Matching Items (19)
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- All Subjects: Biochemistry
- Creators: Levitus, Marcia
Description
Molecular dynamics (MD) simulations provide a particularly useful approach to understanding conformational change in biomolecular systems. MD simulations provide an atomistic, physics-based description of the motions accessible to biomolecular systems on the pico- to micro-second timescale, yielding important insight into the free energy of the system, the dynamical stability of contacts and the role of correlated motions in directing the motions of the system. In this thesis, I use molecular dynamics simulations to provide molecular mechanisms that rationalize structural, thermodynamic, and mutation data on the interactions between the lac repressor headpiece and its O1 operator DNA as well as the ERK2 protein kinase. I performed molecular dynamics simulations of the lac repressor headpiece - O1 operator complex at the natural angle as well as at under- and overbent angles to assess the factors that determine the natural DNA bending angle. I find both energetic and entropic factors contribute to recognition of the natural angle. At the natural angle the energy of the system is minimized by optimization of protein-DNA contacts and the entropy of the system is maximized by release of water from the protein-DNA interface and decorrelation of protein motions. To identify the mechanism by which mutations lead to auto-activation of ERK2, I performed a series of molecular dynamics simulations of ERK1/2 in various stages of activation as well as the constitutively active Q103A, I84A, L73P and R65S ERK2 mutants. My simulations indicate the importance of domain closure for auto-activation and activity regulation. My results enable me to predict two loss-of-function mutants of ERK2, G83A and Q64C, that have been confirmed in experiments by collaborators. One of the powerful capabilities of MD simulations in biochemistry is the ability to find low free energy pathways that connect and explain disparate structural data on biomolecular systems. An extention of the targeted molecular dynamics technique using constraints on internal coordinates will be presented and evaluated. The method gives good results for the alanine dipeptide, but breaks down when applied to study conformational changes in GroEL and adenylate kinase.
ContributorsBarr, Daniel Alan (Author) / van der Vaart, Arjan (Thesis advisor) / Matyushov, Dmitry (Committee member) / Wolf, George (Committee member) / Shumway, John (Committee member) / Arizona State University (Publisher)
Created2011
Description
Natural products that target the DNA of cancer cells have been an important source of knowledge and understanding in the development of anticancer chemotherapeutic agents. Bleomycin (BLM) exemplifies this class of DNA damaging agent. The ability of BLM to chelate metal ions and effect oxidative damage of the deoxyribose sugar moiety of DNA has been studied extensively for four decades. Here, the study of BLM A5 was conducted using a previously isolated library of hairpin DNAs found to bind strongly to metal free BLM. The ability of BLM to effect single-stranded was then extensively characterized on both the 3′ and 5′-arms of the hairpin DNAs. The strongly bound DNAs were found to be efficient substrates for Fe·BLM A5-mediated cleavage. Surprisingly, the most prevalent site of damage by BLM was found to be a 5′-AT-3′ dinucleotide sequence. This dinucleotide sequence and others generally not cleaved by BLM when examined using arbitrarily chosen DNA substrate were found in examining the library of ten hairpin DNAs. In total, 111 sites of DNA damage were found to be produced by exposure of the hairpin DNA library to Fe·BLM A5. Also, an assay was developed with which to test the propensity of the hairpin DNAs to undergo double stranded DNA damage. Adapting methods previously described by the Povirk laboratory, one hairpin was characterized using this method. The results were in accordance with those previously reported.
ContributorsSegerman, Zachary (Author) / Hecht, Sidney M. (Thesis advisor) / Levitus, Marcia (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2011
Description
Conformational changes in biomolecules often take place on longer timescales than are easily accessible with unbiased molecular dynamics simulations, necessitating the use of enhanced sampling techniques, such as adaptive umbrella sampling. In this technique, the conformational free energy is calculated in terms of a designated set of reaction coordinates. At the same time, estimates of this free energy are subtracted from the potential energy in order to remove free energy barriers and cause conformational changes to take place more rapidly. This dissertation presents applications of adaptive umbrella sampling to a variety of biomolecular systems. The first study investigated the effects of glycosylation in GalNAc2-MM1, an analog of glycosylated macrophage activating factor. It was found that glycosylation destabilizes the protein by increasing the solvent exposure of hydrophobic residues. The second study examined the role of bound calcium ions in promoting the isomerization of a cis peptide bond in the collagen-binding domain of Clostridium histolyticum collagenase. This study determined that the bound calcium ions reduced the barrier to the isomerization of this peptide bond as well as stabilizing the cis conformation thermodynamically, and identified some of the reasons for this. The third study represents the application of GAMUS (Gaussian mixture adaptive umbrella sampling) to on the conformational dynamics of the fluorescent dye Cy3 attached to the 5' end of DNA, and made predictions concerning the affinity of Cy3 for different base pairs, which were subsequently verified experimentally. Finally, the adaptive umbrella sampling method is extended to make use of the roll angle between adjacent base pairs as a reaction coordinate in order to examine the bending both of free DNA and of DNA bound to the archaeal protein Sac7d. It is found that when DNA bends significantly, cations from the surrounding solution congregate on the concave side, which increases the flexibility of the DNA by screening the repulsion between phosphate backbones. The flexibility of DNA on short length scales is compared to the worm-like chain model, and the contribution of cooperativity in DNA bending to protein-DNA binding is assessed.
