A. James Clark School of Engineering

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    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.
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    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.
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    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.
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    MATHEMATICAL MODELS AND NOVEL BIOMARKERS TOWARD OPTIMIZATION OF BURN INJURY RESUSCITATION
    (2022) Arabidarrehdor, Ghazal; Hahn, Jin-Oh; Mechanical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Extensive burn injury is not only devastating but also a significant challenge for healthcare providers. Following a chain of inflammatory responses post-burn, significant amounts of plasma shift from the vascular compartment into the tissues, simultaneously posing the risks of hypovolemic shock and edema. Standard burn resuscitation protocols aim to replace the lost blood volume while not exacerbating the edema through hourly-titrated intravenous fluid infusion. Due to the significant variability in treatment efficacy, there is a substantial ongoing effort to optimize and individualize the burn resuscitation protocols. In this work, we aim to contribute to this effort by (i) developing a platform for the virtual evaluation of burn resuscitation protocols and (ii) identifying biomarkers to guide fluid resuscitation effectively. The first part of this work presents a mathematical model of burn injury and resuscitation, which can be used for the development and non-clinical testing of burn resuscitation protocols and algorithms, as well as to garner knowledge and intuition into this complex pathophysiology. Our mathematical model consists of a multi-compartmental model of blood volume kinetics, a hybrid mechanistic-phenomenological model of kidney function, and novel lumped-parameter models of burn-induced perturbations in volume kinetics and renal function. We examined our mathematical model’s prediction accuracy and reliability using a rich dataset from 16 sheep with extensive burn injuries and clinical data from 233 real-world burn patients. The second part of this work presents the expansion of the mathematical model to incorporate the cardiovascular and renin-angiotensin-aldosterone systems, as well as detailed descriptions of the kidney’s mechanisms, particularly regarding its blood volume and blood pressure regulation roles. This expansion was motivated by the importance of cardiovascular monitoring in the critical care of burn injury patients. We trained and validated the expanded mathematical model for three species: nine sheep subjects and 15 swine subjects with rich cardiovascular and volume kinetics data, and 233 human subjects with demographic and urinary output (UO) data. To the best of our knowledge, our mathematical model may be the first of its kind which is extensively validated for use as a digital twin to replicate realistic burn patients and replace standard large animal pre-clinical testing of burn resuscitation protocols. The third part of this work presents the identification of biomarkers capable of guiding, optimizing, and individualizing burn resuscitation. The UO, the most common endpoint used to titrate burn resuscitation fluid doses, has many limitations as a single variable. Hence, this work aimed to find convenient and reliable biomarkers from arterial blood pressure (ABP) waveform to complement UO in guiding burn resuscitation. Pulse pressure variation (PPV), systolic pressure variation (SPV), and stroke volume variation (SVV) are dynamic indices derived from ABP that have shown promise in hemorrhage resuscitation but are not investigated for different resuscitation paradigms for burn injury. We observed the longitudinal behavior of PPV, SPV, and SVV for 21 porcine subjects with 40% burn injury, which were each either under-resuscitated, adequately resuscitated, or deliberately over-resuscitated. We investigated the features' potential in tracking reference cardiac output (CO) and stroke volume (SV) via linear regression and correlation analysis. PPV, SPV, and SVV showed plausible and statistically different trends for different paradigms. While they performed just as well as UO in tracking CO and SV, their inherent advantage of being available in real-time and their disagreement with UO in determining the subject status suggest that they may potentially complement UO in the hemodynamic assessment of burn patients.
