Structural Biology Commons^™

Sec-Saxs And Sans Analyses Of The Talin•Vinculin Complex, Gilbert Reyes

Dissertations, Theses, and Capstone Projects

Focal adhesions (FA) are integrin-based cell-matrix adhesion sites composed of adaptor proteins that facilitate the generation of traction forces important for biological functions such as cell migration, embryo development, wound healing, tissue organization and stability, and immune responses. Mature FA are observed when both autoinhibited multi-domain scaffolding protein talin and membrane-cytoskeletal protein vinculin are activated and interact thus promoting the assembly and function of FA. While research has resolved truncated sections of autoinhibited talin and recruitment of vinculin due to mechanotransduction that exposes cryptic vinculin binding sites (VBS), little is known about the global modifications of full length talin in …

From Geometry To Recognition: A Quantitative Framework For Tcr-Pmhc Structural Analysis In The Age Of Predicted Structures, Kun Hee Kim

Dissertations and Theses (Open Access)

Recognition of antigen by a T cell begins with a single physical event, the engagement of the T cell receptor (TCR) with a peptide-major histocompatibility complex (pMHC), and this interaction is encoded in the geometry with which the receptor docks onto the composite pMHC surface. Whether that docking geometry is biologically organized, and whether the structures now produced at scale by prediction preserve recognition or only reproduce that organization, are the two questions this thesis addresses. Two gaps stand in the way. The first is a gap of measurement, because docking geometry has never been made quantitatively comparable across many …

Developing A Protocol For Non-Cellular Transplantation Of Donor Mitochondria Into Recipient Cells, Andrew Parton

Honors Theses

Tumor innervation has emerged as a critical feature of cancer progression, with increased nerve density correlating with enhanced aggressiveness, metastatic dissemination, and poor clinical outcome. However, the functional contribution of neurons to tumor biology remains incompletely defined. We recently identified the intercellular transfer of mitochondria from neurons to cancer cells as a mechanism that promotes tumor progression. We therefore hypothesized that disruption of this transfer could attenuate tumor aggressivity. To directly investigate this process, we developed an approach to isolate mitochondria from donor cells and transplant them into recipient cancer cells independently of canonical cell-cell interactions. Transferred mitochondria were tracked …

Genome-Wide Phenotypic Characterization Of Mycobacteriophage Mercurio, Tyler Downs

Departmental Honors & Graduate Capstone Projects

Bacteriophages, or phages, are being piloted as a supplemental or alternative therapeutic to antibiotics against drug-resistant Mycobacterium pathogens, such as M. tuberculosis, which pose a global public health crisis.  Characterizing bacteriophage genomes can provide insights into efficacy in phage therapy before implementation or elucidate new targets to manipulate bacterial growth.  Overexpression of individual phage genes is useful to study a gene product’s function independent of others.  Here, the results of phage Mercurio plasmid library overexpression in plate-based assays are described.  Mercurio infects nonpathogenic Mycobacterium smegmatis and is genetically related to bacteriophages infecting pathogenic Mycobacterium species.  Eleven gene products of …

Comparative Biomechanics: Blattodea Antennal Morphology And Physiology, Lucas W. Marsh

All Theses

Equipped with muscle-free antennae, many insects can twist and bend their antennae on demand. We hypothesized that hemolymph circulating through the antennae provides pressure that facilitates twisting and bending and provides a feedback control channel for tactile sensing in insects. To test this hypothesis, we conducted a thorough morphological and physiological analysis of antennae in three Clades of Blattodea. We studied morphology by evaluating the distinguishable features of the body and antenna, as well as the internal structure of the antennal lumen. Then, in a series of experiments on live cockroaches, we measured blood pressure along the antenna. This was …

Membrane Organization Of Rheb And Rhoa: Roles In Function And Allosteric Druggability, Chase M. Hutchins

Dissertations and Theses (Open Access)

Small GTPases of the Ras superfamily are membrane-bound molecular switches that regulate nearly all major cellular processes, and their dysregulation drives cancers, neurological disorders, and metabolic diseases. While anchored to cellular membranes, the catalytic domains of small GTPases undergo orientation dynamics, adopting distinct configurations that can occlude or expose effector-binding surfaces and modulate signaling output. These dynamics have been characterized in the Ras oncoproteins, but whether they extend across the superfamily and influence allosteric druggability has remained unknown.

