Biophysics Commons^™

Study Of Zyomogen Granule Movement Along Actin Filaments Using A Single Beam Optical Trap, Justin James Raupp

Wayne State University Dissertations

Zymogen granules are enzymatic vesicles in the pancreas. The surface of these zymogen granules (ZGs) has several different kinds of myosin molecules, such as myosin 1c, 6, 5c, and 7b. These molecular motors may contribute to ZG transportation in cells. To understand the molecular motors involved in the vesicle trafficking, we observed the in vitro motility of purified ZGs from rat pancreas and examined the stepping behavior and force that is generated using a single beam optical trap. To be involved in trafficking, molecular motors have certain characteristics, a high duty ratio and the ability to move continuously along actin …

Abhd5 Induced Morphological Changes On Model Membrane Systems, Nasser S. Junedi

Honors College Theses

Proper regulation of neutral lipid storage (lipogenesis) and release (lipolysis) are critical molecular processes localized to an organelle called the Lipid Droplet (LD). The LD consists of a core with neutral lipids such as triacylglycerols (TAGs) and sterol esters surrounded by a phospholipid monolayer. Dysregulation of the processes localized to the LD are involved in the pathology of various diseases such as Neutral Lipid Storage Disease, diabetes, stroke and cancer. The non-enzymatic protein ABHD5 (α-β Hydrolase Domain-Containing Protein 5), is thought to play a key role in the process of lipolysis by forming homo-oligomers on the surface of the LD …

Single-Lipid Sorting And Dynamics At Nanoscale Membrane Curvatures: The Effects Of Fluorescence Labeling, Composition, Phase, And Temperature, Xinxin Woodward

Wayne State University Dissertations

Nanoscale membrane curvature on cell plasma membrane assists in the spatial organization and domain formation that are critical for life of eukaryotic cells. Lipids and proteins can sense, be sorted by, and generate both functional domains and membrane curvature. Reveal the relationship between membrane curvature, phase separation, and single-molecule behavior is a key to understanding fundamental processes, such as phagocytosis initiation, cell signaling, and membrane budding. Single lipid dynamic and sorting on engineered membrane curvature is studied to understand the effects of fluorescence labeling, composition, phase separation, and temperature. Single particle tracking was used to find radial averaged diffusion at …

Predicting The Structure And Selectivity Of Coiled-Coil Proteins, Mojtaba Jokar

Wayne State University Dissertations

A coiled-coil protein structure consists of two (in coiled-coil dimers) or more interacting α-helical strands that together form a left-handed supercoil structure. Many coiled-coil proteins are involved in significant biological functions such as the regulation of gene expression, known as transcription factors. Also coiled-coil structures entail unique mechanical properties critical to the function and integrity of various motor proteins, cytoskeletal filaments and extra-cellular matrix proteins. Engineering these transcription factors is also expected to create more efficient and practical solutions to treat neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS) and prion …

Alternative Strategies To Inhibit Lysine Methyltransferases And Deubiquitinases In Human Cancers, Nicholas Spellmon

Wayne State University Dissertations

X-ray crystallography is the gold standard method for imagining macromolecules to atomic resolution. Three dimensional data is central to understanding the molecular mechanism how DNA, RNA and proteins function in biological events. Structural insights into these events provide a molecular window to visualize how biological molecules influence human health. Visualizing the architecture of these molecules set the stage for rational and selective drug design. The following dissertation utilizes biochemical and biophysical tools, including X-ray crystallography, to shed light on poorly understood mechanisms related to SMYD2 activity and regulation, USP10 architecture and function, and PDZ-RhoGEF dimerization. SMYD2 is one member of …

Polarized Localization Microscopy (Plm) Detects Nanoscale Membrane Curvature And Induced Budding By Cholera Toxin Subunit B (Ctxb), Abir Kabbani

Wayne State University Dissertations

The curvature of biological membranes at the nanometer scale is critically important for vesicle trafficking, organelle morphology, and disease propagation. Many proteins and lipids interact with diverse curvature sensing and curvature generating mechanisms. Deciphering the molecular mechanisms of toxin-membrane interactions has been limited by the resolution and drawbacks of conventional experimental techniques. This study reveals the inherent membrane bending capability of cholera toxin subunit B (CTxB) through the development and implementation of Polarized Localization Microscopy (PLM). PLM is a pointillist optical imaging technique for the detection of nanoscale membrane curvature in correlation with single-molecule dynamics and molecular sorting.

