Browsing by Subject "NMR"

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Adaptation of advanced MRI and NMR technique for low magnetic field MRI system.
(2012-07) Li, Yun
Nuclear magnetic resonance techniques, including nuclear magnetic imaging (MRI) and nuclear magnetic resonance (NMR) relaxometry are increasingly gaining interest and acceptance by food scientists and engineers. These techniques are being used to study moisture and fat content, water mobility, and distribution of water, fat, and temperature in foods. They are also used to determine freezing front and viscosity of foods. A unique advantage of NMR techniques is their non-invasive and non-destructive nature, making analysis faster and more reliable than conventional techniques. MRI work in the food area is primarily conducted using expensive and hard to operate large MRI systems. Most food companies, especially small companies, are not willing to use or cannot afford such large systems. Therefore, developing small systems specifically for food samples is an urgent need. NMR relaxometry has been used by food scientists for decades. However, only recently food scientists began to realize that the technique has more to offer. Research on the relationship between NMR relaxation times and physiochemical properties of foods is emerging. A suite of techniques were developed for the acquisition of reliable data and high quality images using low field MRI machines. My research has helped solve some key technical problems by improving the hardware configuration, pulse sequences, and data analysis techniques. In my research, advanced pulse sequences were adapted to our low field MRI machines for MRI imaging work and NMR relaxation work. Pulse sequences were tuned to obtain high quality images. New hardware was developed to accommodate unique samples. New NMR data acquisition schemes and data analysis techniques were developed to obtain additional information for the analysis of food stability. Models were designed for state diagrams. Coding programs were developed to calculate and analyze state diagram related parameters. My work involved both hardware and software improvements to facilitate adaptation and implementation of advanced techniques. It is my hope that these low field MRI machines be improved through my study so that they become an affordable, easy-to-operate, and relatively maintenance free analytical tool for food research and development and quality control labs.
Application of NMR in the characterization of existing and development of new methods for pancreas preservation
(2012-10) Scott III, William Earl
Islet transplantation is an emerging treatment for type-1 diabetes. Pancreas preservation has been identified as an area for improvement since many pancreata are exposed to >6-8 hours of cold ischemia reducing viable islet yields. Traditionally utilized methods of preservation depend on low temperatures and specialized preservation solutions to maintain viability. Oxygenation during preservation has been identified as a potential means to extend preservation and improve isolation outcomes. The two-layer method (TLM) was developed to oxygenate pancreata during preservation. After initial excitement, since it relies on oxygen delivery by surface diffusion alone, and oxygenation of human sized pancreata in this manner is impossible; its use clinically has faded. Persufflation (PSF), gaseous oxygen perfusion, offers an alternate means of actively delivering oxygen to tissue via the vasculature. Due to the inability of small animal models to properly demonstrate oxygen limitations, and the lack of consistent availability of human pancreata a porcine model was developed. In order to aid in the development and proper assessment of new techniques, the use of 31P-NMR spectroscopy to monitor ATP during preservation was investigated. It was able to confirm that while TLM may provide adequate oxygenation for maintenance of ATP levels in smaller pancreata (rat) it indeed is unable to maintain ATP in larger organs such as the pig or human pancreas. In contrast PSF demonstrated the ability to maintain pig or human pancreata for at least 24 hours with only a minimal decay in ATP levels observed. The relevance of these findings was confirmed both histologically as well as by the gold standard, islet isolation. Paired studies demonstrated the ability of PSF to maintain and possibly improve outcomes while extending preservation times to at least 24 hours. The methods developed in this dissertation can be applied to the development and comparison of other novel methods of preservation in the pancreas and other organs.
