Kevin Mayer Senor Editor Genetic Engineering & Biotechnology News
Protein Engineers Recognized for Advances in Rational Design and Directed Evolution
Science, like the film industry, has its award-giving bodies. And with a few notable exceptions, such as the Nobel Committee, scientifically oriented award-giving bodies hasten their celebrations of conspicuous achievement. A good thing, too, as many scientific disciplines are thick with talent that is not only deserving of recognition, but is also in immediate need of it, inasmuch as such recognition can build interest in research programs and help keep them going.
Recognition has not been a problem for the field of protein engineering this year. Protein engineering has been receiving special attention though a number of awards ceremonies and other publicity-friendly events. Although protein engineers will never be called “bankable,” a term reserved for the stars of Hollywood blockbusters, they may still leverage their honorifics to achieve something like career stability. Even better, they may secure the funding they need to see ambitious productions through to completion.
Rational Design versus Directed Evolution
Among protein engineering’s leading performers, methods vary as dramatically as acting techniques. At one extreme is rational design, which could be called the classical approach. It relies on a comprehensive understanding of the structure and function of existing proteins, and extrapolates from there, gauging how selected structural variations will lead to functional modifications.
Rational design, or de novo design, is a computationally intense approach, and it emphasizes the development of predictive rules for phenomena such as protein folding. These rules guide simulations that not only confirm the folding dynamics of existing and well-characterized proteins (the protein-folding problem), but also predict how novel proteins are likely to fold into three-dimensional structures (the inverse protein-folding problem).
In rational design, models loom large, as they do in classical acting, which is, according to Wikipedia, “based on the theories and systems of select classical actors and directors.” This kind of protein engineering is complemented by another approach, one called directed evolution, which is extreme in its own way. It shuns the calculated approach, the conscious emulation of existing protein structures and the systematic tweaking of these structures by means as tentative as site-directed mutagenesis. No, directed evolution, like the method approach to acting, is a bold shortcut to the heart of the matter.
In directed evolution, mutagenesis is not site directed, but random. And, through random mutagenesis—actually multiple rounds of mutagenesis—many protein variants are generated, most of which are deleterious. A few of the variants, however, may exhibit even higher degrees of fitness than rationally designed proteins. These exceptional proteins, however, must be identified somehow. Typically, the isolation of the fit (if poorly understood) variants depends on some sort of screening or selection mechanism.
Overall, directed evolution mimics natural evolution. Like the “method” approach to acting, directed evolution may seem a strenuous and even painful way to achieve something that has the ring of authenticity. Directed evolution, however, takes advantage of robotics (to implement iterative cycles of random mutagenesis) and high-throughput screening technology (to detect fitness differences among numerous protein variants).
Rational design and directed evolution are not mutually exclusive approaches to protein engineering. Yet one approach or the other tends to predominate in the work of individual researchers. At least, that appears to be the case among the researchers who have been receiving accolades of late.
The Magic “If”
In one “method” approach, actors are encouraged to ask multiple “what if” questions of their characters and themselves to flesh out motivations and arrive at scenic truths. Seemingly inconsequential questions may end up being profound. Because the ultimate importance of any particular question is impossible to predict, the method may seem haphazard, but there is still a method to the, um, method.
A similar dynamic holds in directed evolution, if we accept the comments shared by Frances H. Arnold, Ph.D., a professor of chemical engineering, bioengineering, and biochemistry at the California Institute of Technology and the winner of the 2016 Millennium Technology Prize. Upon winning the Millennium Technology Prize, Dr. Arnold said that directed evolution “allows us to circumvent our inability to explain how mutations affect protein behavior, much less to predict beneficial ones.”
“The most beautiful, complex, and functional objects on the planet have been made by evolution,” she continued. “We can now use evolution to make things that no human knows how to design. Evolution is the most powerful engineering method in the world, and we should make use of it to find new biological solutions to problems.”
Dr. Arnold’s innovations have revolutionized the slow and costly process of protein modification, and today her methods are being used in hundreds of laboratories and companies around the world. Modified proteins are used to replace processes that are expensive or that utilize fossil raw materials in the production of fuels, paper products, pharmaceuticals, textiles, and agricultural chemicals.
Because Dr. Arnold’s work emphasizes green technology, it is a good fit for the Millennium Technology Prize, a Finnish prize that is awarded each year in recognition of innovators of technologies that promote sustainable development and a better quality of life.
Dr. Arnold’s research group is pursuing several projects that harness the power of directed evolution or feature the recombination of protein blocks to arrive at to simplified (additive) sequence–function relationships. Current projects include opsin engineering, structure-guided protein recombination, enzyme engineering for fuels and chemicals, expanding the chemistry of the flavocytochrome P450 BM3 through directed evolution, and engineering P450 BM3 for noninvasive imaging of neurotransmitters.
Tops in Their Respective Categories
The Protein Society, which calls itself the premiere international society dedicated to supporting protein research, announced the winners of the 2016 Protein Society Awards back in July. Among the winners were researchers representing both kinds of protein engineering, both rational design and directed evolution.
The winner of the Protein Society’s Christian B. Anfinsen Award, which recognizes “technological achievement or significant methodological advances in the field of protein science,” was awarded to Andreas Plückthun, Ph.D., director of the department of biochemistry at the University of Zurich. To achieve this distinction, Dr. Plückthun combined rigorous biophysical studies with the invention of new combinatorial and evolutionary technologies, which he then applied to the production of synthetic antibodies and G-protein-coupled receptors.
