Browsing by Subject "Biopolymers"
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Item Biopolymer analysis by electrospray ionization and tandem mass spectrometry(2004) Keller, Karin Mia; Brodbelt, Jennifer S.Electrospray ionization was used in conjunction with Fourier-transform ion cyclotron resonance (FTICR) mass spectrometry and quadrupole ion trap (QIT) mass spectrometry to study protein and oligonucleotide ions in vacuo. The results help to identify effective strategies for mass spectral analysis of these macromolecules and provide new insight on their gas-phase behavior. Tandem mass spectrometry experiments were conducted to evaluate different ion activation methods for biopolymer sequencing. Multipole storage-assisted dissociation (MSAD) and sustained off-resonance irradiation collision-activated dissociation (SORI- CAD) were compared for protein analysis in FTICR instrumentation, and infrared multiphoton dissociation (IRMPD) and collisonal activated dissociation (CAD) were compared for oligonucleotide analysis in QIT instrumentation. In both studies, the differences in the observed fragmentation patterns were noted and the underlying reasons for these differences were identified. The relative utility of MSAD vs. SORI-CAD and IRMPD vs. CAD were assessed in terms of their ability to produce diagnostic information that could be used to identify the protein or oligonucleotide under study. Tandem mass spectrometry was also employed to study the dissociation patterns of both DNA/metal and DNA/drug complexes. The preferred fragmentation pathways exhibited by these species were observed to vary with the initial charge state of the precursor. The effect of the oligonucleotide sequence, the identity of the metal ion, and the identity of the drug on these pathways was established and (where possible) interpreted in terms of the specific non-covalent bonding patterns present in the parent complexes. Finally, electrospray ionization was evaluated as a tool for screening molecular recognition in nucleic acid aptamer/small molecule interactions. Gas-phase data for binding stoichiometry and relative binding affinity were compared with the known solution behavior for a series of well-characterized case studies. Any observed discrepancies were rationalized in terms of ligand structure and/or the nature of the intermolecular ligand/aptamer interactions.Item Conformational dynamics of an unfolded biopolymer : theory and simulation(2012-12) Cheng, Ryan; Makarov, Dmitrii E.; Florin, Ernst-Ludwig; Elber, Ron; Henkelman, Graeme; Keatinge-Clay, Adrian T.The conformational dynamics of an unfolded biopolymer such as a polypeptide or DNA has attracted a significant amount of attention in the context of protein folding and the design of biomimetic technologies. To this end, recent advances in single-molecule experiments have allowed for biomolecules to be probed with an unprecedented level of detail, shedding light on their dynamics. Motivated by the need to interpret experimental data and to help guide future studies, we use concepts from polymer physics, computer simulations, and experimental data to study the timescales in which an unfolded biopolymer undergoes conformational rearrangement. First, we examine the end-to-end loop formation time in the experimentally relevant scenario where the dynamics are probed using a fluorescence probe and quencher. We show that the loop formation time in the experimentally relevant case is quantitatively dissimilar from the predictions of previous theoretical studies that neglect the quenching kinetics, which are often used to interpret experimental data. We additionally find that the loop formation times can be re-casted in a simple, universal dependence that is characteristic of random-coils. Furthermore, deviations from this universal dependence can be used as a sensitive tool for detecting structural order in unfolded biopolymers. We also consider a surface-tethered polymer chain and investigate the rate of a reaction between the free end and the surface. We explore this rate in the reaction-controlled limit and the diffusion-controlled limit, providing evidence for near-universal dependences of the rate in the respective limits. Next, we examine the transit time of end-to-end loop formation in a case study. We find that approximating the end-to-end dynamics as diffusion in a 1D potential of mean force fails dramatically to describe the transit time. Furthermore, we find that the transit time is uninfluenced by the average entropic force imposed by the polymer chain and is well described by a simple free-diffusion model. Finally, we explore the role of internal friction in the dynamics of an unfolded protein. Using simple polymer models that incorporate internal friction as an adjustable free parameter, we mimic typical single-molecule experiments that probe the unfolded state dynamics and make several experimentally verifiable predictions.Item Investigation of immobilized biopolymers for metal binding(2004) Malachowski, Lisa Lyn; Holcombe, James A.This research focuses on the utility of immobilized poly amino acids for metal remediation and preconcentration. The biohomopolymer poly-L-histidine (PLHis) was immobilized onto controlled pore glass (CPG) and its metal binding capabilities evaluated