Structural-Functional Studies on PSI-IsiA Super-complex in Synechocystis sp. PCC 6803

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Description
The thylakoid membranes of oxygenic photosynthetic organisms contain four large membrane complexes vital for photosynthesis: photosystem II and photosystem I (PSII and PSI, respectively), the cytochrome b6f complex and ATP synthase. Two of these complexes, PSII and PSI, utilize solar…

The thylakoid membranes of oxygenic photosynthetic organisms contain four large membrane complexes vital for photosynthesis: photosystem II and photosystem I (PSII and PSI, respectively), the cytochrome b6f complex and ATP synthase. Two of these complexes, PSII and PSI, utilize solar energy to carry out the primary reaction of photosynthesis, light induced charge separation. In vivo, both photosystems associate with multiple antennae to increase their light absorption cross section. The antennae, Iron Stress Induced A (IsiA), is expressed in cyanobacteria as part of general stress response and forms a ring system around PSI. IsiA is a member of a large and relatively unexplored antennae family prevalent in cyanobacteria. The structure of the PSI-IsiA super-complex from the cyanobacteria Synechocystis sp. PCC 6803 was resolved to high resolution, revealing how IsiA interacts with PSI as well as the chlorophyll organization within this antennae system. Despite these structural insights, the basis for the binding between 18 IsiA subits and PSI is not fully resolved. Several IsiA mutants were constructed using insights from the atomic structure of PSI-IsiA, revealing the role of the C-terminus of IsiA in its interaction with PSI.
Date Created
2024
Agent

Optimized Preparation of Immunologically Relevant Proteins for Structural Studies by X-ray Crystallography and Cryogenic Electron Microscopy

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Description
The complex network of the immune system defends the human body against infection, providing protection from pathogens. This work aims to improve preparation and structural knowledge of two proteins on opposite sides of the immune system spectrum. The first protein,…

The complex network of the immune system defends the human body against infection, providing protection from pathogens. This work aims to improve preparation and structural knowledge of two proteins on opposite sides of the immune system spectrum. The first protein, secreted autotransporter toxin (Sat) is a class I serine protease autotransporter of Enterobacteriaceae (SPATE) that has cytotoxic and immunomodulatory effects on the host. Previous studies on Sat show its ability to aid in bacterial colonization and evasion of the immune system. This work improves the stability of Sat by making mutations to the active serine protease motif (GDSGS) while inhibiting remaining activity with reversible and irreversible serine protease inhibitors. Characterization of Sat by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and size-exclusion chromatography led to the first structural studies of Sat by x-ray crystallography and cryo-EM. Human leukocyte antigen class I proteins play an important role in the adaptive immune system by presenting endogenous viral peptides at the cell surface for CD8+ T cell recognition. In vitro production of HLA-I proteins is a difficult task without endoplasmic reticulum chaperones as present in vivo. Disulfide bond formation, folded light chain and a peptide bound are all key to refolding the HLA-I heavy chain for complex formation. The work presented in this dissertation represents systematic studies aimed at improving the production of HLA-I proteins in vitro in bacterial expression systems. Optimization of every step of the preparation was investigated providing higher expression yields, quality of inclusion bodies, and refolding improvements. With further improvements in the future, this work forms the basis for a more efficient small and large-scale production of HLA-I molecules for functional and structural studies.
Date Created
2024
Agent

Insulin Receptor Fragmentation by Matrix Metalloprotease 1

Description
Insulin resistance is a hallmark of diabetes, a disease that costs healthcare systems hundreds of billions of dollars annually, and although the exact mechanism behind insulin resistance has not been identified, the reduced tyrosine activity of the integral membrane protein…

Insulin resistance is a hallmark of diabetes, a disease that costs healthcare systems hundreds of billions of dollars annually, and although the exact mechanism behind insulin resistance has not been identified, the reduced tyrosine activity of the integral membrane protein insulin receptor (IR) and its decreased cell surface presentation have been linked to insulin resistance. Moreover, IR shedding, the proteolytic cleavage of extracellular domains of IR releasing soluble fragments into blood, has been correlated with diabetes outcomes. We hypothesized that MMP1 may bind the IR and fragment it, thus inducing insulin resistance and contributing to comorbidities. After introducing MMP1 to the IR and using LC-MS, 21 fragments of the IR were identified. MMP1 and the IR, both active and inactive, were submitted to ClusPro and every ClusPro model was siphoned through to find the closest distance that the MMP1 catalytic site is to the cleavage sites of each fragment. The catalytic site distance varied from under 5 angstroms to more than 30 angstroms. The B-Factor across the IR was also calculated to determine the ability for each fragment to flex in a way that the catalytic site could reach it, but the B-Factors were unexpectedly low. A sequence logo was made for the cleavage sites of each fragment and compared to the MEROPS MMP1 cleavage site sequence logo with little overlap. Results such as distances from catalytic site to cleavage site being under 10 angstroms are suggestive of interaction between MMP1 and the IR, although other results such as there being little similarity between the sequence logos dispute that. This may be due to trace amounts of trypsin found within the LC-MS samples and future works would require validation through inhibiting trypsin within the samples.
Date Created
2024-05
Agent

