Our group, the King Lab at the University of Washington, computationally designs self-assembling protein nanoparticles for therapeutic applications. These nanoparticles make great candidates for therapeutics because of their ability to both encapsulate molecules as “cargo” and display antigens on their surface. One of the problems we face in vaccine and therapeutic design is targeted nanoparticle delivery, or making sure nanoparticles are delivered to a specific location, in vivo. Understanding where certain nanoparticles localize in vivo will be useful for determining what influences a nanoparticle’s interactions inside an organism. One approach our group is taking to examine nanoparticle biodistribution is miniprotein library display on synthetic nucleocapsids. These miniproteins are 20-50 amino acids long and were designed to produce stable, folded structures with surface patches that could facilitate binding to target receptors. Synthetic nucleocapsids, or nanoparticles designed to encapsulate their own RNA genomes, display these miniproteins in the library. The library was injected into healthy and tumor-ridden mice. RNA sequences were then obtained from blood, brain, heart, spleen, kidney, liver, lung, tumor, and dose samples. Using unsupervised and supervised learning algorithms such as Principal Component Analysis, Hierarchical Clustering, and Random Forests, I am building mathematical models that can analyze the biochemical properties of these library variants and determine why certain nanoparticles vary in biodistribution. I will also be analyzing another synthetic nucleocapsid library to answer similar questions about nanoparticle biodistribution. By constructing these models, I hope to provide tools that aid in experiments regarding targeted drug design.

Photosynthetic bacteria are ideal systems for the sustainable production of chemicals, but their chemical production is limited by the efficiency with which the bacteria can capture and transfer energy. For this project, I have designed light-harvesting proteins that bind chlorophyll molecules that absorb light of the wavelength reflected by the photosynthetic bacteria Rhodobacter sphaeroides. The LH1 and LH2 light-harvesting complexes found in R. sphaeroides bind chlorophyll molecules in a manner that promotes excitonic coupling and minimizes the energy lost during transfer from the complexes to the reaction centers. The proteins I have engineered are modeled after LH2 and are designed to bind a symmetric ring of pi-stacking chlorophyll dimers, promoting high-efficiency energy transfer between light-harvesting complexes. After characterizing these light-harvesting proteins, they will be further engineered to enable their expression in R. sphaeroides, which will allow these bacteria to absorb a broader range of the solar spectrum and improve their energy transfer efficiency. Incorporating this de novo designed protein into metabolically engineered R. sphaeroides will enhance these bacteria’s ability to convert carbon dioxide to useful chemicals. These engineered light-harvesting proteins could also later be adapted for use in hydrogel-based organic solar cells and forming a 2-D sheet of light-harvesting proteins. In my presentation, I will discuss how I have used the Rosetta protein modeling suite to design hexameric chlorophyll-binding proteins. These proteins are designed to bind twelve chlorophyll molecules, and their cyclical symmetry simplifies the design process. I will present data from characterization experiments such as circular dichroism and fluorescence spectroscopy, which are methods that allow for quantification of the proteins’ binding affinities towards the ZnPPaM chlorophyll, and small-angle x-ray scattering (SAXS) and Size Exclusion Chromatography/multiple-angle light scattering (SEC-MALS), which provide information about the predominant oligomeric states of the designed proteins.

Following a previously established model, I have attempted to engineer a kinase that is activated upon irradiation with cyan light and deactivated upon irradiation with violet light. The DLK-1 kinase used to make this photosensitive construct is involved in a novel C. elegans mitochondrial retrograde response pathway comprised of DLK-1, SEK-3, and PMK-3 mitogen-activated protein kinases. In C. elegans, activation of this pathway promotes lifespan extension. By expressing this photoswitchable DLK-1 (psDLK-1) kinase in C. elegans, we would create a strain containing a photo-sensitive DLK-1 knock-out/knock-in variant. Such a strain would allow us to investigate this longevity pathway’s developmental dependencies and elucidate the identities of other proteins involved in this pathway. Once this pathway is better understood, researchers can conduct further experiments to determine if the pathway is conserved in other organisms, and, if conserved, potentially design therapeutics to activate the pathway and promote longevity in humans. In my presentation, I will discuss my experimental approach for designing psDLK-1, which, briefly, consisted of inserting a dimeric photodissociable Dronpa (pdDronpa) fluorescent protein into two sites of the DLK-1 kinase domain. The first version of psDLK-1 did not demonstrate photosensitivity in C. elegans. However, we are further engineering psDLK-1 in hopes that several modifications will promote photosensitivity. I will describe the modifications we made to the construct and present experimental data demonstrating the impact of these modifications on the kinase’s function. Once we engineer a functional variant of psDLK-1, we can conduct comparative mass spectrometry experiments to determine the identities of other proteins involved in this novel longevity pathway.

