Physics
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Item Simulating membrane-bound cytoskeletal dynamics(2023) Ni, Haoran; Papoian, Garegin A.; Biophysics (BIPH); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The cell membrane defines the shape of the cell and plays an indispensable role in bridging intra- and extra-cellular environments. The membrane, consisting of a lipid bilayer and various attaching proteins, mechanochemically interacts with the active cytoskeletal network that dynamically self-organizes, playing a vital role in cellular biomechanics and mechanosensing. Comprehensive simulations of membrane-cytoskeleton dynamics can bring insight in understanding how the cell mechanochemically responds to external signals, but a computational model that captures the complex cytoskeleton-membrane with both refined details and computational efficiency is lacking. To address this, we introduce in this thesis a triangulated membrane model and incorporate it with the active biological matter simulation platform MEDYAN ("Mechanochemical Dynamics of Active Networks"). This model accurately captures the membrane physical properties, showing how the membrane rigidity, the structure of actin networks and local chemical environments regulate the membrane deformations. Then, we present a new method for simulating membrane proteins, using stochastic reaction-diffusion sampling on unstructured membrane meshes. By incorporating a surface potential energy field into the reaction-diffusion sampling, we demonstrate rich membrane protein collective behaviors such as confined diffusion, liquid-liquid phase separation and membrane curvature sensing. Finally, in order to capture stretching, bending, shearing and twisting of actin filaments which are not all available with traditional actomyosin simulations, we introduce new finite-radius filament models based off Cosserat theory of elastic rods, with efficient implementation using finite-dimensional configurational spaces. Using the new filament models, we show that the filaments' torsional compliance can induce chiral symmetry breaking in a cross-linked actin bundle. All the new models are implemented in the MEDYAN platform, shedding light on whole cell simulations, paving way for a better understanding of the membrane-cytoskeleton system and its role in cellular dynamics.Item DECIPHERING HOW EGFR-GRB2-SOS1 COMPLEX REGULATES KRAS4B ACTIVATION AND LEADS TO HIPPO SIGNALING THROUGH RASSF5(2021) Liao, Tsung-Jen; Fushman, David; Nussinov, Ruth; Biophysics (BIPH); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Ras is a small GTPase, which regulates cell proliferation and apoptosis. Its bifunctional switch is controlled by the nucleotide state: GTP-bound – switch on; GDP-bound – switch off. Among the three Ras isoforms, HRas, NRas, and KRas (with two splice variants of KRas4A and KRas4B), KRas4B is highly oncogenic, the most frequently mutated in lung, colorectal, and pancreatic cancers. However, Ras was thought to be “undruggable” due to the lack of effective pharmacological inhibitors over the past three decades. Most of the current focus has been directed at inhibiting the activation of Ras signaling. Ras proteins transduce signals between cell surface receptors and multiple intracellular signaling cascades. In response to epidermal growth factor receptor (EGFR) activation, growth factor receptor bound protein 2 (Grb2) establishes the connection between EGFR and Ras-specific nucleotide exchange factor (RasGEF), son of sevenless 1 (SOS1). SOS1 activates Ras by exchanging GDP to GTP. In addition to Ras major effectors and pathways, e.g. MAPK and PI3Kα/Akt, which are cell growth related, GTP-bound Ras associating with RASSF5 activates the Hippo pathway, which acts to suppress cell proliferation. In this serial study, we use NMR measurement and molecular dynamics (MD) simulation to investigate the interactions of Grb2–SOS1, SOS1–KRas4B, KRas4B–RASSF5, and the EGFR effects on the binding of Grb2–SOS1. Our findings successfully uncovered (1) a novel Grb2 binding site PKLPPKTYKREH on SOS1 and the most probable binding mode of Grb2–SOS1, (2) strong SOS1 peptide binders induce a closed conformation of Grb2 nSH3 domain but unchanged conformation of Grb2 cSH3 domain, (3) full length Grb2 performs high affinities for one-site SOS1 peptides, and the EGFR segment may facilitate the binding of Grb2 to the particular two-site SOS1 peptide, (4) KRas4B binding to SOS1 allosteric site induces the conformational changes of catalytic site and accelerate the KRas4B activation cycle, (5) the hypothesized mechanism that