Applications of Fluorine Magnetic Resonance for Small-Molecule Screening, Ligand Development, and Oxygen Sensing
(2017-08) Gee, Clifford
Protein-protein interactions (PPIs) play a vital role in biological processes but are difficult to target therapeutically. However, targeting PPIs is an important challenge because their dysregulation is linked to many various disease states including cancers and neurological disorders. While high throughput screening (HTS) has long been the standard method for drug discovery, fragment-based screening (FBS) has emerged as a promising alternative strategy due to its greater coverage of chemical space with smaller library sizes. Successful cases like Vemurafenib and Venetoclax, continue to bolster FBS efforts. Though many techniques, including X-ray crystallography, surface plasmon resonance, and thermal shift assays, have all been used as screening tools, the central hypothesis of this dissertation is that 19F NMR is a powerful and time efficient FBS tool that is complementary to existing tools and is useful for characterizing proteins and small molecule ligands. Protein-Observed Fluorine NMR Spectroscopy (PrOF NMR) due to its high speed, lack of background signals, environmental sensitivity, is an ideal method to use for both ligand discovery and characterization of ligand-protein interactions. Herein, we describe the application of PrOF NMR to two proteins in particular, the KIX domain of CBP/p300 which is part of a larger transcriptional activation complex, and the first bromodomain of BrdT, an epigenetic “reader” protein that has been validated as a target for male contraception. We demonstrate the use of PrOF NMR as a primary screening tool for KIX, identifying key pharmacophores for KIX binding. We also demonstrate the use of PrOF NMR for characterizing ligand-protein interactions, uncovering a new binding site in KIX, distinct from its two native transcription factor binding sites. Validation of hits from other screening campaigns can also be followed via PrOF NMR, and the quantitative information obtained can be used to guide the structure-activity relationship (SAR) process for further ligand development. Beyond ligand discovery in proteins, fluorine magnetic resonance can also be applied as an imaging and oximetry tool. Given the sensitivity of fluorine and its applications in both biophysical and biomedical contexts, fluorine magnetic resonance serves as a new tool for small-molecule screening, ligand development, and oxygen sensing.
Assessment Of Urinary Metabolites In Risk Prediction Of Acute Kidney Injury
(2020-05) Gisewhite, Sarah
Acute kidney injury (AKI) is the sudden decrease or loss of kidney function caused by direct kidney injury or functional impairment. Many patients do not recover renal function, leading to poor quality of life and high healthcare costs. Previous work has been done to evaluate urinary biomarkers associated with AKI, but these studies have focused on a few proteins with questionable diagnostic ability. Due to the complex pathophysiology of AKI, it may be necessary to create a panel of biomarkers for diagnostic and prognostic assessment of AKI. We explored metabolic biomarkers of AKI in combat casualties using metabolomics. In this study, we used proton nuclear magnetic resonance (1H-NMR) spectroscopy to identify urinary metabolic biomarkers associated with the following outcomes: AKI diagnosis, injury severity score (ISS), AKI stage, or a primary outcome of death or need for renal replacement therapy (RRT).
Binding interaction between PKA-C and RKIP and its mutant P74L
(2022-01-03) Olivieri, Cristina; Veglia, Gianluigi; vegli001@umn.edu; Veglia, Gianluigi; University of Minnesota, BMBB Department, Structural Biology Division, Professor Gianluigi Veglia Lab
Kinetical and structural characterization of the interaction between the RAF kinase inhibitor (RKIP) and the cAMP-dependent protein kinase A (PKA-C) by interferometry and nuclear magnetic resonance (NMR) spectroscopy analysis. Together with the analysis performed by Dr. Marsha Rosner's laboratory (University of Chicago), these studies are part of a paper that has been sent to the PNAS journal.
Characterization of the chemical properties of intermediates in furan metabolism
(2012-09) Phillips, Martin Blake
This work describes the characterization of the chemical properties of intermediates in furan metabolism using techniques derived from the fields of analytical and physical chemistry, toxicology, and cancer biology.
