Former WRF Innovation Fellows
ZACHARY CROOK, PhD – Collaborating PI: Jim Olson, FHCRC
Zach is interested in novel therapeutic applications for cysteine knotted peptides. While the Olson Lab has a strong interest in their potential uses for cancer treatment, Zach comes from a research background in neurodegeneration, and wishes to investigate the means for getting these natural drug-like peptides across the blood-brain barrier. Efforts towards this will make use of Rosetta and several known structures. Both the knotted peptides that the Olson Lab routinely produces, and several receptor proteins that facilitate transcytosis of natural signaling proteins, will be used to identify potential knotted peptides that can bind these receptors and efficiently transport into the brain. This has the potential to make the blood-brain barrier a little less imposing for therapeutic applications, including neurodegenerative diseases and brain cancer.
Zach attended college at the University of Colorado in Boulder, graduating with honors in Molecular, Cellular, and Developmental Biology. He went right into the PhD program at MIT, where he studied mouse models of Huntington’s Disease under David Housman, and developed assays to rapidly and accurately determine the effect of test therapeutics. The idea of drug design using naturally stable, bioavailable knotted peptides and their potential utility in diseases of the central nervous system drew him to the lab of Jim Olson, where he brings his past knowledge of screening assays in mouse disease models to mix with the biochemical and drug-design expertise of the Olson Lab and collaborators at the FHCRC.
Naturally occurring metalloproteins, were adaptively evolved to use earth abundant metals as cofactors to effectively tackle their needs and therefore are limited in their function. On the other hand, non-biological late transition metals possess unparalleled potential to catalyze desirable organic transformation for modern synthetic needs. Today, these paradigms can be combined through de novo design of enzyme active sites that incorporate non-biological metals. Gerard will be working along with Dr. Thomas Spiro and Dr. Karen Goldberg to design a metalloenzyme for photocatalytic reduction of CO2 at a (Ru,Zn)-bimetallic active site. Gerard will strategize design techniques to incorporate Ru ion by itself and incorporation of Ru complex to a specific binding pocket in the protein to form the active site. Ultimately, this project will accelerate the merger of the fields of organometallic catalysis and de novo protein design.
Gerard learnt the basics of electronic structure computation while working with Dr. M. Balakrishnarajan during his Master’s degree in Chemistry at Pondicherry University, India. Later, during his Ph. D. at Virginia Commonwealth University under Dr. Nicholas Farrell, he studied novel zinc binding environments in proteins using computational and biophysical techniques. His interests in artificial photosynthesis and the origin of life directed him to his current interest in de novo design of enzymes with non-biological metals for in vitro catalysis. As a WRF Innovation Fellow, he will be working with Dr. Spiro and Dr. Goldberg on the design of a metalloenzyme for photocatalytic reduction of carbon dioxide.
JASON GILMORE, PhD – Collaborating PI: Michael MacCoss, UW Genome Sciences
Jason is a 2014 recipient of a WRF Innovation fellowship and will be working jointly with Michael MacCoss (UW Genome Sciences) and David Baker (UW Biochemistry and IPD) to develop high-throughput, highly sensitive screens to accelerate experimental validation for de novo protein design. The first aim of this project will use liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) to simultaneously quantify hundreds of protein designs expressed together in pooled cultures, as opposed to the traditional methods that require individual expression and confirmation of new designs. Additionally, this project will extend software and experimental techniques for MS-based protein chemical crosslinking experiments to rapidly evaluate disulfide linkages in small de novo designed proteins. Together, these mass spectrometry methods will improve the productivity of protein design protocols and accelerate the development of novel protein-based therapeutics.
During his undergraduate summers, Jason worked at the Pacific Northwest National Laboratory in Richland, WA where he helped to implement automated quality control metrics for the proteomics core facility. After graduating from the University of Pennsylvania in 2007, Jason returned to PNNL for one year and wrote a software tool for predicting protein-protein interaction probabilities. His dissertation work at Dartmouth College, in the proteomics laboratory of Dr. Scott Gerber, focused on the detection and quantification of phosphopeptide species by mass spectrometry-based shotgun sequencing. This included a publication on sequential digestion by complementary proteases to survey previously inaccessible regions of the proteome in complex biological mixture. Subsequently, he developed a computational technique to improve the sensitivity and precision of peptide quantification in cases where isotopically labeled standards failed to fully complement endogenous peptide profiles.
