Physics Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2800
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Item Understanding Allosteric Communication in Biological Systems using Molecular Dynamics Simulations(2024) Samanta, Riya; Matysiak, Silvina; Biophysics (BIPH); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Allostery is critical to survival in living organisms due to its biological relevance in signal transduction, metabolism, and drug discovery. However, the molecular details of this phenomenon remain unclear. In this thesis, I present my work on two allosteric protein systems, each representative of structure-based (E. coli Biotin Protein Ligase) and dynamics-based (B. taurus S100B) allostery. I examined the structural and dynamic features of the proteins and associated variants subjected to various allosteric triggers (ligand/salt/mutations) to study how external/internal perturbations transmit across large distances using Molecular Dyanmic simulations in conjunction with the experiments carried out by our collaborators. Additionally, I carried out Network analyses on the two systems to characterize communication pathways on the protein/ protein family levels. Together, the structural and dynamic features would help us elucidate the underlying mechanism of allostery. The first chapter introduces the two systems with a brief dive into the history of allostery. In the second chapter, my work on E. coli Biotin Protein Ligase and its variants reveal one possible mechanism by which disorder-to-order transitions at the functional surfaces transmit via local changes around the critical residues in the allosteric network. The third chapter explores how the protein network reconfigures to adopt a new allosteric function by studying the allosteric and non-allosteric Biotin Protein Ligases. The fourth chapter elucidates the structural and dynamical markers in bovine S100B, which help to relay information about an allosteric signal by varying two allosteric triggers - ionic strength and target peptide. The final chapter sums up my conclusions, where I propose additional experiments and computational analyses that could be carried out to further our understanding of allostery.Item MULTISCALE MEASUREMENTS OF ELECTRICAL & MECHANICAL CELLULAR DYNAMICS(2023) Alvarez, Phillip; Losert, Wolfgang; Biophysics (BIPH); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)This dissertation focuses on the study and measurement of coupled electrical and mechanical responses in mammalian cells, tissues, and organs. Cellular biophysics often studies forces and their impact on biochemical pathways. These forces can be electrical, resulting in neuronal action potentials or cardiac cell contractions, or mechanical, driving e.g., a cell’s ability to recognize physical probing or surface texture. These forces and their responses, though, are frequently coupled through interlinked cellular mechanisms which result in emergent responses that take both electrical and mechanical signals into account. One challenge in capturing these emergent responses is that they occur on multiple scales, from the intracellular scale to the organ scale, limiting the ability of commercial microscopes to image these responses simultaneously. In this work I use surface texture, optical imaging, and multiscale-capable image analysis algorithms across these scales to elicit and measure electrical and mechanical responses. To image emergent responses from electrical and mechanical coupling, I developed two custom microscopes that can image at multiple length scales and timescales simultaneously. The Multiscale Microscope can capture slow intracellular mechanical dynamics concurrently with fast tissue scale electrical dynamics, while the BEAMM microscope links fast tissue scale electrical dynamics with both intracellular mechanical dynamics and slower organ-scale mechanical and electrical responses. Finally, I describe ongoing and future studies which exploit these new capabilities for multiscale measurements of electrical and mechanical dynamics.Item PROBING BIOPHYSICAL INTERACTIONS TO UNDERSTAND VIRAL DIFFUSION AND PARTICLE FATE IN THE AIRWAY MUCOSAL BARRIER(2023) Kaler, Logan; Duncan, Gregg A; Biophysics (BIPH); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The mucus barrier in the airway is the first line of defense against inhaled particulates and pathogens. Within the mucus barrier, large, heavily glycosylated gel-forming mucin proteins form a network to trap particles for removal. Influenza A virus (IAV) must first cross the mucus barrier before reaching the underlying airway epithelial cells to cause infection. On the IAV envelope, hemagglutinin (HA) binds sialic acid on the surface of the cell to initiate viral entry. However, HA preferentially binds sialic acid attached to galactose by either an ⍺2,3 or ⍺2,6 linkage. In addition to the cell surface, sialic acid is found on mucins and is thought to act as a decoy receptor to entrap the IAV within the mucus layer. However, neuraminidase (NA) on the envelope of IAV cleaves the bond between HA and sialic acid, releasing the virus. While the mechanism of IAV infection has been characterized, the interplay between mucus biophysical properties and the binding of IAV within the mucus network prior to infection requires further investigation. The overall objective of this dissertation is to understand how IAV moves through the mucosal barrier to subsequently cause infection. We hypothesize the structural features of the mucus gel network are responsible for the changes in IAV movement, rather than the binding and unbinding of the virus. To investigate this, we first analyzed the movement of IAV in ex vivo mucus from human endotracheal tubes. In order to further analyze this movement, we developed a novel analysis to calculate the dissociation constant of IAV-mucus binding in a 3D gel network environment. Using this data, we established a pipeline for estimating the passage of particles, including IAV, through the airway mucosal barrier. A machine learning-based trajectory analysis was employed to classify individual trajectories in order to calculate the percentage of particles able to cross the mucus barrier within a physiologically relevant time frame. Lastly, we investigated the effect of sialic acid binding preference on diffusion of IAV through mucus collected from different in vitro human airway epithelial cell cultures. The combined results of these studies confirmed our hypothesis that the mucus microstructure rather than the adhesive interactions of IAV to the mucins was