Antibodies play a key role in imaging, diagnostics and therapeutics. Antibodies are large molecules, often proteins, produced by our immune system that recognize and bind to specific targets. This project focuses on creating antibody-like recognition proteins that can be triggered to bind and release on command. We propose to create this activatable protein by mutating key regions on an existing protein that would act as a scaffold for the new switchable binding protein. FimH, found on the end of bacteria fimbria, is an ideal protein scaffold because it can be triggered to have an allosteric conformational change that affects binding affinity. To fully explore the different mutation possibilities, we propose to randomize 3 complementary determining region (CDR) loops that are crucial in binding to FimH’s original target. This “library” of proteins will then be expressed on bacteria, filtered to remove improperly folded proteins and screened against targets of interest in order to search for unique binders. The display library size is significant in our work because a larger library increases the probability that there could be more unique binders found while screening the library. We have created a display library of 1.07x10⁵ variants of CDR loop 2 randomized. I have focused on maintaining the diversity of the expressed library by optimizing the library transformation rates while randomizing CDR loops 1 and 3. Through this work, I have found an optimum ratio of cells to library DNA that will express a sufficient sample of the library at an affordable cost. We predict that this technology could improve the specificity of diagnostics and imaging by exacting control through triggering binding and unbinding of targets in these systems.

My project is to reveal mechanisms that regulate the abundance and the distribution of dopamine receptors in C. elegans. The neurotransmitter dopamine carries out its modulatory activities though transmembrane dopamine receptors. These receptors that reside on plasma membranes bind extracellular dopamine and propagate activities through G-protein coupled signaling pathways. The abundance and location of dopamine receptors are highly dynamic, which permits dopamine to modulate brain activities in response to physical changes such as stress and ageing. Here, I propose to image fluorescently tagged dopamine receptors in living worms. I will use CRISPR/Cas9-mediated gene editing approaches to insert fluorescent tags into genes encoding dopamine receptors, which results transgenes located in near native gene environment (such as promoters and 3' UTR regions). To quantify the abundance of dopamine receptors on plasma membranes, I will tag a pH-sensitive green fluorescent protein (pHluorin) to extracellular domains of dopamine receptors. When dopamine receptors are in transporter vesicles, the pHluorin fluorescence is quenched because extracellular domains of dopamine receptors reside in the acidic vesicle lumen. Once dopamine receptors are delivered onto plasma membrane, the pHluorin fluorescence becomes visible as pH is neutralized. To monitor the total amount of dopamine receptors, I will introduce a pH-insensitive red fluorescent protein to the intracellular domain of dopamine receptors. The ratio between green and red fluorescence will indicate the surface fraction of dopamine receptors. As a control, I will conduct pharmacological and behavior assays to test whether the engineered dopamine receptors are functional in vivo. Results from this study will allow us to visualize how dopamine receptors are regulated to support brain functions.

Because a protein’s three-dimensional structure defines its function, improved methods for resolving structure are an important objective in molecular biology. For example, the structures of many pharmaceutically relevant proteins are difficult to characterize with current experimental approaches. Computational techniques that predict structures from amino acid sequences obviate problematic physical manipulation of proteins, but are unreliable. Computational prediction improves, however, when supplemented with limited structural data. We propose generating data that describes spatial constraints with deep mutational scanning, a method we developed to measure the functional consequences of hundreds of thousands of variants simultaneously. I will use large-scale mutagenesis to create single- and double-mutant variants of two essential yeast proteins, cdc42 and guk1. These constructs will be transformed into Tet-Off yeast, in which the endogenous cdc42 or guk1 promoter is replaced with a repressible tet promoter. I will then employ high-throughput DNA sequencing to track variant frequencies before and after competitive growth in doxycycline. Stable, functioning variants should rescue growth and increase in frequency while deleterious variants will decrease in frequency. From these frequencies, I will derive functional scores. We hypothesize that functional scores given by two single mutations will predict the functional score of those mutations combined in a double mutant; double mutants with unexpectedly high or low scores would suggest interaction between the mutated positions. Interacting pairs associate amino acids in space, revealing spatial constraints that may enhance computational approaches for determining otherwise intractable three-dimensional protein structures.