ContributorsSpiriti, Justin Matthew (Author) / van der Vaart, Arjan (Thesis advisor) / Chizmeshya, Andrew (Thesis advisor) / Matyushov, Dmitry (Committee member) / Fromme, Petra (Committee member) / Arizona State University (Publisher)
Created2011
Description
V(D)J recombination is responsible for generating an enormous repertoire of immunoglobulins and T cell receptors, therefore it is a centerpiece to the formation of the adaptive immune system. The V(D)J recombination process proceeds through two steps, site-specific cleavage at RSS (Recombination Signal Sequence) site mediated by the RAG recombinase (RAG1/2) and the subsequent imprecise resolution of the DNA ends, which is carried out by the ubiquitous non-homologous end joining pathway (NHEJ). The V(D)J recombination reaction is obliged to be tightly controlled under all circumstances, as it involves generations of DNA double strand breaks, which are considered the most dangerous lesion to a cell. Multifaceted regulatory mechanisms have been evolved to create great diversity of the antigen receptor repertoire while ensuring genome stability. The RAG-mediated cleavage reaction is stringently regulated at both the pre-cleavage stage and the post-cleavage stage. Specifically, RAG1/2 first forms a pre-cleavage complex assembled at the boarder of RSS and coding flank, which ensures the appropriate DNA targeting. Subsequently, this complex initiates site-specific cleavage, generating two types of double stranded DNA breaks, hairpin-ended coding ends (HP-CEs) and blunt signal ends (SEs). After the cleavage, RAG1/2 proteins bind and retain the recombination ends to form post-cleavage complexes (PCC), which collaborates with the NHEJ machinery for appropriate transfer of recombination ends to NHEJ for proper end resolution. However, little is known about the molecular basis of this collaboration, partly attributed to the lack of sensitive assays to reveal the interaction of PCC with HP-CEs. Here, for the first time, by using two complementary fluorescence-based techniques, fluorescence anisotropy and fluorescence resonance energy transfer (FRET), I managed to monitor the RAG1/2-catalyzed cleavage reaction in real time, from the pre-cleavage to the post-cleavage stages. By examining the dynamic fluorescence changes during the RAG-mediated cleavage reactions, and by manipulating the reaction conditions, I was able to characterize some fundamental properties of RAG-DNA interactions before and after cleavage. Firstly, Mg2+, known as a physiological cofactor at the excision step, also promotes the HP-CEs retention in the RAG complex after cleavage. Secondly, the structure of pre-cleavage complex may affect the subsequent collaborations with NHEJ for end resolution. Thirdly, the non-core region of RAG2 may have differential influences on the PCC retention of HP-CEs and SEs. Furthermore, I also provide the first evidence of RAG1-mediated regulation of RAG2. Our study provides important insights into the multilayered regulatory mechanisms, in modulating recombination events in developing lymphocytes and paves the way for possible development of detection and diagnotic markers for defective recombination events that are often associated immunodeficiency and/or lymphoid malignancy.