responsible for the differences in IAV diffusion. This work provides further insight into role of the mucosal barrier in IAV infection and identifies the mucus gel network microstructure as a target for the development of therapeutics against IAV.Item Non Traditional Solvent Effect On Protein Behavior(2022) Lee, Pei-Yin; Matysiak, Silvina; Chemical Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Protein preservation has been a long lasting research topic due to its importance in many bio-pharmaceutical applications. A ”cold chain” is a commonplace solution to protein preservation, which stores biochemical products at a refrigerated temperature. A big advantage of cold chain is that the storing process is straightforward, without many further processes before the use of stored bio-products. However, it can also experience malfunction of the cooling system and results in economic lost and health care crisis. Ionic liquids (ILs), as a type of non traditional solvents, consist only of ions and are reported to be a potential candidate to replace the use of cold chain. The advantages of ILs include low flammability, high conductivity and less toxicity compared to some organic solvents. The most interesting feature of ILs is their extremely large number of cation-anion combinations, that can be tailored for specific use according to different needs. This thesis aims to investigate specific mechanism behind how ILs modulate protein behavior, specifically, how ILs affect protein stability, activity, and aggregation. We approach the research questions through the lens of molecular dynamics (MD) simulations and complement with experimental findings. In the first part of the thesis we first investigate the effects of two imidazolium based ILs (1-ethyl-3-methylimidazolium ethylsulfate, [EMIM]+[EtSO4]− and 1-ethyl-3-methylimidazolium diethylphosphate, [EMIM]+[Et2PO4]−) on lysozyme stability and activity. We collaborate with an experiment group at the University of Massachusetts (Bermudez lab) to complement our simulation results. Both ILs are found to destabilize lysozyme stability. In addition, both the cation and anions lower the stability of lysozyme, but in a different fashion. [EMIM]+ interacts with an Arg-Trp-Arg bridge that is critical in lysozyme stability through π–π and cation–π interactions, leading to a local induced destabilization. On the other hand, both anions interact with the whole protein surface through short-range electrostatic interactions, with [Et2PO4]− having a stronger effect than [EtSO4]−. Lysozyme activity is also reduced by the presence of the two ILs, but can be recovered after rehydration. It is found that the protein-ligand complex is less stable in the presence of ILs. In addition, a dense cloud of [EMIM]+ is found in the vicinity of the lysozyme active site residues, possibly leading to a competition with the sugar ligand. A fast leaving of these [EMIM]+ is observed after rehydration, which explains the reappearance of the active site and the recover of lysozyme activity. Although classical all-atom MD simulations can provide us with a great deal of microscopic information, they are often limited by the temporal-spatial scale of the simulated systems. For example, systems with high viscosity solvents or systems involving large number of atoms will be difficult to reach convergence for all-atom MD. In this case, coarse grained (CG) MD can come into play to achieve the desired time- and length- scales. The faster sampling obtained from CG MD is achieved by reducing the degree of freedom of the system and by removing local energetic barriers. In CG MD, similar atoms are grouped to functional groups and thus the free energy landscape is smoothen. We develop a novel CG MD named ”Protein Model with Polarizability and Transferability (ProMPT)”. The novelty of this model is the inclusion of the charged dummies that can result in change of dipoles. These dipoles can reflect the change of environments and thus allow the model to respond to different environmental stimulus. We validate ProMPT with several benchmark proteins: Trp-cage, Trpzip4, villin, ww-domain, and β-α-β. ProMPT is able to simulate folding-unfolding and secondary structure transformation with minimal constraints, which is not feasible with previous CG models. In addition, ProMPT can also reproduce the experimental results for the dimerization of glycophorin A (GpA) with different point mutations. Here we demonstrate the ability of the model to capture the change of conformational space caused by point mutation. In the last part of this thesis, we combine ProMPT and an in-house CG IL model to study the effects of [TEA]+[Ms]− on amyloid beta 16-22 (Aβ16−22) aggregation. Aβ16−22 is the hydrophobic core region and is the smallest fragment of Aβ that can fibrilize. Aβ has been extensively linked to the pathogenesis of the Alzheimer’s disease. [TEA]+[Ms]− is reported to suppress the formation of β-sheets and induce helices at high concentration. From our results, both β-sheet content and the aggregate size decrease with the increase of IL concentration, which are in agreement with experiments. Aggregates can form in both water and IL, but with different morphologies. In water, a nice hydrophobic core involving Phe-Phe interactions can form as well as intact β-sheet contacts. In addition, a cross β-sandwich structure is also observed, as seen from previous literature. However, the same hydrophobic core can not persist in the presence of IL. Aggregate structures in IL are not stable over time due to the [TEA]+-Phe interaction. Helicity is also computed for Aβ16−22 in water and in IL at different concentrations and a positive correlation is found. The increase in helicity at high [TEA]+[Ms]− concentration can be explained by the reduction of the inter-peptide contacts, which then increases the opportunity for the peptides to form helical structures. Single peptide studies also reveal that [TEA]+[Ms]− increases the helicity, possibly through cation-induced dipole enhancement. In this thesis, a series of detailed investigations on the effects of ILs on protein behavior is performed. Specific interactions between IL functional groups and protein local/global structures are examined. The mechanisms we studied here will help constructing a holistic view for the design of IL-protein pair applications. The construction of the new CG protein/IL model provides another tool for the scientific community to study secondary structure transformation, folding- unfolding, and other biochemical processes that are sensitive to the environment with CG MD.