RASSF5 is a tumor suppressor in vivo but opposite in vitro, and (6) the dynamic mechanism of RASSF5 auto-inhibition. Our effort in elucidating the mechanism of Ras and Ras effectors results in 8 publications and offers a new venues for future therapeutic strategies.Item LIPID FORCE FIELD PARAMETERIZATION FOR IMPROVED MODELING OF ION-LIPID INTERACTIONS AND ETHER LIPIDS, AND EVALUATION OF THE EFFECTS OF LONG-RANGE LENNARD-JONES INTERACTIONS ON ALKANES(2019) Leonard, Alison N; Klauda, Jeffery B; Biophysics (BIPH); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Chemical specificity of lipid models used in molecular dynamics simulations is essential to accurately represent the complexity and diversity of biological membranes. This dissertation discusses contributions to the CHARMM36 (C36) family of lipid force fields, including a revised model for the glycerol-ether linkage found in plasmalogens and archaeal membranes; interaction parameters between ions and lipid oxygens; and evaluation of the effects of long-range Lennard-Jones parameters on alkanes. Long-range Lennard-Jones interactions have a significant impact on structural and thermodynamic properties of systems with nonpolar regions such as membranes. Effects of these interactions on properties of alkanes are investigated. Implementation of the Lennard-Jones particle-mesh Ewald (LJ-PME) method with the C36 additive and Drude polarizable force fields improves agreement with experiment for thermodynamic and kinetic properties of alkanes, with Drude outperforming the additive FF for nearly all quantities. Trends in the temperature dependence of the density and isothermal compressibility are also improved. Phospholipids containing an ether linkage between the glycerol backbone and hydrophobic tails are prevalent in human red blood cells and nerve tissue. Ab initio results are used to revise linear ether parameters and develop new parameters for the glycerol-ether linkage in lipids. The new force field, called C36e, more accurately represents the dihedral potential energy landscape and improves solution properties of linear ethers. C36e allows more water to penetrate an ether-linked lipid bilayer, increasing the surface area per lipid compared to simulations carried out with the original C36 parameters and improving structural properties. In addition to modulating membrane structure, lipid-ion interactions influence protein-ligand binding and conformations of membrane-bound proteins. Interaction parameters are introduced describing Be2+ affinity for binding sites on lipids. Experimental binding affinities reveal that Be2+ strongly binds to phosphoryl groups. Revised interaction parameters reproduce binding affinities in solution simulations. In a separate effort, experimental results for the radius of gyration (R_g) of polyethylene glycol (PEG) in various concentrations of KCl reveal that, while C36e parameters reproduce experimental R_g of PEG in the absence of KCl, adding salt results in underestimation of〖 R〗_g. It is found that the water shell around PEG affects R_g calculated from neutron scattering experiments, and K+-PEG interactions increase the gauche character of PEG.Item UNCOVERING THE BIOPHYSICAL MECHANISMS OF HISTONE COMPLEX ASSEMBLY(2018) Zhao, Haiqing; Papoian, Garegin A.; Biophysics (BIPH); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)At the most basic level, inheritance in living beings occurs by passing the genomic information such as the DNA sequences from the parent generation to the offspring generation. Hence, it is a fundamental goal for every generation to efficiently express the genomic information and safely pass it on to the next generation. In human and other eukaryotic species, this mission is mediated via chromatin, a macromolecule with intricate hierarchical structure. The fundamental unit of chromatin is called a nucleosome, a complex of histone proteins wrapped around with DNA. To carry out diverse biological functions such as transcription and DNA replication, the DNA-protein complex must dynamically transition between more compact, closed states and more accessible, open ones. To fully understand the chromatin structure and dynamics, it is essential to comprehend the basic structural unit of chromatin, nucleosome. In this dissertation, I present my doctoral research in the exploration of the nucleosome dynamics problem, focusing on the assembly process of histone proteins. From histone monomer to dimer, then to tetramer, octamer, and nucleosome, I used different computational modeling theories and techniques, together with different experimental