HANNAH GELMAN, PhD – Collaborating PI: Doug Fowler, Department of Genome Sciences
Guiding protein design with comprehensive maps of mutant function
Computational protein design — in which new protein sequences are developed to perform a specific function — promises efficient generation of biological molecules that can carry out novel functions. Designed proteins could be ideal for the treatment of rare or emerging diseases and potentially mitigate the side effects of more broadly acting classes of drugs. This method is hampered by the difficulty of predicting the effect of mutations on protein function, especially if the mutation affects protein stability or structure, and by our incomplete understanding of how protein physical properties like thermodynamic stability affect protein function.
The efficiency of a designed protein can be significantly enhanced with deep mutational scanning (DMS), in which a library of mutants based on the designed sequence is expressed and subjected to weak functional selection (e.g., binding to the targeted ligand). The change in the distribution of sequences over the selection — enrichment of some sequences and depletion of others — is measured and used to determine the relative function of every sequence. This strategy refines the sequence found by computational design to find a better performing variant, but does not address the underlying mismatch between the performance predicted by the design algorithm and that measured in the real world. Integration of DMS and computational design can be pushed even further so that data from DMS is used to improve design algorithms at the outset instead of to refine the algorithm’s output.
Incorporating the large-scale DMS data into the development of protein design algorithms will require us to quantitatively and accurately measure specific biophysical and biochemical properties rather than rely on the more easily obtained relative measurements of mutant function that are currently used. A limited number of assays have been developed to measure these properties in a high throughput manner, but each is highly targeted to a specific protein target. We will expand and combine them in a unified platform that can reproducibly characterize libraries of protein mutants across many of the properties that may be correlated to overall function. For each mutant we will analyze how the measured physical properties correlate with each other and with mutant function. We can then compare these measurements to the predictions of the design algorithm and use the quantitative data obtained to improve the algorithm’s ability to accurately predict physical properties and how these properties affect protein function.
Our method can guide the implementation of additional design constraints that better represent the key
contributions to a protein’s ability to function. A more complete and accurate design algorithm will streamline the design process as it will reduce the need for multiple rounds of computational design and functional screening. In addition, more accurate design algorithms will open the door for more ambitious targets for protein design — for example, the design of novel functions or modes of action — that are currently out of reach.
BENJAMIN GROVES, PhD – Collaborating PI: Georg Seelig, UW Electrical Engineering
Signal processing is central to biology. The response of cells to internal and external cues is critical to many fundamental biological phenomena, from multicellular development, to the efficiency of microbial metabolism, to how diseases progress. In nature, cells process information in many ways, but common to all cells is protein-based signaling. Proteins are the nuts and bolts of the cell, and protein-based signaling offers speed, versatility and dynamic properties that are not seen in other types of signaling pathways. A large number of protein signaling pathways are based on kinases, which by catalyzing the addition of a phosphate moiety onto specific target proteins are able to effect a conformational change, trigger an interaction with yet another protein or even activate another kinase. Being able to build such pathways would deepen our understanding of them and would be very powerful. However, two hurdles stand in the way:
(1) There are only a small number of well-characterized parts. Many proteins are modular, with certain functions being confined to a sub-region of the protein. Moreover, many of these protein modules continue to operate even in the context of a different protein, making it possible to mix and match parts to make new functional proteins. Such an approach can be used to control protein signaling. In much the same way as a relay race, one signaling protein must recognize the next in order to transmit the signal. This can be accomplished by using complementary protein interaction domains. Along these lines, Ben is collaborating with Dr. Daniel Adriano-Silva in David Baker’s lab to develop methods to facilitate the large-scale design and high-throughput functional testing of novel protein interaction domains. (2) We don’t always know which parts are necessary to make a functional kinase. Though the addition of a protein interaction domain can direct a kinase towards a novel target, it does not always mean that the target is phosphorylated in the desired fashion, this is particularly true if the target is another kinase.
Ben is investigating how to direct these versatile signaling proteins to novel targets, either by using them to control the degradation of another protein, or the activity of another kinase. Ben joined Georg Seelig’s group as a postdoc in 2011 and became an Institute for Protein Design post-doctoral Fellow in 2014. He is interested in expanding our understanding of how cells control their behavior through the construction of novel signaling pathways.