In this dissertation, I address these questions through Rheb and RhoA, two small GTPases with complementary differences in subfamily lineage, membrane localization, …

Understanding The Role Of Toxs As A Bile Sensing Component Of The Toxrs Virulence Regulatory System In Vibrio Spp., Minje Kim

Dartmouth College Ph.D Dissertations

Pathogenic Vibrio bacteria, such as V. cholerae and V. parahaemolyticus, colonize in the human small intestine to cause severe gastrointestinal disease. During the infection, these bacteria encounter bile salts, antimicrobial cholesterol metabolites secreted into the intestine. Pathogenic Vibrio species have evolved to utilize bile salts as signals to regulate virulence. The signaling depends in part on ToxRS, a conserved co-component transmembrane transcription regulator. ToxRS consists of the transcription factor ToxR and its membrane-tethered binding partner ToxS. ToxS is required for ToxR stability and full transcriptional activity. Although bile salts are known to influence ToxRS-dependent virulence gene expression, the molecular …

Protein Allostery Probed By Ligands Across Sites, Virgil A. Woods

Dissertations, Theses, and Capstone Projects

Allostery is a pervasive regulatory principle throughout biology, yet the structural pathways by which distal inputs alter active‑site chemistry within protein structures remain incompletely defined and exploited. This dissertation uses protein tyrosine phosphatase 1B (PTP1B) as a tractable model to map those pathways with complementary experimental and computational tools. I integrate high‑resolution hydrogen-deuterium exchange mass spectrometry (HDX-MS), room-temperature crystallography, NMR, steady-state kinetics, crystallographic pseudo-ensembles, and machine-learning-guided ligand discovery. The working premise is that regulation reflects redistribution within a conformational ensemble rather than a binary switch. That orthogonal perturbations by small molecules, mutations, and protein partners can be used to both …

Computational And Functional Characterization Of Terebrid Venom Peptides, Favour T. Achimba

Dissertations, Theses, and Capstone Projects

Venom peptides represent a natural repertoire of bioactive compounds that are potent modulators of ion channels. Teretoxins, venom peptides from terebrid snails, remain underexplored despite their structural and functional diversity. This thesis is the first to comprehensively explore the structure and function of teretoxins using a comparative computational sequence and structure model followed by experimental assays. The majority of the functional focus has been on identifying the ion channel molecular targets of uncharacterized teretoxins. Teretoxins, like other venom peptides, are hypothesized to manipulate the cellular function of a diversity of ion channels. Dysregulation of ion channels leads to several …

Comparing The Differences Of Hsp90Α And Hsp90Β In Native Protein Complex Incorporation, Daryna Serediuk

Honors Theses and Capstones

Epichaperomes are long-lasting, stable assemblies of chaperones, co-chaperones, and associated factors that facilitate cell survival in maladaptive cellular states. Their inhibition has emerged as an attractive therapeutic strategy for the treatment of cancer and neurodegenerative diseases (Rodina et al., 2016). Epichaperomes are hallmarks of robust proliferation in cancer cell populations across many cancer types and are abundantly present in several stem cell varieties, including induced pluripotent stem cells, human embryonic kidney cells, and cancer stem cells (Kishinevsky et al., 2018).

