Solid-state NMR (ssNMR) has matured into a versatile method to provide structural information, probe protein dynamics and detect small molecule binding and -protein interaction of a variety of biomolecular assemblies including amyloid fibrils, viral particles and membrane proteins. Membrane proteins embedded in liposomes are natural targets for ssNMR as their native states are solids. Magic angle spinning (MAS) ssNMR studies using moderate spinning frequencies provide detailed structural information and probe subtle conformational change. Development of fast magic angle spinning ssNMR enables proton-detection which increases sensitivity and facilitates protein dynamics measurements. In this dissertation, we applied moderate and fast MAS ssNMR to study potassium ion channel and protein dynamics Chapter 1 will introduce concepts and theory of solid-state NMR pulse sequences and experiments. Chapter 2 will discuss the application and perspectives of solid-state NMR to membrane protein systems. In Chapter 3, we test an allostery mechanism for inactivation using a KcsA mutant (H25R/E118A) that exhibits an open pH gate across a broad range of pH values. We present solid-state NMR measurements of this open mutant at neutral pH to probe the affinity for potassium at the selectivity filter. This result strongly supports our assertion that the open pH gate allosterically affects the potassium binding affinity of the selectivity filter. In this mutant the protonation state of a glutamate residue (E120) in the pH sensor is sensitive to potassium binding, suggesting that this mutant also has flexibility in the activation gate and is subject to transmembrane allostery. In Chapter 4, I optimize protein expression, purification and reconstitution into native environment protocols of a bacterial potassium transporter, KtrB. In chapter 5, methods and experimental details of setting up 60 and 40 kHz fast MAS ssNMR are discussed. With fast MAS ssNMR setup, multidimensional NMR experiments with higher sensitivity could be collected on a perdeuterated sample with less sample mass required. In Chapter 6, we employ fast MAS ssNMR to measure bulk and residue site-specific 15N and carbonyl 13C relaxation of microcrystalline ubiquitin. Carbonyl R1ρ relaxation profiles provide additional information on protein backbone dynamics.

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Alzheimer's Disease (AD) is a devastating neurodegenerative disease that is characterized by brain atrophy, neuronal and synaptic loss, cognitive decline, trouble handling activities of daily life, and ultimately leads to death. Worldwide, at least 30 million people suffer from AD, with 5.8 million suffering in the US alone. Despite extensive basic and clinical research, the underlying molecular mechanisms behind AD remain largely unknown. There are four FDA-approved compounds are used for alleviating symptoms but have no curative potency. The first potentially disease-modifying AD drug, aducanumb, was approved by FDA in June 2021. The main histopathological traits of AD are the Amyloid-beta (Aβ) peptide and the tau protein. Aβ aggregates to form extracellular plaques in brain parenchyma and vasculature while tau forms intraneuronal tangles. Aβ is produced by enzymatic cleavage of the amyloid precursor protein (APP) in the brain. Once APP cleavage occurs, Ab monomers either aggregate extracellularly to form buildups of sticky plaque or embed themselves within the neuronal cell membrane to form pores, causing homeostatic dysregulation and eventually cell death. The mechanism of membrane pores formed by Ab and the pore structure remain to be characterized. This study aims to analyze the structure of four Aβ species in lipid membranes. These are the most abundant form of Aβ, Aβ1-40, and the more cytotoxic form, Aβ1-42, as well as their pyroglutamylated counterparts, pEAβ3-40 and pEAβ3-42, which are hypertoxic. These peptides have been studied using biophysical approaches, i.e., circular dichroism, fluorescence spectroscopy, and Fourier transform infrared spectroscopy. Elucidation of the structure of Aβ membrane pores provides valuable insight into the mechanism of Aβ toxicity and may help develop novel therapies for the lethal mystery that is AD.