Combined Application of Density Functional Theory and Molecular Mechanics Sampling Techniques to study Chemical Systems, from Intramolecular Rearrangements to Polymerization Reactions
(2023-05) Chiniforoush, Sina
Modern techniques in computational chemistry have allowed for the investigation of a diverse array of problems in chemistry and material sciences. However, one of the main challenges in the use of these techniques is the trade-off between computational cost and chemical accuracy. Methods like density-functional theory (DFT) are often accurate, but at the expense of higher computational resources. Methods like molecular mechanics (MM) are less computationally expensive, but fail to describe important features of chemical systems. While the study of chemical systems of relatively small size can often be carried out using methods like DFT, some of these systems have a high number of conformational degrees of freedom despite their relatively small size, and it’s often not possible to accurately describe important characteristics of these systems without capturing these all possible conformers. In this case, using MM-based sampling methods followed by DFT computations can allow for a relatively accurate description of these systems. This work contains three studies. In chapter 2, the mechanistic details of Newman-Kwart rearrangement under oxidative conditions is explored using DFT, and using theoretical predictions, modifications to the Newman-Kwart substrate are proposed to increase reactivity. In chapter 3, a combination of MM sampling methods and DFT are used to evaluate the temperature sensitivity of 19F chemical shifts in a library of organofluorine compounds screened for temperature sensing, and computations were used to successfully predict the chemical shift temperature sensitivity of these compounds, and finally used to guide the synthesis of more temperature sensitive compounds. In chapter 4, the same combination of DFT and MM techniques were used to describe two Aluminum-based ring-opening transesterification polymerization (ROTEP) catalysts, and a variety of the features of these catalysts, including the origin of their stereoselectivity, the mechanism of the inversion of catalyst chirality, and the relative stereoselectivity of the catalysts in the initiation stage, and the mechanism of stereoselectivity in the propagation stage, were described.
Cyclic AMP dependent Protein Kinase A (PKA) mutant associated with Fibrolamellar Hepatocellular carcinoma (FLHCC): Structure, dynamics and in cell studies.
(2020-03) Karamafrooz, Adak
The overall goal of this thesis is to gain a better understanding of the structure, dynamics and function of PKA-DNAJB1 in vitro and in vivo, using human PKA-WT as reference for the studies. I have utilized NMR for most of structural and dynamics studies, ITC for binding experiments and Phosphoproteomics for in vivo studies.
Data for AI-designed NMR spectroscopy RF pulses for fast acquisition at high and ultra-high magnetic fields
(2023-06-15) Veliparambil Subrahmanian, Manu; Veglia, Gianluigi; vegli001@umn.edu; Veglia, Gianluigi; Veglia Lab
The data contains RF shapes for NMR spectroscopy designed using an evolutionary algorithm and artificial intelligence. These new RF pulses cover significantly broader bandwidths for 1H and 15N nuclei and allow rapid spectra acquisition. We also re-engineered the basic transverse relaxation optimized spectroscopy experiment (RAPID-TROSY) which can enhance the spectral sensitivity of well-folded proteins up to 180 kDa molecular weight.
Development of peptide-based 19F MRI agents and BPTF-bromodomain inhibitors
(2019-07) Kirberger, Steven
Molecular imaging is the process of using targeted probes to detect abnormalities at the molecular level by observing interactions to specific biomarkers. Magnetic resonance imaging (MRI) presents an interesting avenue with respect to development of probes for the early detection of disease. In particular, 19F MRI shows promise for this development as the fluorine nucleus possesses many similar characteristics as the conventionally used proton but has the distinction of a lack of background signal found natively in biological systems. State of the art 19F MRI agents involve the use of perfluorinated compounds that often suffer from stability issues, bioaccumulation, as well as persistence within the environment. The first part of this dissertation describes the design and optimization of a peptide-based 19F MRI agent. These peptide scaffolds show promise for future use as 19F MRI probes due to their high signal, water solubility, and facile degradation in vivo to prevent bioaccumulation. The resultant byproducts have also been shown to be environmentally benign. This work is the focus of Chapters 2 and 3 of this document. In a second project, the development of a small molecule inhibitor of an epigenetic protein target is described. AU1, the first reported small molecule inhibitor of the bromodomain of a protein called BPTF, was discovered in the Pomerantz lab in 2015. As BPTF is a relatively understudied protein, there exists a need to improve the potency of AU1 as a probe for the various functions of its bromodomain, as it has been implicated in numerous diseases including: pancreatic cancer, melanoma, colorectal cancer, hepatocellular carcinoma, breast cancer, bladder cancer, and lung cancer. Structural analogs have been developed and analyzed in an attempt to improve upon AU1 in terms of its potency, solubility, and reduction of potential off-target binding. The work described in Chapter 4 of this document shows progress toward these goals, and the development of our 19F NMR assays for the analysis of protein ligands. A collaborative effort involving the (S)-enantiomer of AU1 is briefly described in Chapter 1. In a third section, the collaborative work between our lab and that of Ratmir Derda to develop peptide auxiliaries to improve therapeutic life-time in vivo is described. Chapter 5 details the use of 19F NMR to analyze the binding strength and location of numerous fluorinated peptides designed in the Derda lab.
