Biophysics Commons^™

Functional Effects Of Calcium Regulation Of Thin Filaments At Single Particle Resolution, Christopher Solis-Ocampo

Electronic Theses and Dissertations

Heart disease is the leading cause of death in the United States. Understanding heart function at the molecular level is critical for developing of more effective treatments. In the cardiac muscle, the thin filament is composed by troponin (Tn), tropomyosin (Tm), and F-actin. It provides Ca2+-dependent regulation of contraction by modulating myosin attachment and force generation in a cooperative scheme. However, this mechanism remains unclear. To understand thin filament activation, we studied the binding and functional properties of Tn and Tm to F-actin at single particle resolution by employing fluorescence image colocalization, in vitro motility assays, and Förster resonance energy …

Two-Photon Excitation Based Photochemistry And Neural Imaging, Kevin Andrew Hatch

Open Access Theses & Dissertations

Two-photon microscopy is a fluorescence imaging technique which provides distinct advantages in three-dimensional cellular and molecular imaging. The benefits of this technology may extend beyond imaging capabilities through exploitation of the quantum processes responsible for fluorescent events. This study utilized a two-photon microscope to investigate a synthetic photoreactive collagen peptidomimetic, which may serve as a potential material for tissue engineering using the techniques of two-photon photolysis and two-photon polymerization. The combination of these techniques could potentially be used to produce a scaffold for the vascularization of engineered three-dimensional tissues in vitro to address the current limitations of tissue engineering. Additionally, …

Probing Allosteric, Partial Inhibition Of Thrombin Using Novel Anticoagulants, Stephen S. Verespy Iii

Theses and Dissertations

Thrombin is the key protease that regulates hemostasis; the delicate balance between procoagulation and anticoagulation of blood. In clotting disorders, like deep vein thrombosis or pulmonary embolism, procoagulation is up-regulated, but propagation of clotting can be inhibited with drugs targeting the proteases involved, like thrombin. Such drugs however, have serious side effects (e.g., excessive bleeding) and some require monitoring during the course of treatment. The reason for these side effects is the mechanism by which the drugs’ act. The two major mechanisms are direct orthosteric and indirect allosteric inhibition, which will completely abolish the protease’s activity. Herein we sought an …

Photoacoustic Calorimetry Studies Of The Earliest Events In Horse Heart Cytochrome-C Folding, Tarah A. Word

USF Tampa Graduate Theses and Dissertations

The protein folding problem involves understanding how the tertiary structure of a protein is related to its primary structure. Hence, understanding the thermodynamics associated with the rate-limiting steps for the formation of the earliest events in folding is most crucial to understanding how proteins adopt native secondary and tertiary structures. In order to elucidate the mechanism and pattern of protein folding, an extensively studied protein, Cytochrome-c (Cc), was chosen as a folding system to obtain detailed time-resolved thermodynamic profiles for the earliest events in the protein folding process. Cytochrome-c is an ideal system for understanding the folding process for several …

Computational Modeling Of Rna-Small Molecule And Rna-Protein Interactions, Lu Chen

Dissertations and Theses (Open Access)

The past decade has witnessed an era of RNA biology; despite the considerable discoveries nowadays, challenges still remain when one aims to screen RNA-interacting small molecule or RNA-interacting protein. These challenges imply an immediate need for cost-efficient while predictive computational tools capable of generating insightful hypotheses to discover novel RNA-interacting small molecule or RNA-interacting protein. Thus, we implemented novel computational models in this dissertation to predict RNA-ligand interactions (Chapter 1) and RNA-protein interactions (Chapter 2).

Targeting RNA has not garnered comparable interest as protein, and is restricted by lack of computational tools for structure-based drug design. To test the potential …

Designing Fret Assays To Study Electrostatic Interactions Pertaining To The Binding Of Intrinsically Disordered Proteins, Ashley Ann Howard

Graduate Theses and Dissertations

Fibroblast growth factor receptor plays a major role in several biological processes. Without FGFR, a human cannot live. FGFR is involved in cell differentiation and wound healing. Of course, if FGFR signaling becomes unregulated, it causes severe distress in the body. Several cancers are contributed to high signaling levels, as well as developmental conditions like rickets and Kallmann’s syndrome. FGFR is thought to undergo an auto-inhibition (or self-regulatory) process in order to try to facilitate regulation. The exact method of this inhibition is currently unknown, but is proposed to involve the unstructured acid box region of FGFR. We developed a …

