Biochemistry, Biophysics, and Structural Biology Commons^™

Detecting And Accounting For Multiple Sources Of Positional Variance In Peak List Registration Analysis And Spin System Grouping, Andrey Smelter, Eric C. Rouchka, Hunter N. B. Moseley

Molecular and Cellular Biochemistry Faculty Publications

Peak lists derived from nuclear magnetic resonance (NMR) spectra are commonly used as input data for a variety of computer assisted and automated analyses. These include automated protein resonance assignment and protein structure calculation software tools. Prior to these analyses, peak lists must be aligned to each other and sets of related peaks must be grouped based on common chemical shift dimensions. Even when programs can perform peak grouping, they require the user to provide uniform match tolerances or use default values. However, peak grouping is further complicated by multiple sources of variance in peak position limiting the effectiveness of …

Real-Time Sensing Of Single-Ligand Delivery With Nanoaperture-Integrated Microfluidic Devices, W. Elliott Martin, Ning Ge, Bernadeta R. Srijanto, Emily Furnish, C. Patrick Collier, Christine A. Trinkle, Christopher I. Richards

Chemistry Faculty Publications

The measurement of biological events on the surface of live cells at the single-molecule level is complicated by several factors including high protein densities that are incompatible with single-molecule imaging, cellular autofluorescence, and protein mobility on the cell surface. Here, we fabricated a device composed of an array of nanoscale apertures coupled with a microfluidic delivery system to quantify single-ligand interactions with proteins on the cell surface. We cultured live cells directly on the device and isolated individual epidermal growth factor receptors (EGFRs) in the apertures while delivering fluorescently labeled epidermal growth factor. We observed single ligands binding to EGFRs, …

Use Of Crispr To Develop A Follicle Stimulating Hormone Receptor Knockout In Human Granulosa Cells To Study Lipid Raft Residency, Jenna Pradhuman

Honors Theses

Human follicle stimulating hormone (hFSH) is a protein hormone responsible for stimulating the gonads and is necessary for regulating reproductive systems in both females and males. The actions of hFSh are carried out by the hFSH receptor (hFSHR), a seven transmembrane receptor that belongs to the G protein-coupled receptor family. Once FSH activates its receptor, the G protein inside the cell that is associated with the receptor is activated and starts a cascade of signaling that results in activation of protein kinase A (PKA) and the p44/42 MAP kinase (MAPK). The activation of these secondary proteins is responsible for follicular …

Study Of Biologically Important Macromolecules By Nuclear Magnetic Resonance, Christopher Michael Demott

Legacy Theses & Dissertations (2009 - 2024)

Intrinsically disordered proteins or unstructured segments within proteins play an important role in cellular physiology and pathology. A combination of peptide aptamers selected by using the yeast-two-hybrid scheme, and in-cell NMR identified high affinity binders to a transiently structured intrinsically disordered proteins (IDP). This method was validated using the prokaryotic ubiquitin-like protein, Pup, of the Mycobacterium proteasome. We discover two peptide aptamers that bind to opposite sites of a transient helix in Pup, an intrinsically disordered protein, that have vastly different effects on the survival of Mycobacterium bovis BCG.

Purification, Optimization, And Growth Of New Delhi Metallo-Β-Lactamase-1 Protein Crystals Mixed With Nz218 Inhibitor, Brandon M. Wills

Celebration of Learning

New Delhi metallo-β-lactamase-1 is a problematic gene found in certain strains of bacteria that cause them to become antibiotic resistant to nearly all known antibiotics. While some antibiotics are available to treat patients with a bacterial infection, most are toxic or do not have 100% success rates. With that being said, it is imperative that we search for a molecule that is successfully able to inhibit the effects of this gene every time. Such a discovery would help tremendously with new antibiotic drug development and also prevent further damage by these dangerous bacteria. In this presentation, I will describe the …

Using Simple Self-Assembling Peptides To Attain Novel Protein-Like Functions, Tyler Smith

Honors Capstone Projects - All

Proteins carry out many extremely efficient functions, including catalysis and biomolecule recognition. Underlying this efficiency is their extraordinary complexity and ability to fold into unique three-dimensional structures. Attempts to replicate this efficiency through de novo design have only shown moderate success, and it is unclear how modern-day proteins may have evolved. However, short peptides that alternate hydrophobic and hydrophilic residues can self-assemble into amyloid fibrils to achieve well-defined secondary structure. These aggregates may have served as a template from which the first proteins were derived. We designed self-assembling seven-residue peptides that are able to act as Zn²⁺-dependent esterases. …