Proteases have a fundamental role in regulating diverse biological processes and are important in the biotechnological and medical fields. Therapeutic proteases have great potential but have been limited due to the lack of high throughput protein engineering methods. In this thesis, a method was developed for high throughput screening of protease libraries based on competitive growth in selective media. A proof-of-principle method using the Tobacco Etch Virus protease (TEVp) was developed. TEVp and the reporter consisting of an aggregation-prone peptide amyloidbeta42 (Aβ42) genetically fused to the antibiotic resistance enzyme chloramphenicol acetyltransferase (CAT), were co-expressed in Escherichia coli. The CAT enzyme makes the cells resistant to Chloramphenicol (Cml). Two different reporters containing different cleavage sites situated between the Aβ42 and CAT were used for which TEVp has distinguishable proteolysis efficiencies. Cleavage of the fusion protein would give the cell a growth advantage in media with Cml since the CAT enzyme would avoid misfolding due to Aβ42. The method demonstrates that cells with different substrates can be differentiated based on their survival. A 100-fold enrichment of clones expressing the efficient substrate was also demonstrated from a background of 1:1000 of clones expressing the inefficiently cleaved substrate. Moreover, a semi-rational TEVp library was successfully cloned and co-electroporated with the reporter into E. coli for future selection.

Neuronal culture systems have many applications, such as basic research into neuronal structure, function, and connectivity as well as research into diseases, conditions, and injuries affecting the brain and its components. In vitro dissociated neuronal systems have typically been derived from embryonic brain tissue, most commonly from the hippocampus of E18 rats. This practice has been motivated by difficulties in supporting regeneration, functional recovery and long-term survival of adult neurons in vitro. The overall focus of this dissertation research was to develop a dissociated neuronal culture system from human and animal adult brain tissue, one more functionally and developmentally correlative to the mature brain. To that end, this work was divided into five interrelated topics: development of an adult in vitro neuronal culture system comprised of electrically functional, mitotically stable, developmentally mature neurons from the hippocampus of adult rats; creation of stable two-cell neuronal networks for the study of synaptic communication in vitro; coupling of electrically active adult neurons to microelectrode arrays for high-throughput data collection and analysis; identification of inadequacies in embryonic neuronal culture systems and proving that adult neuronal culture systems were not deficient in similar areas; augmentation of the rat hippocampal culture system to allow for the culture and maintenance of electrically active human neurons for months in vitro. The overall hypothesis for this dissertation project was that tissue engineered in vitro systems comprised of neurons dissociated from mature adult brain tissue could be developed using microfabrication, defined medium formulations, optimized culture and maintenance parameters, and cell-cycle control. Mature differentiated glutamatergic neurons were extracted from hippocampal brain tissue and processed to purify neurons and remove tissue debris. Terminally differentiated rat hippocampal neurons recovered in vitro and displayed mature neuronal morphology. Extracellular glutamate in the culture medium promoted neuronal recovery of electrical function and activity. After recovery, essential growth factors in the culture medium caused adult neurons to reenter the cell cycle and divide multiple times. Only after reaching confluence did some neurons stop dividing. Strategies for inhibition of neuronal mitotic division were investigated, and manipulation of the cdk5 pathway was ultimately found to prevent division in vitro. Prevention of mitotic division as well as optimization of culture and maintenance parameters resulted in a neuronal culture system derived from adult rats in which the neuronal morphology, cytoskeleton and surface protein expression patterns, and electrical activity closely mirrored mature, terminally differentiated adult neurons in vivo. Improvements were also made to the growth surfaces on which neurons attached, regenerated, and survived long-term. Culture surfaces, in this case glass cover slips, were modified with the chemical substrate N-1 (3-(trimethoxysilyl) propyl)-diethylenetriamine (DETA) to create a covalently modified interface with exposed cell-adhesive triamine groups. DETA chemical surfaces were also further modified to create high-resolution patterns, useful in creating engineered two-cell networks of adult hippocampal neurons. Adult hippocampal neurons were also coupled to microelectrode array systems (MEAs) and recovered functionally, fired spontaneously, and reacted to synaptic antagonists in a manner consistent to adult neurons in vivo. Last, neurons from the brains of deceased Alzheimer's disease (AD) patients and from brain tissue excised during surgery for Parkinson's disease (PD), Essential Tremor (ET), and brain tumor were isolated and cultured, with these neurons morphological regenerating and electrically recovering in vitro.