Influenza A viruses have two main surface proteins that usually have opposing functions. Hemagglutinin (HA) is traditionally thought of as the receptor-binding protein which binds to sialic acid on the surface of the target cell, while neuraminidase (NA) is a sialidase that removes sialic acid receptors to allow for viral release. However, recently, it was discovered that a mutant NA can function as a receptor-binding protein when a single amino-acid substitution (G147R) is made near the active site. Thus, to study the effects of a mutant receptor-binding NA in a controlled fashion, we sought to produce virus-like particles (VLPs), which express only NA, but not HA on their surface. Initially VLPs were made by co-transfecting 293T cells with a plasmid expressing the mutant NA and a plasmid expressing the influenza matrix genes. This method of producing VLPs gave a lower yield than what was required for further experiments. In order to optimize VLP production, I hypothesized that I could instead use the HIV Gag polyprotein which alone can cause membrane budding and scission. By comparing the VLP production efficiency of particles which contain either the influenza matrix genes or the HIV capsid protein, I hope to develop a method which allows us to produce large quantities of VLPs for receptor-binding studies. If using HIV Gag polyprotein does prove to be more reliable, I will begin using this technique to produce VLPs in order to identify the receptor for the NA-binding mutant previously discovered. I will then begin testing the NAs of contemporary human viruses to determine if the phenomenon of NA-binding occurs in nature. Overall, this work aims to better understand a newly recognized mode of influenza infection.

In the Hague Lab we study a class of proteins called G-protein coupled receptors (GPCR's). GPCR's are seven-pass transmembrane proteins that are involved in cell signaling. One GPCR we study is called alpha-1D adrenergic receptor. Adrenergic receptors bind the endogenous catecholamines adrenaline and noradrenaline. An important functional role of the alpha-1D GPCR in the body is to regulate blood pressure by affecting blood vessel diameter.  Our lab recently discovered that the alpha-1D adrenergic receptor's relatively long N-terminal domain is cleaved in human cells. We are currently elucidating the specific cleavage site - which we suspect is a GL matrix metalloprotease domain - and the physiological purpose of the cleavage. Over the last few months we have narrowed down the region of cleavage to approximately 15 amino acids. We also have a working theory that the N-terminal cleavage of the protein allows for better cell surface localization. We are currently examining how the cleavage event effects the signaling characteristics of the protein.

It has been shown that the chemical nor-binaltorphimine (norBNI) inactivates the k-opioid receptor (KOR) for days or weeks in vivo, and also results in downstream activation of c-Jun N-terminal kinase (JNK). If JNK is blocked in vivo by SP610025 or by knocking out the JNK1 gene, norBNI inactivates the KOR for just hours. We hypothesize that there is a JNK mediated inactivation of the KOR. To further investigate this, I will create constructs that will allow us to study the interactions of JNK and arrestin, two proteins that are involved in the transduction of KOR signaling. I aim to express a version of each JNK1 splice variant tagged with luciferase and arrestin3 tagged with Venus in human embryonic kidney (HEK293) cells that have been transfected with rat KOR. Venus and luciferase are two bioluminescent molecules that emit different wavelengths of light depending on their distance from one another. After treating these cells with norBNI, I can dtermine the level of their interaction by using a bioluminescence resonance energy transfer (BRET) assay. These experiments will provide useful data in determining the extent of the interactions between JNK1 and arrestin3. This data will help us understand the protein cascade that results in the deactivation of the KOR, and if certain drugs activate certain splice variants. Understanding this pathway has implications in creating analgesics with smaller potential for abuse and addiction, and creating pharmaceuticals that could help with stress related depression and addiction relapse.

Most important biological interactions with the environment occur at interfaces such as a lipid cellular membrane. These interactions are often dynamic and show time-dependent changes in material properties (e.g, viscoelastic properties). A detailed understanding of biomechanical properties at molecular and subcellular level is vital for basic understanding of normal/abnormal biological processes. Indeed, abnormal changes in the mechanics of biological systems are often indicators of pathophysiological states. For example, amyloid beta peptide (Aβ), a protein present in neural plaques formed in Alzheimer’s disease and cholesterol are reported to alter cell membrane mechanical properties leading to neuronal degeneration. Atomic force microscopy (AFM) provides an ideal tool to image the structure and examine the mechanics in physiologically relevant medium. We have created a new cantilevered probe for AFM for the study of nano-to-microscale mechanics of biological interfaces. We used it to examine the role of Aβ1-42 insertion in DPPC lipid monolayers and its effect on the viscoelastic properties that underlie altered membrane fluidity. Real-time analysis of mechanical properties of the model membrane monolayer in the presence of Aβ1-42 shows a decrease in the viscosity of the monolayers over time, consistent with increased membrane fluidity. The finding is consistent with changes in membrane permeability due to Aβ-inserted pores.