Chemistry & Biochemistry
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Item ESOTAXIS: IDENTIFYING THE FACTORS THAT INFLUENCE NANOTOPOGRAPHIC GUIDANCE OF THE DYNAMICS AND ORGANIZATION OF THE ACTIN CYTOSKELETON AND OTHER MOLECULES INVOLVED IN DIRECTED CELL MIGRATION(2024) Hourwitz, Matt; Fourkas, John T.; Losert, Wolfgang; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Directed migration is a crucial capability of cells in developmental and immunological processes. Defects in cell migration can lead to negative health outcomes. Cell motion depends on the organization and dynamics of internal components, especially the actin cytoskeleton, and the extracellular environment. Microscale and nanoscale topographical cues, with at least one dimension that is much smaller than most cells, can bias cell motion over long distances, due to the guidance of the organization and dynamics of the cytoskeleton and other molecules and assemblies within the cell. In this work, I describe a technique to reproduce patterned nanotopographic substrates for use in the study of esotaxis, the guided organization and dynamics of the actin cytoskeleton and other cellular components in response to nanotopographic cues. The guidance of actin drives directed cell motion along a pattern with dimensions much smaller than the cell. The dimensions of the nanotopography determine the extent to which cellular components are guided. Differences in the physical properties of the plasma membrane and the actin cytoskeleton among cell lines will influence the extent of guidance by nanotopography. Asymmetric patterns can accentuate the distinctions in esotactic responses among cell lines and drive contact guidance in different directions. The cytoskeletal response to nanotopography is a local phenomenon. A cell in contact with multiple nanotopographic cues simultaneously will show distinct organization of actin in the different regions of the cell. The importance of local actin dynamics requires an analysis method, optical flow, that can identify and track the distinct cytoskeletal motions in different parts of the cell. The formation of adhesions attached to the extracellular matrix is a characteristic of the migratory behavior of many types of cells and these adhesions are credited with allowing the cell to sense and interact with the underlying substrate. Actin can sense nanotopographic cues without the widespread availability of adhesive ligands. Although adhesion to the substrate strongly increases the extent of cell spreading and migration on nanoridges, epithelial cells can align with and migrate along nanotopography even with a dearth of adhesive cues. Therefore, actin is a supreme sensor of nanotopography that can drive directed cell migration.Item UNRAVELING THE ROLE OF LASSA VIRUS TRANSMEMBRANE DOMAIN IN VIRAL FUSION MECHANISM(2024) Keating, Patrick Marcellus; Lee, Jinwoo; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Lassa virus (LASV) is the most prevalent member of the arenavirus family and the causative agent of Lassa fever, a viral hemorrhagic fever. Although there are annual outbreaks in West Africa and recently isolated cases worldwide, no current therapeutics or vaccines pose LASV as a significant global public health threat. One of the key steps in LASV infection is the delivery of its genetic material by fusing its viral membrane with the host cell membrane. This process is facilitated by significant conformational changes within glycoprotein 2 (GP2), yielding distinct prefusion and postfusion structural states. However, structural information is missing to understand the changes that occur in the transmembrane domain (TM) during the fusion process. Investigating how the TM participates in membrane fusion will provide new insights into the LASV fusion mechanism and uncover a new therapeutic target sight to combat the dangerous infection. Here, we describe our protocols for expressing and purifying the isolated TM and our GP2 constructs which we use to probe the relationship between the structure of the TM and its influence on the function of GP2.We express TM as a fusion protein with a Hisx9 tag and a TrpLE tag using E. coli bacterial cells. We purify the TM using Nickel affinity chromatography and enzymatic cleavage to remove the tags. Since the TM is prone to aggregation, we must use a strong denaturant, trichloroacetic acid (TCA), to remove the TM from the resin. The isolated TM is then buffer exchanged to a detergent solution for structural studies. Using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy, our structural studies revealed a pH-dependent structural change resulting in an N-terminal extension of the alpha helix in the postfusion state. To test the importance of this structural change, we used the GP2 construct to perform a modified lipid mixing fusion assay. Our results from the fusion assay and a combined mutational study revealed that this structural change is important for the fusion efficiency of GP2. Loss of this extension resulted in lower fusion activity. To further understand these structural changes and to probe the TM’s environmental interactions, we turned to fluorine NMR. This method gives us a unique and highly sensitive probe to monitor changes