SCIENCE & TECHNOLOGY
PROTEIN CONTROL AND MODIFICATION At 20th Protein Society symposium, researchers show how to poke, perturb, and redesign proteins STU BORMAN, C&EN WASHINGTON
Francis of the University ofCalifornia, Berkeley. He and coworkers recently developed a new reaction that introduces a single reactive ketone or aldehyde at the N-terminus of a protein when the protein is mixed with pyridoxal phosphate (Angew. Chem. Int. Ed. 2006,45,5307). "It's a really mild reaction, it's easy to carry out, and the reactive group can be further derivatized using aldehydeor ketone-specific reactions, such as oxime formation," Francis said. The researchers also recently developed a palladium-catalyzed allylic alkylation that attaches long lipid tails to proteins, a process that can be used to customize the solubility of enzymes, antibodies, viral capsids, and other proteins (J. Am. Chem. Soc. 2006,128,1080).
ize proteins "was almost a pipe dream." But for the Protein Society, an organi these techniques are now beginning to see more routine use by researchers who are not zation of protein scientists that's particularly knowledgeable about protein headquartered in Bethesda, Md, but has members and meetings all synthesis but nevertheless want to use it to over the world. The year was unique in that it study their own favorite systems. marked the 20th anniversary of die society's MODIFYING PROTEINS with exquisite con Muir and coworkers carry out their own annual symposium, which alternates between studies by introducing targeted changes into trol can also be accomplished by coaxing Boston and San Diego and was held in the peptide fragments and then combining the cells into incorporating nonnatural amino latter location last month. fragments into novel proteins. They recently acids into their proteins. "For 3 billion or What was not unique this year was the ex used such an approach to make proteins that 4 billion years, life on this planet has been traordinarily broad range ofprotein research contain nonnatural amino acids and probes, constrained by a 20-amino-acid code," said which permitted them to track movements presented at the meeting, because that Peter G. Schultz, director of the Genomics kind of breadth is typical of Institute of the Novartis Protein Society symposia. | Research Foundation, La ΗγΟ Topics of meeting sessions | Jolla, Calif. "I think we can 2 -O 3 PO-^AS^ O H ranged from synthesis of ;= safely say that that restricproteins with precise modi 1 tion is gone. Now it's up to Β * ΜΓ Λ M fications, optimizing protein 2 the chemist's imagination, Pyridoxal phosphate • f drugs, and one-molecule£ and one can think about at-a-time protein sensing to § fashioning proteins and 0 ^ t designing proteins from the maybe whole organisms Myoglobin bottom up, analyzing the dy that are not limited by the namics of protein function, existing code." CH30>^0>\/ONH2 CH3CK//\0>^0 and characterizing protein In work carried out at η 7? aggregates. Scripps Research Institute, where Schultz is a professor Dramatic advances are THE TERMINALATOR A reactive group of chemistry, he and his cooccurring in these and oth derived from the N-terminus of a protein can 0 % workers developed a mutaer areas of protein science. be used as a chemical handle for attaching genesis method in which a In the synthetic realm, for interesting compounds to the end of the protein, such as the transfer RNA (tRNA) and example, "there have truly polymer shown. tRNA synthetase are gebeen enormous strides made netically engineered to bring a nonnatural in the area of bringing tools of organic of the proteins between different parts of amino acid into a protein at a single site. chemistry to proteins," said protein chemist living cells. "We used a photo-triggering Schultz noted that the group has already Tom W. Muir of Rockefeller University, in system to follow the kinetics of movement used this type of approach to expand the New York City, who chaired a session on the of two différent forms of a signaling protein genetic code by over 35 nonnatural amino exquisitely controlled synthesis of proteins. simultaneously, one phosphorylated and one acids. "I don't think there are a whole lot of Two key goals of synthesizing proteins with not, and showed that one goes faster than constraints left right now" in the types of precise modifications are to study how they another," Muir said. "That's sort of state customized amino acids that can be incorwork and to develop tailored versions with of the art in terms of what you can do to a porated into proteins, he said. Ambrx, a La new functions. protein with synthesis." Muir noted that when he entered the field Another way to leverage organic diemistiy Jolla-based company Schultz cofounded, is of synthetic protein chemistry in the early to create bespoke proteins was described by further developing the technology and plans to use it commercially. 1990s, using organic reactions to custom assistant professor of chemistry Matthew B.
