University of Utah biochemists have reported an advance in the production of functional mirror-image proteins. According to a groundbreaking study published in this week's online edition of the Proceedings of the National Academy of Sciences, they have chemically synthesized a record-length mirror-image protein and used this protein to demonstrate that a cellular chaperone, which helps "fold" large or complex proteins into their functional state, has a previously unappreciated talent -- the ability to fold mirror-image proteins. These findings will greatly facilitate mirror-image protein production for applications in drug discovery and synthetic biology.
Proteins are "handed" -- with both left- (L-) and mirror-image right-handed (D-) forms. Life on Earth standardized on only L-proteins; since D-proteins are not found in nature, organisms have not evolved the ability to digest them. Therefore, mirror-image proteins have tremendous potential as a new class of long-lived drugs. However, a major limitation in developing mirror-image therapeutics is that they must be chemically synthesized -- an arduous task for larger proteins. An additional challenge is that large proteins often require the assistance of cellular "chaperones" to fold into their functional state, and D-chaperones are not available.
"Recent advances in chemical protein synthesis make it more feasible to produce larger synthetic D-proteins," says Michael S. Kay, M.D., Ph.D., professor of biochemistry at the University of Utah School of Medicine and senior author of the study. However, chemical synthesis is not the only challenge. These proteins must also be folded into their proper three-dimensional shapes. Dr. Kay continues, "Larger, more complex proteins often rely on chaperones to prevent misfolding and produce functional protein. The fundamental question we sought to answer was whether natural L-chaperones could 'crossover' to fold mirror-image D-proteins."
Kay, together with co-authors Matthew T. Weinstock, Ph.D. and Michael T. Jacobsen (former and current graduate students) synthesized the D-form of the bacterial protein DapA, a 312-amino acid protein that requires the assistance of a chaperone known as GroEL/ES to fold under physiologic conditions.
"We wondered if we could make and fold larger proteins to greatly expand the reach of mirror-image drug discovery," adds Dr. Weinstock (now a scientist at Synthetic Genomics, La Jolla, CA). Prior to this study, the longest synthetic D-protein reported was the 204-residue D-VEGF dimer. Using their synthetic D-DapA, the Utah group demonstrated that natural GroEL/ES was capable of folding the D-protein into its active conformation (i.e., it is 'ambidextrous', folding both L- and D-proteins). This discovery provides proof-of-concept for the use of natural GroEL/ES to fold D-proteins for a variety of mirror-image drug discovery and synthetic biology applications.
"Future research will focus on pushing the size limits of chemical protein synthesis even further and developing methods to improve the efficiency, quality, and yield of the synthetic process," says Jacobsen.
These capabilities will accelerate and enable further mirror-image drug discovery programs being pursued by the Kay lab in collaboration with their commercialization partner Navigen (a biotech startup in Salt Lake City). Mirror-image proteins may also be useful for the study of dangerous protein toxins. Mirror-image versions of such proteins would have the identical chemical characteristics as the natural protein (enabling detailed biochemical studies), but will generally be non-toxic to natural organisms.
Kay and his colleagues are also focused on long-term applications of their work. According to Kay, an intriguing potential application of chemical D-protein synthesis is the construction of a mirror-image ribosome, the protein-making machine of the cell. "A mirror-image ribosome would provide a dramatically easier and more cost-effective method to produce usable quantities of diverse mirror-image proteins, and would be the heart of a fully synthetic mirror-image cell," says Kay.
These ambitious goals remain years away, but Dr. Weinstock adds, "As a result of this work, we are now ready to start considering these possibilities."
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