Dissecting the Molecular Mechanism of Temperature Sensing and Regulation in TRP Channels

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Description
Receiving signals and responding to the environment is crucial for survival for every living organism. One of those signals is being able to detect environmental and visceral temperatures. Transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential melastatin 8…

Receiving signals and responding to the environment is crucial for survival for every living organism. One of those signals is being able to detect environmental and visceral temperatures. Transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential melastatin 8 (TRPM8) are ion channels within cells that allow higher organisms to detect hot and cold temperatures, respectively. These TRP channels are also implicated in diverse physiological roles including pain, obesity, and cancer. As a result, these channels have garnered interest as potential targets for therapeutic interventions. However, the entanglement of TRPV1 and TRPM8 polymodal activation where it responds to a variety of different stimuli has caused adverse side effects of body thermal dysregulation and misregulation when antagonizing these channels as drug targets. This dissertation will dissect the molecular mechanism and regulation of TRPV1 and TRPM8. An in-depth look into the complex and conflicting results in trying to find the key area for thermosensation as well as looking into disentangling the polymodal activation modes in TRPV1. The regulatory mechanism between TRPM8 with phosphoinositide interacting regulator of TRPs (PIRT) and calmodulin will be examined using nuclear magnetic resonance (NMR). A computational, experimental, and methodical approach into ancestral TRPM8 orthologs using whole-cell patch-clamp electrophysiology, calcium mobilization assay, and cellular thermal shift assay (CETSA) to determine whether these modes of activation can be decoupled. Lastly, smaller studies are covered like developing a way to delivery full-length and truncated protein using amphipols to artificial and live cells without the biological regulatory processes and the purification of the TRPM8 transmembrane domain (TMD). In the end, two successful methods were developed to study the polymodal activation of proteins.
Date Created
2023
Agent

Highly Sensitive and Multiplexed Single Cell In-situ Protein Imaging with Cleavable Fluorescent Probes

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Description
Without a doubt, protein is the most crucial biomolecule performing life and biological functions of any living cell. Profiling various protein expression in individual cells has raised a great interest for scientist and researchers over decades in attempts to reveal…

Without a doubt, protein is the most crucial biomolecule performing life and biological functions of any living cell. Profiling various protein expression in individual cells has raised a great interest for scientist and researchers over decades in attempts to reveal cell-to-cell variation, which used to be masked in many previous population average measurement methods. Immunofluorescence (IF) has been a well-established single cell protein analysis technique as for its fast and high-resolution detection and localization, simple and adaptable workflows, and affordable instrumentation. However, inadequate detection sensitivity and multiplexing capability are the two limitation of this platform that remain incompletely addressed in many decades. In this work, several improvements have been proposed and demonstrated to improve existing drawbacks of conventional immunofluorescence. An azide-based linker featured in the novel fluorescent probes synthesis has enable iterative protein staining on the same tissue sample, which subsequently increase the multiplex capacity of IF. Additionally, the multiple fluorophore introduction to the proteins target via either layer by layer biotin-cleavable fluorescent streptavidin or tyramide signal amplification (TSA) have significantly increase the detection sensitivity of the platform. With these advances, IF has the potential to detect, image and quantify up to 100 protein targets in single cell in the tissue sample. In addition of desirable features of IF, these improvements have further turned the technique into a powerful proteomic study platform for not only research setting but also clinical study setting. It is anticipated this highly sensitive and multiplexed, renovated IF method will soon be translated into biomedical studies.
Date Created
2023
Agent

Investigating Suspected Light Sensitivity of Potential Opsin Proteins in Pocillopora damicornis