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"Why design proteins? We want to be able to build onp modify, improve, and change these wonderful nanoscale machines that nature has made." WWW.CEN-0NLINE.ORG
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SCIENCE & TECHNOLOGY Caltech, who chaired the session. "Over techniques his group developed earlier this At California Institute of Technology, 200 modifications have been identified so year for observing gene expression in living professor of chemistry and chemical enfar, and we're just beginning to understand cells in real time on a one-molecule-at-agineering David A. Tirrell and coworkers time basis {Science 2006,311,1600; Nature their biological roles." also specialize in nonnatural amino acid 2 0 0 6 , 4 4 0 , 358). A dramatic movie the modifications. "Until a couple of years ago, Hsieh-Wilson discussed her group's regroup created with one of the techniques the field was largely a curiosity,,, Tirrell said. cent discovery that glycosylation of CREB shows yellow fluorescent "But Pete's lab, my group, and others have (cyclic AMP-response flashes emerging from now shown a broad enough range of amino element binding proeach molecule of proacids incorporated in several different ways tein) inhibits the protein tein at the instant of its that the feasibility is thoroughly established, under hyperglycemic creation from ribosomes and the need to establish feasibility has conditions and that this, of living cells (bernstein. changed to a focus on utility." in turn, leads to inhibiharvard.edu/images/ tion or downregulation Tirrell and coworkers specialize in a 1119623clip.mov). of a gene important for technique to incorporate nonnatural amino survival of pancreatic acids in which they deplete the supply of a Single-molecule techcells that make insulin. natural amino acid in a cell and then feed niques like Xie's could This study revealed a it an amino acid analog. The cell incorpobe particularly useful previously unknown sigrates the analog into newly synthesized for measuring the pronaling pathway that could have implications proteins either at a single site or at many duction of scarce proteins, such as tranfor a better understanding of diabetes. sites at which the natural amino acid would scription factors. "We're viewing bursts of have appeared. Allozyne, a company Tirrell individual protein expression in real time," Some researchers just can't be satisfied cofounded in Seattle, is trying to develop Xie said, "making it possible to study tranwith proteins as they are and instead dream and exploit this approach commercially scription, translation, and other processes of designing proteins as they might be—with to develop medicines. "If either Ambrx or in a quantitative way." novel structures and new types of functions. Allozyne succeeds in commercialization ''Why design proteins?" asked associate proof protein therapeutics, people will have to OTHERS PREFER to focus on ways proteins fessor ofpharmacology Rama Ranganathan take nonnatural amino acid methods more of the University of Texas Southwestern are modified after they are created on riboseriously,,, Tirrell said. Medical Center, in Dallas, who chaired a somes. Such "posttranslational" modificasymposium session on protein design. "We tions include glycosylation, acetylation, Tirrell and coworkers recently extended want to be able to build on, modify, improve, hydroxylation, and methylation. "Proteins this approach by labeling newly synthe_, and change these wonderful sized proteins in a selected ë nanoscale machines that natime window (after neuronal Newly synthesized £ ture has made." stimulation, pathogen infecazide-funtionalized Alkyne1 Caltech professor of bioltion, heat or cold shock, drug Proteins ° ogy and chemistry Stephen Proteins tagged N administration, or another proteins £ L. Mayo and professor of stimulus) in such a way that Azide-functionalized § biochemistry David Baker of they can be subsequently 1. Lyse cells amino acid • the University of Washingisolated and identified (Proc. 2. Conjugate ton, Seattle, both of whom Natl. Acad. Set. USA 2 0 0 6 , functionalized proteins with spoke at the symposium, 103,9482). In this technique, alkyne tag Cell are among those who have called BONCAT (bioorthoghad notable success in the onal noncanonical amino acid past few years at designing tagging), nonnatural amino novel proteins. They and acids with side chains (such Digest tagged proteins with protease others have created proteins as azides) that can undergo a that catalyze reactions, bind specific conjugation reaction specific small-molecule subare incorporated into some strates, act as biosensors, and fraction of positions where a Identify newly synthesized bind to targets different from corresponding natural amino proteins with tandem the native versions. Baker and acid normally occurs in newly mass spectrometry Affinity coworkers recently used comsynthesized proteins. The side chromatography putational methods to design chains are conjugated with a TAG TEAM A technique called a protein fold that had never functional group that makes BONCAT uses functionalized amino been seen in nature (C&EN, the proteins easy to isolate, acid analogs to label specific types of protein in a specific Nov. 24,2003, page 11). and they are then analyzed time window after neuronal stimulation, pathogen infection, by mass spectrometry. Protein design methods heat or cold shock, drug administration, or another stimulus "are getting to the point so that the proteins can be easily isolated and identified. Whereas Tirrell likes to where they could be used as create customized proteins, tools to ask interesting questions in chemiothers prefer to watch individual proteins can be extensively modified, expanding cal biology," Mayo said, "but not enough of spectroscopicalry while they're actually beupon the structural and functional diverthat is happening yet. There hasn't been ing created. At the symposium, professor of sity encoded in the genome," said associenough focus on what can be done with chemistry and chemical biology X. Sunney ate professor of chemistry and chemical these methodologies, such as designing Xie of Harvard University discussed two engineering Linda C. Hsieh-Wilson of
In the early 1990s, using organic reactions to customize proteins "was almost a pipe dream."