The Application Of Hydrogen/Deuterium Exchange And Covalent Labeling Coupled With Mass Spectrometry To Examine Protein Structure, Nicholas B. Borotto

Doctoral Dissertations

Thorough insight into a protein’s structure is necessary to understand how it functions and what goes wrong when it malfunctions. The structure of proteins, however, is not easily analyzed. The analysis must take place under a narrow range of conditions or risk perturbing the very structure being probed. Furthermore, the wide diversity in size and chemistry possible in proteins significantly complicates this analysis. Despite this numerous methods have been developed in order to analyze protein structure. In this work, we demonstrate that mass spectrometry (MS)-based techniques are capable of characterizing the structure of particularly challenging proteins. This is done through …

Engineered Protein Polymer-Gold Nanoparticle Hybrid Materials For Small Molecule Delivery, Min Dai, Ja Frezzo, E Sharma, R Chen, N Singh, C Yuvienco, E Caglar, S Xiao, A Saxena, Jk Montclare

Publications and Research

We have fabricated protein polymer-gold nanoparticle (P-GNP) nanocomposites that exhibit enhanced binding and delivery properties of the small hydrophobic molecule drug, curcumin, to the model breast cancer cell line, MCF-7. These hybrid biomaterials are constructed via in situ GNP templated-synthesis with genetically engineered histidine tags. The P-GNP nanocomposites exhibit enhanced small molecule loading, sustained release and increased uptake by MCF-7 cells. When compared to the proteins polymers alone, the P-GNPs demonstrate a greater than 7-fold increase in curcumin binding, a nearly 50% slower release profile and more than 2-fold increase in cellular uptake of curcumin. These results suggest that P-GNP …

A Survey Of The Current Drug Screening Techniques To Obtain Rational Design And Study Drug-Target Interactions, Stephen Dansereau

Legacy Theses & Dissertations (2009 - 2024)

Different techniques have been developed over the years for the purpose of studying proteins and understanding their functions. Early techniques typically employed bioluminescence or fluorescence such such as the firefly protein luciferase and the jellyfish green fluorescent protein (GFP), respectively, to localize proteins within the cell. X-ray crystallography has also provided valuable structural details of many different proteins in vitro. Yet, nuclear magnetic resonance (NMR) spectroscopy offers the most realistic insight into proteins' physiologic structures and how proteins function in their native, cellular environments.

Cell Type–Dependent Mechanisms For Formin-Mediated Assembly Of Filopodia, Lorna E. Young, Ernest G. Heimsath, Henry N. Higgs

Dartmouth Scholarship

Filopodia are finger-like protrusions from the plasma membrane and are of fundamental importance to cellular physiology, but the mechanisms governing their assembly are still in question. One model, called convergent elongation, proposes that filopodia arise from Arp2/3 complex-nucleated dendritic actin networks, with factors such as formins elongating these filaments into filopodia. We test this model using constitutively active constructs of two formins, FMNL3 and mDia2. Surprisingly, filopodial assembly requirements differ between suspension and adherent cells. In suspension cells, Arp2/3 complex is required for filopodial assembly through either formin. In contrast, a subset of filopodia remains after Arp2/3 complex inhibition in …

Binding Interactions Of (R)- And (S)-Hydroxypropyl-Com Dehydrogenases And The Zinc Knuckle Proteins Air1 And Air2, Jeremy W. Bakelar

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

A thorough understanding of protein function requires knowledge of how proteins interact with their substrates and with other proteins. The work entailed in this dissertation describes the binding interactions of proteins from two different model systems: (1) the dehydrogenase enzymes R- and S-HPCDH and (2) the zinc knuckle proteins Air1 and Air2.

R- and S-HPCDH are highly similar enzymes (42% identical) that function in a unique metabolic pathway found in the soil bacterium Xanthobacter autotrophicus. The bacterium produces R- and S-HPCDH simultaneously to facilitate the transformation of two different forms of the organic …

Distinguishing Macrophage Activation States By Mass Spectrometry, Matthias Manfred Knust

Graduate Theses and Dissertations

Macrophages are versatile and highly adaptive cells that are involved in a wide range of physiological processes including host defense, homeostasis or regeneration, as well as pathogenesis. They react to their microenvironment, assuming various roles based on chemical and/or physical cues, and can reversibly shift between these so-called activation states. Concurrently, the technique of immunohistochemistry is used to gain spatial information on activated macrophages on tissue sections. The aim of this work was to find mass spectral biomarkers that allow the differentiation of activation states, and establish conditions that can be used in imaging mass spectrometry (IMS) experiments to investigate …

