattering for sub-nanometre resolution imaging“. Although the image in the press release (above) of the gold particles is too low a magnification to see the rows of atoms, the high resolution version of the image in the research paper clearly shows the gold atoms, with a spacing of 0.236 nm between atomic planes. Additional information is available on the authors’ project web site “Welcome to the ΠΦ project“.

—James Lewis