As the world population is aging, the cases of Alzheimer’s Disease (AD) are increasing. AD is a disorder of the brain which is characterized by the aggregation of amyloid beta (Aβ) plaques. This leads to the death of numerous brain cells thus affecting the cognitive and motor functions of the individual. Till date, no cure for the disease is available. Aβ are peptides with 40/42 amino acid residues but, their exact mechanism(s) of action in AD is under debate. Having different amino acid residues makes them susceptible to form hydrogen bonds. Dendrimers with sugar units are often referred to as glycopolymers and have been shown to have potential anti-amyloidogenic activity. However, they also have drawbacks, the synthesis involves multiple tedious steps, and dendrimers themselves offer only a limited number of functional units. Pseudodendrimers are another class of branched polymers based on hyperbranched polymers. Unlike the dendrimers, they are easy to synthesize with a dense shell of functional units on the surface. One of the main goals in this dissertation is the synthesis and characterization of pseudodendrimers and dendrimers based on 2,2-bis(hydroxymethyl)-propionic acid (bis-MPA), an aliphatic polyester scaffold, as it offers biocompatibility and easy degradability. Furthermore, they are decorated with mannose units on the surface using a ‘click’ reaction forming glyco-pseudodendrimers and glyco-dendrimers. A detailed characterization of their structures and physical properties was undertaken using techniques such as size exclusion chromatography, asymmetric flow field flow fractionation (AF4), and dynamic light scattering. The second main focus of this work has been to investigate the interaction of synthesized glyco-pseudodendrimers and glyco-dendrimers with Aβ 40 peptides. For this task, five different concentrations of the synthesized glycopolymers were tested with Aβ 40 using the Thioflavin T assay. The results of the synthesized polymers which produced the best results of showing maximum anti-aggregation behavior against Aβ 40 were confirmed with circular dichroism spectroscopy. AF4 was also used to investigate Aβ 40-glycopolymer aggregates, which has never been done before and constitutes the highlight of this dissertation. Atomic force microscopy was used to image Aβ 40-glycopseudodenrimer aggregates. A basic but important step in the development of drug delivery platforms is to evaluate the toxicity of the drugs synthesized. In this work, preliminary studies of the cytotoxicity of glyco-pseudodendrimers were performed in two different cell lines. Thus, this study comprises a preliminary investigation of the anti-amyloidogenic activity of glyco-pseudodendrimers synthesized on an aliphatic polyester backbone.:Abstract List of Tables List of Figures Abbreviations 1 Introduction 1.1 Objectives of the work 1.2 Thesis overview 2 Fundamentals and Literature 2.1 Alzheimer’s Disease and its impact 2.1.1 Neurological diagnosis of AD 2.1.2 Histopathology of AD 2.1.3 Amyloid precursor protein (APP) and its role in AD 2.2. Amyloid Beta (Aβ) peptide 2.2.1 Aβ peptide 2.2.2. Location and function 2.2.3 Amyloid hypothesis 2.2.4 The mechanism of Aβ aggregation 2.2.5 Amyloid fibrils 2.2.6 Toxicity of Aβ 2.3 Research methods to study Aβ aggregates 2.3.1 Models to study the mode of action of aggregates 2.3.2 Endogenous Aβ aggregates and synthetic aggregates 2.3.3 Strategies to alter aggregation of amyloids 2.4 Treatment and therapeutics 2.4.1 Current therapeutics 2.4.2 Current therapeutic research 2.4.2.1 Reduction of Aβ production 2.4.2.2 Reduction of Aβ plaque accumulation 2.4.2.2.1 Anti-amyloid aggregation agents 2.4.2.2.2 Metals 2.4.2.2.3 Immunotherapy 2.4.2.2.4 Dendrimers as potential anti-amyloidogenic agent 2.6 Dendrimers 2.6.1 Definition 2.6.2 Structure Table of Contents 2.6.3 Synthesis 2.6.4 Properties 2.7 Pseudodendrimers - a sub-class of hyperbranched polymer 2.7.1 Definition 2.7.2 Structure 2.7.3 Synthesis 3 Analytical Techniques 3.1 Size Exclusion Chromatography Coupled to Light Scattering (SEC-MALS) 3.2 Asymmetric Flow Field Flow Fractionation (AF4) 3.3 Dynamic Light Scattering 3.4 Molecular Dynamics Simulation 3.5 Nuclear Magnetic Resonance Spectroscopy 3.6 Thioflavin T fluorescence 3.6.1 Kinetic analysis 3.7 Circular Dichroism Spectroscopy 3.8 