The core protein of the eukaryotic heat shock protein machinery, Heat Shock Protein 90 (Hsp90), is a central chaperone that …

A Framework For Characterizing The Peripheral Immune Isonome Using Long-Read Single-Cell Rna Sequencing And Its Relevance To Neurological Disease, Patricia Hayes Doyle

Theses and Dissertations--Neuroscience

Long-read single-cell RNA sequencing provides an opportunity to understand human health and disease at isoform resolution, revealing cellular diversity and disease mechanisms difficult to resolve with bulk or short-read methodologies.

Using a modified PIPseq workflow and computational pipeline adapted for Oxford Nanopore (ONT) sequencing, we profiled isoform usage across immune cells, integrating marker expression and isoform discovery, generating the largest long-read single-cell dataset of human immune cells from a single individual to date. We identified non-canonical protein-coding variants of GZMB and CD3G enriched in unexpected cell types. We also discovered novel transcripts from CMC1 and LYAR with cell-type-specific signatures that …

Exploring The Limitations Of Substrate Scope In Dimethylallyltryptophan Synthases, Evan T. Miller

Theses and Dissertations--Pharmacy

Natural products (NPs), are the largest source of bioactive compounds in Nature, and are essential to the treatment of human disease. Nearly 50% of drugs approved for use from 1981 to 2019 owe some aspect of their development to NPs, either in terms of their structure, pharmacophore, or mechanism of action. However, NPs are difficult to structurally optimize. Chemoenzymatic methods for NP derivatization have been increasingly seen as practical alternatives to traditional synthetic methods. Prenyltransferases (PTs) are involved in the primary and secondary metabolism of plants, bacteria, and fungi, and they are key enzymes in the biosynthesis of many clinically …

Seeing Through The Fuzz: Understanding Conformational Diversity In The Fuzzy Coat Of Amyloids Across Fibril Polymorphs Using Solid-State Nmr, Carah K. Allen

Scripps Senior Theses

The aggregation of α-synuclein (α-syn) into amyloid fibrils is a central pathological hallmark of Parkinson’s disease and other related synucleinopathies. Although monomeric α-syn is intrinsically disordered, the protein assembles into multiple fibril polymorphs that differ in their β-sheet core structures. Surrounding each polymorph is the “fuzzy coat,” which is a highly dynamic, intrinsically disordered region (IDR) that is not resolved by high-resolution structural techniques, such as Cryo-EM, but is thought to influence fibril interactions, propagation, and toxicity. Understanding whether different polymorphs maintain distinct fuzzy-coat conformational ensembles is therefore essential for connecting amyloid structure to biological function. To address this question, …

Context Dependent Regulation Of Dicer Function During Oocyte-To-Embryo Transition, Nick A. Newkirk

Dissertations and Theses (Open Access)

Over two decades ago, Dicer was discovered as the primary enzyme responsible for the generation of small RNAs known as the microRNAs (miRNAs) and short interfering RNAs (siRNAs). Spatiotemporal regulation of Dicer-dependent small-RNA biogenesis affects many aspects of biology, including aging, disease progression, fertility, and cancer. Interestingly, a distinct population of Dicer-dependent small RNAs, known as the endogenous siRNAs (endo-siRNAs), are uniquely abundant in the germ cells and gametes, and are critical for normal fertility. Despite their function regulating new life, the mechanisms controlling their biogenesis and subsequent germ cell functions remain poorly defined. Work to parse these mechanisms in …

From Sequence To Allostery: How Amino Acid Differences Rewire Coupled Conformational Heterogeneity In The Ptp Enzyme Family, Shivani Sharma

Dissertations, Theses, and Capstone Projects

Protein Tyrosine Phosphatases (PTPs) are critical regulators of cellular signaling, and their activity is often modulated through allosteric mechanisms. Allostery is defined as a chemical or conformational change in one site of the protein due to a binding event at another site. Although several studies have investigated aspects of allostery in PTPs, the mechanistic foundations underlying allosteric regulation remain poorly understood. Despite a highly conserved catalytic domain across PTPs, regions with low or no conservation suggest potential "rewiring" of allosteric networks. This raises key questions about the universality versus individuality of allosteric mechanisms across the PTP family.

