The integrin Mac-1 (αMβ2, CD11b/CD18) is an important adhesion receptorexpressed on macrophages and neutrophils. It plays a crucial role in phagocytosis, cell-cell fusion, and cell migration. αMβ2 is also the most promiscuous integrin with over 100 known ligands that span a broad range of physical and chemical attributes, many of which bind to the inserted (I) domain from the αM subunit. The interaction of αMI-domain with cytokine pleiotrophin (PTN) were determine. PTN is a cationic protein known to induce Mac-1- mediated adhesion and migration in cells. The data showed that PTN’s interaction with αMI-domain contains both divalent cation-dependent and independent mechanisms. In particular, PTN’s N-terminal domain has weak interactions with the N/C-termini side of αMI-domain using a metal-independent mechanism. However, stronger interaction is achieved through the chelation of the divalent cation in the metal ion-dependent adhesion site of active αMI-domain by PTN’s acidic residues. Although many acidic residues in PTN can act as the chelator, active αMI-domain’s interaction with PTN’s E98 plays an especially important role. NOE, chemical shift perturbation (CSP) data, and mutagenesis studies showed residues near E98 are at the binding interface and the E98 mutation greatly reduced binding affinity between two proteins. Interestingly, the CSP and MD simulation data showed the binding interface can be supported by the interaction of PTN’s H95 with the acidic clusters D242, E244, and D273 from αMI-domain, while PTN’s E66 form electrostatic interaction with R208 and K245 from αMI-domain. The determined recognition motif of αMI-domain for its ligands is (H/R/K)xxE. The ability to accommodate the longer distance between E and (H, R, K) compared to the zwitterionic motif RGDii explained how αMβ2 can interact with a large repertoire of ligands and be versatile in its functional portfolio.

G protein coupled receptors (GPCRs) mediate various of physiologicalactivities which makes them significant drug targets. Determination of atomic level structure of GPCRs facilitates the structure-based drug design. The most widely used method currently for solving GPCR structure is still protein crystallography especially lipidic cubic phase (LCP) crystallization. LCP could mimic the native environment of membrane protein which stable the membrane proteins. Traditional synchrotron source requires large size large size protein crystals (>30 micron) due to the radiation damage during data collection. However, acquiring large sized protein crystals is challenging and not guaranteed practically. In this study, a novel method was developed which combined LCP technology and micro-electron diffraction (MicroED) technology. LCP-MicroED technology was able to collect complete diffraction data sets from serval submicron protein crystals and deliver high resolution protein structures. This technology was first confirmed with soluble protein crystals, proteinase K and small molecule crystals, cholesterol. Furthermore, this novel method was applied to a human GPCR target, Î22- adrenergic receptor (Î22AR). The structure model was successfully built which proved the feasibility of applying LCP-MicroED method to GPCRs and other membrane proteins. Besides, in this research, a novel human GPCR target, human histamine 4 receptor(H4R) was studied. Different constructs were expressed, purified, and characterized. Some key residuals that affect ligand binding were confirmed.