Development Of Experimental And Instrumental Systems To Study Biological Systems, Amanda J Hemphill

Open Access Dissertations

Chapters 1-4 of this thesis describes the development of an experimental system to measure diffusion-limited reaction kinetics in a biological environment. About 100 years ago, the relationship between reaction rate and diffusion in homogenous solution, ie water or buffer, was described as a linear relationship by Smoluchowski. Applying this theory naively would suggest that since the diffusion coefficients drop by factors of 4-100 then the rates of reaction would drop by the same amount. However, recent theory and simulations suggest that this does not hold. Even though biological diffusion coefficients drop to 0.1-20% of that in buffer, these recent studies …

The Exchanged Ef-Hands In Calmodulin And Troponin C Chimeras Impair The Ca2+-Induced Hydrophobicity And Alter The Interaction With Orai1: A Spectroscopic, Thermodynamic And Kinetic Study, Drake Jensen, Nicole Reynolds, Ya-Ping Yang, Shubha Shakya, Zhi-Qiang Wang, Dennis J. Stuehr, Chin-Chuan Wei

Chemistry Faculty Research, Scholarship, and Creative Activity

Background

Calmodulin (CaM) plays an important role in Ca²⁺-dependent signal transduction. Ca²⁺ binding to CaM triggers a conformational change, forming a hydrophobic patch that is important for target protein recognition. CaM regulates a Ca²⁺-dependent inactivation process in store-operated Ca²⁺entry, by interacting Orai1. To understand the relationship between Ca²⁺-induced hydrophobicity and CaM/Orai interaction, chimera proteins constructed by exchanging EF-hands of CaM with those of Troponin C (TnC) are used as an informative probe to better understand the functionality of each EF-hand.

Results

ANS was used to assess the context of the induced …

Structure And Stability Of Amyloid Fibrils Studied By Advanced Vibrational Spectroscopy, Marudachalam Shanmugasundaram

Legacy Theses & Dissertations (2009 - 2024)

Protein misfolding often leads to the formation of refractory protein aggregates like amyloid fibrils. These fibrils possess a highly ordered structure and are implicated in over 25 severe diseases including Alzheimer’s, Parkinson’s and prion diseases. This work was focused on understanding the morphology and conformation of amyloid fibrils and their stability after formation. The deconstruction of fibrils as well as other aggregates like inclusion bodies under mild conditions was also investigated using Archaeal chaperones.

New Experimental And Theoretical Tools For Studying Protein Systems With Elements Of Structural Disorder, Tairan Yuwen

Open Access Dissertations

Disordered proteins are one class of proteins which do not possess well-folded three-dimensional structures as their native conformations. Many eukaryotic proteins have been found to be fully disordered or contain certain disordered regions. Disordered proteins usually display several characteristic properties, such as increased motional freedom and the conformational heterogeneity caused by that. The elements of structural disorder are commonly involved in many important biological functions and are implicated in many diseases. Therefore, the study of disordered proteins has become one of the most important research topics in recent years. This thesis presents results from three different research projects; the common …

Quantum Mechanics In Complex Systems, Ross Douglas Hoehn

Open Access Dissertations

This document should be considered in its separation; there are three distinct topics contained within and three distinct chapters within the body of works. In a similar fashion, this abstract should be considered in three parts. Firstly, we explored the existence of multiply-charged atomic ions by having developed a new set of dimensional scaling equations as well as a series of relativistic augmentations to the standard dimensional scaling procedure and to the self-consistent field calculations. Secondly, we propose a novel method of predicting drug efficacy in hopes to facilitate the discovery of new small molecule therapeutics by modeling the agonist-protein …

Interactions Of Eukaryotic Translation Initiation Factors And 3' Untranslated Region Of Barley Yellow Dwarf Virus Mrna During Protein Synthesis: A Study Of Equilibrium Binding, Kinetics And Thermodynamics, Bidisha Banerjee

Dissertations, Theses, and Capstone Projects

Eukaryotic initiation factor (eIF) 4F binding to mRNA is the first committed step in cap-dependent protein synthesis. Barley Yellow Dwarf Virus (BYDV) employs a cap-independent mechanism of translation initiation which is mediated by a structural element BTE (BYDV translation element) located in the 3’ UTR of its mRNA. eIF4F bound the BTE and a translational inactive mutant with high affinity; thus questioning the role of eIF4F in translation of BYDV. To examine the effects of eIF4F in BYDV translation initiation, BTE mutants with widely different in vitro translation efficiencies ranging from 5-164% compared to WT were studied. Using fluorescence anisotropy …