PLM combines …

Exploring The Physics Of Proteins At Molecular Level By Neutron And X-Ray Scattering, Utsab Raj Shrestha

Wayne State University Dissertations

The protein structures revealed by the crystallographic studies have provided the valuable information over the years regarding their biological functions. However, such snapshots of protein fluctuations averaged over time may not be enough to fully capture the underlying biological phenomena. A deeper understanding of the protein dynamics is crucial for elucidating the structural pathways or the transition mechanism from the initial state to the final state necessary for regulating the physical and chemical processes. Hence, the biological activities and functions are mainly governed by the protein conformational dynamics. However, the direct correlation of a wide range of protein dynamics to …

Structural Basis Of Membrane Protein Scaffolding And Signaling In Human Disease, Joshua Holcomb

Wayne State University Dissertations

Protein structural elucidation by means of X-ray crystallography is a powerful approach for both insight into a proteins biophysical properties and function. To date X-ray crystallography remains the gold standard in high resolution structural determination and serves as the basis for rational drug design for the purpose of combating a number of human diseases. Such an approach also allows for the exploitation of how various proteins interact with their substrates providing a molecular basis for their physiological function. In this dissertation, using X-ray crystallographic analysis along with other biophysical characterization methods, we seek to understand the mechanistic foundation for which …

The Effects Of The N-Terminal Extensions Of Cardiac Troponins On The Ca2+ Regulation Of Myosin Atpase Kinetics In Cardiac Myofibrils, Laura Gunther

Wayne State University Dissertations

Contraction of cardiac muscle is the basis of heart function. Heart failure, i.e., weakened contraction of cardiac muscle is the most common cause of morbidity and mortality of heart diseases. Cardiac muscle contraction is regulated by calcium via the function of troponin, a protein complex associated with the myofilaments in muscle cells. The cardiac troponin subunits T (cTnT) and I (cTnI) have unique N-terminal extensions that can be selectively removed by restrictive proteolysis during cardiac adaptation to physiological and pathological stresses, indicating a role of these proteins in modulating cardiac contraction. This study aims to understand the effects of the …

Interaction Forces And Reaction Kinetics Of Ligand-Cell Receptor Systems Using Atomic Force Microscopy, Anwesha Sarkar

Wayne State University Dissertations

Atomic Force Microscopy (AFM) provides superior imaging resolution and the ability to measure forces at the nanoscale. It is an important tool for studying a wide range of bio-molecular samples from proteins, DNA to living cells. We developed AFM measurement procedures to measure protein interactions on live cells at the single molecular level. These measurements can be interpreted by using proper statistical approaches and can yield important parameters about ligand-receptor interactions on live cells. However, the standard theory for analyzing rupture force data does not fit the experimental rupture force histograms. Most of the experimental measurements of rupture force data …

Dynamics Of Biopolymers On Nanomaterials Studied By Quasielastic Neutron Scattering And Mdsimulations, Gurpreet Dhindsa

Wayne State University Dissertations

Neutron scattering has been proved to be a powerful tool to study the dynamics of biological systems under various conditions. This thesis intends to utilize neutron scattering techniques, combining with MD simulations, to develop fundamental understanding of several biologically interesting systems. Our systems include a drug delivery system containing Nanodiamonds with nucleic acid (RNA), and two specific model proteins, β-Casein and Inorganic Pyrophosphatase (IPPase).

RNA and nanodiamond (ND) both are suitable for drug-delivery applications in nano-biotechnology. The architecturally flexible RNA with catalytic functionality forms nanocomposites that can treat life-threatening diseases. The non-toxic ND has excellent mechanical and optical properties and …

Hiv-1 Rna Dimerization At Single Molecule Level, Hansini R. Mundigala

Wayne State University Dissertations

The Dimerization Initiation Sequence (DIS) is a conserved hairpin-loop motif on the 5' UTR of the HIV-1 genome. It plays an important role in genome dimerization through formation of a "kissing complex" intermediate between two homologous DIS sequences. This bimolecular kissing complex ultimately leads to the formation of an extended RNA duplex. Understanding the kinetics of this interaction is key to exploiting DIS as a possible drug target against HIV. We wish to report a novel study that makes an important contribution to understanding the dimerization mechanism of HIV-1 RNA in vitro. Our work has employed single-molecule fluorescence resonance energy …

Characterization Of Initial Iron Binding Location And The Structure/Iron Binding Site On S.Cerevisiae Isu And On D.Melanogaster Frataxin, Andria V. Rodrigues