Glycans are complex biological sugar polymers that are commonly found covalently attached to proteins, lipids, and lipoproteins. About 50% of all mammalian proteins are glycosylated. Aberrant glycosylation is a hallmark of most types of cancer, and glycosylation changes that occur in this disease are known to facilitate tumor development. In this dissertation, a bottom-up approach to glycomics, “glycan node analysis”, which is a method based on glycan linkage analysis that quantifies unique glycan features, such as “core fucosylation”, “α2-6 sialylation”, “β1-6 branching”, and “bisecting GlcNAc”, as single analytical signals by gas chromatography-mass spectrometry (GC-MS), was applied to cancer cell lines, antibodies, extracellular vesicles, and low density lipoproteins to understand the mechanisms leading to aberrant glycosylation in cancer, and to understand the role of blood plasma glycan sialylation in cancer immunity. Specific tumor antigens such as β1-6-branching, β1-4-branching, bisecting GlcNAc, antennary fucosylation, and Tn antigen (GalNAc-Ser/Thr), were found to be regulated by IL-6 in HepG2 cells; fewer glycan features were regulated by IL-1β. Additionally, neuraminidase enzyme treatment of alpha-1 antitrypsin IgG demonstrates how glycan node analysis can be used to detect relative changes in “α2-6-sialylation” along with corresponding increases in terminal galactose. Extracellular vesicles (EVs) derived from metastatic and non-metastatic cancer cell lines displayed upregulated or downregulated expression of several specific glycan nodes, particularly 3-GlcNAc, which represents hyaluronic acid. EVs displayed several glycan features that distinguished them from the whole blood plasma glycome. These results were promising for developing new diagnostic strategies in cancer. A “liquid phase permethylation” procedure for glycan node analysis that does not require spin columns was applied for the first time to whole biological specimens, and it demonstrated potential clinical utility in detecting specific tumor antigens. Significantly different glycan node profiles were found among three cancer cell lines and in peripheral blood mononuclear cells from healthy donors. Changes in glycosylation and mechanisms regulating glycan changes were studied extensively in cancer cells. Subsequently, it is reported how glycosylation changes can have an impact in cancer immunity. A novel role for oxidized-desialylated low density lipoprotein in cancer immunity is reported, and its implications in cancer and atherosclerosis are discussed.

The transient receptor potential channel subfamily V member 1 (TRPV1) functions as the heat and capsaicin receptor. It can be activated by heat, protons, pungent chemicals, and a variety of other endogenous mediators of nociception. TRPV1 is a non-selective cation channel consisting of 6 transmembrane domains (S1-S6), with helices S1-S4 forming the sensing domain and the S5-S6 helices forming the pore domain. Understanding the TRPV1 channel is imperative due to its relation to a variety of human diseases, including cancer, type II diabetes, hyper and hypothermia, and inflammatory disorders of the airways and bladder. Although TRPV1 is the best-studied thermosensitive-TRP channels of all the 28 family members, the molecular underpinning and the contributions of the human TRPV1 pore domain in thermo-sensing remains elusive. Recently, the human TRPV1 sensing domain was found to contribute to heat activation. It was found to undergo a non-denaturing temperature-dependent conformational change. This finding triggered interest in studying the function and the role of the human TRPV1 pore domain in the heat activation process. Specifically, to identify whether heat activation is intrinsic to the pore domain. This thesis paper explores and optimizes the purification protocol of the human TRPV1 pore domain through three different methods. The first method was using a denaturant, the second method was increasing the length of the histidine tags through Q5 insertion, and the third method was incorporating the protein construct into nanodiscs. In addition to the above three methods, size exclusion chromatography and ion-exchange chromatography were utilized after thrombin cleavage to separate the human TRPV1 pore domain from the cleaved MBP deca-histidine tags as well as the impurities.

The small mitogenic cytokine Pleiotrophin (PTN) is well-known for its roles in

tissue growth, development, and repair. First isolated from neuronal tissues, much interest in this protein resides in development of the central nervous system and neuronal regeneration. Owning to its role in growth, development and its ability to promote angiogenesis and metastasis, PTN’s overexpression in cancers such as glioblastoma, has become the focal point of much research. Many of the receptors through which PTN acts contain glycosaminoglycans (GAGs), through which PTN binds. Thus, understanding the atomistic detail of PTN’s architecture and interaction with GAG chains is of significant importance in elucidating its functional role in growth and malignancy of biological tissues, as well as in neural development and progression of other diseases. Herein the first solution state structure of PTN was solved via nuclear magnetic resonance (NMR), with extensive characterization of its ability to bind GAG. Structurally, PTN consists of two -sheet domains connected by a short flexible linker, and flanked by long flexible termini. Broad distribution of positively charged amino acids in the protein’s sequence yields highly basic surfaces on the -sheet domains as well as highly cationic termini. With GAG chains themselves being linear anionic polymers, all interactions between these sugars and PTN are most exclusively driven through the electrostatic interactions between them, with no discernable specificity for GAG types. Moreover, this binding event is coordinated mostly through basic patches located in the C-Terminal domain (CTD). Although the flexible C- terminus has been shown to play a significant role in receptor binding, data here also reveal an adaptability of PTN to maintain high affinity interactions through its structured domains