Nonenzymatic Glycosylation Of Erythrocyte Membrane Proteins. Relevance To Diabetes, J A. Miller, Ellen M. Gravallese, H F. Bunn

Ellen M. Gravallese

Nonenzymatic glycosylation of proteins of the erythrocyte membrane was determined by incubating erythrocyte ghosts with [3H]borohydride. The incorporation of tritium into protein provides a reliable assay of ketoamine linkages. The membrane proteins from 18 patients with diabetes incorporated twice as much radioactivity as membrane proteins from normal erythrocytes. After acid hydrolysis, amino acid analysis showed that the majority of radioactivity was localized to glucosyllysine. Autoradiograms showed that all of the major proteins of the erythrocyte membrane, separated by electrophoresis on sodium dodecyl sulfate gels, contained ketoamine linkages. No protein bands in either normal or diabetic erythrocytes showed significant preferential labeling. …

Liver Perilipin 5 Expression Worsens Hepatosteatosis But Not Insulin Resistance In High Fat-Fed Mice, Michelle B. Trevino, David Mazur-Hart, Yui Machida, Timothy King, Joseph Nadler, Elena V. Galkina, Arjun Poddar, Sucharita Dutta, Yumi Imai

Mathematics & Statistics Faculty Publications

Perilipin 5 (PLIN5) is a lipid droplet (LD) protein highly expressed in oxidative tissues, including the fasted liver. However, its expression also increases in nonalcoholic fatty liver. To determine whether PLIN5 regulates metabolic phenotypes of hepatosteatosis under nutritional excess, liver targeted overexpression of PLIN5 was achieved using adenoviral vector (Ad-PLIN5) in male C57BL/6J mice fed high-fat diet. Mice treated with adenovirus expressing green fluorescent protein (GFP) (Ad-GFP) served as control. Ad-PLIN5 livers increased LD in the liver section, and liquid chromatography with tandem mass spectrometry revealed increases in lipid classes associated with LD, including triacylglycerol, cholesterol ester, and phospholipid classes, …

Novel Nmr Based Technologies To Study Macromolecular Structures, Subhabrata Majumder

Legacy Theses & Dissertations (2009 - 2024)

Nuclear Magnetic Resonance Spectroscopy (NMR) is one of the principle tools in structural biology to probe macromolecular structures and interactions. The atomic resolution afforded by this technique has been widely used to probe protein-protein, and protein-ligand interactions in-vitro. However, the natural milieu of the proteins is the living cell and the cellular cytoplasm is extremely heterogeneous. The NMR studies of folded protein in-cell, till now, have been limited by non-specific interactions of the cytosol. This thesis outlays a general methodology to study protein structure/interactions inside the living cells using NMR. In a closely related objective, it also describes the use …

The Formin Fmnl3 Assembles Plasma Membrane Protrusions That Participate In Cell–Cell Adhesion, Timothy J. Gauvin, Lorna E. Young, Henry N. Higgs

Dartmouth Scholarship

FMNL3 is a vertebrate-specific formin protein previously shown to play a role in angiogenesis and cell migration. Here we define the cellular localization of endogenous FMNL3, the dynamics of GFP-tagged FMNL3 during cell migration, and the effects of FMNL3 suppression in mammalian culture cells. The majority of FMNL3 localizes in a punctate pattern, with >95% of these puncta being indistinguishable from the plasma membrane by fluorescence microscopy. A small number of dynamic cytoplasmic FMNL3 patches also exist, which enrich near cell–cell contact sites and fuse with the plasma membrane at these sites. These cytoplasmic puncta appear to be part of …

Automatic Animation Of Molecular Motion Using Python And Cinema 4d, Diana Zajac, Nathaniel Smith, Dan Gurnon

Annual Student Research Poster Session

No abstract provided.