Atomic Force Microscopy 3.9 Cytotoxic assay 3.9.1 MTT assay 3.9.2 Determining the level of reactive oxygen species 3.9.3 Changes in mitochondrial transmembrane potential 3.9.4 Flow cytometric detection of phosphatidyl serine exposure 4 Experimental Details and Methodology 4.1 Details of chemicals/components used 4.1.1 Other materials 4.1.2 Peptide preparation 4.1.3 Buffer preparation 4.1.4 Fibril growth conditions 4.2 Synthesis and characterization of polymers 4.2.1 Synthesis and characterization of pseudodendrimers and dendrimers 4.2.1.1 Synthesis of hyperbranched polymer (1) 4.2.1.2 Synthesis of protected monomer 4.2.1.2.1 bis-MPA acetonide (2) 4.2.1.2.2 bis-MPA-acetonide anhydride (3) 4.2.1.3 Synthesis of protected pseudodendrimers (4, 6 and 8) and protected dendrimers (10, 12, and 14) 4.2.1.4 Deprotection of pseudodendrimers (5,7, and 9) and dendrimers (11,13 and 15) 4.2.2 Synthesis of glyco-pseudodendrimers and glyco-dendrimers 4.2.2.1 Pentynoic anhydride (16) 4.2.2.2 Synthesis of pentinate modified pseudodendrimers (17, 18 and 19) and dendrimers (20, 21 and 22) 4.2.2.3 3-Azido-1-propanol (23) 4.2.2.4 Mannose propyl azide tetraacetate (24) Table of Contents 4.2.2.5 Mannosepropylazide (25) 4.2.2.6 Glyco-pseudodendrimers (Gl-P) (26, 27 and 28) and glyco- dendrimers (Gl-D) (29, 30 and 31) 4.3 Analytical techniques and their general details 4.3.1 SEC-MALS - Instrumentation, software and analysis 4.3.2 AF4 - Instrumentation, software and analysis 4.3.2.1 Sample preparation 4.3.2.2 Method development for analysis of Gl-P and Gl-D 4.3.2.3 Method development for analysis of Aβ 40 and its interaction with Gl-P and Gl-D 4.3.3 Batch DLS - Instrumentation, software and analysis 4.3.3.1 Sample preparation 4.3.4 Theoretical calculations and molecular dynamics simulations 4.3.4.1 Ab-initio calculations 4.3.4.2 Modelling of the polymer structures 4.3.4.2.1 Pseudodendrimers 4.3.4.2.2 Dendrimers 4.3.4.2.3 Modification of the polymers with special end groups 4.3.4.2.4 Preparing of the THF solvent box 4.3.4.2.5 Solvation of the polymer structures 4.3.4.3 Molecular dynamics simulations 4.3.4.3.1 Evaluation of the simulation trajectories 4.4 Investigation of interaction of Gl-P and Gl-D with amyloid beta (Aβ 40) 4.4.1 ThT Assay - Instrumentation and software 4.4.1.1 Sample preparation 4.4.1.2 Kinetics based on ThT assay- software and data analysis 4.4.2 CD spectroscopy - Instrumentation and software 4.4.2.1 Sample preparation 4.4.3 AFM - Instrumentation and software 4.4.3.1 Substrate and sample preparation 4.4.3.2 Height determination and counting procedures 4.4.3.3 Topography and diameter 4.5 Cytotoxicity 4.5.1 Zeta potential 4.5.2 Cell culturing 4.5.3 Sample preparation 4.5.4 MTT assay 4.5.5 Changes in mitochondrial transmembrane potential (JC-1 method) 4.5.6 Flow cytometric detection of phosphatidyl serine exposure (Annexin V and PI method) 5 Results and Discussion 5.1 Synthesis and characterization of glyco-pseudodendrimers and glyco- dendrimers 5.1.1 Synthesis and characterization of hyperbranched polyester Table of Contents 5.1.2 Synthesis and characterization of pseudodendrimers P-G1-OH, P-G2-OH and P-G3-OH 5.1.3 Synthesis and characterization of dendrimers D-G4-OH, D-G5-OH and D-G6-OH 5.1.4 Synthesis and characterization of Gl-P and Gl-D 5.1.4.1 Molecular size determination of Gl-P and Gl-D using SEC 5.1.4.2 Particle size determination using batch DLS 5.1.4.3 Apparent densities 5.1.4.4 Molecular size determination of Gl-P and Gl-D using AF4 ..... 5.1.5 Molecular dynamics simulation 5.2 Investigation of interaction of Gl-P and Gl-D with amyloid beta (Aβ 40) ...... 5.2.1 ThT Assay 5.2.1.1 Kinetics based on ThT assay 5.2.2 CD spectroscopy 5.2.3 Time dependent AF4 5.3.2.1 Separation of Aβ 40 by AF4 5.3.2.2 Aβ 40 amyloid aggregation in the presence of Gl-P and Gl-D 5.2.4 AFM 5.2.4.1 Height 5.2.4.2 Topography and diameter 5.2.4.3 Length 5.2.4.4 Morphology 5.2.5 Cytotoxicity 5.2.5.1 MTT assay 5.2.5.2 Changes in mitochondrial transmembrane potential 5.2.5.3 Flow cytometric detection of phosphatidyl serine exposure 6 Conclusions and Outlook 7 Bibliography Appendix Acknowledgements