when termini are removed. Additionally, analysis of binding information revealed for the first time the presence of a secondary GAG binding site within PTN. It is shown that PTN’s CTD constitutes the major binding site, while the N-terminal domain (NTD) contains the much weaker secondary site. Finally, compilation of high-resolution data containing the atomistic detail of PTN’s interaction with GAG provided the information necessary to produce the highest accuracy model to date of the PTN-GAG complex. Taken together, these findings provide means for specific targeting of this mitogenic cytokine in a wide array of biological applications.

Coronaviruses are the causative agents of SARS, MERS and the ongoing COVID-19 pandemic. Coronavirus envelope proteins have received increasing attention as drug targets, due to their multiple functional roles during the infection cycle. The murine coronavirus mouse hepatitis virus strain A59, a hepatic and neuronal tropic coronavirus, is considered a prototype of the betacoronaviruses. The envelope protein of the mouse hepatitis virus (MHV-E) was extensively screened with various membrane mimetics by solution state nuclear magnetic resonance spectroscopy to find a suitable mimetic, which allowed for assignment of ~97% of the backbone atoms in the transmembrane region. Following resonance assignments, the binding site of the ion channel inhibitor hexamethylene amiloride (HMA) was mapped to MHV-E using chemical shift perturbations in both amide and aromatic transverse relaxation optimized spectroscopy (TROSY) spectra, which indicated the inhibitor binding site is located at the N-terminal opening of the channel, in accord with one of the proposed HMA binding sites in the envelope protein from the related SARS (severe acute respiratory syndrome) betacoronavirus. Structure calculation of residues M1-K38 of MHV-E, encompassing the transmembrane region, is currently in progress using dihedral angle restraints obtained from isotropic chemical shifts and distance restraints obtained from manually assigned NOE cross-peaks, with the ultimate aim of generating a model of the MHV-E viroporin bound to the inhibitor HMA. This work outlines the first NMR studies on MHV-E, which have provided a foundation for structure based drug design and probing interactions, and the methods can be extended, with suitable modifications, to other coronavirus envelope proteins.

There is increasing interest and demand in biology studies for identifying and characterizing rare cells or bioparticle subtypes. These subpopulations demonstrate special function, as examples, in multipotent proliferation, immune system response, and cancer diagnosis. Current techniques for separation and identification of these targets lack the accuracy and sensitivity needed to interrogate the complex and diverse bioparticle mixtures. High resolution separations of unlabeled and unaltered cells is an emerging capability. In particular, electric field-driven punctuated microgradient separations have shown high resolution separations of bioparticles. These separations are based on biophysical properties of the un-altered bioparticles. Here, the properties of the bioparticles were identified by ratio of electrokinetic (EK) to dielectrophoretic (DEP) mobilities.

As part of this dissertation, high-resolution separations have been applied to neural stem and progenitor cells (NSPCs). The abundance of NSPCs captured with different range of ratio of EK to DEP mobilities are consistent with the final fate trends of the populations. This supports the idea of unbiased and unlabeled high-resolution separation of NSPCs to specific fates is possible. In addition, a new strategy to generate reproducible subpopulations using varied applied potential were employed for studying insulin vesicles from beta cells. The isolated subpopulations demonstrated that the insulin vesicles are heterogenous and showed different distribution of mobility ratios when compared with glucose treated insulin vesicles. This is consistent with existing vesicle density and local concentration data. Furthermore, proteins, which are accepted as challenging small bioparticles to be captured by electrophysical method, were concentrated by this technique. Proteins including IgG, lysozyme, alpha-chymotrypsinogen A were differentiated and characterized with the ratio factor. An extremely narrow bandwidth and high resolution characterization technique, which is experimentally simple and fast, has been developed for proteins. Finally, the native whole cell separation technique has also been applied for Salmonella serotype identification and differentiation for the first time. The technique generated full differentiation of four serotypes of Salmonella. These works may lead to a less expensive and more decentralized new tool and method for transplantation, proteomics, basic research, and microbiologists, working in parallel with other characterization methods.

