Research in chemical and synthetic biology: Fall 2015
Our lab studies questions that span the combined interfaces of chemistry, biology, medicine, biophysics, and biotechnology. Overall, we seek to understand the fundamental chemical mechanisms that control and regulate protein and small molecule interactions in cells and apply this knowledge to exploit and manipulate cell function and drive the development of next-generation therapeutics. Sometimes our goals demand the development of new chemical biology tools, while other times we apply state-of-the-art chemical, genetic, structural, or biophysics tools developed by others in new ways. Graduate students hail from all over Yale, including the departments of Chemistry, Cell Biology, Molecular, Cellular, and Developmental Biology and Molecular Biophysics and Biochemistry. But no matter what their background, all students become expert in tools and techniques that span the chemistry-biology-bio-engineering continuum, from organic synthesis to structure determination and from cell biology to genetic engineering. And they end up in leadership positions in academics, the pharmaceutical and biotechnology industries, and venture capital/consulting. More information about our science and past and present lab members can be found at http://www.schepartzlab.yale.edu/warmbodies.html.
Chemical biology. We are interested in understanding how proteins control the flow of molecules and molecular information through cellular membranes. This interest required the development of two new tools–bipartite tetracysteine display and a strategy for labeling cellular organelles–and their application to characterize how coiled coils encode and decode growth factor identity in receptor tyrosine kinases and how the Golgi apparatus manages its natural cargo. We are also interested in how the endocytic pathway can be repurposed to traffic peptides, proteins, and their mimetics to desired cellular locale.
Development and application of bipartite tetracysteine display
Fluorogenic molecules–those that glow only upon interaction with a prescribed protein, lipid, saccharide, or nucleic acid–are essential tools for localizing and monitoring events in live cells, sometimes even in real time. Bis-arsenical dyes such as FlAsH and ReAsH are one class of fluorogenic molecules: they are not fluorescent when coordinated through arsenic to two ethanedithiol ligands (EDT), but can glow brightly when EDT is exchanged for four proximal Cys thiols on a target protein, an arrangement termed a tetracysteine (Cys4) motif. As shown by Nathan Luedtke, if the thiols of a Cys4 motif are distant in primary sequence but close by virtue of association or conformation, the fluorogenicity of ReAsH provides a read-out of protein-protein interactions or conformational changes in living cells (Goodman et al., 2009; Lowder et al., 2011; Luedtke et al., 2007; Scheck and Schepartz, 2011). As described below, in recent years we have applied bipartite tetracysteine display to understand how protein-protein interactions in the receptor tyrosine kinase EGFR encode and decode chemical information.
EGFR is mutated in >20% of all lung cancer, and lung cancer accounts for >30% of all cancer deaths. Although targeted EGFR therapies exist, they inevitably fail as the cancer mutates to develop resistance; drug-resistant (DM) EGFR is considered untreatable. We have exploited the virtues of bipartite tetracysteine display to provide new insights into how EGFR communicates information across the plasma membrane; our work has identified a new potential therapeutic strategy for DM EGFR (A. Doerner et al., 2015; Lowder et al., 2015; Scheck et al., 2012). Rebecca Scheck and Amy Doerner learned that the binding of most EGFR-specific growth factors to the extracellular domain (ECD) induces one of two antiparallel coiled coils (EGF-type or TGF-a-type) in the cytoplasmic juxtamembrane segment (JM) (Scheck et al., 2012), and that coiled coil identity predicts growth-factor dependent signaling (Doerner et al., 2015). Julie Sinclair designed cell permeable, hydrocarbon-stapled peptides that inhibit EGFR allosterically by blocking JM coiled coil formation (Sinclair et al., 2014; Sinclair and Schepartz, 2014). Most recently, Melissa Lowder discovered that WT and drug-resistant, double mutant (DM) EGFR contain different JM coiled coils, and that the DM EGFR-selectivity of certain tyrosine kinase inhibitors (TKIs) correlates with long-range changes in JM structure (Lowder et al., 2015). Currently, we are studying the role these JM conformations play in dictating specific downstream oncogenic signaling pathways. Our studies of EGFR seek to illuminate one of the most elusive of all protein functions–allostery–in the context of one of most important human oncogene families–EGFR.
