Chemical and Biomolecular Engineering Theses and Dissertations
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Item EXPERIMENTAL INVESTIGATION OF THE LIPID-BINDING MECHANISM OF OSH4 PROTEIN(2024) Konakbayeva, Dinara; Karlsson, Amy; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Recent findings show that intracellular lipid traffic between organelles primarily occurs through a non-vesicular pathway involving lipid transport proteins (LTPs) and is facilitated by areas of close apposition between two organelles so called membrane contact sites (MCS). Oxysterol-binding homologue (Osh) proteins in the yeast Saccharomyces cerevisiae serve as examples of LTPs. Osh proteins are crucial for transporting signaling lipids and are believed to form MCSs. In this study, we examined the binding mechanism of the Osh4 protein, aiming to gain a better understanding of its explicit membrane-binding mechanism.The Osh4 protein possesses an α-helical binding domain known as the amphipathic lipid-packing sensor (ALPS)-like motif. Our approach involved utilizing experimental methods to examine the biophysical interactions of both the ALPS peptide and the full-length Osh4 protein. To investigate the binding interactions of ALPS with membranes of different lipid compositions, we examined its interactions with three different mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC; has a zwitterionic head group) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS; has a negatively charged head group)—1:1 POPC-POPS, 4:1 POPC-POPS, and 9:1 POPC-POPS—as well as pure POPC. To understand the structural changes in ALPS and model membranes during peptide-membrane interactions, we performed a series of experimental studies. Circular dichroism (CD) was used to study the changes in the secondary structure of ALPS in different environments. The CD data indicated that the α-helical conformation of the ALPS peptide was more pronounced in the presence of POPC-POPS liposomes, especially with a higher content of POPS lipid, compared to liposomes composed entirely of POPC. This observation underscores the significant influence of anionic lipids in the facilitation of peptide folding at the membrane-water interface. X-ray diffraction was utilized to study the changes in membrane structure upon ALPS binds to it. The X-ray diffraction results showed that the ALPS peptide caused thinning of the multilayer with an increased POPS lipid ratio. This could be due to the electrostatic interaction of the positively charged Lys residue in the ALPS sequence with the anionic POPS lipid. We also studied the binding of the peptide to membranes by observing changes in the Trp fluorescence emission spectrum of ALPS upon the addition of liposomes. We observed a blue shift in the fluorescence emission maximum of Trp with higher POPS content. This suggests that the ALPS peptide was experiencing a more hydrophobic and less polar environment in the presence of the liposomes, indicating possible penetration of the peptide into the hydrocarbon region of the bilayer. The blue shifts of Trp emission in the presence of POPS liposomes were higher than those observed with POPC liposomes and suggest that the ALPS peptide binds better to charged POPS lipids, which is consistent with the X-ray diffraction data. We also conducted Trp fluorescence titration and ITC experiments to gain deeper insights into the binding affinity of the ALPS peptide to a model membrane. Using fluorescence data, we estimated the binding constant for the binding of ALPS to liposomes by performing titration measurements of vesicles with the ALPS peptide. Our analysis demonstrated that ALPS binding to 4:1 POPC-POPS lipid membranes had a Kd of 1.88 ± 0.47 μM, which corresponds to a free energy change (ΔG) of -7.82 ± 0.15 kcal/mol. Additionally, the ITC experiments performed with the same vesicles yielded a ΔG of -4.41± 0.04 kcal/mol. This result is slightly less than the ΔG value of -7.82 ± 0.15 kcal/mol obtained from fluorescence spectroscopy titration. The observed discrepancy of -3.41 kcal/mol may indicate the energy associated with the folding of the ALPS peptide. In order to understand how Osh4 forms MCSs between two membranes, we need to examine how the membranes interact with the full-length protein. The first step to achieve this is to produce the protein through recombinant protein production methods. After evaluating two different fusion tags, glutathione S-transferase (GST) and small ubiquitin-related modifier (SUMO), it was found that the SUMO tag resulted in higher protein yield and greater protein purity. Our work lays the foundation for future experiments with the full-length Osh4 protein to improve our understanding of the mechanisms of lipid transport between membranes. Our results emphasize the ALPS peptide’s selectivity for specific lipid environments, particularly its affinity for anionic lipids. We demonstrated that the presence of anionic lipids is crucial for the motif's ability to induce conformational changes upon binding to a membrane, and these conformational changes likely play a critical role in intracellular lipid trafficking and membrane organization.Item Study of Membrane Binding Proteins and Related Signaling Molecules(2023) Allsopp, Robert James; Klauda, Jeffery B; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The membrane contact site theory is a critical theory to understanding lipid transport. The Osh protein is a yeast lipid transport protein theorized to form membrane contact sites. We investigated the contact site theory by identifying a second binding domain and studying the Osh Amphipathic Lipid Packing Sensor (ALPS) to explain better why each protein might target different organelles. The α6- α7 domain appears more charged and prefers lipids with oppositely charged inositol sugars, making it ideal for binding to the Trans Golgi Network (TGN) and the plasma membrane. The ALPS peptide is another dedicated binding domain bound in several membrane types with varied Phosphatidylcholines (PC) tails to vary the lipid packing. If the force field was valid, the results indicate that Osh4 ALPS prefers the loose packing of POPC, and Osh5 ALPS prefers the tighter packing of DMPC. More input from the wet lab is needed before researchers can make predictions from the force field. Another vital area of research is antimicrobial peptides (AMPs) that disrupt the membrane. Part of the dissertation focused on determining the dual placement of the AMPs on the surface and inserted into the membrane. For the first time, the membrane properties of bilayers with AMPs were studied, using the combination of all-atom simulation informed by x-ray scattering. The surface tension was a critical parameter that enabled us to compare the simulation to the wet lab results and became vital in allowing the peptide to be inserted into the membrane and remain stable. The 5-HT3A project simulated predicted structures of toxins with computational tools. Our work simulated these toxins for the first time, and we observed the unbiased binding of σ-GVIIIA conotoxin to the allosteric binding pocket. In the first trajectories, the ion channel pore remained closed, similar enough to the native apo crystal structure that water could form a partially water-filled channel for a few microseconds. In one example, the 5-HT3A had serotonin in all of the binding pockets for close to 1 µs. The long simulation of the conotoxin showed that the extracellular domain (ECD) was deformed by more than a nanometer compared to a control. This deformation was the first indication that such a conformation is possible and might be related to the presence of the toxin. Finally, traumatic brain injury was studied by identifying new molecules that activate fibroblast growth factor (FGF) and toll-like receptor (TLR) proteins. The focus on FGF resulted in identifying a critical conformational change and potential new binding sites (previously unknown) that activate FGF without activating damaging inflammatory TLR responses.Item Effect of Protein Folding State and Conformational Fluctuations on Hydrogel Formation and Protein Aggregation(2022) Nikfarjam, Shakiba; Woehl, Taylor J; Anisimov, Mikhail; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In this thesis we investigate the role of protein unfolding on protein aggregation and hydrogel formation in two different systems. In the context of designing protein-based hydrogels as biomaterials, we investigate how protein unfolding affects the formation dynamics of hydrogels in response to temperature changes, denaturation, and chemical reactions. In a second context we establish how microsecond to millisecond fluctuations in an amyloid forming protein, beta-2-microglobulin, correlate to the amyloid forming propensity of the protein, with an emphasis on understanding how conformational changes in the native folded state provide thermodynamic driving forces for amyloid nucleation.The work on protein hydrogel yielded two key results. First, we observed that the lifetime of dissipative hydrogels decreased and their mechanical stiffness increased with increasing denaturant concentration and constant fuel concentration. At a higher denaturant concentration, the concentration of solvent-accessible cysteines increases the stiffness of the hydrogel at the cost of a faster consumption of H_2 O_2, which is the cause of the shorter gel lifetime. This work utilizing biological macromolecules in kinetically controlled dissipative structures opens the door to future applications of such systems in which the biomolecules' structures can control the reaction kinetics. Another substantial outcome of our work is to uncover mechanisms underlying the initiation of nucleation in the initial stages of amyloid aggregate formation. The study of conformational fluctuations in the structure of the amyloid-forming protein beta 2-microglobulin (β_2 M) yielded three key results. First, β_2 M variants' aggregation propensity correlates with their conformational fluctuations rate. A longer-lived misfolded subpopulation increases the chance of aggregation initiation by increasing the collision chance of the protein's sticky regions. Second, the observed millisecond interconversions agree with the timescales required for the interconversion of a protein's structure between its subpopulations. Third, the fluctuations themselves could be a driving force for the nucleation of aggregates by decreasing the lag-time of nucleus formation by a sudden large fluctuation.Item Advancements in Label-free Biosensing