Development of Protein-Observed Fluorine Nuclear Magnetic Resonance Spectroscopy as a Ligand Discovery Technique
(2017-04) Urick, Andrew
Fragment-based drug design (FBDD) has been rapidly gaining traction in the drug discovery process. A central tenant of fragment-based molecular screening is to use less sophisticated small molecules to sample chemical space more efficiently. With Vemurafenib and Venetoclax as FDA approved therapeutics from FBDD and several others in Phase III clinical trials, FBDD is becoming a validated technique for drug discovery. However, because of their small size these fragments are likely to bind to their target with a low affinity, necessitating more sensitive methods to detect protein-ligand interactions during a screen. Nuclear magnetic resonance spectroscopy has emerged as one of several powerful biophysical techniques for conducting fragment screens. In this thesis, a 19F protein-observed NMR method for detecting bromodomain−ligand interactions using fluorine-labeled aromatic amino acids due to the conservation of aromatic residues in the bromodomain binding site is described. Therein, we test the sensitivity, accuracy, and speed of this method with small molecule ligands. Experiment times on the order of a few minutes and the simplicity of the NMR spectra obtained make this approach well-suited to the investigation of small- to medium-sized proteins, as well as the screening of multiple proteins in the same experiment. Simplified 19F NMR spectra allowed for simultaneous testing of multiple bromodomains to assess selectivity and identification of a new BPTF ligand. Fluorine labeling only modestly affected the Brd4 structure and function assessed by isothermal titration calorimetry, circular dichroism, and X-ray crystallography. To benchmark its potential as a ligand discovery tool, we compare the protein-observed 19F NMR screening method with the well-characterized ligand-observed 1H CPMG NMR screen. We selected the first bromodomain of Brd4 as a model system because of the high ligandability of Brd4 and the need for small molecule inhibitors of related epigenetic regulatory proteins. We conclude that for the protein class understudy here, protein-observed 19F NMR and 1H CPMG have similar sensitivity, with both being effective tools for ligand discovery. The speed, ease of interpretation, and low concentration of protein needed for binding experiments affords a new method to discover and characterize both native and new ligands.
Eco-Friendly Boron Complex Addition to Aldehydes
(2019-08) Merritt, Marcy
A series of aldehyde ligands were synthesized to increase yield and decrease resource use and waste production. Boron diphenyl complexes were then separately added to each of the ligands greatly increasing and shifting their intensity and fluorescence wavelength. The complexes were characterized via NMR and GCMS. The stability of these luminescent boron complexes and ability to be vacuum sublimated onto a glass surface makes them suitable for OLED application.