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    EFFECT OF MISMATCHED BASE PAIRS ON DNA PLECTONEMES
    (2022) Desai, Parth Rakesh; Das, Siddhartha; Mechanical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Base pair mismatches in DNA occur during replication and can result in mutations and certain types of cancer. The exact mechanism by which mismatch repair proteins recognize mismatches is still not well understood. Structures of mismatch recognition proteins bound to a mismatch indicate that the process involves introducing a sharp bend in the DNA and flipping out the mismatched base. Under external torsional stress, an elastic rod with a defect would buckle at the defect, provided the defect reduces the local bending stiffness. In vivo, if the same energetic scenario prevails, it could localize (or pin) the mismatch at the plectoneme end loop (plectoneme refers to a structure formed by the DNA when it buckles, and its helical axis wraps or writhes around itself in the presence of a critical torsional stress) and make the mismatched base pair more accessible to the mismatch repair protein. In genomic DNA, however, the entropic cost associated with plectoneme localization could make pinning unfavorable. Magnetic-tweezers-based studies of DNA supercoiling, performed at high salt concentrations, have shown that in DNA harboring a single mismatch, the plectoneme will always localize at the mismatch. Theoretical studies have predicted that under physiological salt concentrations, plectoneme localization becomes probabilistic, i.e., the plectoneme does not always localize at the mismatch. Plectoneme localization under physiological salt conditions is dependent on the number of mismatches and tension applied to the DNA. However, both experimental and theoretical approaches are currently limited to positively supercoiled DNA. In the current dissertation, we aim to study plectoneme localization, in physiologically relevant conditions, using state-of-the-art molecular dynamics (MD) simulations and single molecule magnetics tweezers-based experiments.In order to simulate plectoneme localization we first develop a framework using the widely available sequence and salt dependent OxDNA2 model. We verify that the OxDNA2 model can quantitively reproduce a reduction in bending rigidity due to the presence of the mismatch(es), similar to all-atom MD simulations. We then verify that the current framework can reproduce the experimentally observed plectoneme pinning (at the location of the mismatches). Next, we simulate plectoneme pinning under physiologically relevant conditions. We find that the plectoneme pinning (at the location of the mismatches) becomes probabilistic and this probability of plectoneme pinning increases with an increase in the number of mismatches. We also simulate a longer 1010 base pair long DNA to study the influence of entropic effects on plectoneme pinning. Next, we extend the simulation framework to simulate a negatively supercoiled, i.e., under-wound, DNA molecule. In vivo, DNA is maintained in a negatively supercoiled state. Negative supercoiling can result in local melting at the mismatched base pairs: this local melting would further reduce the local bending rigidity at the mismatched base pairs and could enhance plectoneme pinning. We find that negative supercoiling significantly enhances plectoneme pinning in comparison with equivalent levels of positive supercoiling. We also find that the mismatched base pairs are locally melted and the plectoneme end loop is bent significantly more as compared to the positive supercoiling case. Additionally, we simulate the 1010 base pair long DNA under two different negative super-helical densities, i.e., two different degrees of unwinding. We find that the super helical density does not affect the plectoneme pinning probabilities. We also conduct simulations of DNA under different stretching forces (0.3 pN, 0.4 pN and 0.6 pN). Negatively supercoiled DNA under relatively high stretching force (~0.6 pN) absorbs tortional stress by locally melting instead of supercoiling. Simulations of DNA under different forces allow us to study the effect of mismatches on the competition between supercoiling and local melting in a negatively supercoiled DNA. We find that higher stretching forces, up to a maximum set by the onset of melting, increase plectoneme pinning at the location of mismatch. Finally, we propose and develop a single molecule assay to validate the simulations results presented in the previous chapters. Previous single-molecule magnetic tweezers measurements of mismatch DNA buckling and pinning were limited to the high force (~2 pN) – high salt (>0.5 M NaCl) regime. We propose to overcome this limitation by attaching a small gold nano-bead via a di-thiol group close to the mismatched base pairs, which permits direct observation of transient DNA buckling at the mismatch. We generate a DNA substrate that can be used to directly observe plectoneme pinning at the mismatch. We perform single-molecule magnetic tweezers measurements to verify that the presence of the di-thiol group does not result in anomalous pinning in an intact DNA molecule.
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    Brillouin confocal microscopy in off-axis configuration
    (2021) Fiore, Antonio; Scarcelli, Giuliano; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Three-dimensional Brillouin confocal microscopy is an imaging modality that correlates with mechanical properties in biological media from subcellular to tissue level. Over the years we developed new approaches to this technique that improve the spectral performance and can measure directly the local refractive index as well as the complex modulus of the sample; to achieve this goal, we probed two co-localized Brillouin scattering geometries. The confocal microscopy setting ensures three-dimensional mapping with high resolution, while the back scattering configuration allows access to the sample from the same side. For these reasons, such an instrument constitutes a new approach in investigating biological phenomena providing both local index of refraction and mechanical information with a single measurement. This technique has been improved in speed and spatial resolution in order to be applied to some specific challenging material characterization such as liquid-liquid phase separation.