Using Field-Effect Transistors and Aided by Molecular Dynamics Simulations(2019) Guros, Nicholas; Klauda, Jeffery B; Balijepalli, Arvind; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Biosensors are used to characterize or measure concentrations of physiologically or pathologically significant biomarkers that indicate the health status of a patient, for example, a biomarker associated with a specific disease or cancer. Presently, there is a need to improve the capabilities of biosensors, which includes their rate of detection, limit of detection, and usability. With respect to usability, it is advantageous to develop biosensors that can detect a biomarker that is not labeled, such as with a conventional fluorescent, magnetic, or radioactive label, prior to characterization or measurement by that biosensor. Such biosensors are known as label-free biosensors and are the primary focus of this work. Biosensors are principally evaluated by two standards: their sensitivity to detect a target biomarker at physiologically relevant concentrations and their specificity to detect only the target biomarker in the presence of other molecules. The elements of biosensing critical to improving these two standards are: biorecognition of the biomarker, immobilization of the biorecognition element on the biosensor, and transduction of biomarker biorecognition to a measurable signal. Towards the improvement of sensitivity, electrostatically sensitive field-effect transistors (FET) were fabricated in a dual-gate configuration to enable label-free biosensing measurements with both high sensitivity and signal-to-noise ratio (SNR). This high performance, quantified with several metrics, was principally achieved by performing a novel annealing process that improved the quality of the FET’s semiconducting channel. These FETs were gated with either a conventional oxide or an ionic liquid, the latter of which yielded quantum capacitance-limited devices. Both were used to measure the activity of the enzyme cyclin-dependent kinase 5 (Cdk5) indirectly through pH change, where the ionic-liquid gated FETs measured pH changes at a sensitivity of approximately 75 times higher than the conventional sensitivity limit for pH measurements. Lastly, these FETs were also used to detect the presence of the protein streptavidin through immobilization of a streptavidin-binding biomolecule, biotin, to the FET sensing surface. To study the biomolecular factors that govern the specificity of biomarker biorecognition in label-free biosensing, molecular dynamics (MD) simulations were performed on several proteins. MD simulations were first performed on the serotonin receptor and ion channel, 5-HT3A. These simulations, which were performed for an order of magnitude longer than any previous study, demonstrate the dynamic nature of serotonin (5-HT) binding with 5-HT3A. These simulations also demonstrate the importance of using complex lipid membranes to immobilize 5-HT3A for biosensing applications to adequately replicate native protein function. The importance of lipid composition was further demonstrated using MD simulations of the ion channel alpha-hemolysin (αHL). The results of these simulations clearly demonstrate the lipid-protein structure-function relationship that regulates the ionic current though a lipid membrane-spanning ion channel. Finally, to demonstrate the impact of MD simulations to inform the design of FET biosensing, a strategy to use FETs to measure the ultra-low ionic currents through the ion channel 5-HT3A is outlined. This strategy leverages critical elements of 5-HT biorecognition and ion channel immobilization extracted from MD simulations for the design of the proposed FET sensing surface interface.Item COMPUTATIONAL STUDIES OF LIPID BILAYERS AND TRANSMEMBRANE PROTEINS(2017) Khakbaz, Pouyan; Klauda, Jeffery B; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Deep understanding of lipid bilayers in three phases at the molecular level could potentially lead us to design a novel artificial membrane. Molecular modeling of bacterial membranes is important as they are cheap and an environmentally friendly candidate to produce fuels. Molecular investigation of transmembrane proteins is crucial as mutations in them were observed in multiple diseases including cancer. The inner membrane of Escherichia coli (E. coli) was modeled to include lipid diversity and demonstrate that this is needed to properly probe the interaction of lipids and transmembrane proteins. Molecular dynamics (MD) simulations were used with the all-atom CHARMM36 (C36) force field. Lipid diversity affects the properties of the E. coli inner membrane and indicated the importance of including lipids with different head groups and acyl chains. The effect of the growth stage of the E.coli colony significantly influenced thicknesses and bulk properties of the membrane. Phase transitions of fully saturated lipid bilayers, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dipalmitoyl-sn-glycero-phosphocholine (DPPC) and their mixtures were probed for the first time using MD simulations. The phase transitions from fluid (Lα) phase to ripple (Pβ) phase and to the gel (Lβ) phase were obtained within temperature range in good agreement with experimental phase transition