ContributorsWang, Guannan (Author) / Chang, Yung (Thesis advisor) / Levitus, Marcia (Committee member) / Misra, Rajeev (Committee member) / Anderson, Karen (Committee member) / Arizona State University (Publisher)
Created2012
Description
Transient Receptor Potential Vanilloid-1 (TRPV1) is an integral membrane polymodal cation channel involved in various essential biological functions, including thermosensing, thermoregulation, and nociception. Discrete TRPV1 activation modes such as ligand, heat, and proton have been challenging to disentangle. However, dissecting the polymodal nature of TRPV1 is essential for therapeutic development. The human TRPV1 (hTRPV1) voltage-sensing like domain (VSLD; transmembrane helices S1-S4) contains the canonical vanilloid ligand binding site and significantly contributes to thermosensing. Nuclear magnetic resonance (NMR)-detected studies probe the role of the hTRPV1-VSLD in TRPV1 polymodal function. The hTRPV1-VSLD is identified as an allosteric hub for all three primary TRPV1 activation modes and demonstrates plasticity in chemical ligand modulation. The presented results underscore molecular features in the VSLD that dictate TRPV1 function, highlighting important considerations for future therapeutic design.
ContributorsOwens, Aerial M. (Author) / Van Horn, Wade D. (Thesis advisor) / Levitus, Marcia (Committee member) / LaBaer, Joshua (Committee member) / Arizona State University (Publisher)
Created2023
Description
For cold chain tracking systems, precision and versatility across varying time intervals and temperature ranges remain integral to effective application in clinical, commercial, and academic settings. Therefore, while electronic and chemistry/physics based cold chain tracking mechanisms currently exist, both have limitations that affect their application across various biospecimens and commercial products, providing the initiative to develop a time temperature visual indicator system that resolves challenges with current cold chain tracking approaches. As a result, a permanganate/oxalic acid time temperature visual indicator system for cold chain tracking has been proposed. At thawing temperatures, the designed permanganate/oxalic acid reaction system undergoes a pink to colorless transition as permanganate, Mn(VII), is reduced to auto-catalytic Mn(II), while oxalate is oxidized to CO2. Therefore, when properly stored and vitrified or frozen, the proposed visual indicator remains pink, whereas exposure to thawing conditions will result in an eventual, time temperature dependent, designed color transition that characterizes compromised biospecimen integrity. To design visual indicator systems for targeted times at specific temperatures, absorbance spectroscopy was utilized to monitor permanganate kinetic curves by absorbance at 525 nm. As a result, throughout the outlined research, the following aims were demonstrated: (i) Design and functionality of 1x (0.5 mM KMnO4) visual indicator systems across various time intervals at temperatures ranging from 25°C to -20°C, (ii) Design and functionality of high concentration, 5x, visual indicator systems across varying targeted time intervals at temperatures ranging from 25°C to 0°C, (iii) Pre-activation stability and long-term stability of the proposed visual indicator systems.
ContributorsLjungberg, Emil (Author) / Borges, Chad (Thesis advisor) / Levitus, Marcia (Committee member) / Williams, Peter (Committee member) / Arizona State University (Publisher)
Created2024
Description
Since understanding the nature of proteins, it has been a long held belief that protein sequence dictated structure which then determined function. As such, all proteins contained structure and those that did not must not serve a purpose. For the last 25 years, scientists have begun to understand that disordered proteins, lacking structure, did not lack function. Their unique ability to undergo liquid-liquid phase separation served a cellular purpose, most involving nucleic acids. As more is uncovered, these unique proteins are being used to build new systems. Phase separated disordered proteins were used to design a functional organelle using the enzyme horseradish peroxidase and its chromatic substrate ABTS. Upon doing so, it was discovered that disordered proteins are highly susceptible to chemical modification through radical reactions with tyrosine. The increased frequency of tyrosine in disordered proteins provides multiple sites of conjugation by the ABTS radical and other substrates. These modifications then alter the physical properties of the proteins. The phase separated system was also incorporated with shell proteins from bacterial microcompartments in an attempt to limit access to the droplets. Through expression with truncations of the disordered sequence, shell proteins were able to interact with the droplets. Despite the appearance of complete coatings, they were found to be permeable to their surroundings, though much more stable than uncoated droplets. Just as disordered proteins are considered outside the traditional structures, so too are many students entering higher education. Non-traditional students are becoming more prevalent in the undergraduate population, though they are woefully underrepresented in the natural sciences. The benefits these students bring to their programs is highlighted and the circumstances that drive them away from STEM is explored. Non-traditional students contribute to the diversity of the scientific population, though many pursue education in non-STEM fields. To support these students, focus is put on andragogy (the teaching of adults), rather than pedagogy (the teaching of children). Non-traditional students face isolation and discrimination that is not being addressed by higher education institutions, hindering their ability to succeed. Through infrastructure designed for adult learners, STEM fields can be diversified in non-traditional ways.