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    PHOSPHOLIPID BEHAVIOR AND DYNAMICS IN CURVED BIOLOGICAL MEMBRANES
    (2020) JING, HAOYUAN; Das, Siddhartha SD; Mechanical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Curvature in biological membranes defines the morphology of cells and organelles and serves key roles in maintaining a variety of cellular functions, enabling trafficking, recruiting and localizing shape-responsive proteins. For example, the bacterial protein SpoVM is a small amphipathic alpha-helical protein that localizes to the outer surface of a forespore, the only convex surface in the mother bacteria. Understanding several of these membrane curvature dependent events rely on a thorough understanding of the properties, energetics, and interactions of the constituent lipid molecules in presence of curvatures. In this dissertation, we have used molecular dynamics (MD) simulations to explore how the curvature of the lipid bilayer (LBL), a simplified mimic of the cell membrane, affects the packing fraction and diffusivity of lipid molecules in the LBL, energetics of lipid flip flop in the LBL, and lipid desorption from the LBLs. We have also investigated the interaction between LBLs and a small bacterial protein, SpoVM, which was previously shown to preferentially embed in positively curved membranes. Our work started with simulating convex surface, represented by the nanoparticle supported lipid bilayers (NPSLBLs) in MD. We first quantified the self-assembly, structure, and properties of a NPSLBL with a diameter of 20 nm and showed how the type of the nanoparticle (NP) affects the properties of the NPSLBLs. Second, we studied the energetics of lipid flip flop and desorption from LBLs for the cases of planar substrate supported lipid bilayer (PSSLBL) and NPSLBL. Finally, we investigated the energetics of SpoVM desorption from the PSSLBL and the NPSLBL providing clues to the fundamental driving forces dictating the curvature sensing of SpoVM. In Chapter 1, we discuss the motivation, methods, biological relevance, and the overall structure of this thesis. In Chapter 2, the structure and properties of a pre-assembled NPSLBL were studied. In Chapter 3, we report the MD simulation results on the structure and properties, such as diffusivity, of the lipid molecules within the LBLs of the NPSLBLs formed through the self-assembly route. We compare our findings with that of unsupported lipid bilayer nanovesicles (NVs). Our results show that the structure of the NPSLBLs, although affected by the type of the NPs, is still similar with the free NV consisting of identical number and species of lipid. On the other hand, the properties such as the diffusivity of the lipid molecules within the LBL are significantly different between the cases of NPSLBL and the free vesicle. Results are provided for different combinations of the lipid molecules and the NP materials. The findings described in Chapters 2 and 3 will be eventually useful in long-term for designing new generation of NPSLBLs as drug carrier. In Chapter 4, we focus on the lipid flip-flop and desorption from the LBLs for NPSLBLs and PSSLBLs. We investigated the energetics of a lipid molecule traversing through the lipid bilayer (from inner-to-outer and outer-to-inner leaflet) as a function of the position of the hydrophilic head group of the lipid within the LBL. We obtained the potential of mean force (PMF) by using umbrella sampling. Most importantly, we observed little effect of the curvature in the variation of the lipid flip-flop PMF, establishing that the energetics of lipid migration within the supported bilayer, which implies that energy changes associated with bilayer fluctuations, is independent of the shape of the supported bilayer. The conclusion is supported by the reported experimental results. Next, in Chapter 5, MD simulations are carried out to reveal the energetics of a single SpoVM protein undergoing desorption from LBLs of NPSLBLs and PSSLBLs. The free energy comprises of five different contributions: 1) the free energy change for deforming the protein in the bilayer with respect to the conformation of the protein in the membrane, 2) the free energy change for reorienting the protein in the bilayer about the first Euler angle with the conformation of the protein restrained, 3) the free energy change for reorienting the protein in the bilayer about the second Euler angle with the conformation and the first Euler angle restrained, 4) the free energy change for changing the position of the center of the protein from the membrane to the bulk water with conformation and both Euler angles restrained, and 5) the free energy change for deformation of the protein in the bulk water with respect to the conformation of the protein in the membrane. Through these simulations, we confirmed that SpoVM prefers NPSLBLs rather than PSSLBLs, indicating by a lower free energy change. Additionally, we revealed that the SpoVM membrane sensing is based on the interplay between the packing of the hydrophilic head groups of the lipids and the packing of the acyl chains of the lipids. Our findings reported in Chapter 5 might be helpful in the development of diagnosis and treatment of diseases associated with protein mislocalization.
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    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.