through the use of a flow injection analysis - flame atomic absorption system (FIA-FAAS). The metal binding capability of PLHis-CPG was determined through the analysis of the generated breakthrough curves. The polymer likely coordinates cationic metals through the imidazole side chain (pKa ≈ 6) present on each histidine residue with both strong and weak binding sites for Cu2+, Cd2+, Co2+, and Ni2+. It has also been shown that the protonated imidazole side chain present in acidic conditions is capable of binding metal oxyanions such as chromates, arsenates, and selenites; although oxyanion binding currently exhibits interferences from competing anions in solution, such as sulfate and nitrate. Poly-L-Aspartic Acid (PLAsp) and Poly-L-Glutamic Acid (PLGlu) were also individually immobilized onto controlled pore glass (CPG) and compared using their metal binding capabilities. Elemental combustion analysis was used to yield polymer coverage approximations. Formation constants and site capacities of both polymers for Cd2+ were determined through equilibrium and breakthrough studies. Additionally, the metal selectivity of PLAsp and PLGlu was evaluated when breakthrough curves were run with several metals present in solution at one time. Both polymers exhibited similar binding trends and binding strengths for all of the metals studied. This likely reflects the absence of a predetermined tertiary structure of the polymers on the surface and the relatively high residue-per-metal ratio (~20:1), which places less stringent requirements on the steric hindrance between the side chains and the resultant ìwrappingî of the peptide around the metal. Initial attempts at determining formation constants of PLAsp and PLGlu through competitive binding experiments with either EDTA or oxalate present were unsuccessful due to complications caused by the current immobilization procedure. Therefore, alternate immobilization procedures were investigated utilizing an epoxide linker. These methods eliminate the formation of an amine functionality on the surface. Additionally, a combinatorial approach was used in an attempt to elucidate an optimal copolymer primary structure for successful binding of a target metal. This approach included screening the library for successful binding with micro x-ray fluorescence (MXRF) and obtaining the sequence of the successful copolymer through Edman Degradation. A considerable amount of the metal binding experiments conducted in this research used the analysis of breakthrough curves generated through flow injection-flame atomic absorption spectrometry. Solution flow rate is a critical parameter in breakthrough analysis. Due to the absence of an inexpensive, on-line flow meter for flow injection analysis systems, an electronic flow meter was constructed to measure the flow rate during the FIAAS measurements. Thus, flow rates can be measured while collecting breakthrough data, and continuous monitoring of flow rates is possible.Item Physical characterization of bacterial biofilm polymer networks to determine the role of mechanics in infection and treatment(2018-11-29) Kovach, Kristin N.; Gordon, Vernita Diane; Florin, Ernst-Ludwig; Marder, Michael P; Smyth, Hugh D; Lynd, NathanielBiofilms are communities of microorganisms that produce a matrix of extracellular polymers to surround and protect themselves from external forces in their environment. This communal lifestyle is incredibly beneficial for microorganism survival. Characterization of the mechanical properties of biofilms is a vital and understudied component of fully understanding these biological systems. In this dissertation, we break down the mechanical response of the Pseudomonas aeruginosa biofilm by its constituent polymers. These bacteria produce unique polymers to resist a variety of stresses. In the first part of this dissertation, using oscillatory bulk rheology, we characterize the viscoelasticity of biofilm polymer networks. Using genetically manipulated lab strains of P. aeruginosa, we isolate the mechanical response of each polymer by analyzing biofilms comprised primarily of one type of polymer. We find that the polymers have unique mechanical properties: some increase the yield strain and others increase elastic modulus. In strains of P. aeruginosa isolated from chronic infections, we find that the bacteria evolve to increase production of polymers that maximize the energy required to yield the matrix. In the second part of this dissertation, we work to mechanically compromise each of the polymers in the matrix. By attacking different matrix components, we learn more about the structural properties that give rise to mechanical properties as well as identify the most promising therapeutic treatments to break down biofilm infections. We find that specific enzymes are useful for decreasing yield strain of biofilms and increasing the diffusivity of the matrix. Decrease in yield strain means that biofilms will take less deformation before losing mechanical integrity, and the increase in matrix diffusivity means that current treatments such as antibiotics are more effective as the antibiotics can more easily reach the bacteria in the matrix to effectively kill them. This dissertation treats biofilms as polymer networks, divorcing the analysis from biological