Factors Affecting Recognition and Chemical Reactivity in Macromolecular Systems
(2015-07) Isley III, William
As chemists characterize molecular systems in greater detail, it becomes clear that some observables can only be properly studied at the macromolecular scale. However, elucidating the physical principles behind such phenomena as molecular recognition or chemical reactivity can be difficult when moving into the macromolecular regime. The objective of this work is to provide insights and predictions to complement experimental undertakings. The work is divided into two categories: 1) modeling molecular recognition through prediction of intermolecular interactions with highly accurate methods and 2) the modeling of chemical reactivity. The separation of N2 and CH4 is particularly pertinent for the natural gas industry, and improved materials for performing this separation would provide an enormous cost savings. This work is focused on the prediction of a new material capable of performing this separation. Through application of multiple tiers of quantum chemical methods, and comparison to similar known experimentally synthesized materials, a novel material is predicted to effect the separation of N2 and CH4 through selective binding of N2 to an open vanadium metal site. A particularly valuable tool for monitoring target delivery or guest encapsulation in macromolecular systems is an easily observed signal that indicates the status of a host-guest complex. Guest complexation can alter the observable properties of the host, including the spin crossover properties of a host macromolecule. Particular care was taken to correlate guest recognition to changes in paramagnetic NMR chemical shifts induced in the host system. The monitoring of subtle changes in a protein's environment is a challenging and complex problem; however, the observation of ligand complexation in biomolecular systems is extremely important in the design of new medicinal therapeutic drugs. This work aims to develop a quantum chemical method to assist experimental assignment of challenging 19F NMR spectra in proteins. The importance of accurately modeling the hydration environment is extremely critical for accurate comparison to experimental measurements. Selective detection of chemical impurities is an attractive capability to have for any chemical process. A key impurity in industrially synthesized explosive TNT is DNT. Given the high prevalence of DNT in TNT, detection of DNT through electrochemistry is a useful sensor for explosives. This work characterizes the mechanism of DNT electrochemical reduction A new material was found to rapidly catalyze the decomposition of extremely toxic chemical warfare agents. The macromolecular metal organic framework NU-1000 was demonstrated to be extremely effective in catalyzing the hydrolysis of phosphoester based chemical warfare agents. Predictive computations were performed on a nerve agent simulant DMNP, and toxic nerve agents GD (Soman) and VX agents, uncovering the key role that the metal nodes of NU-1000 play in activation of the phosphoester bonds for hydrolytic attack
Galectin-1 Interactions with complex glycans and Alpha-linked carbohydrates
(2013-01) Miller, Michelle C.
Galectin-1 (gal-1) is defined as an alpha-galactoside-binding protein, which crosslinks apposing glycans to mediate cell-cell and cell-extracellular matrix interactions. In this way, gal-1 facilitates cellular adhesion and migration and induces signaling pathways for growth and regulation. Gal-1 is overexpressed in an array of human cancers and correlates with poor patient prognosis. Reports on the role of gal-1 in malignant growth provide evidence that gal-1 is involved in tumor immune escape and is essential for angiogenesis as well as promoting metastasis. For these reasons, gal-1 is considered a good target for novel cancer therapeutics. Towards this goal, we used NMR spectroscopy to probe the binding of gal-1 to complex carbohydrates such as davanat, a galactomannan poised to enter into phase III clinical trials for the treatment of metastatic colorectal cancer, and GRG, a galactorhamnogalacturonate being developed as an oncological therapeutic. We found that davanat, an alpha-galactoside, interacts with a novel region on gal-1, outside of its conventional carbohydrate binding site, while GRG interacts at an area that includes and extends out from the carbohydrate binding site. Additionally, we found that gal-1 affects the hydrodynamic behavior of these glycans, and based on these findings, we hypothesized that gal-1 has a role in decongesting cell-surface glycans, which may assist in the formation of plasma-membrane microdomains. These results spurred additional studies with alpha-linked disaccharides and a series of galactomannans with varying Man/Gal ratios. Overall, this work contributes to an expanded understanding of gal-1/glycan interactions, which will be useful towards the design and optimization of gal-1 targeting drugs.
