Biochemistry Commons^™

A Novel Transmembrane Ligand Inhibits T Cell Receptor Activation, Yujie Ye

Doctoral Dissertations

T lymphocytes (T cells) play essential roles in the adaptive immune system. Each mature T cell expresses one type of functional T cell receptor (TCR). The TCR recognizes antigens bound to the major histocompatibility complex (MHC) in antigen presenting cells. The resulting stimulation signal crosses the transmembrane domain of TCR and initiates downstream signaling cascades. The human immune system relies on TCRs to recognize a variety of pathogens. Normally, TCR can distinguish the self-antigens from pathogenic antigens. However, dysfunction or aberrant expression of TCRs causes different inflammatory and autoimmune diseases, which afflict millions of people annually (Chapter I). Current treatments …

Investigation Of Kinase Conformational Dynamics And Analytes Detection With Protein Nanopore, Fanjun Li

Doctoral Dissertations

Protein nanopores are pore-forming proteins which have been developed as single-molecule biosensors. Due to the high sensitivity, selectivity, label-free and real-time detection methodology, protein nanopores have been used for a wide variety of applications. In this dissertation, we use ClyA nanopore to investigate kinase conformational dynamics and develop a kinase/nanopore system for the specific detection of kinase allosteric inhibitors. Besides, we engineer OmpG nanopore to be a sensor for nucleic acid detection. Protein kinases play essential roles in cellular regulation by catalyzing the phosphorylation of target proteins and are promising drug targets. The conformational dynamics are critical for kinase functions. …

Controlling Myosin’S Function Via Interactions Between The Substrate And The Active Site, Mike K. Woodward

Doctoral Dissertations

Molecular motors, such as myosin, have evolved to transduce chemical energy from ATP into mechanical work to drive essential cellular processes, from muscle contraction to vesicular transport. Dysfunction in these motors is a root cause of many pathologies necessitating the application of intrinsic control over molecular motor function. We hypothesized that altering the myosin’s energy substrate via minor positional changes to the triphosphate portion of the molecule will allow us to control the protein and affect its in vitro function. We utilized positional isomers of a synthetic non-nucleoside triphosphate, azobenzene triphosphate, and assessed whether myosin’s force- and motion-generating capacity could …

Investigating Structures And Functions Of Apoptotic Caspases, Ishankumar V. Soni

Doctoral Dissertations

Caspases are cysteine aspartate proteases involved in various cellular pathways including apoptosis, inflammation, and neurodegeneration. Caspase-9 is classified as an initiator apoptotic caspase that is activated upon intrinsic stress. Caspase-9 is composed of two domains: an N- terminal CARD domain and a catalytic core domain. We have employed hydrogen deuterium exchange mass spectrometry (H/DX-MS) to determine the 1) dynamics of the full-length caspase- 9, 2) dynamic impacts on caspase-9 upon substrate-induced dimerization, and 3) regions involved in the CARD: catalytic core domains interactions. Upon intrinsic stress, caspase-9 activates executioners, procaspase-3 and -7 but not procaspase-6. We have employed site-directed mutagenesis …

Deciphering Protein Higher-Order Structure And Interactions Via Diethylpyrocarbonate Labeling-Mass Spectrometry, Xiao Pan

Doctoral Dissertations

The study of protein higher-order structures is vital because it is closely related to the investigation of protein folding, aggregation, interaction and protein therapeutics. Consequently, numerous biochemical and biophysical tools have been developed to study protein higher-order structures in many different situations. The combination of covalent labeling (CL) and mass spectrometry (MS) has emerged as a powerful tool for studying protein structures and offers many advantages over other traditional techniques, such as better structural coverage, high throughput, high sensitivity, and the ability to study proteins in mixtures. This dissertation focuses on diethylpyrocarbonate (DEPC) as an effective CL reagent that can …

Quantitative Imaging Of Tensile Forces At Cell-Cell Junction With Dna-Based Probes, Puspam Keshri

Doctoral Dissertations

Mechanical forces are an integral part in biology, they regulate several cellular properties, such as morphology, proliferation, migration. These forces are also involved in receptor signaling and the differentiation of different cell types. Different proteins and biomolecules such as cadherin, integrin, notch proteins are essential elements of these processes. Measuring these intercellular forces are challenging considering the minimal intensity (piconewton-level) of these molecular forces. In our lab, we have developed a membrane DNA tension probe (MDTP) that uses a DNA hairpin module to sense tensile forces and has a lipid anchor to modify onto live-cell membranes. The programmability of DNA …

Design And Synthesis Of Purine Based Neuroprotectors And Novel Synthetic Methods For The Trifluoromethylation Of Aldehyde Hydrazones, Puspa Aryal

Doctoral Dissertations

"Purine-derived compounds are widely investigated as cyclin-dependent kinase inhibitors that have broad applications in the design of pharmaceuticals for treating diseases, such as diabetes, atherosclerosis, and cancers. Towards the goal of effective AGE-inhibitors, and neuroprotector compounds we have synthesized a series of novel purine-based triazoles and investigated their neuroprotective effects, using SHSY-3Y human neuroblastoma cell line. Through these studies, we have identified purine-based neuroprotector compounds that favorably modulate oxidative stress induced by the Fenton reaction-generated reactive oxygen species (ROS).

The C(sp²−H)-trifluoromethylation of hydrazones would give access to the αtrifluoromethylated hydrazones that can serve as intermediates in the synthesis …