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protein therapeutics or understanding usually thought of as bad actors because for catalysis, signaling, or other functions," how some naturally occurring biological their formation is associated with neurodeexplained associate professor of biochemisproteins work." generative conditions such as Alzheimer's try Dorothée Kern of Brandeis University, disease. In a Protein Society presentation Waltham, Mass., who chaired a session on One challenge for the field is that comon the origins of such debilitating disorders, putational design programs currently tend the topic. An enzyme can't function if it chemistry professor Christopher to be hard to use. "It's unlikely your | M. Dobson of the University of average protein chemist is going to i Cambridge, in England, noted that be able to download one of these | amyloid fibrils are not only probprograms and use it in the way its | lematic agents in biology but also developers intended," Mayo said. S fascinating nanostructures. "It's going to be a while before there are software packages out CREB -Xthere that are user-friendly and HE OUTLINED experiments on Ι Ι ΡΙ^^ΜΕ^^^βίΙΙΐΚΐ:';V .Si:-:': | CRE really help you with your project." amyloid fibrils carried out in con junction with colleagues in the There is but a short concepNanoscience Centre and the SUGAR SWITCH CREB protein bound to the tual leap between designing proCavendish Laboratory at the Uni gene promoter CRE becomes glycosylated when teins from scratch and improving versity of Cambridge. In these the sugar level in blood is high. This in turn leads therapeutic proteins by design. A studies, fibrils were probed by a to downregulation of IRS-2 (insulin receptor new feature of this year's Protein series of atomic force microscopy substrate-2), a gene involved in survival of insulin Society symposium was a session techniques, allowing some of their producing pancreatic cells. on proteins as commercial drugs, mechanical properties to be mea highlighting "how decades of prosured and interpreted. tein chemistry research have made it possible to improve commercial "The fibrils turn out really to protein therapeutics," explained be extraordinary," Dobson said. session cochair Christine E. Smith, "On a weight-for-weight basis, they senior director of genomics and have the same strength as steel, biotechnology at Pfizer Global and their toughness is close to Research & Development, Chesthat of silk. These results support terfield, Mo. the view that some amyloid forms of proteins might be useful as ad Research fellow Michael R. Devanced materials, rather than just Felippis of Lilly Research Laboragiving rise to unpleasant diseases. tories, Indianapolis, discussed how They're infinitely 'functionizable' basic research on proteins has led nanostructures with dramatic to an important practical result: properties, and what's more, they designer insulins that work better can self-assemble in water at room than conventional human insulin temperature." for diabetic patients. For example, ONE AT A TIME Each flash of light (yellow) signals the Lilly's Humalog is a designed insu- ribosomal biosynthesis of one molecule of fluorescent Knowledge of the mechanical lin with "a sequence inversion in protein in bacterial cells. properties of amyloid fibrils also the C-terminus of one chain that has extremely important conse makes it faster acting," DeFelippis said. quences for understanding how and why stays in just a single state. It has to take in "This makes it possible to take it closer to they are sometimes associated with patho substrates, catalyze reactions, and release meal time, whereas regular human insulin logical conditions. "The new study helps us products, and it must move to do this. A sig is supposed to be taken 30 to 60 minutes see why amyloid deposits can be so difficult naling protein has to go from an inactive to before meals. More important, the pharto degrade by the biological mechanisms an active state, so a conformational change macological properties of Humalog more that would normally clear this type of un is absolutely essential there as well. closely match physiological insulin secretion. wanted material from the body," Dobson Researchers have recently begun to mea From a patient outcome and convenience said. "In addition, it provides real insight sure those kinds of motions, Kern said. standpoint, some of the new designer ininto the way amyloid can proliferate in tis Their findings indicate that protein mo sulin formulations are a major advance in sue, leading to the spread of disease." tions happen on a timescale of picoseconds diabetes treatment." to seconds, depending in part on how many Amyloid fibrils are thus turning out to atoms are involved in the transitions. Local have surprising significance in disciplines Efforts to rationally redesign proteins dynamics, involving small protein regions, ranging from nanoscience to neuroscience, would benefit from a better understandare very fast, whereas collective dynamics, Dobson noted. And in view of the extraor ing of protein behavior, and many groups involving larger conformational changes, dinary diversity of research discussed at are attempting to achieve just that. For inare slower, because different protein regions this year's Protein Society sessions, that's stance, researchers have been studying how have to be in the right state for these transi something that one could say not just about the dynamics of proteins—their tendency tions to occur. amyloids but about all proteins. For proteins to constantly move and change conformathat are useful or just interesting, leave it up tion—contribute to and underlie protein Another aspect of protein behavior gar to protein scientists to poke, perturb, and function. nering lots of attention these days is aggrega redesign them in everyway they can pos tion. Proteins sometimes aggregate to form "Proteins have evolved so they move sibly imagine. • species known as amyloid fibrils. These are between substates that will be productive WWW.CEN-0NLINE.ORG
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