Dr. Plückthun’s laboratory combined the Escherichia coli platform with ribosome display, a method of cell-free selection and evolution from very large libraries of whole proteins, to create a true in vitro protein evolution technology. Over the last few years, this technology has facilitated the development of repeat proteins as an alternative to antibodies.
“By using consensus engineering, libraries of repeat proteins have been designed from which specific, high-affinity binding proteins can be selected,” notes the laboratory’s website. “Most of our work so far has concentrated on designed ankyrin repeat proteins (DARPins).” These antibody mimetic proteins “are very stable and do not have disulfide bonds and therefore, unlike most antibodies, also work inside the cell. They can be prepared in very large amounts from E. coli, and show affinities up to the picomolar range.”
The directed evolution technology has also led to highly stable G-protein-coupled receptors that can be used for structural studies and in drug screening. Several engineered therapeutics, developed on the basis of his research, are now in late-phase clinical development.
The winner of the Protein Society’s Stein and Moore Award, reserved for “leaders in protein science who have made sustained, high-impact research contributions to the field,” was Jane Clarke, Ph.D., a professor of molecular biophysics at the University of Cambridge. Her research is multidisciplinary, combining single-molecule and ensemble biophysical techniques with protein engineering and simulations to investigate protein folding, misfolding, and assembly.
Dr. Clarke’s group studies families of proteins using a multidisciplinary approach, to shed light on the folding of related proteins, the folding of multidomain proteins, and intrinsically disordered proteins (IDPs). For example, Dr. Clarke’s group compares the folding of a number of related proteins from large structural families to advance her investigations of the relationship between amino acid sequence and topology and protein stability.
Most proteins, her group points out, consist of a number of independently folding domains. Consequently, pertinent questions include the following: How do domain:domain interactions modulate the properties of the protein? How do larger, multidomain proteins avoid misfolding?
Finally, the many proteins that have large disordered segments are often involved in important signaling pathways. IDPs fold upon binding to a target. Going forward, Dr. Clarke’s group will develop tools to study globular protein folding and investigate IDP folding mechanisms.
Film lore has it that Dustin Hoffman, true to his “method” ways, prepared to play a sleep-deprived character by actually neglecting to sleep. Hoffman’s co-star, the more classically oriented Laurence Olivier, was moved to ask, “Why not try acting? It’s much easier.”
If the story is true, the question was probably asked in jest, since classical acting, despite appearances, might actually require more work than method acting. (According to Wikipedia, classical acting integrates “the expression of the body, voice, imagination, personalizing, improvisation, external stimuli, and script analysis.”) Similarly, rational design may be more effortful than directed evolution. It’s just that in rational design, much of the work is carried out by a computer. In fact, there can be so much work that even the most powerful computers can be overtasked.
Computers may be used to calculate all the atomic-level attractions and repulsions between a protein’s constituent parts, as well as between the protein’s constituent parts and the water molecules in the protein’s vicinity. All the pushes and pulls, all the deflections and rebounds, and all the stretches and contractions, in aggregate, account for how a protein can start as an extruded sequence of linked amino acids and end as a warped, coiled, and folded three-dimensional structure, fit for a particular role.
This solution to the “protein-folding problem,” however, is itself a problem. It is a brute-force approach for which no amount of available computational power may suffice to see it through—at least not until computers become considerably more powerful, or until the experimental data that is incorporated into computer models becomes much more finely grained, or both.
Rather than wait for such improvements, rational design is looking for computational shortcuts. Actually, it has already found one big shortcut, one that bypasses structural possibilities that are inconsistent with the co-evolution of amino acid pairs. This sort of co-evolution became apparent only after high-quality genome sequence data started to became available, making it possible to identify amino acid pairs that are distant from each other when the protein is unfolded, but are near to each other when the protein is in its active, three-dimensional form.
Amino acid co-evolution has been exploited in the work of David A. Baker, Ph.D., director of the Institute for Protein Design at the University of Washington. Dr. Baker might qualify as the Laurence Olivier of protein engineering since he is, in addition to being classically oriented, in possession of a mantle full of awards. Dr. Baker received young investigator awards from the National Science Foundation and the Beckman Foundation. He has also received and the Packard Foundation fellowship in Science and Engineering, the Irving Sigal Young Investigator award from the Protein Society, and the Overton Prize from the International Society of Computational Biology. He is a recipient of the Feynman Prize from the Foresight Institute, the AAAS Newcomb Cleveland prize, the Sackler prize in biophysics, and the Centenary award from the Biochemical Society.
His most recent distinction? His laboratory’s work was recognized on the cover of the July 22 issue of Science magazine. This issue included a feature, “Rules of the Game,” which suggested that Dr. Baker’s team had all but solved the protein-folding problem, and a report, “Accurate Design of Megadalton-Scale Two-Component Icosahedral Protein Complexes,” which described how Dr. Baker’s team designed and assembled large protein nanocages that are similar to viral capsules and are, potentially, capable of serving as drug delivery platforms.
The Science feature indicated that Dr. Baker looks forward to going beyond “a strategy he refers to as ‘Neandertal protein design,’ tweaking the genes for existing proteins to get them to do new things.” He was quoted directly as follows: “We were limited by what existed in nature. … We can now short-cut evolution and design proteins to solve modern-day problems.”
Dr. Baker expressed similar sentiments in his commentary on his team’s nanocage work: “As we pursue applications, we can design protein structures specifically for those applications, [without having to deal with] a lot of evolutionary baggage, of things evolving for one reason and then trying to adapt them for a new purpose.” In addition to pursuing applications, future work, he said, will focus on designing “more dynamic structures that undergo structural transitions in response to environmental changes.”