ContributorsCostantino, Michele (Author) / Ghirlanda, Giovanna (Thesis advisor) / Mills, Jeremy (Committee member) / Matyushov, Dmitry (Committee member) / Arizona State University (Publisher)
Created2024
Description
Rubisco activase (Rca) from higher plants is a stromal ATPase essential for reactivating Rubiscos rendered catalytically inactive by endogenous inhibitors. Rca’s functional state is thought to consist of ring-like hexameric assemblies, similar to other members of the AAA+ protein superfamily. However, unlike other members, it does not form obligate hexamers and is quite polydisperse in solution, making elucidation of its self-association pathway challenging. This polydispersity also makes interpretation of traditional biochemical approaches difficult, prompting use of a fluorescence-based technique (Fluorescence Correlation Spectroscopy) to investigate the relationship between quaternary structure and function. Like cotton β Rca, tobacco β Rca appears to assemble in a step-wise and nucleotide-dependent manner. Incubation in varying nucleotides appears to alter the equilibrium between varying oligomers, either promoting or minimizing the formation of larger oligomers. High concentrations of ADP seem to favor continuous assembly towards larger oligomers, while assembly in the presence of ATP-yS (an ATP analog) appears to halt continuous assembly in favor of hexameric species. In contrast, assembly in the “Active ATP Turnover” condition (a mixture of ATP and ADP) appears to favor an almost equal distribution of tetramer and hexamer, which when compared with ATPase activity, shows great alignment with maximum activity in the low µM range. Despite this alignment, the decrease in ATPase activity does not follow any particular oligomer, but rather decreases with increasing aggregation, suggesting that assembly dynamics may regulate ATPase activity, rather than the formation/disappearance of one specific oligomer. Work presented here also indicates that all oligomers larger than hexamers are catalytically inactive, thus providing support for the idea that they may serve as a storage mechanism to minimize wasteful hydrolysis. These findings are also supported by assembly work carried out on an Assembly Mutant (R294V), known for favoring formation of closed-ring hexamers. Similar assembly studies were carried out on spinach Rca, however, due to its aggregation propensity, FCS results were more difficult to interpret. Based on these findings, one could argue that assembly dynamics are essential for Rca function, both in ATPase and in regulation of Rubisco carboxylation activity, thus providing a rational for Rca’s high degree of polydispersity.
ContributorsSerban, Andrew J (Author) / Wachter, Rebekka M. (Thesis advisor) / Levitus, Marcia (Thesis advisor) / Redding, Kevin E (Committee member) / Van Horn, Wade D (Committee member) / Arizona State University (Publisher)
Created2018
Temperature and polarizability effects on electron transfer in biology and artificial photosynthesis
Description
This study aims to address the deficiencies of the Marcus model of electron transfer
(ET) and then provide modifications to the model. A confirmation of the inverted energy
gap law, which is the cleanest verification so far, is presented for donor-acceptor complexes.
In addition to the macroscopic properties of the solvent, the physical properties of the solvent
are incorporated in the model via the microscopic solvation model. For the molecules
studied in this dissertation, the rate constant first increases with cooling, in contrast to the
prediction of the Arrhenius law, and then decreases at lower temperatures. Additionally,
the polarizability of solute, which was not considered in the original Marcus theory, is included
by the Q-model of ET. Through accounting for the polarizability of the reactants, the
Q-model offers an important design principle for achieving high performance solar energy
conversion materials. By means of the analytical Q-model of ET, it is shown that including
molecular polarizability of C60 affects the reorganization energy and the activation barrier
of ET reaction.
The theory and Electrochemistry of Ferredoxin and Cytochrome c are also investigated.
By providing a new formulation for reaction reorganization energy, a long-standing disconnect
between the results of atomistic simulations and cyclic voltametery experiments is
resolved. The significant role of polarizability of enzymes in reducing the activation energy
of ET is discussed. The binding/unbinding of waters to the active site of Ferredoxin leads
to non-Gaussian statistics of energy gap and result in a smaller activation energy of ET.
Furthermore, the dielectric constant of water at the interface of neutral and charged
C60 is studied. The dielectric constant is found to be in the range of 10 to 22 which is
remarkably smaller compared to bulk water( 80). Moreover, the interfacial structural
crossover and hydration thermodynamic of charged C60 in water is studied. Increasing the
charge of the C60 molecule result in a dramatic structural transition in the hydration shell,
which lead to increase in the population of dangling O-H bonds at the interface.