Integrins are a family of αβ heterodimeric transmembrane receptors. As an important class of adhesion receptors, integrins mediate cell adhesion, migration, and transformation through bidirectional signaling across the plasma membrane. Among the 24 different types of integrins, which are notorious for their capacity to recognize multiple ligands, the leukocyte integrin αMβ2 (Mac-1) is the most promiscuous member. In contrast to other integrins, Mac1 is unique with respect to its preference for cationic ligands. In this thesis, a new Mac-1 cationic ligand named pleiotrophin (PTN) is uncovered. PTN is an important cytokine and growth factor. Its activities in mitogenesis and angiogenesis have been extensively researched, but its function on immune cells was not widely explored. In this research, the cell biology and biochemical evidences show that PTN can regulate various Mac-1-expressing cells functions through the activation of the extracellular signal regulated kinases. Direct interactions between PTN and the αM I-domain, the major ligand-binding domain of Mac-1, has been shown using biolayer interferometry analyses and confirmed by solution NMR spectroscopy. The binding epitopes and the binding mechanism of PTN and αM I-domain interaction were further revealed by peptide array analysis and microscale thermophoresis. The data suggested that PTN’s thrombospondin type-1 repeat (TSR) domains and αM I-domain metal-ion-dependent adhesion site (MIDAS) are the major binding sites. In addition, this interaction followed a novel metal-ion independent binding mechanism which has not been found in other integrins. After a series of characterizations of αM I-domain using both experimental and computational methods, it showed that activated αM I-domain is significantly more dynamic than inactive αM I-domain, and the dynamics seem to modulate the effect of Mg2+ on its interactions with cationic ligands. To further explore the PTN induced Mac-1 structure rearrangement, intact Mac-1 was studied by negative stain electron microscopy. The results showed that the Mac-1 exhibited a very heterogeneous conformation distribution in detergents. In contrast, the Mac-1 adopted predominantly the bent conformation in phospholipid nanodisc condition. This Mac-1 nanodisc model provides a new platform for studying intact Mac-1 activation mechanism in a more physiologically relevant manner in the future.

Transient receptor potential vanilloid member 1 (TRPV1) is a membrane protein ion channel that functions as a heat and capsaicin receptor. In addition to activation by hot temperature and vanilloid compounds such as capsaicin, TRPV1 is modulated by various stimuli including acidic pH, endogenous lipids, diverse biological and synthetic chemical ligands, and modulatory proteins. Due to its sensitivity to noxious stimuli such as high temperature and pungent chemicals, there has been significant evidence that TRPV1 participates in a variety of human physiological and pathophysiological pathways, raising the potential of TRPV1 as an attractive therapeutic target. However, the polymodal nature of TRPV1 function has complicated clinical application because the TRPV1 activation mechanisms from different modes have generally been enigmatic. Consequently, tremendous efforts have put into dissecting the mechanisms of different activation modes, but numerous questions remain to be answered.

The studies conducted in this dissertation probed the role of the S1-S4 membrane domain in temperature and ligand activation of human TRPV1. Temperature-dependent solution nuclear magnetic resonance (NMR) spectroscopy for thermodynamic and mechanistic studies of the S1-S4 domain. From these results, a potential temperature sensing mechanism of TRPV1, initiated from the S1-S4 domain, was proposed. Additionally, direct binding of various ligands to the S1-S4 domain were used to ascertain the interaction site and the affinities (Kd) of various ligands to this domain. These results are the first to study the isolated S1-S4 domain of human TRPV1 and many results indicate that the S1-S4 domain is crucial for both temperature-sensing and is the general receptor binding site central to chemical activation.