In this work, …

Role Of Nucleic Acids As Chaperones In Protein Folding, Zijue Huang

Electronic Theses and Dissertations

Many proteins have slow folding times in vitro that are physiologically untenable. To combat this challenge, ATP-dependent chaperonins are thought to possess the unique ability to catalyze protein folding. Performing quantitative model selection using protein folding and unfolding data, we here show that short nucleic acids containing Gquadruplex (G4) structure can also catalyze protein folding. Performing the experiments as a function of temperature demonstrates that the G4 reshapes the underlying driving forces of protein folding. To understand the structural basis of this catalytic activity, we introduce NMR method to solve the structures, at base-level resolution, of a multiconformer G4 with …

Claudin-1-Mediated Signaling And Therapy Resistance In Colorectal Cancer: From Mechanism To Translation, Mark W. Primeaux

Theses & Dissertations

Colorectal cancer (CRC) remains a leading cause of cancer-related mortality, with metastatic cases showing poor response to chemotherapy due to both intrinsic and acquired therapy resistance. Claudin-1 (CLDN1), a tight junction protein, is overexpressed and mislocalized outside of tight junctions in CRC, where it contributes to an aggressive, metastatic phenotype. Although this causal relationship has been well established, the mechanisms by which CLDN1 promotes tumor progression were unclear due to its lack of intrinsic enzymatic activity. This dissertation investigates the molecular mechanisms through which CLDN1 drives oncogenic signaling, its role in therapy resistance, and its translational potential as both a …

Structural Functional Investigation Of Hap40, Amanda M. Solbach

Dissertations and Theses (Open Access)

STRUCTURAL FUNCTIONAL INVESTIGATION OF HAP40

Amanda Marie Solbach

Dissertation Advisor: Sheng Zhang, Ph.D.

Huntington’s disease (HD) is a devastating neurodegenerative disorder caused by an expanded CAG trinucleotide repeat in the Huntingtin (HTT) gene, resulting in a mutant huntingtin (mHTT) protein with an elongated polyglutamine tract. While the genetic basis of HD is well established, the mechanisms underlying mHTT toxicity and its impact on cellular function remain incompletely understood. Recent discoveries implicate Huntingtin-associated protein 40 (HAP40) as a critical regulator of HTT stability and intracellular function. However, the physiological and pathological significance of this interaction has not been fully explored.

This …

Elucidating The Roles Of Eukaryotic Initiation Factors Involved In Dap5 Mediated Translation, Jacob Nk Quartey

Dissertations, Theses, and Capstone Projects

Translation initiation in eukaryotes is a highly regulated process essential for accurate protein synthesis. It is a dynamic process that involves a complex interplay between messenger RNAs (mRNAs), ribosomal subunits, and a host of initiation factors, ensuring precise start codon selection and the subsequent assembly of the translation machinery. This process has well been known to be mediated by the eukaryotic Initiation Factor (eIF4F), which consists of the cap binding protein eIF4E, the scaffolding protein eIF4GI, and the helicase factor eIF4A.The recognition and binding of eIF4E to the m⁷G cap structure of the mRNA is essential for the …

Biophysical Analysis Of S100a12 And The Nucleosome Core Particle, Christopher Diforte Jr.

Dissertations, Theses, and Capstone Projects

Calcium signaling and chromatin remodeling are fundamental regulatory processes that control protein activity and gene expression, respectively. This thesis investigates the molecular mechanisms by which local ligand binding and structural perturbations propagate allosteric effects across larger protein systems, using two distinct yet mechanistically analogous models: the calcium binding protein S100A12 and the nucleosome core particle. These studies are unified by a shared emphasis on how physical forces govern biological function.