Alzheimer’s Disease (AD) is a neurodegenerative disease marked by progressive neuronal cell death, leading to dementia. AD is the most common disease that results in dementia and largely affects the elderly, with five million people in the United States diagnosed with the disease as of 2015 and approximately 35 million people worldwide. Diseases resulting in dementia cost the US healthcare system an estimated $172 billion in 2010 and that cost is expected to increase as the population ages and as diagnostic techniques improve so that more people are treated (Holtzman, 2011). The disease was first reported by psychiatrist Alois Alzheimer at the onset of the 20th century, when one of his patients “suffered memory loss, disorientation, hallucinations and delusions and died at the age of 55,” then was found to have severe brain atrophy post-mortem (Cipriani, Dolciotti, Picchi, & Bonuccelli, 2011). There are palliative treatments available that marginally slow disease progression but there is currently no cure for the disease (Awasthi, Singh, Pandey, & Dwivedi, 2016). More research is needed to develop effective therapeutic strategies to combat the disease. Currently, AD cytotoxicity is believed to be caused by increased amyloid β (Aβ) peptide plaque deposition in the brain, as described by the amyloid cascade hypothesis (Barage & Sonawane, 2015). The current understanding is that oligomers of Aβ peptide lead to neuronal death through multiple mechanisms, most notably hyper-phosphorylation of the tau protein. Having a better understanding of the structural changes in the fibrillization process of Aβ will provide a broader insight into mechanisms of cell death and open new possibilities for pharmacological treatments, which is what this research intends to provide.

Protein misfolding and the accumulation of insoluble aggregates is a hallmark of several neurodegenerative disorders, including Alzheimer’s (AD) and Parkinson’s disease (PD). In AD and PD patients, extracellular protein deposits consisting of amyloid beta (Aβ) and intraneuronal inclusions composed of alpha synuclein (αS) are observed, respectively. Notably, the spatiotemporal patterning of soluble protein oligomers of αS and Aβ closely follow disease progression, giving support to an emerging role of soluble oligomers in PD and AD pathogenesis. However, the structural features underlying the toxicity of Aβ and αS oligomers remain elusive. This doctoral dissertation aims at elucidating the structural determinants of oligomer toxicity by focusing on the development and application of multidisciplinary approaches based primarily on solution NMR in combination with electron microscopy, multi-angle light scattering, fluorescence microscopy, wide-angle x-ray diffraction and cellular biophysics. Using this interdisciplinary approach, in chapters 2 and 3, we identify at atomic resolution the key structural elements that facilitate the colocalization, interaction and subsequent insertion of soluble Aβ oligomers into membranes, which ultimately result in the loss of membrane integrity. Notably, we show that small molecules, such as green tea catechins, remodel these structural features and effectively perturb the interactions with membranes. In chapter 4, we extend these analyses to αS and identify how the chaperone, Human Serum Albumin (HSA), remodels toxic αS oligomers into non-toxic species and breaks the catalytic cycle that generates new toxic oligomers. Lastly, in chapter 5, we describe a novel solution NMR approach to map at atomic resolution the sites of early self-association, with minimal bias from monomer dynamics, an effect that frequently dominates residue-dependent variations in solution NMR measurements. Overall, given that Aβ and αS are archetypical amyloidogenic proteins, we anticipate that the structure – toxicity relationships established herein, and the related experimental approaches may be transferrable to other amyloidogenic systems. / Dissertation / Doctor of Philosophy (PhD)