Expression In Yeast Of The Metacaspase Scp3 From The Fungus Schizophyllum Commune, Anne Kaminski

Honors Theses

Metacaspases are caspase homologs that are found in plants, fungi, and protists. They are involved in programmed cell death, a structured and regulated process to break down cells. There is limited information available on metacaspases and further research is necessary to understand how they work. One reason metacaspases are studied is that they are considered to be a potential drug target for pathogenic microorganisms. Five metacaspases scp1-5 were identified in S. commune through their similarity to the yeast metacaspase Yca1. The overall goal of the lab is to study and characterize these proteins. This thesis outlines the path taken to …

Stoichiometries And Affinities Of Interacting Proteins From Concentration Series Of Solution Scattering Data: Decomposition By Least Squares And Quadratic Optimization, Himanshu Chandola, Tim E. Williamson, Bruce A. Craig, Alan M. Friedman, Chris Bailey-Kellogg

Dartmouth Scholarship

In studying interacting proteins, complementary insights are provided by analyzing both the association model (the stoichiometry and affinity constants of the intermediate and final complexes) and the quaternary structure of the resulting complexes. Many current methods for analyzing protein interactions either give a binary answer to the question of association and no information about quaternary structure or at best provide only part of the complete picture. Presented here is a method to extract both types of information from X-ray or neutron scattering data for a series of equilibrium mixtures containing the initial components at different concentrations. The method determines the …

Primary Microrna Processing Assay Reconstituted Using Recombinant Drosha And Dgcr8., Ian Barr, Feng Guo

Natural Sciences and Mathematics | Faculty Scholarship

In animals, the Microprocessor complex cleaves primary transcripts of microRNAs (pri-miRNAs) to produce precursor microRNAs in the nucleus. The core components of Microprocessor include the Drosha ribonuclease and its RNA-binding partner protein DiGeorge critical region 8 (DGCR8). DGCR8 has been shown to tightly bind an Fe(III) heme cofactor, which activates its pri-miRNA processing activity. Here we describe how to reconstitute pri-miRNA processing using recombinant human Drosha and DGCR8 proteins. In particular, we present the procedures for expressing and purifying DGCR8 as an Fe(III) heme-bound dimer, the most active form of this protein, and for estimating its heme content.

Clustering And Classification Of Multi-Domain Proteins, Neethu Shah

Department of Computer Science and Engineering: Dissertations, Theses, and Student Research

Rapid development of next-generation sequencing technology has led to an unprecedented growth in protein sequence data repositories over the last decade. Majority of these proteins lack structural and functional characterization. This necessitates design and development of fast, efficient, and sensitive computational tools and algorithms that can classify these proteins into functionally coherent groups.

Domains are fundamental units of protein structure and function. Multi-domain proteins are extremely complex as opposed to proteins that have single or no domains. They exhibit network-like complex evolutionary events such as domain shuffling, domain loss, and domain gain. These events therefore, cannot be represented in the …

Gene Expression Studies For The Analysis Of Domoic Acid Production In The Marine Diatom Pseudo-Nitzschia Multiseries, Katie Boissonneault, Brooks M. Henningsen, Stephen S. Bates, Deborah L. Robertson, Sean Milton, Jerry Pelletier, Deborah A. Hogan, David E. Housman

Dartmouth Scholarship

Pseudo-nitzschia multiseries Hasle (Hasle) (Ps-n) is distinctive among the ecologically important marine diatoms because it produces the neurotoxin domoic acid. Although the biology of Ps-n has been investigated intensely, the characterization of the genes and biochemical pathways leading to domoic acid biosynthesis has been limited. To identify transcripts whose levels correlate with domoic acid production, we analyzed Ps-n under conditions of high and low domoic acid production by cDNA microarray technology and reverse-transcription quantitative PCR (RT-qPCR) methods. Our goals included identifying and validating robust reference genes for Ps-n RNA expression analysis under these conditions.

Utilizing Nmr Spectroscopy And Molecular Docking As Tools For The Structural Determination And Functional Annotation Of Proteins, Jaime Stark

Department of Chemistry: Dissertations, Theses, and Student Research

With the completion of the Human Genome Project in 2001 and the subsequent explosion of organisms with sequenced genomes, we are now aware of nearly 28 million proteins. Determining the role of each of these proteins is essential to our understanding of biology and the development of medical advances. Unfortunately, the experimental approaches to determine protein function are too slow to investigate every protein. Bioinformatics approaches, such as sequence and structure homology, have helped to annotate the functions of many similar proteins. However, despite these computational approaches, approximately 40% of proteins still have no known function. Alleviating this deficit will …

Thermal And Sodium Dodecylsulfate Induced Transitions Of Streptavidin, Mark J. Waner, Irina Navrotskaya, Amanda Bain, Edward D. Oldham, David P. Mascotti