collaborations, to investigate the overall thermodynamics and specific mechanistic details of nucleosome dynamics at different levels. My work has shed light on the fundamental principles governing the histone protein folding and histone complex assembly, in particular, highlighting similarities and differences between the canonical and variant CENP-A histones.Item Structural biology of GroEL assisted protein folding(2014) Fei, Xue; Lorimer, George H; Biophysics (BIPH); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)GroEL/ES is the classical example of molecular chaperone that assists the re-folding of many misfolded proteins (SP). Recent kinetic analyses revealed a new paradigm of how GroEL/ES uses ATP to assist protein folding. Following these pioneering biochemical studies, I address two fundamental questions related to GroEL-assisted protein folding using structural biology methods. First, how does GroEL capture SP and how does SP change the kinetics of ADP release? Second, how does GroEL/ES encapsulate SP and control the duration of SP encapsulation? Chapter 1 summarizes the ATPase cycle of GroEL revealed by systematic biochemical studies, and identifies knowledge gaps in the GroEL-assisted protein folding. Chapter 2 describes general methods of protein purification and computational approaches, used to analyze conformational differences between two GroEL structures. Chapter 3 and 4 are focused on the capturing of substrate protein by GroEL. Crystal structures of GroELD83AR197A-ADP14 and GroELD83AR197A show for the first time, ADP binding breaks seven-fold symmetry in the apical and intermediate domains. Such asymmetry provides the structural basis for GroEL to capture heterogeneous SPs and for SP to regulate the release of ADP. In chapter 5, I described how GroEL/ES encapsulates substrate protein. Two crystal structures of the predominate SP encapsulation complexes: GroEL-GroES2 "football" complex were reported. One of the complexes is SP free and the other encapsulates two Rubisco molecules simultaneously. From the conformational rearrangement of the inter-ring interface, we proposed "football" complex transmits ATP asymmetry between the rings through an electrostatic interaction between K105 and A109. Chapter 6 summarized the new knowledge gained by determining these four crystal structures. This chapter ends with a discussion on how chaperonin machine like GroEL promotes the correct folding of various proteins.Item Methods of single-molecule energy landscape reconstruction with optical traps(2012) de Messieres, Michel Escande; La Porta, Arthur; Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Optical traps facilitate measurement of force and position as single molecules of DNA, RNA, or protein are unfolded and refolded. The effective energy landscape of a biomolecule can be reconstructed from the force and position data, providing insight into its structure and regulatory functions. We have developed new experimental and analytical methods to reconstruct energy landscapes by taking advantage of the harmonic constraint of an optical trap. We demonstrate the effectiveness of these methods using a model DNA hairpin and then apply these methods to study problems of practical biophysical interest. CCR5 mRNA has been demonstrated to stimulate -1 programmed ribosomal frameshifting and we measure its structural properties. We measure the binding energy of a GA/AG tandem mismatch, one of many mismatches with unusual properties. We use our single-molecule methods to reproduce bulk measurements of the nearest-neighbor DNA base-pair free energy parameters and we consider possible refinements to the model. We also study an alternative method of measuring energy landscapes, Dynamic Force Spectroscopy (DFS), and conduct experiments on DNA quadruplexes to demonstrate the effectiveness of DFS with optical traps. Finally, we develop theory to elucidate the role of noise in optical trap measurements of energy landscapes.Item USING SINGLE MOLECULE TECHNIQUES TO DETERMINE THE MECHANISM OF DNA TOPOLOGY SIMPLIFICATION BY TYPE IIA TOPOISOMERASES(2011) Hardin, Ashley Harris; Thirumalai, Devarajan; Neuman, Keir C; Chemical Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Type IIA topoisomerases are essential, universally conserved proteins that modify DNA topology by passing one segment of duplex DNA (the transfer, or T-segment) through a transient double strand break in a second segment of DNA (the gate, or G-segment) in an ATP-dependent reaction. Type IIA topoisomerases decatenate, unknot, and relax supercoiling in DNA to levels below equilibrium, resulting