Description

Model organisms like Homo sapiens, Drosophila, and E. coli, while useful to various fields of study, present a problem to the scientific community: many other organisms’ proteins, metabolic processes, and biochemical mechanisms are not as well understood by comparison. Pocillopora…

Model organisms like Homo sapiens, Drosophila, and E. coli, while useful to various fields of study, present a problem to the scientific community: many other organisms’ proteins, metabolic processes, and biochemical mechanisms are not as well understood by comparison. Pocillopora damicornis (Pdam), like many other coral organisms, faces environmental stresses and threats to its survival in ocean ecosystems with limited understanding of its biochemical mechanisms, making it difficult to help preserve. However, upon analyzing the symbiotic relationship of Pdam and photosynthetic algae, it was reasoned that the coral organism is capable of detecting light. Following up with results of prior bioinformatics analysis courtesy of Kumar, L., Klein-Seetharaman, J., Et. Al, it was proposed that light sensitive proteins in corals are the following four candidates: 2270, 12246, 629, 19775. If chromophores form and their opsin shifts can be visualized in the case in any of the coral candidate opsin genes, it supports the hypothesis that the proteins are indeed a light sensitive opsin protein. If a light sensitive opsin protein is identified, it provides a direction by which efforts can be directed towards to understand corals at the biochemical level for their preservation in the face of unprecedented threats to sustainability.

Date Created
2023-05
Agent

Regulated Intramembrane Proteolysis of the p75 neurotrophin receptor by γ-secretase in the context of the lipid bilayer

Description

Alzheimer’s disease (AD) is a common neurodegenerative disorder affecting approximately 10% of people aged 65 and up and 30-50% over 85. In pathological AD representations, a way to recognize early onset AD is the increased levels of pro-NGF in BFCNs…

Alzheimer’s disease (AD) is a common neurodegenerative disorder affecting approximately 10% of people aged 65 and up and 30-50% over 85. In pathological AD representations, a way to recognize early onset AD is the increased levels of pro-NGF in BFCNs that come from the downregulation of NGF with age. Pro-NGF has a higher affinity for p75NTR, which binds and participates in the pro-NGF-p75NTR-sortilin complex sequentially cleaved by α- and γ-secretase. Pro-NGF triggers apoptosis through the cleavage of the intracellular membrane by γ-secretase. Since γ-secretase physically cleaves off the intramembrane portion that promotes TNF- and Fas-dependent apoptotic signaling pathways, it has a crucial role in AD and must be better understood. This research aims to understand better and visualize γ-secretase and its actions, specifically with its interactions with the substrate p75NTR in the RIP process. To analyze γ-secretase function, the proteins must be produced and analyzed through the protein expression protocol. During protein production, DNA, cell concentrations, and optical density measurements were difficult to produce due to the incompetency of e. coli cells (DH5α), contamination of the Sf9 insect cell culture, and decreased viability of aged insect cells. We identified the problems and improved the conditions for future project development.

Date Created
2023-05
Agent

Serial Crystallographic Studies for Therapeutic Drug Advancement

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Description
Macromolecular structural biology advances the understanding of protein function through the structure-function relationship for applications to scientific challenges like energy and medicine. The proteins described in these studies have applications to medicine as targets for therapeutic drug design. By understanding…

Macromolecular structural biology advances the understanding of protein function through the structure-function relationship for applications to scientific challenges like energy and medicine. The proteins described in these studies have applications to medicine as targets for therapeutic drug design. By understanding the mechanisms and dynamics of these proteins, therapeutics can be designed and optimized based on their unique structural characteristics. This can create new, focused therapeutics for the treatment of diseases with increased specificity — which translates to greater efficacy and fewer off-target effects. Many of the structures generated for this purpose are “static” in nature, meaning the protein is observed like a still-frame photograph; however, the use of time-resolved techniques is allowing for greater understanding of the dynamic and flexible nature of proteins. This work advances understanding the dynamics of the medically relevant proteins NendoU and Taspase1 using serial crystallography to establish conditions for time-resolved, mix-and-inject crystallographic studies.
Date Created
2022
Agent

Cryo-electron Microscopy Enables Structural Studies of Protein Complexes Relevant to Neurodegeneration

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Description
The understanding of protein functions in vivo is very important since the protein is the building block of a cell. Cryogenic electron microscopy (cryo-EM) is capable of visualizing protein samples in their near-native states in high-resolution details. Cryo-EM…