temperature. DMPC and DPPC bilayers in Lβ phase resulted in fatty acid chains tilted relative to bilayer normal and with average tilt angle in agreement with experiment. MD simulations revealed molecular-level structural details of the pure DMPC bilayer in Pβ phase at a temperature to compare to experimental X-Ray diffraction measurements. The structure of the major and minor arm is in agreement with experiment when enough lipids are used to model this phase. The final two topics involved collaborations with experimental labs to provide insight into experimental observables. First, MD simulations successfully showed that improved tolerance and production of biorenewables of a metabolically engineered E.coli strain is the result of increased bilayer thickness. Secondly, MD simulations of the homodimerization of plexin A3 were used to probe the association of the transmembrane (TM) and juxtamembrane (JM) domains of this important cell-signaling membrane protein. These simulations indicated multiple dimerization conformations, and suggested importance of extracellular domain residues on strong TM interactions.Item Computational Studies of Membrane Models and their Interaction with a Peripheral Protein in Yeast, and Disruption of the Water-Oil Interface by a Hydrotrope(2017) Monje-Galvan, Viviana; Klauda, Jeffery B; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Biological and non-biological interfaces were studied using all-atom molecular dynamics simulations to understand the interaction between different molecules at the atomic level. Simulation were run to analyze the dynamics and structure of cell membrane models and their interaction with a specific protein. Additionally, the effect of a small alcohol at the water-oil interface was examined as a model for amphiphilic molecules, which are relevant in chemistry and biology. Previously developed organelle-specific membrane models for yeast S. cerevisiae (Biochem. 54:6852-6861) were improved to reflect leaflet asymmetry of the trans-Golgi network (TGN) and plasma membranes. Each model was built based on experimental trends to study interleaflet coupling and lipid clustering. The (previous) symmetric endoplasmic reticulum (ER) and TGN models were further used to study the effect of sterol type in the structural properties of the membrane, and lipid-protein interactions with a lipid transport protein in yeast, Osh4. The protein’s phenylalanine loop was determined to have the strongest interaction with the bilayer among the protein’s six binding regions (BBA-Biomemb. 1858:1584-1593). The protein’s lid, the ALPS-like motif (Amphipathic Lipid Packing Sensor), was also simulated with simple (2-lipid) bilayers and with the symmetric ER and TGN models. Key residues for peptide-membrane interaction were identified based on their interaction energy, and a time scale of ~1µs determined for stable peptide binding. The interfacial dynamics between water and cyclohexane were examined in the presence of a hydrotrope - an amphiphilic molecule that reduces the interfacial tension between two liquids. Simulations were run for water-cyclohexane systems and all butanol isomers separately to understand the effect of this hydrotrope’s chemical structure on the interface. The results reproduced experimental data trends, showing that a hydrotrope concentration of as little as 0.6mol% in the aqueous phase reduces the interfacial tension to nearly half the value of a binary water-cyclohexane mixture. Tert-butanol was further compared with experimental studies showing that at low concentrations (< 10mol%) the simulations accurately reproduce experimental data. In addition, theoretical correlations from simulation data show the system follows van der Waals theory of smooth interfaces, and describe the crossover behavior of this hydrotrope from surfactant-like to co-solvent based on its concentration in solution, and describe the crossover behavior of this hydrotrope from surfactant-like to co-solvent based on its concentration in solution.Item COMPUTATIONAL STUDIES ON ORGANELLE-SPECIFIC YEAST MEMBRANE MODELS(2014) Monje-Galvan, Viviana; Klauda, Jeffery B; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Computational models were built for the endoplasmic reticulum (ER), trans-Golgi network (TGN), and plasma membranes (PM) of yeast Saccharomyces cerevisiae. Based on experimental data, ergosterol, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol lipids were included. Lipid packing, order parameters (SCD), electron density profiles (EDPs), and lipid rotation were studied for each model. The average surface area per lipid decreased from 63.82±0.03 Å2 in the ER to 47.09±0.12 Å2 at the PM; while the compressibility modulus (KA) varied in opposite direction (PM>TGN>ER). The SCD values were higher (more ordered) for the PM lipids than the ER and TGN membranes by a factor of 1.5. The bilayer thickness estimated from EDPs was larger for the PM (43.9±0.1 Å) than the ER or TGN (37.6±0.1 Å). These properties followed expected experimental trends and were compared against a previous model built by Jo et al. (Biophys J. 2009, 97:50-58).Item Membrane models of E. coli containing cyclic moieties in the aliphatic lipid chain(2012) Pandit, Kunal; Klauda, Jeffery B; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Most molecular dynamics (MD) simulations of bacterial membranes simplify the membrane by composing it of only 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) or in some cases with1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) as well. However, an important constituent of bacterial membranes are lipids with a cyclopropane ring in the acyl chain. We developed a complex membrane that reflects the diverse population of lipids within E. coli cytoplasmic membranes, including cyclic lipids. Differences between the deuterium order profile of cyclic and monounsaturated lipids are observed. Furthermore, inclusion of the ring decreases the surface density of the bilayer and produces a more rigid membrane as compared to POPE/POPG membranes. Additionally, the diverse acyl chain length creates a thinner bilayer which better matches the hydrophobic thickness of E. coli transmembrane proteins. We believe the complex membrane is more accurate and suggest the use of it in MD simulations rather than simple membranes.Item Computational studies of lactose permease of E. coli as a model for membrane transport proteins(2013) Pendse, Pushkar; Klauda, Jeffery; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Membrane transport proteins actively transport ions, metabolites, drug molecules, and others across the amphiphilic cell membrane. The Major Facilitator Superfamily (MFS) is an important class of membrane transporters whose members are found in almost all types of organisms. The MFS proteins transport a diverse range of molecules including sugars, peptides, and drug molecules. In this work, lactose permease (LacY) of E. coli, which transports galactosides across the plasma membrane by the proton symport mechanism, is studied as a model for the MFS proteins. First, a hybrid two-step simulation approach was developed to determine the unknown periplasmic-open structure of LacY starting with the cytoplasmic-open x-ray crystal structure. A periplasmic-open model for LacY that agrees with several indirect experimental measurements was obtained. Sugar binding and protonation of Glu269 were found to be the triggers for LacY's structural change from the cytoplasmic- to the periplasmic-open state. Mutations in residues Asn245, Ser41, Glu374, Lys42 and Gln242 to prevent cross-domain hydrogen bonding were proposed that might aid in crystallizing LacY in the periplasmic-open state. The second focus of this dissertation was a comprehensive study on the binding of high- and low-affinity binders as well as non-binders to LacY in its cytoplasmic- and periplasmic-open states. A possible pathway for the substrate translocation, which involves aromatic stacking interactions of substrates with Phe354 and Tyr350, was suggested. Binding free energy values calculated using the alchemical free energy perturbation method agree with the experimental data. The differences in binding affinities result from dissimilarities in the binding structures of different sugars. The binding free energy values as well as the binding structures in the cytoplasmic- and the periplasmic-open states of LacY provide a quantitative proof of the alternating access mechanism in LacY. Lastly, hybrid quantum mechanics/molecular mechanics (QM/MM) studies on LacY were performed, which are the first QM/MM studies of proton transport in an MFS protein. These QM/MM studies suggest an important role of water molecules in proton transfer. A hydronium ion intermediate was observed during proton transfer from Glu325 to His322, which is consistent with the experimental hypothesis. The transfer of proton from His322 to Glu269 was found to be the rate-determining reaction. More extensive QM/MM calculations on LacY are proposed to fully probe proton translocation in this protein. This dissertation has touched all the important aspects of LacY's transport cycle and results from this study can be beneficial for understanding the mechanism of other MFS proteins such as the drug efflux protein EmrD, which shares its structural and functional motif with LacY.Item COMPUTATIONAL STUDIES ON THE BINDING AND DYNAMICS OF THE OSH4 PROTEIN OF YEAST AND A MODEL YEAST MEMBRANE SYSTEM(2010) Rogaski, Brent Joseph; Klauda, Jeffery B; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Osh4 is an oxysterol binding protein homologue found in yeast that is essential for the intracellular transport of sterols. It has been proposed that Osh4 acts as a lipid transport protein, binding a single sterol residue and transporting it from the endoplasmic reticulum to the plasma membrane. The dynamics of Osh4 as well as ergosterol binding was observed using molecular dynamics simulations. Blind docking of several model lipid head group moieties was used to detect potential binding regions along the Osh4 surface favorable towards phospholipid interaction. Models frequently docked to a lysine-rich region on the side of the protein's β-barrel. A model ergosterol-containing membrane system for yeast was also constructed and simulated using molecular dynamics, and an improvement to the deuterium order parameters was observed over previous models. Understanding how Osh4 attaches to cellular membranes will lead to a clear understanding of how this protein transports sterols in vivo.