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    BILAYER MEMBRANE ELECTROSTATICS AND CHARGE-REGULATED MEMBRANE-NANOPARTICLE INTERACTIONS
    (2018) Sinha, Shayandev; Das, Siddhartha; Mechanical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Nanoparticle (NP) driven targeted drug delivery and NP driven imaging of cells, tumors etc. have been one of the most investigated areas in interfacial and biomedical engineering in recent years involving a massive amount interdisciplinary efforts cutting across disciplines like physics, chemistry, material science, biology, pharmaceutics, and engineering. Drug delivery or imaging with the NPs invariably require the NPs to first adhere to the surface of a cell, which is bound by a cell membrane (also known as plasma membrane or PM). All of these processes occur in an electrolyte medium as the fluids present inside and outside the cell have ions inside them. There have been significant amount of studies on adhesion of nanoparticles but until today, there has been very less number of investigations on the role of the ionic environment on such systems of adhesion. The ions present in the intracellular and the extracellular space produce an electric double layer (EDL) on both sides of the PM. The PM is also a semipermeable membrane i.e it does not let all kinds of ions to pass through it. The moieties that it lets to pass through it is completely dependent on the ion channels present across it and such semi-permeable action dictates the ion distribution around the PM, which in turn would regulate the NP-PM interactions. The main aim of this dissertation is to look into the influence of this ionic environment and the role that it can play on adhesion of NPs. In order to look deeply we first look into the electrostatics of the PMs. We develop a continuum model to investigate the role of the ionic environment or the EDL on the electrostatics present across the membrane. This investigation led us to a very important aspect of membrane electrostatics. We found out charge-inversion (CI) like characteristics on the cytosol side (fluids present inside the cell) of the membrane. There has been no previous reports of such CI like characteristics in either the PM electrostatics or more importantly, in a system consisting of only monovalent electrolyte ions (as is the case we consider). In the next step, we looked into the role of the the surface charge density of the membrane and the concentration of the ions in influencing this PM electrostatics. This led to more interesting results. We found out that for biologically relevant conditions and for standard membrane surface charges, there is a possibility of having the location of CI on the surface of the membrane itself. This is a most remarkable result establishing a positive zeta potential on the surface of the negatively charged PM and we explored the phase-space where such situation of opposite signs of membrane zeta potential and membrane surface charge persists. This electrostatics definitely influences various measurable properties of the membrane. One such very important measurable property of a membrane is the membrane capacitance. It has been widely reported that the ionic environment does not influence the capacitance much. However, with exploration of this phase-space through our continuum simulations we were able to pinpoint a domain where the capacitance can be influenced by as much as 15%. This also stems from the fact that the electrostatics of the system is itself very interesting to study under various conditions. We then move on to explore the effect of this electrostatics on the adhesion of NP on the membranes. Most of these adhesive processes occur through the receptor-ligand (R-L) mechanism. Therefore, until and unless a ligand is able to physically influence a receptor and can get bonded to it, the process of adhesion will never begin. The electrostatics can cause a hindrance to this phenomenon. The main reason is the electrostatic osmotic or disjoining pressure, which causes a repulsion between the ligand-bearing NP and the receptor-bearing cell membrane, and forbids the NP to come to significant proximity of the PM for ensuring that the ligands start to interact with the receptors. Through our analysis, we calculated such repulsion and calculated the distance up to which this repulsion remains strong and can overcome the influence of other attractive effects (e.g., van der Waals forces or thermal forces) that drive the NP closer to the PM. We hypothesize that if the length of the ligand-receptor complex is not larger than this distance up to which the electrostatic repulsion effects remain dominant then the process of adhesion will not even begin. Next, we study what is the role of this ionic environment for the case where the NP adhere to the PMs non-specifically. Such non-specific adhesion (NSA) refers to the adhesion of the NP to the PM by actual physical attachment without involving R-L interactions. Understanding such NSA is vital to gauge the side effects of the NP-based drug delivery -- the dug carrying NP will invariably adhere (non-specifically) to the healthy cells causing damages to the healthy cells. Therefore the current practice necessitates uses of those NPs that demonstrate least cytotoxicity post adhesion and internalization in healthy cells. We show that when metallic NPs non-specifically adhere to the PMs, the resulting destruction of the surface charge effects of PMs would lead to a favorable energy change, which in turn drives the NP NSA to even stiffer membranes (e.g., cell membranes rich in cholesterol). Subsequently, we show that one can use biomimetic NPs (namely NPs encapsulated in PM-derived lipid bilayers) to ensure that electrostatic interactions between the biomimetic NPs and the PM can usher in the most coveted scenario where one can simultaneously ensure the promotion of specific adhesion and prevention of NSA. Finally we address the future directions of this work and how this work can start the discussion about the role of other kinds on nanoparticles in drug delivery and therapy.
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    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.