(ET) and then provide modifications to the model. A confirmation of the inverted energy
gap law, which is the cleanest verification so far, is presented for donor-acceptor complexes.
In addition to the macroscopic properties of the solvent, the physical properties of the solvent
are incorporated in the model via the microscopic solvation model. For the molecules
studied in this dissertation, the rate constant first increases with cooling, in contrast to the
prediction of the Arrhenius law, and then decreases at lower temperatures. Additionally,
the polarizability of solute, which was not considered in the original Marcus theory, is included
by the Q-model of ET. Through accounting for the polarizability of the reactants, the
Q-model offers an important design principle for achieving high performance solar energy
conversion materials. By means of the analytical Q-model of ET, it is shown that including
molecular polarizability of C60 affects the reorganization energy and the activation barrier
of ET reaction.
The theory and Electrochemistry of Ferredoxin and Cytochrome c are also investigated.
By providing a new formulation for reaction reorganization energy, a long-standing disconnect
between the results of atomistic simulations and cyclic voltametery experiments is
resolved. The significant role of polarizability of enzymes in reducing the activation energy
of ET is discussed. The binding/unbinding of waters to the active site of Ferredoxin leads
to non-Gaussian statistics of energy gap and result in a smaller activation energy of ET.
Furthermore, the dielectric constant of water at the interface of neutral and charged
C60 is studied. The dielectric constant is found to be in the range of 10 to 22 which is
remarkably smaller compared to bulk water( 80). Moreover, the interfacial structural
crossover and hydration thermodynamic of charged C60 in water is studied. Increasing the
charge of the C60 molecule result in a dramatic structural transition in the hydration shell,
which lead to increase in the population of dangling O-H bonds at the interface.
ContributorsWaskasi, Morteza M (Author) / Matyushov, Dmitry (Thesis advisor) / Richert, Ranko (Committee member) / Heyden, Matthias (Committee member) / Beckstein, Oliver (Committee member) / Arizona State University (Publisher)
Created2019
Description
Rapid and reliable separation and analysis of proteins require powerful analytical methods. The analysis of proteins becomes especially challenging when only small sample volumes are available, concomitantly with low concentrations of proteins. Time critical situations pose additional challenges. Due to these challenges, conventional macro-scale separation techniques reach their limitations. While microfluidic devices require only pL-nL sample volumes, they offer several advantages such as speed, efficiency, and high throughput. This work elucidates the capability to manipulate proteins in a rapid and reliable manner with a novel migration technique, namely dielectrophoresis (DEP). Since protein analysis can often be achieved through a combination of orthogonal techniques, adding DEP as a gradient technique to the portfolio of protein manipulation methods can extend and improve combinatorial approaches. To this aim, microfluidic devices tailored with integrated insulating obstacles were fabricated to create inhomogeneous electric fields evoking insulator-based DEP (iDEP). A main focus of this work was the development of pre-concentration devices where topological micropost arrays are fabricated using standard photo- and soft lithographic techniques. With these devices, positive DEP-driven streaming of proteins was demonstrated for the first time using immunoglobulin G (IgG) and bovine serum albumin. Experimentally observed iDEP concentrations of both proteins were in excellent agreement with positive DEP concentration profiles obtained by numerical simulations. Moreover, the micropost iDEP devices were improved by introducing nano-constrictions with focused ion beam milling with which numerical simulations suggested enhancement of the DEP effect, leading to a 12-fold increase in concentration of IgG. Additionally, concentration of β-galactosidase was observed, which seems to occur due to an interplay of negative DEP, electroosmosis, electrokinesis, diffusion, and ion concentration polarization. A detailed study was performed to investigate factors influencing protein DEP under DC conditions, including electroosmosis, electrophoresis, and Joule heating. Specifically, temperature rise within the iDEP device due to Joule heating was measured experimentally with spatial and temporal resolution by employing the thermosensitive dye Rhodamine B. Unlike DNA and cells, protein DEP behavior is not well understood to date. Therefore, this detailed study of protein DEP provides novel information to eventually optimize this protein migration method for pre-concentration, separation, and fractionation.
ContributorsNakano, Asuka (Author) / Ros, Alexandra (Thesis advisor) / Hayes, Mark (Committee member) / Levitus, Marcia (Committee member) / Arizona State University (Publisher)
Created2014