Identification and Characterization of Phosphorus Composition in Lake Superior and St. Louis River Estuary Sediments Using Phosphorus Nuclear Magnetic Resonance Spectroscopy
(2017-12) Schoechert, Hannah
Phosphorus has been a contaminant of concern for many freshwater lakes for decades. Excessive bioavailable phosphorus often leads to the eutrophication of a particular body of water. Information on the specific chemical composition of phosphorus in sediment is fundamental to understanding its bioavailability and eutrophication potential to a lake ecosystem. A single-step sodium hydroxide-ethylenediaminetetraacetic acid (NaOH-EDTA) extraction and a phosphorus nuclear magnetic resonance (31P NMR) spectroscopy protocol were developed and subsequently performed on St. Louis River Estuary (SLRE) and Chequamegon Bay (CB) sediment samples. Results show the presence of phosphorus-containing compounds comparable to other oligotrophic waterbodies, and compounds typically detected in sediment samples from eutrophic lakes were not detected in any sample. For the CB samples, as the water depth increased, so did the number of peaks identified. Similarly, as the number of peaks increased, there was an increase in relative abundance of different phosphorus. For the SLRE samples, it was observed that the phosphorus composition in the sediment mirrored the phosphorus sediment composition from the Chequamegon Bay samples, suggesting there are similar hydrological conditions between the two sites.
Nuclear Magnetic Resonance and Circular Dichroism Spectroscopy to study the folding state of artificial, primordial-like proteins
(2024-07-25) Blascyk, Eli
Proteins are the primary molecular machinery for all known life. All proteins are made from the standard 20 amino acids in accordance to the universal genetic code of life; however, prior to the last universal common ancestor, this code was not set in stone and had to evolve to include each of the amino acids. The Seelig lab has previously generated libraries of random proteins using reduced sets of amino acids in order to simulate possible early protein functionality. The reduced sets of amino acids were based on the consensus chronology of incorporation into the genetic code and consisted of 5, 9, 16, and 20 of the amino acids used in modern proteins. This research aimed to characterize the structures of ATP binding proteins from each mRNA display library, primarily focusing on the 5AA and 9AA alphabet libraries. HSQC NMR and CD spectroscopy were used to characterize the structural and folding state of these proteins. Measurements from these spectroscopy experiments reveal secondary structure characteristics of up to 45% alpha helicity for individual variants. Further characterization efforts for these proteins are ongoing, including additional NMR and CD experiments as well as attempts to crystallize the proteins for X-ray crystallography.
On the Role of Allosteric Cooperativity in the Regulation of Protein Kinase A and its Implications in Disease
(2021-05) Walker, Caitlin
First articulated half a century ago, allostery has remained a universal phenomenon and is essential in understanding processes beyond the molecular level, such as cellular signaling and disease. Allostery also referred to as allosteric regulation, is a process by which biological macromolecules transmit the effect of binding at one site to an often distal, functional site, allowing for regulation. To facilitate the modulation of function between sites, allosteric signal is propagated through conserved amino acid residues, often comprising various structural elements of a protein. In general, allosteric communication is of fundamental interest and potentially of high relevance for drug design and protein engineering. Furthermore, the dysfunction of allosteric networks has been implicated in the etiology of human diseases. However, defining these networks of residues that mediate crosstalk between distal sites remains experimentally challenging and thus, poorly characterized. Since allosteric signal propagation relies on subtle conformational rearrangements, nuclear magnetic resonance (NMR) has emerged as an instrumental tool in investigating allosteric communication. This thesis aims to map allosteric networks at atomic resolution to understand how mutations in protein kinase A (PKA) influence allosteric communication to elicit the progression of various disease states. In this work we demonstrate how disease mutations associated with Cushing’s Syndrome and Fibrolamellar Hepatocellular Carcinoma attenuate the allosteric network of PKA, thereby disrupting the finely tuned regulation, specificity, and activation of PKA to generate dysfunctional signaling. The findings of this thesis provide critical insights into the importance of intramolecular allostery in facilitating functional signaling, directly showing how changes in allosteric networks of proteins lead to dysfunction.