responses, in an attempt to well-characterize the understudied mechanical properties of biofilms. By approaching these systems from a physical standpoint, we are able to learn more about biofilms by breaking the mechanical response into constituent components, as well as learn about how enzymatic treatments alter biofilm properties.Item Simulation studies of biopolymers under spatial and topological constraints(2008-05) Huang, Lei, 1978-; Makarov, Dmitrii E.The translocation of a biopolymer through a narrow pore exists in universal cellular processes, such as the translocations of nascent proteins through ribosome and the degradation of protein by ATP-dependent proteases. However, the molecular details of these translocation processes remain unclear. Using computer simulations we study the translocations of a ubiquitin-like protein into a pore. It shows that the mechanism of co-translocational unfolding of proteins through pores depends on the pore diameter, the magnitude of pulling force and on whether the force is applied at the N- or the C-terminus. Translocation dynamics depends on whether or not polymer reversal is likely to occur during translocation. Although it is of interest to compare the timescale of polymer translocation and reversal, there are currently no theories available to estimate the timescale of polymer reversal inside a pore. With computer simulations and approximate theories, we show how the polymer reversal depends on the pore size, r, and the chain length, N. We find that one-dimensional transition state theory (TST) using the polymer extension along the pore axis as a reaction coordinate adequately predicts the exponentially strong dependence of the reversal rate on r and N. Additionally, we find that the transmission factor (the ratio of the exact rate and the TST rate) has a much weaker power law dependence on r and N. Finite-size effects are observed even for chains with several hundred monomers. If metastable states are separated by high energy-barriers, transitions between them will be rare events. Instead of calculating the relative energy by studying those transitions, we can calculate absolute free energy separately to compare their relative stability. We proposed a method for calculating absolute free energy from Monte Carlo or molecular dynamics data. Additionally, the diffusion of a knot in a tensioned polymer is studied using simulations and it can be modeled as a one-dimensional free diffusion problem. The diffusion coefficient is determined by the number of monomers involved in a knot and its tension dependence shows a maximum due to two dominating factors: the friction from solvents and “local friction” from interactions among monomers in a compact knot.Item Synthesis and characterization of short-chain peptides for use in metal remediation and preconcentration(2006) Stair, Jacqueline Leslie; Holcombe, James A.Designing materials for metal remediation and preconcentration based on naturally occurring metal binding proteins has become of growing interest due to their inherent selective and strong binding, ease of synthesis and available amino acid building blocks, and environmental innocuity. One approach is through the use of immobilized synthetic biohomopolymers which can provide the selectivity based on the amino acid side chain moiety with strong binding, easy on-demand release, and reusability. An attempt to increase metal binding selectivity of these biohomopolymers was done though cross-linking at specific locations as to effectively “lock” in place the preferential binding cavity for a particular metal. The cross-linking of these materials resulted in decreased metal capacities with little to no increases in the targeted metal selectivity. This was likely due to the loss of bound metal during cross-linking and to a lack of rigidity in the overall cross-linked polymer. Short composite peptides synthesized on a commercially available resin, TentaGel were also examined as a means to increase metal selectivity. These peptides showed surprisingly high metal binding capacities, strong binding, and residue per metal binding ratios which were an order of magnitude better than results previously reported for longer chain poly-amino acids (50 – 70 residues) attached to porous glass supports. Metal binding selectivity’s were altered by changing only one amino acid and metal release under acidic conditions was surprisingly rapid for these shorter peptides. As a result of these findings, the metal binding and conformational changes between TentaGel immobilized short and long peptide chains during metal binding and release were monitored using Raman microscopy. These results indicated that metal release occurred via conformational changes in addition to proton displacement. Lastly, a method to screen multi-metal binding capabilities of combinatorial peptide libraries was developed using electrothermal vaporization inductively coupled plasma mass spectrometry (ETV-ICP-MS). With the exception of metals that are bound tightly to the peptide, acid stripping of the metals in a single bead into a small volume appears as a viable quantitative analytical approach when using this method with instrumental precisions of better than ±10% for most metals when larger polymer beads (~250 µm) were employed.