in the structure and membrane environment. We describe our incorporation protocol of fluorine into the TM and our method for incorporating the TM into a lipid bilayer system. We describe preliminary results showing sensitive changes in the structure of the TM and the implications this method has to enhance our understanding of the LASV membrane fusion mechanism.Item Structural and Biophysical Explorations of Protein Degradation Tags(2023) Bonn, Steven Michael; Fushman, David; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Ubiquitin is a 76-amino acid, well-folded and highly stable protein that is highly conserved across all eukaryotes. It is a post-translational modifier of other proteins through itsattachment via an isopeptide bond to a substrate lysine sidechain. Multiple ubiquitin units can be stacked to form a polyubiquitin chain, with chain topologies and cellular outcomes varying based on which of ubiquitin’s lysine residues they are attached to. The most common and well-studied outcome of polyubiquitin attachment is degradation by the proteasome, a massive barrel-shaped protease complex responsible for general protein quality control as well as cell cycle progression. Proteasomes have been discovered in archaea and bacteria, and are controlled by the small archaeal modifier protein (SAMP) and the disordered prokaryotic ubiquitin-like protein (Pup), respectively. Recently, a second bacterial proteasome operon was discovered with a new putative signaling protein, ubiquitin bacterial (UBact). Here, the first investigation of the UBact proteasomal operon is presented. Using nuclear magnetic resonance (NMR) spectroscopy and a variety of biophysical techniques, UBact is demonstrated to be disordered in solution and interact with its putative proteasomal receptor. This sets the groundwork for further studies of the UBact system. Additionally, NMR is used to explore the activity and directionality of various deubiquitinase enzymes responsible for breaking down polyubiquitin chains, and for exploring small molecule binding to ubiquitin chains themselves. This likewise provides a groundwork for further studies of the ubiquitin system, whose dysregulation is responsible for many diseases and is an area of intense therapeutic development.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.Item BIOPHYSICAL STUDIES OF UBIQUITIN: FROM FOLDING TO PROTEIN ENGINEERING(2021) Camara, Christina M; Fushman, David; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The signaling protein ubiquitin is known for its ubiquity — existing in nearly all cel- lular compartments, holding a prominent role in major cellular signaling pathways and serving as a model system for protein folding. Herein, we honor this stature by exploring several aspects of the ubiquitin system form biophysical, structural, and computational per- spectives. Our efforts begin from the standpoint of protein engineering, where we extend ubiquitin’s function by installing a transition–metal binding motif and elevate it to the sta- tus of a metalloprotein. In doing so, we introduce novel spectroscopic behaviors, reactive propensities, and the capability to form non–canonical polyubiquitin chains — with appli- cations that span from molecular nanotechnology to synthetic biology. We then shift to foundational investigations of ubiquitin’s fold. By characterizing local degrees of freedom, we demonstrate how conformational motions of ubiquitin’s C–terminus can be controlled by the cellular microenvironment. This response, in turn, can regulate molecular recog- nition within the ubiquitination cascade. Finally, we approach global aspects of ubiquitin folding — exploring how a motif containing the C–terminus and the β5 strand might assem- ble into ubiquitin’s β –grasp architecture — with general lessons for ubiquitin–like proteins and other systems with an apparent two–state folding mechanism.Item BIOCHEMICAL AND STRUCTURAL CHARACTERIZATION OF NUSG PARALOG LOAP(2021) Elghondakly, Amr; Winkler, Wade; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The NusG family of transcription factors is the only universally conserved family of transcription elongation regulators in all three domains of life. NusG proteins exert ubiquitous genetic regulatory effects by reversibly binding RNA-polymerase (RNAP) during transcription elongation and modulate its function. A phylogenetic analysis of the NusG family of proteins identified several distinct subfamilies of NusG paralogs that are widespread amongst bacterial species. These different NusG paralogs are likely to exert regulatory control over distinct subsets of genes. Yet, despite the importance of the genes they regulate, most of the subfamilies of NusG paralogs (e.g., UpxY, TaA, ActX and LoaP) have not been investigated in depth. Additionally, the regulatory mechanisms that these transcription elongation factors employ are likely to differ between one another to allow for specific recruitment to target operons and prevent competition with the housekeeping NusG factor. The LoaP subfamily of NusG proteins is primarily encoded by Actinobacteria, Firmicutes and Spirochaetes. While regulons for the LoaP subfamily have only been identified in a few organisms, the loaP gene is oftentimes