In the first part, we explore calcium mediated allostery in S100A12, focusing on the distinct roles of its two EF-hand calcium binding loops (EF-I and EF-II). Through a combination of …

Kinetic Analysis Reveals T6a-Protein Specificity For T6a And Hn6a Trna Modifications, Cassandra Mae Harker

University Honors Theses

Transfer RNA (tRNA) is essential for accurate translation of genetic information and plays a role in cellular processes such as lipid aminoacylation and bacterial conjugation. Base modifications help tRNA fold correctly and function properly. An important base modification, N6-threonylcarbamoyladenosine (t6A) occurs at position A37 in the anticodon-stem-loop of tRNA. This modification is crucial for decoding ANN codons. Interestingly, an Archeal organism, Methanocaldococcus jannaschii, can have either t6A and hydroxynorvalylcarbamoyl-6- adenosine (hn6A) modification at the same position on tRNAs. As Tsa proteins catalyze the formation of both t6A and hn6A modifications, this raises the question of whether discrimination between these …

Structural Studies Towards The Mechanism Of Scanning And Initiator Trna Recruitment During Eukaryotic Translation Initiation, Da Cui

Dissertations, Theses, and Capstone Projects

The central dogma, proposed by Francis Crick in 1958, marks the direction of the flow of genetic information: from DNA to DNA (DNA replication), DNA to RNA (transcription), and RNA to protein (translation). Each of these is tightly regulated and important for cells and organisms to function correctly, grow, and reproduce.

In the eukaryotic system, the initiation stage of translation is the first, the rate-limiting, and the most regulated stage. It involves direct regulation of multiple eukaryotic initiation factors (eIFs), scanning factors such as DHX29, and other factors such as Poly-A binding protein (PABP). It starts with the small subunit …

Fire Fossils: Biomineral Infrastructures In Untangling Climates, Tanmayee More

Masters Theses

Diatoms drift, sediments sink, wildfires generate their own weather. Lightning strikes can blast sand into glass, and microscopic organisms do the same in the cold recesses of the ocean. What alchemies and metabolisms catalyze space, time, and energy? What are the thermodynamics of infrastructure in Climate Change?

Places, materials, and beings are afterimages. Through alchemical experiments, and multi-scalar imaging, I develop simulations of wildfires, and their profound, reverberating effects. In this thesis, a glass-meets-landscape experimental process enters these obscured conditions and renders new material relations for the built environment.

Tree Story, Jia Hu

Masters Theses

What is Nature?

Nature is a system of intelligence. It means designing for efficiency—often by learning from strategies that have evolved over time. In my research, I use patterns to interpret and decode nature.

To explore nature, I began with the red cedar tree, aiming to simulate and predict its growth patterns—forms shaped by both internal biology and external forces. By analyzing its geometry, I sought to understand how trees embody the dynamic relationship between organism and environment. These patterns reveal the adaptive logic of life.

Patterns are central to understanding nature. While tree geometry may appear chaotic, it follows …

Sequence Conservation Of Suppressor Of Ikk Epsilon Phosphorylation Sites, Kaydence M. Isaacs

Undergraduate Honors Theses

Suppressor of IKK epsilon (SIKE) is a protein that is implicated in immune response signaling pathways. SIKE interaction networks suggest roles in cell motility and macromolecular complexes mediating kinase/phosphatase activity. In this project, we aim to better understand the function of SIKE by analyzing amino acid sequences across different species. In human SIKE, viral infection leads to phosphorylation of SIKE at defined serine residues. Comparing SIKE sequences, and specifically these phosphorylation sites, allows us to discover its ancestral connections to innate immune function. This has particular applications in bridging the evolutionary gap between fungi and metazoa SIKE sequences. Multiple sequence …

Biochemical And Structural Insights Into The Yersinia Effector Yopm And Its Negative Regulation Of The Pyrin Inflammasome, Bethany Wairimu Mwaura