Combination Of The Computational Methods: Molecular Dynamics, Homology Modeling And Docking To Design Novel Inhibitors And Study Structural Changes In Target Proteins For Current Diseases, Katherine Cristina Parra

USF Tampa Graduate Theses and Dissertations

In this thesis, molecular dynamics simulations, molecular docking, and homology modeling methods have been used in combination to design possible inhibitors as well as to study the structural changes and function of target proteins related to diseases that today are in the spotlight of drug discovery. The inwardly rectifying potassium (Kir) channels constitute the first target in this study; they are involved in cardiac problems. On the other hand, tensin, a promising target in cancer research, is the second target studied here.

The first chapter includes a brief update on computational methods and the current proposal of the combination of …

Conformational Features Of The Human U2-U6 Snrna Complex, Ravichandra Bachu

Dissertations, Theses, and Capstone Projects

The splicing of precursor messenger (pre-m) RNA, during which noncoding intervening sequences are excised and flanking coding regions ligated, is an integral reaction of gene expression. In eukaryotes, it is carried out by a dynamic RNA-protein complex called the spliceosome, in which five small nuclear (sn) RNA components are actively involved in recognition and chemical aspects of the process. A complex formed between U2 and U6 snRNAs is implicated in the chemistry of pre-mRNA splicing. The catalytic activity of the U2-U6 snRNA complex is dependent on the presence of Mg2+ ions, and the complex has been shown to have several …

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 …

Influence Of Cholesterol And Bilayer Asymmetry On Membrane Protein Distribution In Polymer-Tethered Raft-Mimicking Lipid Membranes, Noor Fueza Hussain

Open Access Dissertations

It is now widely recognized that lipid rafts, which are membrane domains enriched in cholesterol (CHOL) and sphingolipids (SL), play a significant functional role in the plasma membrane. Raft domains particularly affect membrane functionality by causing sequestering of membrane proteins. Underlying mechanisms of raft-associated membrane protein sequestration remain elusive, due to the complexity, transient nature, and small size of raft domains in cellular membranes. To address these challenges, this dissertation unveils the relationship between lipid raft composition and membrane protein sequestration and function using raft-mimicking model membrane mixtures comprised of coexisting liquid-ordered (lo) and liquid-disordered (ld) domains with reconstituted membrane …

Structure-Activity Relationship Of 2,3-Benzodiazepin-4-Ones As Noncompetitive Ampa Receptor Antagonists, Mohammad Shaban Qneibi

Legacy Theses & Dissertations (2009 - 2024)

2,3-Benzodiazepin-4-one (BDZ-11) derivatives are a special group of 2,3-benzodiazepine compounds. These compounds have been previously synthesized as AMPA receptor inhibitors, and they are also potential drugs for the treatment of various neurological diseases involving excessive AMPA receptor activity. AMPA receptors are a subtype of glutamate ion channel proteins and are responsible for the majority of excitatory neurotransmission in the mammalian central nervous system. Specifically, they are critically involved in neuronal development and brain activities, including learning and memory. Overstimulation of AMPA receptors is associated with some neurological diseases such as ALS, stroke and Alzheimer's disease. Despite the fact that hundreds …

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 …

Reaction Enthalpy And Volume Profiles For Excited State Reactions Involving Electron Transfer And Proton-Coupled Electron Transfer, William Antonio Maza

USF Tampa Graduate Theses and Dissertations

Electron transfer, ET, and proton-coupled electron transfer, PCET, reactions are central to biological reactions involving catalysis, energy conversion and energy storage. The movement of electrons and protons in either a sequential or concerted manner are coupled in a series of elementary reaction steps in respiration and photosynthesis to harvest and convert energy consumed in foodstuffs or by absorption of light into high energy chemi-cal bonds in the form of ATP. These electron transfer processes may be modulated by conformational dynamics within the protein matrix or at the protein-protein interface, the energetics of which are still not well understood. Photoacoustic calorimetry …

"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 …

Protein-Lipid Interactions: Influence Of Anchoring Groups And Buried Arginine On The Properties Of Membrane-Spanning Peptides, Vitaly V. Vostrikov