in global topology simplification. The mechanism underlying non-equilibrium topology simplification remains speculative, though several plausible models have been proposed. This thesis tests two of these, the bend angle and kinetic proofreading models, using single-molecule techniques. The bend angle model postulates that non-equilibrium topology simplification scales with the bend angle imposed on the G-segment DNA by a type IIA topoisomerase. To test this model, we used atomic force microscopy and single molecule Förster resonance energy transfer to measure the extent of bending imposed on DNA by three type IIA topoisomerases that span the range of topology simplification activity. We found that all proteins bent DNA, but the imposed bends are similar and cannot account for the differences among the enzymes. These data do not support the bend angle model and suggest that DNA bending is not the sole determinant of non-equilibrium topology simplification. Based on the assumption that the rates of collision between DNA segments is higher in knotted, linked, and supercoiled DNA than in topologically free or relaxed DNA, the kinetic proofreading model proposes that two successive binding events between a G-segment bound topoisomerase and a putative T-segment are required to initiate strand passage. As a result of the two step process, the overall rate of strand passage should scale with the square of the collision probability of two DNA segments. To test this model, we used magnetic tweezers to manipulate a paramagnetic bead tethered to the surface by two DNA molecules. By rotating the bead, we varied the proximity, and thus collision rate, of the two molecules to determine the relationship between collision probability and rate of strand passage. Our data indicate that the strand passage rate scales linearly with the collision probability, which is inconsistent with the kinetic proofreading model.Item The Yin and Yang of Amyloids: Insights from alpha-Synuclein and the Pmel17 Repeat Domain(2011) Pfefferkorn, Candace Marie; Lee, Jennifer C; Thirumalai, Devarajan; Chemical Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)While amyloidogenic proteins are commonly associated with human diseases such as Alzheimer's and Parkinson's disease, it is intriguing that amyloid fibrils also are utilized for essential biological processes. A key question then is why many amyloids are harmful whereas some serve essential functional roles. To begin to address this question, the environmental factors regulating the conformational changes in the Parkinson's disease-related protein, alpha-synuclein (alpha-syn), and a critical polypeptide fragment, the repeat domain (RPT) of Pmel17, the protein required for melanin formation are examined. The role of membranes in modulating alpha-syn conformation is investigated because membranes are ubiquitous in vivo and affect alpha-syn aggregation in vitro. Using single tryptophan-containing alpha-syn variants (F4W, Y39W, F94W, Y125W) as site-specific fluorescent probes, distinct phospholipid vesicle and SDS micelle interactions have been identified and membrane binding equilibria measured. The role of specific N-terminal residues in membrane binding also has been assessed. Specifically, environments of the highly sensitive Trp4 probe in alpha-syn polypeptide fragments (residues: 1 - 4, 1 - 6, 1 - 10, and 1 - 15) upon membrane binding were characterized using steady-state fluorescence and time-resolved anisotropy. The penetration depths of alpha-syn and N-terminal peptides into the lipid bilayer also were determined using brominated lipids as heavy-atom quenchers. To simultaneously monitor alpha-syn and bilayer structure, neutron reflectometry (NR) and a sparsely-tethered bilayer lipid membrane (stBLM) were employed. Using NR and an stBLM, alpha-syn concentration dependent effects on both protein structure and membrane properties were measured. To begin to address biophysical and biochemical differences between pathological and functional amyloid, a systematic investigation of the effects of solution pH (4→7) on RPT aggregation was performed since melanosomes, acidic organelles where Pmel17 fibrils are formed, change pH during maturation. Using intrinsic tryptophan fluorescence, circular dichroism spectroscopy, and transmission electron microscopy, local, secondary, and fibril morphological structure were monitored, respectively. Notably, RPT fibril morphology can be transformed directly by changing solution pH, suggesting that pH is a natural regulatory mechanism for Pmel17 amyloid formation and its subsequent dissolution in vivo.