Dartmouth College Ph.D Dissertations

Bacteria utilize sophisticated secretion systems to inject effectors into host cells in order to facilitate their survival and replication in the host. Some pathogenic species of Gram-negative bacteria use a conserved contact-dependent T3SS to inject a wide array of effectors. While the effectors diverge in their structures, functions and cell-localization, the T3SS is well conserved. Several effectors discussed in this thesis target key host inflammasome responses. The focus of this work is how the Yersinia effector YopM inhibits the pyrin inflammasome. During Yersinia infection, two effectors, YopE and YopT inadvertently activate the pyrin inflammasome while targeting RhoA to avoid phagocytosis. …

The Role Of Secondary And Tertiary Structure In The Cap-Independent Translation Of Fgf-9 And Hif-1-Alpha, Amanda Michelle Whittaker

Dissertations, Theses, and Capstone Projects

Under normoxic conditions, eukaryotes initiate translation of RNA through eIF4E recognition of the 5’ cap. However, under cellular stress, eukaryotic translation must be initiated through a 4E-independent, or “cap-independent” mechanism, involving eukaryotic initiation factor 4G (eIF4G) binding directly to the 5’ untranslated regions (5’ UTR) of the RNA. eIF4G binding then recruits the ribosome to the transcript. While this mechanism is useful for translation of apoptotic transcripts and transcripts involved in cell survival, cap-independent translation is also utilized by oncogenic RNA for tumorigenesis. Previous work by our lab and others has categorized this recruitment and initiation mechanism as either internal-ribosome-entry-site …

Characterizing And Targeting The Non-Catalytic Functions Of Phosphatase Of Regenerating Liver 3 (Prl-3) In Oncogenesis And Cancer Progression, Jeffery T. Jolly

Theses and Dissertations--Molecular and Cellular Biochemistry

Phosphatase of Regenerating Liver 3 (PRL-3) is frequently upregulated in various cancers and is associated with poor patient prognosis. Although traditionally studied for its phosphatase activity, PRL-3 also interacts with the CNNM family of magnesium transporters through its catalytic site, and these two functions are mutually exclusive at any given time. Most previous studies relied on a commonly used PRL-3 mutation that disrupts both phosphatase activity and CNNM binding, making it challenging to determine which function drives its oncogenic effects. To address this gap in the field, I utilized a panel of PRL-3 mutants that selectively disrupt either phosphatase activity …

Functional Analysis Of Ligand Binding Domain 2 Of Outer Surface Protein C Of Borreliella Burgdorferi, Gavin Z. Chambers

Theses and Dissertations

Lyme disease, caused by the spirochete Borreliella burgdorferi, is the most common vector-borne infection in North America. A critical determinant of early mammalian infection is outer surface protein C (OspC), a highly variable lipoprotein that is indispensable for spirochete survival but whose precise function remains unresolved. OspC has been proposed to mediate diverse roles, including complement evasion, adhesion, tissue dissemination, and antiphagocytic activity, supporting the view that it is a multifunctional virulence factor. Structural studies have identified two putative ligand-binding domains (LBD1 and LBD2), with LBD2 forming a solvent-exposed crown at the membrane-distal end of the OspC dimer.

To …

Development Of Cyan And Blue Thermostable Fluorescent Proteins: Characterization And Structural Analysis, Acacia J. Jurkowski

Graduate Theses/Dissertations

Fluorescent proteins are commonly used as cell markers in living organisms. Modifications by mutation can be used to improve the qualities of these proteins including fluorescence, thermostability, pH stability, and chemical stability. The goal of this project is to use mutagenesis to improve the fluorescence of thermostable cyan and blue proteins derived from the thermal green protein (TGP). The first cyan protein developed by the DeVore lab (CTP 0.0) shifted the fluorescence to cyan but decreased the quantum yield (Φ_F) to 0.056. Further mutations were incorporated to increase the quantum yield through incorporating hydrogen bonding interactions to the …