Mark J. Waner

The strong specific binding of streptavidin (SA) to biotin is utilized in numerous biotechnological applications. The SA tetramer is also known to exhibit significant stability, even in the presence of sodium dodecylsulfate (SDS). Despite its importance, relatively little is known about the nature of the thermal denaturation pathway for SA. This work uses a homogeneous SA preparation to expand on the data of previous literature reports, leading to the proposal of a model for temperature induced structural changes in SA. Temperature dependent data were obtained by SDS and native polyacrylamide gel electrophoresis (PAGE), differential scanning calorimetry (DSC), and fluorescence and …

New Tools To Study Amyloid Fibrils And Intrinsically Disordered Proteins In Vitro And In Vivo, Jacqueline D. Washington

Legacy Theses & Dissertations (2009 - 2024)

Amyloid fibrils are β-sheet-rich protein aggregates commonly found in the organs and tissues of patients with various amyloid-associated diseases. The structure of insulin fibrils was characterized by deep ultraviolet resonance Raman and Nuclear Magnetic Resonance spectroscopy combined with hydrogen-deuterium exchange. Our new approach of combining NMR and Raman spectroscopy with molecular dynamic simulations for characterizing amyloid fibrils provided exclusive knowledge about fibril structure at amino acid residue resolution.

Protein Structures Under Physiological Conditions, Karl Michael Bertrand

Legacy Theses & Dissertations (2009 - 2024)

My research focused on the evaluation of protein structures and protein dynamics inside eukaryotic cells under physiological conditions. The primary analyses of my research involved the use of in-cell Nuclear Magnetic Resonance spectroscopy using Heteronuclear Single Quantum Coherence experiments. This allowed me to visualize protein structures at an atomic resolution level, as well as, study the interactions of these proteins with small molecules.

Secondary Structure, A Missing Component Of Sequence- Based Minimotif Definitions, David P. Sargeant, Michael R. Gryk, Mark W. Maciejewsk, Vishal Thapar, Vamsi Kundeti, Sanguthevar Rajasekaran, Pedro Romero, Keith Dunker, Shun-Cheng Li, Tomonori Kaneko, Martin Schiller

Life Sciences Faculty Research

Minimotifs are short contiguous segments of proteins that have a known biological function. The hundreds of thousands of minimotifs discovered thus far are an important part of the theoretical understanding of the specificity of protein-protein interactions, posttranslational modifications, and signal transduction that occur in cells. However, a longstanding problem is that the different abstractions of the sequence definitions do not accurately capture the specificity, despite decades of effort by many labs. We present evidence that structure is an essential component of minimotif specificity, yet is not used in minimotif definitions. Our analysis of several known minimotifs as case studies, analysis …

Quantitative Comparison Of Errors In 15n Transverse Relaxation Rates Measured Using Various Cpmg Phasing Schemes, Wazo Myint, Yufeng Cai, Celia Schiffer, Rieko Ishima

Celia A. Schiffer

Nitrogen-15 Carr-Purcell-Meiboom-Gill (CPMG) transverse relaxation experiment are widely used to characterize protein backbone dynamics and chemical exchange parameters. Although an accurate value of the transverse relaxation rate, R(2), is needed for accurate characterization of dynamics, the uncertainty in the R(2) value depends on the experimental settings and the details of the data analysis itself. Here, we present an analysis of the impact of CPMG pulse phase alternation on the accuracy of the (15)N CPMG R(2). Our simulations show that R(2) can be obtained accurately for a relatively wide spectral width, either using the conventional phase cycle or using phase alternation …

Methyl- And Normal-Cytosine Deamination By The Foreign Dna Restriction Enzyme Apobec3a, Michael Carpenter, Ming Li, Anurag Rathore, Lela Lackey, Emily Law, Allison Land, Brandon Leonard, Shivender Shandilya, Markus-Frederik Bohn, Celia Schiffer, William Brown, Reuben Harris

Celia A. Schiffer

Multiple studies have indicated that the TET oxidases and, more controversially, the AID/APOBEC deaminases have the capacity to convert genomic DNA 5-methylcytosine (MeC) into altered nucleobases that provoke excision repair and culminate in the replacement of the original MeC with a normal cytosine (C). We show that human APOBEC3A (A3A) efficiently deaminates both MeC to thymine (T) and normal C to uracil (U) in single-stranded DNA substrates. In comparison, the related enzyme APOBEC3G (A3G) has undetectable MeC-to-T activity and 10-fold less C-to-U activity. Upon 100-fold induction of endogenous A3A by interferon, the MeC status of bulk chromosomal DNA is unaltered …

Minireview: Protein Interactions, Jessica Child

Honors Theses and Capstones

No abstract provided.