GLENNA FOIGHT, PhD – Collaborating PI: Dustin Maly, UW Chemistry
Cellular signaling pathways are complex networks of protein and enzymatic interactions. Understanding their contributions to disease states is an important goal. The development of protein inhibitors through computational design and directed evolution is a powerful approach for specifically targeting individual elements of signaling pathways. Small-molecule inhibitors offer temporal control and cell permeability, but the development of small molecules that specifically target only one protein in the cell is a difficult and lengthy process. The Maly lab has combined the power of protein and small-molecule inhibitors in a system known as a chemical genetic switch. This involves the fusion of two components of a proteinprotein interaction to a protein of interest such that a small molecule that disrupts the interaction will allow activation of the protein. The first part of my research will involve creating a new chemical genetic switch system by designing a protein interaction partner for an existing protein and small-molecule inhibitor. The new system will use components that are foreign to mammalian cells, thus offering compatibility with future studies of mammalian signaling pathways. My second project will focus on developing inhibitors of the oncogenic protein Ras. Ras is an important signaling protein in numerous cellular processes, and the Ras family is the most frequently mutated protein family in human cancers. I will design protein inhibitors that specifically target individual oncogenic mutants and family members of Ras. These inhibitors will aid in dissecting the complex functional differences between different mutants and variants of Ras. Furthermore, I will use the successfully engineered chemical genetic switch developed in my first project to confer small-moleculebased, temporal control over these Ras inhibitors in cells. I did my undergraduate degree in biochemistry at North Carolina State University where I performed Xray crystallography research, coincidentally, on structures of oncogenic Ras mutants in the lab of Dr. Carla Mattos. My interest in protein structure led me to the lab of Dr. Amy E. Keating at MIT, where I studied the determinants of protein-protein interaction specificity in my graduate work. My interest in studying signaling processes controlled by protein-protein interactions led me to the lab of Dr. Dustin Maly at the UW. Designing specific protein interactions and using my designs to disrupt cellular interaction networks will be an exciting combination of my expertise and interests.
ZHIZHI WANG, PhD – Collaborating PI: Wenxing Xu, UW Biological Structure
As a WRF Innovation Fellow at the Institute for Protein Design, Zhizhi is working on designing bio-orthogonal E3 ubiquitin ligases that can be specifically activated by cell-permeable agonists using RNF146-iso-ADPR as the template. Zhizhi will further convert the design into inducible protein knockout system in cells and model organisms. This system may allow for knocking down a protein in an inducible and tunable manner. Development and optimization of such systems will not only provide a powerful tool for biological research, but may also have profound implications in therapeutics.
Zhizhi graduated with B.S in Biological Sciences from Peking University, Beijing, China in 2007, and then joined the Biological Physics, Structure and Design Graduate Program at the University of Washington. His thesis work was performed in the lab of Dr. Wenqing Xu in the Department of Biological Structure where he studied how a protein post-translational modification called poly(ADP-ribosyl)ation is recognized by WWE domains and how it is degraded by poly(ADP-ribose) glycohydrolase using X-ray crystallography. After graduation in 2012, Zhizhi continued working in Dr. Wenqing Xu’s lab. Recently, in close collaboration with Dr. Rachel Klevit’s lab in the Department of Biochemistry at the UW, he identified the first small molecule (iso-ADPR) inducible E3 ubiquitin ligase (RNF146), whose activity is switchable by ligand binding.
YALAN XING, PhD – Collaborating PI: Hannele Ruohola-Baker, UW ISCRM
Yalan plans to extend her research interest to the understanding of the molecular mechanism underlying early hESC developments: the regulation of epigenetic changes during naïve to primed hESC transition. Yalan has collaborated with Dr. James Moody at the IPD to design and synthesize polycomb EED binding inhibitors, which mimic the binding between EED and EZH2 (methyltransferase for H3K27me3) and will be used to disrupt the endogenous EED-EZH2 binding. This inhibitor will be used to test if the EED/EZH2 interaction is essential for epigenetic changes during the naïve to primed hESC transition. In the long run, Yalan will design and synthesize respective binding proteins at the IPD to disrupt multiple interfaces potentially required for EZH2 activity.
During her PhD study with Prof. Willis Li in University of Rochester, Yalan investigated the transgenerational epigenetic inheritance in model organisms, and characterized the requirement for heterochromatin modification in the germline stem cells maintenance, which is through interaction with JAK/STAT signaling. Her post-doctoral training with Prof. Hannele Ruohola-Baker lab in University of Washington has granted her the opportunity to further pursue her interest in stem cell biology. Using the model organism Drosophila, Yalan conducted genetic screens for essential factors in germline stem cell self-renewal and later on elucidated the molecular mechanism of how stem cells are protected against apoptosis through signals from apoptotic neighbor cells. Meanwhile, Yalan used the human iPSCs to demonstrate the differential roles of the Hypoxia-inducible factors (HIFs) and metabolic shifts during the reprogramming process. She plans to bring her expertise in these fields to her WRF project.