Neurotoxic aggregates of amyloid beta (Aβ) peptide contribute to the etiology of Alzheimer's disease (AD). Aβ1-42 forms oligomeric structures that undergo further aggregation into protofibrils and fibrils. Oligomeric Aβ1-42 is more toxic than monomers or mature fibrils. In this work, we used two distinct approaches to inhibit Aβ1-42 oligomerization and toxicity. First, seven distinct but overlapping Aβ fragments were used to identify their individual aggregation propensities and their effects on Aβ1-42 oligomerization and cytotoxicity. Studies on suppression of Aβ1-42 cytotoxicity by peptides, including those derived from Aβ1-42, have been conducted before, but peptides encompassing the whole Aβ1-42 sequence have not been systematically analyzed. Aβ1-42 was allowed to aggregate and form oligomeric assemblies in aqueous buffer for 4 h in the absence or presence of 2-fold molar excess of an Aβ fragment. Cytotoxicity analysis then recorded the impact of each fragment on Aβ1-42 cytotoxicity as well as the toxicity of the fragments themselves. An enzyme-linked immunosorbent assay that detects oligomeric Aβwas used to determine the effect of each fragment on Aβ1-42 oligomerization after 4 h of aggregation. Four fragments of Aβ1-42 inhibited the toxicity of oligomeric Aβ1-42 to various degrees, while two others conferred no cellular protection against Aβ1-42 toxicity. Interestingly, one fragment enhanced Aβ1-42 toxicity after 4 h of aggregation. Three of the four fragments that blocked Aβ1-42 toxicity partially disrupted oligomer formation, showing correlation between the inhibition of Aβ1-42 aggregation and the inhibition of cellular toxicity. Second, we examined whether protein disulfide isomerase (PDI), a chaperone mainly found in the endoplasmic reticulum, could reverse the oligomeric state of aggregated Aβ1-42 and thus its toxicity. Previous work has demonstrated that PDI inhibits Aβ1-42 aggregation at sub-stoichiometric concentrations. To assess PDI's effect on Aβ1-42 toxicity, Aβ1-42 was allowed to aggregate for 2 h before the addition of PDI at a 1:10 molar ratio of PDI to Aβ1-42 and then allowed to aggregate for another 2 h. MTS cytotoxicity assays using PC-12 cells showed that adding PDI 2 h after the start of aggregation improves cell survival. Through a differential centrifugation assay followed by Western blot, we qualitatively illustrated that PDI can reverse a 2 h aggregate of Aβ1-42 to the monomeric state. Overall, in this project we have learned that inhibiting the oligomeric assembly of Aβ1-42 directly decreases the effect of Aβ1-42 toxicity. Inhibition of Aβ1-42 toxicity was seen with both fragments derived from Aβ1-42 and PDI, shedding light into two novel approaches as possible therapeutics for AD.

Solid State Nuclear Magnetic Resonance (ssNMR) spectroscopy can be a powerful tool for investigating the atomic-level structures and dynamics of biological macromolecules, including proteins. In this dissertation, I present an ssNMR study of three diverse proteins, revealing insights into their respective secondary structures, conformational variations, and intermolecular interactions. Additionally, I introduce novel computational methods to facilitate the assignment of chemical shifts of ssNMR spectra. The first of the proteins is the capsid protein of the Rous Sarcoma Virus. In previous research, the structure of the hexameric lattice of the in-vitro tubular assembly of the capsid protein was determined. In this study, chemical shift assignments were completed and the structure of the T=1 capsid assembly (comprising entirely of a pentameric lattice) of the I190V mutant variant of the capsid was determined, providing the missing component of the in-vivo capsid structure. The second protein studied was amyloid-beta 42, a particularly cytotoxic variant of the main component of amyloid plaques in the brains of Alzheimer's disease patients. Chemical shift assignments were made on ssNMR data from samples aggregated in cholesterol-containing phosphatidylcholine (POPC) lipid vesicles, and secondary structure and molecular distance information was obtained. Lastly, preliminary chemical shift assignments, statistics, and structural analysis was done on the polypeptide Ref-2Cx4, derived from the conserved domain of the Hawaiian bobtail squid reflectin protein. The reflectin protein, used in the squid's camouflage mechanism, possesses optically reflective and proton-conductive properties. The final part of the dissertation addresses a major bottleneck in ssNMR studies—the assignment of chemical shifts. I introduce Visual Assist, a suite of computational tools designed to streamline and expedite the assignment process. The developed computational methods are validated on the diverse set of proteins above, demonstrating their general applicability and efficiency.