Improving bipartite display
Despite wide utility for tagging proteins and their assemblies, the mechanism by which ReAsH becomes fluorescent upon protein association is unknown (Scheck and Schepartz, 2011). Using sophisticated computational techniques, Allison Walker has shown that ReAsH fluorescence is rotamer-restricted, and depends on the relative orientation of the aryl chromophore and the appended arsenic chelate. They do not support a mechanism in which fluorogenicity arises from the relief of ring strain. The calculations identify those As-aryl rotamers that support fluorescence; binding sites whose conformation locks ReAsH into a fluorescent rotamer upon binding should be highly fluorescent. By providing a higher resolution view of the structural basis for fluorogenicity, these findings should aid both the design of more selective bis-arsenicals and the identification of optimal protein targets. Future work will focus on developing a search algorithm for ideal ReAsH binding sites that can be applied to any protein or protein-protein interaction with a known structure.
Controlling molecular trafficking: discovery and application of penta-arg proteins
The inefficient delivery of proteins and peptide mimetics into the mammalian cell cytosol limits their potential as therapeutics and research tools. Previous strategies to deliver proteins to cells relied on proteins that contained four to six arginines (Daniels and Schepartz, 2007; Smith et al., 2008), however Jacob Appelbaum demonstrated that few of these proteins are actually trafficked to the cytosol (Appelbaum et al., 2012). To solve this problem Jacob developed ZF 5.3 and aPP 5.3, which contain a precise array of five arginines on an a-helix backbone—a "penta-arg motif" that allows for efficient delivery to the cytosol (Appelbaum et al., 2012). We showed further that proteins containing a penta-arg motif are taken up by endocytosis, and that a single penta-arg motif specifies release of the associated protein from vesicles that form early along the endosomal pathway—in particular, those endosomes characterized by the guanosine triphosphatase (GTPase) Rab5 (Appelbaum et al., 2012).
In more recent work, Jonathan LaRochelle worked with Garrett Cobb in Liz Rhoades' laboratory to apply fluorescence correlation spectroscopy (FCS) to provide a precise, accurate, and direct measure of the relative efficiencies with which different potentially cell-permeable molecules traffic to the cell interior (LaRochelle et al., 2015). Using FCS, we discovered that molecules containing a "penta-arg motif" reach the cytosol intact, with exceptionally high efficiencies—in certain cases exceeding 50%. The transport efficiency of the most efficient penta-arg molecule, ZF 5.3 is at least ten-fold higher than that observed for the widely studied cationic sequence derived from HIV Tat or polyarginine Arg8, and equals that of hydrocarbon stapled peptides that are active in cells and animals. Current work is focused on a genomic strategy to identify the molecular machinery that supports penta-arg trafficking, and the application of this technology to develop peptides, proteins, enzymes, antibodies, and peptide mimetics that predictably and efficiently reach the interior of mammalian cells.
Visualizing cellular compartments at super-resolution
Super-resolution 'nanoscopes' dramatically increase the resolving power of light microscopes, revealing new and rich details of protein and organelle structure, function, and dynamics in live cells. The application of these powerful nanoscopy techniques, including single molecule switching (SMS) and stimulated emission depletion (STED) methods–are severely limited by a lack of compatible dyes. Both methods demand fluorophores that are bright, photostable, non-toxic, and live cell-compatible; SMS methods also demand a dye that "blinks" in a very specific way. As nanoscopes push the resolution envelope beyond tens of nanometers, there is a critical need for ever more bright and photostable fluorophores that support multi-color imaging and whose blinking remains coherent with increasingly rapid cameras. The design and synthesis of such dyes pose real challenges for chemistry.