Mitosis produces two genetically identical daughter cells, each inheriting their own nucleus and a full set of replicated chromosomes from the parent cell. Inaccurate chromosome segregation can result in severe consequences like cancer and developmental defects. Microtubules are dynamic cytoskeletal components that provide the forces necessary to segregate chromosomes into their respective daughter cells during mitosis. The kinetochore is an assembly of proteins and protein complexes located on the centromere that binds to microtubule ends to attach chromosomes to the force-generating microtubules. The accurate segregation of chromosomes relies on the ability of the kinetochore to strongly bind chromosomes to microtubule ends. Ndc80 complex is an outer kinetochore component that binds microtubule ends and is required for proper chromosome segregation. Emerging cellular data suggests that multiple Ndc80 complexes interact with one microtubule end to facilitate chromosome separation. In vivo data suggests multiple Ndc80 complexes are arranged around microtubules. To closely model the native kinetochore-microtubule interface, we have begun to assemble a structured particle of multiple Ndc80 complexes in vitro using designed proteins that form oligomers. The particles have different geometries and stoichiometries. A method to couple Ndc80 complex to the designed protein was developed. We then tested the coupling efficiency under different temperatures and concentrations to optimize the reaction and ensure complete particle assembly. We found that the reaction goes nearly to completion with a 3:1 ratio of Ndc80 complex to designed protein at room temperature with a reaction time of thirty minutes. We will now measure the stoichiometries of the particles, which are designed to have four, five, or six Ndc80 complexes. Successful formation of these organized particles will allow us to measure the effect of geometry and stoichiometry on the ability of the Ndc80 complex to make strong attachments to microtubules.

During mitosis, chromosomes are organized and separated by the dynamic mitotic spindle. The kinetochore plays a central role in proper chromosome segregation. It is recruited by specialized sites on the chromosome, known as centromeres, and forms attachments to the spindle microtubule. The Dam1 and Ndc80 complexes are essential microtubule-binding elements of the outer kinetochore in budding yeast. The Ndc80 complex bridges the kinetochore to the dynamic microtubule tip, while the Dam1 complex enhances the Ndc80 complex’s residence time on microtubules and load bearing ability. It was previously reported that swapping yeast histone proteins with human histone proteins resulted in poor complementation, in higher frequencies of aneuploidy, and impaired adaption to novel nutrient environments. Several mutations in the Dam1 complex and one mutation In the Ndc80 complex were found to independently rescue these defects. To determine how these mutations rescue human histone swapping in budding yeast, we purified mutant versions of the Dam1 and Ndc80 complexes. We used an optical trap to measure if the mutations alter the amount of force required to break attachments to a dynamic microtubule. A difference in rupture force between wild type and mutant proteins tells us that the mutations rescue histone swapping by changing the load bearing ability of the kinetochore-microtubule interface. Further study is needed to determine the mechanism for rescue. A null result tells us that further research is required, and a novel interaction may be present. This research will uncover differences in human and yeast kinetochore structure and function.

In response to various stimuli, proteins undergo changes in shape (conformational changes) that are essential to their function in cell signalling, enzyme activity and many other crucial biological processes. Previous studies have shown that intramolecular distance changes associated with the conformational dynamics of proteins can be probed using transition-metal-ion-fluorescence-resonance-energy-transfer (tmFRET), in which a donor fluorophore transfers light energy to an acceptor metal ion in a steeply distance-dependent manner. tmFRET is both highly sensitive and highly specific: each unique donor-acceptor pair can only measure a limited range of distances reliably. Here, we expand the utility of tmFRET by employing new strategies to create a library of unique donor-acceptor pairs each sensitive to a different range of distances.To showcase the accuracy and precision of our measurements, we use a maltose-binding-protein (MBP) model system, which undergoes a well-studied conformational change upon the binding of maltose. In order to label MBP with Cu(II) ions that act as tmFRET acceptors, we utilize macrocyclic chelator-maleimides, which simultaneously coordinate Cu(II) and covalently modify a cysteine introduced into MBP. Using amber codon suppression, we introduce fluorescent non-canonical amino acids (ncAA’s) into MBP to act as tmFRET donors. Our fluorescence measurements show that tmFRET between the ncAA donor and chelated-Cu(II) acceptor accurately reports MBP’s intramolecular distance changes. We also show that by varying the exact structure of the molecule that chelates Cu(II) we can tune the sensitivity of tmFRET to distances ranging from 0.8-1.8 nanometers. Thus, this chelator-maleimide approach to labelling proteins with tmFRET acceptors could prove to be a powerful tool for researchers studying conformational motions of that scale.

 Fluorescence resonance energy transfer (FRET) is a method used to elucidate changes in a protein’s structure (conformational changes). FRET works by measuring distances between amino acids in the presence and absence of ligand. The distance is measured by the highly distance dependent degree of energy transfer between an acceptor and a donor, typically two fluorophores which overlap in absorption and emission spectra. FRET is limited by the range of distances at which it is accurate, incomplete incorporation of acceptors, and its ease of use. The technique we will use to improve FRET is click chemistry. Click chemistry is a set of highly specific reactions that allow for the selective addition of desired functional groups to a target of interest. Our goal is to determine if FRET will be more sensitive to smaller ranges with click chemistry, which would increase measurement accuracy of conformational changes. To determine if click chemistry can be used with FRET, we first isolate maltose binding protein (MBP) with a non-canonical amino acid (ncAA) introduced at our site of interest from cells. The role of the ncAA is to “click”, or undergo a linking reaction with a specific functional group, with our selected fluorophore to ensure its attachment to the site of interest. Our modified protein will be incubated with our fluorophore, and FRET will be done on the protein under different conditions in order to test if our modified FRET works. If this method works, we expect to see distance dependent quenching of the fluorophore which would indicate that 1) the method works and 2) would serve to establish a benchmark for future experiments with different proteins. Combining click chemistry with FRET could be a powerful tool for measuring conformational dynamics in proteins.