The understanding of protein functions in vivo is very important since the protein is the building block of a cell. Cryogenic electron microscopy (cryo-EM) is capable of visualizing protein samples in their near-native states in high-resolution details. Cryo-EM enables the visualization of biomolecular structures at multiscale ranging from a cellular structure to an atomic structure of protein subunit.Neurodegenerative diseases, like Alzheimer’s disease and frontotemporal dementia, have multiple dysregulated signaling pathways. In my doctoral studies, I investigated two protein complexes relevant to these disorders: one is the proNGF- p75 neurotrophin receptor (p75NTR)- sortilin neurotrophin complex and the other is the p97R155H mutant complex. The neurotrophins are a family of soluble basic growth factors involved in the development, maintenance, and proliferation of neurons in the central nervous system (CNS) and peripheral nervous system (PNS). The ligand for the neuronal receptors dictates the fate of the neuronal cells. My studies focused on understanding the binding interfaces between the proteins in the proNGF-p75NTR-sortilin neuronal apoptotic complex. I have performed the biochemical characterization of the complex to understand how the complex formation occurs. Single amino-acid mutation of R155H on the N-domain of p97 is known to be the prevalent mutation in 40% patients suffering from neurodegenerative disease. The p97R155H mutant exhibits abnormal ATPase activity and cofactor dysregulation. I pursued biochemical characterization in combination with single-particle cryo-EM to explore the interaction of p97R155H mutant with its cofactor p47 and determined the full-length structures of the p97R155H-p47 assemblies for the first time. About 40% p97R155H organizes into higher order dodecamers, which lacks nucleotide binding, does not bind to p47, and closely resembles the structure of p97 bound with an adenosine triphosphate (ATP)-competitive inhibitor, CB-5083, suggesting an inactive state of the p97R155H mutant. The structures also revealed conformational changes of the arginine fingers which might contribute to the elevated p97R155H ATPase activity. Because the D1-D2 domain communication is important in regulating the ATPase function, I further studied the functions of the conserved L464 residue on the D1-D2 linker using mutagenesis and single-particle cryo-EM. The biochemical and structural results suggested the torsional constraint of the D1-D2 linker likely modulates the D2 ATPase activity. Our studies thus contributed to develop deeper knowledge of the intricate cellular mechanisms and the proteins affected in disease pathways.
Date Created
2022
Agent

Biophysical Characterization and Structural Studies of Proteins in Pursuit of Vaccinations Against Infectious Diseases

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Description
Infectious diseases are the third leading cause of death in the United States and the second leading cause of death in the world. This work aims to advance structural studies of vital proteins involved in the infection process of both…

Infectious diseases are the third leading cause of death in the United States and the second leading cause of death in the world. This work aims to advance structural studies of vital proteins involved in the infection process of both a bacterial and a viral infectious disease in hopes of reducing infection, and consequently, fatality rates. The first protein of interest is OspA, a major outer surface protein in Borrelia burgdorferi – the causative bacterium of Lyme disease. Previous functional studies of OspA allude to both a role in colonization of B. burgdorferi in the tick vector and in evasion of the human immune system. This work describes the first ever structural studies of OspA as it is seen by the immune system: in the outer membrane. OspA was expressed in and purified from the outer membrane of Escherichia coli prior to characterization via circular dichroism (CD), native polyacrylamide gel electrophoresis, and electron microscopy. Characterization studies of OspA provide the first evidence of multimeric formation of OspA when translocated to the outer membrane, which presents a new perspective from which to build upon for the design of vaccinations against Lyme disease. The second protein of interest is nonstructural protein 15 (Nsp15), a protein responsible for facilitating immune system evasion of SARS-CoV-2 – the virus responsible for the COVID-19 pandemic. Nsp15 functions to enzymatically cleave negative sense viral RNA to avoid recognition by the human immune system. The work described in this dissertation is dedicated to the electron microscopy work utilized to reveal structural information on an inactive variant of Nsp15 bound to RNA sequences. Negative stain electron microscopy was used to verify Nsp15 structural integrity, as well as reveal a low-resolution image of structural deviation when RNA is bound to Nsp15. Cryo-electron microscopy was performed to solve structural density of Nsp15 without RNA to a resolution of 3.11 Å and Nsp15 bound to 5-nucleotides of RNA to a resolution of 3.99 Å. With further refinement, this structure will show the first structural data of Nsp15 bound to a visible RNA sequence, revealing information on the binding and enzymatic activity of Nsp15.
Date Created
2022
Agent
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