Over the past two years we contributed to a 4-way collaboration with Joerg Bewersdorf, Jim Rothman, and Derek Toomre in Yale Cell Biology. The collective goal is to apply state-of-the-art chemical biology and instrumentation to visualize Golgi dynamics and trafficking. Last year, Roman Erdmann and Alex Thompson reported a strategy to visualize the Golgi in live cells using two novel reagents: a trans-cyclooctene-containing ceramide lipid (Cer-TCO) and a reactive, tetrazine-tagged, SiR dye (SiR-Tz) (Erdmann et al., 2014). These reagents assemble via a rapid 'tetrazine-click' reaction into Cer-SiR, which enables (highly) prolonged live cell imaging by 3D confocal and STED microscopy. Cer-SiR is non-toxic and does not perturb Golgi function. It is exceptionally photostable when compared to commercial lipid dyes and even compared to a protein labeled with SiR. Yet, Cer-SiR photostability is not infinite (16% decrease over 500 3D stacks), and there is no doubt that increased photostability correlates directly with increased resolution by STED. Current work is therefore focused on the design and synthesis of new dyes with increased photostability or whose photophysical properties are optimized for rapid cameras.
Synthetic biology. We are interested in the design and evolution of non-natural polymers that fold into stable, defined, three-dimensional shapes, bind small molecules, and promote chemical reactions. This interest led to the first (and still only) example of a protein containing not a single α-amino acid and our efforts to repurpose the ribosome for the synthesis of all manner of exotic polymers.
Proteins with alternative backbones and improved function
Natural biopolymers fold with fidelity, can exist as oligomers or discrete complexes, and possess kinetic and thermodynamic signatures that distinguish them from most non-biological polymers and smaller molecules. In 2007, Doug Daniels and James Petersson reported that certain oligomers of β3-amino acids (β-peptides) fold into bundles of defined stoichiometry that resemble natural proteins in many respects (Daniels et al., 2007; Petersson et al., 2007; Petersson and Schepartz, 2008). β-peptide bundles represent the first–and still only–example of a non-biological polymer with protein-like functions. More recently, Cody Craig applied rational and computational methods to improve β-peptide bundle stability and increase diversity within the core and learned how to control both quaternary structure and diversity (Craig et al., 2011). Matt Molski made significant progress adapting these structures to a membrane environment (Molski et al., 2010) and remodeling the core (Molski et al., 2013), and Pam Wang, Jon Miller and Michael Melicher developed bundles with catalytic (Wang et al., 2014) and allosteric metal ion binding activity (Miller et al., 2014) and the capacity to recognize sugars (Melicher et al., 2013). Together, these properties should allow us to recruit substrates to the bundle surface for selective catalysis, and future work will focus squarely on this goal.
Repurposing the ribosome to synthesize exotic polymers
Biological materials, catalysts, and nanomachines power all living organisms and many technologies that interrogate and advance science and technology. But today, our ability to rationally design and evolve new macromolecules is limited to the few polymers for which Nature has provided a genetic code. This limitation precludes the rapid development of next generation materials, therapeutics, catalysts, fuels, and chemicals. With Allison Walker and Wes Robertson, we are leading a multi-institutional effort to expand the genetic code in an unprecedented way, by repurposing the bacterial protein-synthesizing machinery–the ribosome–to generate new, non-natural classes of sequence-defined, evolvable polymers bearing only a glancing resemblance to natural α-peptides. Working in vitro and then in vivo, this team will interweave and advance state-of-the-art evolutionary, genetic engineering, biophysical, cell biological, statistical, and computational technologies to enable, for the first time, the sequence-templated biosynthesis of new molecules containing backbone-modified D-α-, β- and y-amino acids and the β-keto acid monomers used to assemble polyketide natural products. Our work seeks to monitor, interrogate, and understand translation biology, and with this knowledge evolve and engineer valuable promiscuity into the ribosome, its associated translation factors, and the host organisms.