On the Role of Conformational Dynamics in Allostery and Cooperativity in Protein Kinase A
(2017-02) Li, Geoffrey
Protein kinases are a large class of enzymes that regulate a wide array of vital cellular processes. Their dysregulation has been associated with fatal diseases including cancer, cardiovascular, and metabolic diseases. Hence, they have been important drug targets for years. While an enormous wealth of information about the structure and functions of kinases is available to date, a comprehensive mechanism of allosteric regulation of activity remains elusive. This thesis aims to investigate the role of conformational dynamics in the allosteric regulation and binding cooperativity of kinases using the cAMP-dependent protein kinase (PKA) as a model system. In this work, we demonstrated how allostery in PKA is propagated by changes in the hydrogen bond network between residues. We showed that different nucleotides and inhibitors modulate the allosteric cooperativity of PKA to different extent. Using NMR spectroscopy, we established how ligands influence substrate binding affinity by altering the kinase’s conformational dynamics through suppression and formation of sparsely-populated high-energy states. The findings of this work provide a new paradigm for designing more effective therapeutic agents that can steer the conformational landscape of kinases to better fine-tune their activity and functions.
Proteasome activation by the 19S regulatory particle: structural dynamics of 26S assembly and substrate recognition
(2013-06) Ehlinger, Aaron Christopher
Since its discovery in the late 1970s, the ubiquitin-proteasome system (UPS) has become recognized as the major pathway for regulated cellular proteolysis. Processes ranging from cell cycle control, pathogen resistance, and protein quality control rely on selective protein degradation at the proteasome for homeostatic function. Perhaps as a consequence of the importance of this pathway, and the genesis of severe diseases upon its dysregulation, protein degradation by the UPS is highly controlled from the level of substrate recognition to proteolysis. Technological advances over the last decade have created an explosion of structural and mechanistic information that has underscored the complexity of the proteasome and its upstream regulatory factors. Significant insights have come from study of the 19S proteasome regulatory particle (RP) responsible for recognition and processing of ubiquitinated substrates destined for proteolysis. Established as a highly dynamic proteasome activator, a large number of both permanent and transient RP components with specialized functional roles are critical for proteasome function. This research investigates the dynamic nature of protein-protein interactions involved in proteasome assembly and substrate recruitment, and how they provide context to our current understanding of proteasome activation by the RP.
Structural and dynamics analysis of pathogenic modifications in cardiac sarcoplasmic reticulum proteins involved in Ca2+ transport
(2018-01) Nelson, Sarah
Calcium signaling pathways are essential for the coordination of contraction and relaxation in cardiac muscle. Disruption of cardiac calcium cycling by pathogenic modifications in calcium transport proteins leads to a variety of cardiomyopathies including dilated cardiomyopathy and arrhythmias. The following thesis summarizes the structural and dynamic characterization of key regulatory proteins involved in calcium release and reuptake in the sarcoplasmic reticulum (SR). Calmodulin (CaM), a calcium-sensing protein that regulates its cellular targets based on the level of calcium in the cell, mediates calcium release from the SR via the homotetrameric calcium release channel, ryanodine receptor (RyR). The CaM-RyR complex has been a challenging structural target due to the size and complexity of the RyR. By applying a combination of solution and solid-state NMR techniques we have begun to develop a molecular model for CaM’s regulation of the RyR and how this regulation is disrupted by pathogenic modifications such as oxidation and mutation. Disruptions in calcium reuptake to the SR due to mutations in the small transmembrane protein, phospholamban (PLN), result from dysregulation of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA). These PLN mutations are primarily associated with the development of dilated cardiomyopathy and by applying solution and solid-state NMR techniques we have begun to develop a model for how changes in PLN’s structure and dynamics correlate to the dysregulation of SERCA. Together, the structural and dynamic studies outlined in this thesis provide further insights into the correlations between protein structure and function and the crucial roles CaM and PLN play in cardiac function.

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