Wayne State University Dissertations

Iron-induced free radical damage has been implicated in the pathology of diseases of iron overload such as Friedreich's Ataxia, a genetic disorder characterized by an accumulation of iron in actively metabolizing tissues ultimately leading to cardio- and neuro- degeneration and cell death. It is caused by an inability to synthesize the mitochondrial protein, frataxin. Frataxin has been shown by numerous groups to be a part of the iron-sulfur cluster (ISC) multicomplex, where it functions in the capacity of a potential iron provider and an allosteric modulator of both the cysteine desulfurase and scaffold protein ISU. My research has been focused …

The Nh2-Hypervariable Region Modulates The Binding Affinity Of Troponin T For Tropomyosin, Chinthaka Kaushalya Amarasinghe

Wayne State University Theses

The troponin complex plays a central role in the allosteric function of sarcomeric thin filaments by enacting conformational changes during the Ca2+-regulated contraction and relaxation of striated muscle. The troponin subunit T (TnT) has two binding sites for tropomyosin (Tm) and is responsible for anchoring the troponin complex to the thin filament. Although the C-terminal and middle regions of the TnT polypeptide chain are highly conserved among the three muscle type isoforms, the hypervariable N-terminal region has evolutionarily diverged significantly among isoforms. Previous studies have shown that the N-terminal variable region fine-tunes Ca2+ regulation of muscle contractility via modulation of …

How Atomic Level Interactions Drive Membrane Fusion: Insights From Molecular Dynamics Simulations, Navendu Bhatnagar

Wayne State University Dissertations

This project is focused on identifying the role of key players in the membrane fusion process at the atomic level with the use of molecular dynamics simulations. Membrane fusion of apposed bilayers is one of the most fundamental and frequently occurring biological phenomena in living organisms. It is an essential step in several cellular processes such as neuronal exocytosis, sperm fusion with oocytes and intracellular fusion of organelles to name a few. Membrane fusion is a frequent process in a living organism but is still not fully understood at the atomic level in terms of the role of various factors …

Elucidation Of The Functional Role Of Pcbp-The Cytosolic Iron Chaperone Protein Family, In Cellular Iron Homeostasis, Poorna Subramanian

Wayne State University Dissertations

Biological Role of Ferritin - Iron is essential for life and often utilized as a cofactor in many proteins. In humans, iron accumulation causes cirrhosis, arthritis, cardiomyopathy and diabetes mellitus, and it is associated with increased risk of cancer and heart disease. In contrast, decreased brain iron content results in permanent neurocognitive and motor impairment. Intracellular iron content must be maintained within a narrow range to avoid the adverse effects of iron depletion or excess, and this function is performed by the protein ferritin. Ferritins are iron storage proteins that are ubiquitously expressed in animals, plants and bacteria. They serve …

Single-Stranded Dna Scanning And Deamination With Single-Molecule Resolution, Padhuk Gamarala Senavirathne

Wayne State University Dissertations

SINGLE-STRANDED DNA SCANNING AND DEAMINATION WITH SINGLE-MOLECULE RESOLUTION

by

GAYAN SENAVIRATHNE

December 2013

Advisor: Dr. David Rueda & Dr.Ashok Bhagwat

Major: Chemistry (Analytical)

Degree: Doctor of Philosophy

APOBEC3G (Apo3G) and Activation-Induced cytidine Deaminase (AID) are the most notable members of APOBEC enzymes, a family of cytidine (C) deaminases with crucial functions in the immune system. In T cells, Apo3G deaminates C in viral cDNA to halt the replication of HIV 1 strains lacking viral infectivity factor (vif). In B cells, AID is required for the diversification of antibodies through initiation of somatic hypermutation (SHM), and class switch recombination (CSR), by …

"Fine-Tuning" Of Ribosomal Structure And Functions By Pseudouridylation And Rna-Protein Interactions, Jun Jiang

Wayne State University Dissertations

ABSTRACT

"Fine-tuning" of ribosomal structure and functions by pseudouridylation and RNA-protein interactions

by

JUN JIANG

AUGUST 2012

Advisor: Prof. John SantaLucia Jr.

Major: Chemistry (Biochemistry)

Degree: Doctor of Philosophy

Ribosomal structure and functions appear to be "fine-tuned" by pseudouridylation and RNA-protein interactions. Pseudouridylation may promote base stacking interactions by mediating the base stacking between residues on both sides. In the RNA duplex region, this enhanced stacking interaction contributes to stabilization of duplex folding. In the loop region, enhanced stacking in one structural motif may destabilize the conformation of adjacent structural residues. This hypothesis is supported by both UV-melting experiments, where …

The Spliceosomal Protein Prp8 Stabilizes A Compact Conformation Of The U2-U6 Complex, Subasinghe Appuhamilage Lemintha Imali Subasinghe