found adjacent to long operons encoding for biosynthesis of secondary metabolites suggesting a regulatory relationship with these pathways. In Bacillus velezensis, LoaP promotes transcription antitermination of two long biosynthetic operons which encode for two different polyketide antibiotics: difficidin and macrolactin. Intriguingly, the cis-determinants for LoaP antitermination include a small RNA hairpin (~26 nts) located within the 5’ leader region of target operons. LoaP associates with the RNA hairpin in vitro with nanomolar affinity and high specificity via basic residues that are highly conserved within the C-terminal KOW domain, in contrast to other well-characterized bacterial NusG proteins which do not exhibit RNA-binding activity. These data indicate that LoaP employs a distinct regulatory mechanism to achieve targeted regulation of large biosynthetic operons in bacteria. Furthermore, this discovery expands the repertoire of macromolecular interactions exhibited by bacterial NusG proteins during transcription elongation to include an RNA ligand. Crystallographic studies of LoaP-RNA complex are in progress, and recent results will be discussed.Item Structural and Biophysical Characterization of Homo Sapiens RIOK2 in Complex with Selective Prostate Cancer Inhibitors and its Transition State Complex(2021) Seraj, Nishat; LaRonde, Nicole; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Homo sapiens Riok2 (HsRiok2) is an atypical, Ser/Thr protein kinase that has been implicated in a number of cancers, including Prostate Cancer. More recently, evidence has shown that HsRiok2 plays a biological role in the upregulation of ERG-positive cancer cells, thus presenting itself as an attractive drug target. The first known prostate cancer inhibitor against HsRiok2 has been identified, as well as select derivative inhibitors and has been shown both in vitro and in vivo to target HsRiok2. Here, the first ever Homo sapiens Riok2 in complex with two known Prostate Cancer inhibitors at 1.75 Å resolution and 2.70 Å resolution is reported, respectively. To evaluate conformational changes upon inhibitor binding, the first ever Homo sapiens Riok2 transition state complex is reported, capturing a snapshot of the kinase poised for phosphoryl transfer, at 2.80 Å resolution. These structural insights reveal the influence of the phosphate-binding loop (P-loop) in HsRiok2’s dimerization and potentially a catalytically inactive state, mediated by the presence of the drug candidate. Its dimerization interface is prevented from interacting with the Pre-40S ribosome, preventing HsRiok2 from carrying out its function as an ATPase/Kinase to further mature the pre-40S particle. These findings provide the first blueprint for a structure-based drug design approach to facilitate the development of more selective prostate cancer inhibitors.Item EPIGENETICS TUNE CHROMATIN MECHANICS, A COMPUTATIONAL APPROACH(2021) Pitman, Mary; Papoian, Garegin A; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The base unit of DNA packaging in eukaryotes, the nucleosome, is adaptively modified for epigenetic control. Given the vast chemical space of chromatin and complexity of signaling and expression, much of our knowledge about genetic regulation comes from a biochemical or structural perspective. However, the architecture and function of chromatin also mechanically responds to non-equilibrium forces. Mechanical and biochemical properties are not independent of one another and the interplay of both of these material properties is an area of chromatin physics with many remaining questions. Therefore, I set out to determine how the material properties of chromatin are altered by biochemical variations of nucleosomes. All-atom molecular dynamics is employed coupled with new computational and theoretical tools. My findings and predictions were collaboratively validated and biologically contextualized through multiscale experimental methods. First, I computationally discover that epigenetic switches buried within the nucleosome core alter DNA accessibility and the recruitment of essential proteins for mitosis. Next, using new computational tools, I report that centromeric nucleosomes are more elastic than their canonical counterparts and that centromeric nucleosomes rigidify when seeded for kinetochore formation. We conclude that the material properties of variants and binding events correlate with modified loading of transcriptional machinery. Further, I present my theoretical approach called Minimal Cylinder Analysis (MCA) that uses strain fluctuations to determine the Young's modulus of nucleosomes from all-atom molecular dynamics simulations. I show and explain why MCA achieves quantitative agreement with experimental measurements. Finally, the elasticity of hybrid nucleosomes in cancer is measured from simulation, and I implicate this oncogenic variant in potential neocentromere formation. Together, these data link the physics of nucleosome variations to chromatin states' plasticity and biological ramifications.Item EMERGENT NETWORK ORGANIZATION IN LINEAR AND DENDRITIC ACTIN NETWORKS REVEALED BY MECHANOCHEMICAL SIMULATIONS(2021) Chandrasekaran, Aravind; Papoian, Garegin A; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Cells