Graduate Theses and Dissertations

Designed transmembrane peptides were employed for investigations of protein-lipid interactions by means of oriented solid-state deuterium NMR spectroscopy using isotope-enriched alanine residues. Using the model GWALP23 sequence (GGALW(LA)6LWLAGA) as a host peptide having single interfacial tryptophan anchor residues, the effects of different guest mutations were explored. Replacements of glycine residues 2 and 22 to positively charged lysine or arginine on both termini had little influence on the peptide average orientation. Conversely, glycine to tryptophan substitutions had profound effects, manifested in the increased dynamics and altered tilt direction of the peptide. While the charged residues at the peptide termini did not …

Bioinformatics, Thermodynamics And Kinetics Analysis Of An All Alpha Helical Protein With A Gree-Key Topology, Hai Li

Chemistry & Biochemistry Theses & Dissertations

Computational and experimental studies focusing on the role of conserved residues for folding and stability is an active and promising area of research. To further expand our understanding we present the results of a bioinformatics analysis of the death domain superfamily. The death domain superfamily fold consists of six α-helices arranged in a Greek-key topology, which is shared by the all β-sheet immunoglobulin and mixed α/β-plait superfamilies. Our sequence and structural studies have identified a group of conserved hydrophobic residues and corresponding long-range interactions, which we propose are important in the formation and stabilization of the hydrophobic core and native …

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 …

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.

Homologous And Heterologous Crystallin Protein Interactions As The Molecular Basis Of Inherited Cataract, Priya Ranjan Banerjee

Legacy Theses & Dissertations (2009 - 2024)

Age-related cataract is the most common cause of blindness worldwide. Nearly fifty percent of Americans above the age of 75 are diagnosed with this disease (http://www.nei.nih.gov/eyedata/pbd_tables.asp), and surgical intervention is the sole method of treatment at present (http://www.nei.nih.gov/healthyvision/objective/cataracts.asp). In the developing world, even this treatment is not readily available. These are compelling reasons to search for better treatments to delay, prevent or arrest cataract formation. Recent evidence suggests that age-related cataracts also have a genetic component. Therefore, determining the mechanisms underlying genetic cataracts with a known association to a protein-mutation is one important strategy towards understanding the molecular basis for …

Probing Molecules In Confined Space, Carissa Marie Vetromile

USF Tampa Graduate Theses and Dissertations

Despite the plethora of information regarding cellular crowding and its importance on modulating protein function the effects of confinement on biological molecules are often overlooked when investigating their physiological function. Recently however, the encapsulation of biomolecules in solid state matrices (NafionTM, sol-gels, zirconium phosphate,etc.) has increased in importance as a method for examining protein conformation and dynamics in confined space as well as novel applications in biotechnology. Biotechnological applications include, but are not limited to, bioremediation, biosensors, biocatalysts, etc. In order to better utilize solid state materials as substrates for biological molecules an understanding of the effects of encapsulation on …

Systematic Approach To Electrostatically Induced 2d Crystallization Of Nanoparticles At Liquid Interfaces, Sumit Kewalramani, Suntao Wang, Yuan Lin, Huong Giang Nguyen, Qian Wang, Masafumi Fukuto, Lin Yang

Faculty Publications

We report an experimental demonstration of a strategy for inducing two-dimensional (2D)crystallization of charged nanoparticles on oppositely charged fluid interfaces. This strategy aims to maximize the interfacial adsorption of nanoparticles, and hence their lateral packing density, by utilizing a combination of weakly charged particles and a high surface charge density on the planar interface. In order to test this approach, we investigated the assembly of cowpea mosaic virus (CPMV) on positively charged lipid monolayers at the aqueous solution surface, by means of in situX-ray scattering measurements at the liquid–vapor interface. Theassembly was studied as a function of the solution …

Conformational Change And Topological Stability Of Proteins, Jeffrey Andrew Tibbitt

Chemistry & Biochemistry Theses & Dissertations

The conformation and topology of a protein changes when stabilizing forces are absent, but the mechanisms by which these changes occur remains elusive. This dissertation aims to broaden the understandings. On the conformational level, the M20 loop conformers of E. coli dihydrofolate reductase are interrogated to identify factors responsible for their stability as well as to determine how one conformer might change into another. Molecular dynamics is used to simulate the open, closed and occluded conformers (observed in X-ray crystal structures) under a series of different single ligand conditions. Analysis shows that all open conformers move to a similar new …