Wayne State University Theses

The spliceosome is a large, RNA-protein complex that catalyzes pre-mRNA splicing during mRNA maturation. The RNA components (small nuclear RNA; snRNAs) of the spliceosome have been well studied and are believed to be involved in the splicing catalysis. Although proteins are essential for splicing, they may not be directly involved in catalysis. Among hundreds of proteins, Prp8 is the only protein that interacts with all of the catalytically important snRNAs. Therefore, it is hypothesized that Prp8 may catalyze splicing either by directly participating in catalysis or by stabilizing the conformation of the catalytically active spliceosome. In order to test whether …

Artificial And Natural Nucleic Acid Self Assembling Systems, Marcus Wood

Wayne State University Dissertations

Nucleic acids are good candidates for nanomachine construction. They participate in all the processes of life, and so can function as structural building blocks and dynamic catalysts. However, to use nucleic acids as nanomachines, a better understanding of their material properties, how to design structures using them, and their dynamics is needed. We have tried to address these issues, in a small way, with nucleic acid force field development, an attempt at nanostructural design and synthesis using DNA, and a study of the RNA/protein regulatory dynamics of the tryptophan regulatory attenuation protein.

Mechanical Forces And Tumor Cells: Insight Into The Biophysical Aspects Of Cancer Progression, Indrajyoti Indra

Wayne State University Dissertations

Mechanical forces play an important role in the regulation of cellular behavior and physiological processes including adhesion, migration, proliferation, tissue repair, embryogenesis and development. In addition, a number of diseases including cancer, have been linked to changes in cellular and extracellular mechanical properties. However, whether a correlation exists between the progression of cancer towards metastasis and mechanical factors has not been clearly defined. Additionally, how a cell responds to changes in extracellular mechanical cues as it gains metastatic abilities is poorly understood. To address these questions, we have utilized a panel of murine breast cancer cell lines with progressive metastatic. …

Characterization Of Splicing Mechanisms By Single-Molecule Fluorescence, Krishanthi Sanjeewani Karunatilaka

Wayne State University Dissertations

Group II introns rank amongst the largest self-splicing ribozymes found in bacteria and organellar genomes of various eukaryotes. Despite the diversity in primary sequences, group II introns posses highly conserved secondary structures consisting of six domains (D1-D6). To perform its function, the large multidomain group II intron RNA must adopt the correctly folded structure. As a result, in vitro splicing of these introns requires high ionic strength and elevated temperatures. In vivo, this process is mainly assisted by protein cofactors. However, the exact mechanism of protein-mediated splicing of group II intron RNA is still not known.

In order to …

Laser-Assisted Single-Molecule Refolding, Rui Zhao

Wayne State University Dissertations

Non-coding RNAs must fold into precise secondary and tertiary structures in order to perform the biological functions. Due to the flexibility of RNA, the RNA folding energy landscape can be rugged and full of local minimum (kinetic trap). To provide a means to study kinetically trapped RNAs, we have developed a new technique combining single-molecule FRET detection with laser induced temperature jump. We have calibrated the magnitude of the temperature jump with 1˚C accuracy using gold micro-size sensor. The accuracy of temperature calibration was confirmed by close agreement between single-molecule and bulk DNA duplex melting experiments.

HIV 1 DIS RNAs …

Investigating The Metal Binding Sites In Znta, A Zinc Transporting Atpase, Sandhya Muralidharan

Wayne State University Dissertations

ZntA from Escherichia coli is a member of the P_IBtype ATPase family of transporters. The P_IB-type ATPase pumps maintain cellular homeostasis of heavy metals such as Zn²⁺, Co²⁺, Cu²⁺, Cu⁺, and mediate resistance to toxic metals such as Pb²⁺, Cd²⁺ and Ag⁺. ZntA confers resistance to Pb²⁺, Cd²⁺, and Zn²⁺ by pumping these ions out of the cytoplasm. ZntA has two metal binding sites, one in the hydrophilic N-terminal domain and the other in the transmembrane region. The …

Single Molecule Studies Of Spliceosomal Snrnas U2-U6, Zhuojun Guo

Wayne State University Dissertations

Spliceosomes catalyze the maturation of precursor mRNAs in organisms ranging

from yeast to humans. Their catalytic core comprises three small nuclear RNAs (U2, U5

and U6) involved in substrate positioning and catalysis. It has been postulated, but never

shown experimentally, that the U2-U6 complex adopts at least two conformations that

reflect different activation states. We have used single-molecule fluorescence to probe the

structural dynamics of a protein-free RNA complex modeling U2-U6 from yeast and

mutants of highly conserved regions of U2-U6. Our data show the presence of at least

three distinct conformations in equilibrium. The minimal folding pathway consists of …