employ networks of filamentous biopolymers to achieve shape changes and exert migratory forces. As the networks offer structural integrity to a cell, they are referred to as the cytoskeleton. Actin is an essential component of the cellular cytoskeleton. The organization of the actin cytoskeleton is through a combination of linear and branched filaments. Despite the knowledge of various actin-binding proteins and their interactions with individual actin filaments, the network level organization that emerges from filament level dynamics is not well understood. In this thesis, we address this issue by using advanced computer simulations that account for the complex mechanochemical dynamics of the actin networks. We begin by investigating the conditions that stabilize three critical bundle morphologies formed of linear actin filaments in the absence of external forces. We find that unipolar bundles are more stable than apolar bundles. We provide a novel mechanism for the sarcomere-like organization of bundles that have not been reported before. Then, we investigate the effect of branching nucleators, Arp2/3, on the hierarchical organization of actin in a network.By analyzing actin density fields, we find that Arp2/3 works antagonistic to myosin contractility, and excess Arp2/3 leads to spatial fragmentation of high-density actin domains. We also highlight the roles of myosin and Arp2/3 in causing the fragmentation. Finally, we understand the cooperation between the linear and dendritic filament organization strategies in the context of the growth cone. We simulate networks at various concentrations of branching molecule Arp2/3 and processive polymerase, Enabled to mimic the effect of a key axonal signaling protein, Abelson receptor non-tyrosine kinase (Abl). We find that Arp2/3 has a more substantial role in altering filament lengths and spatial actin distribution. By looking at conditions that mimic Abl signaling, we find that overexpression mimics are characterized by network fragmentation. We explore the consequence of such a fragmentation with perturbative simulations and determine that Abl overexpression causes mechanochemical fragmentation of actin networks. This finding could explain the increased developmental errors and actin fragmentation observed in vivo. Our research provides fundamental self-assembly mechanisms for linear and dendritic actin networks also highlights specific mechanochemical properties that have not been observed earlier.Item PLASTICITY IN PROTEIN SEQUENCE-FUNCTION RELATIONSHIPS(2021) He, Chenlu; Beckett, Dorothy; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Allostery is defined as the functional regulation at one site in a protein by activity at a distant site. Because of the ubiquitous occurrence and diverse cellular roles of allosteric proteins, designing novel allosteric proteins is of great interest for applications in synthetic biological and disease treatment. However, the engineering of allostery is often hindered by our limited understanding of the protein sequence- function relationship, especially at residue positions that are distant from functional sites or evolutionarily nonconserved. In this dissertation, the sequence-function relationship was investigated in the Escherichia coli biotin protein ligase (BirA) system, which serves as both an essential metabolic enzyme and a transcription regulator. In its repressor function, binding to the vitamin biotin allosterically activates BirA dimerization and the resulting repression complex assembly on the biotin operator sequence. Although the allosteric regulation is conserved among bifunctional biotin protein ligases such as BirA, their sequences, even those of functional importance, are highly divergent. The in vitro characterization of BirA super repressor variants reveals that the sensitivity of transcription repression response to input biotin concentration can be altered solely through substitution-perturbed dimerization. These single amino acid substitutions are located at sites scattered throughout the protein structure including some that are distal from the BirA dimerization surface. Computational simulations indicate that the long-range effect of substitutions on dimerization results from rearrangement of a residue network that contributes to the allosteric activation in BirA. Several loops on the BirA dimerization surface were characterized for their roles in the corepressor-induced dimerization. The study of nonconserved amino acid positions spanning these surface loops reveals that a broad range of functional response in dimerization and transcription repression can be achieved by sequence variations at the nonconserved residues. Surprisingly, the substitution outcomes poorly correlate with amino acid chemistry or evolutionary frequencies, which deviates from canonical expectations based on conserved residues. Combined, these results illustrate the plastic nature of protein sequence-function relationship and provide insight into how this plasticity functions in the mechanism and evolution of allostery in BirA. Our deepened understanding of allostery in BirA and in general may facilitate the development of synthetic allosteric proteins in the future.