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University of Colorado
1.
Edelmaier, Christopher.
Computational Modeling of Mitosis in Fission Yeast.
Degree: PhD, 2018, University of Colorado
URL: https://scholar.colorado.edu/phys_gradetds/237 
► Mitosis ensures the proper segregation of chromosomes into daughter cells, which is accomplished by the mitotic spindle. During fission yeast mitosis, chromosomes establish bi-orientation as…
(more)
▼ Mitosis ensures the proper segregation of chromosomes into daughter cells, which is accomplished by the mitotic spindle. During fission yeast mitosis, chromosomes establish bi-orientation as the bipolar spindle assembles, meaning that sister kinetochores become attached to microtubules whose growth was initiated by the two sister poles. This process includes mechanisms that correct erroneous attachments made by the kinetochores during the attachment process. This thesis presents a 3D physical model of spindle assembly in a Brownian dynamics-kinetic Monte Carlo simulation framework and a realistic description of the physics of microtubule, kinetochore, and chromosome dynamics, in order to interrogate the dynamics and mechanisms of chromosome bi-orientation and error correction. We have added chromosomes to our previous physical model of spindle assembly, which included microtubules, a spherical nuclear envelope, motor proteins, crosslinking proteins, and spindle pole bodies (centrosomes). In this work, we have explored the mechanical properties of kinetochores and their interactions with microtubules that achieve amphitelic spindle attachments at high frequency. A minimal physical model yields simulations that generate chromosome attachment errors, but resolves them, much as normal chromosomes do.
Advisors/Committee Members: Meredith D. Betterton, Matthew A. Glaser, Loren Hough, Thomas Perkins, Richard McIntosh.
Subjects/Keywords: computational; fission; mitosis; nonequilibrium; yeast; Biophysics; Cellular and Molecular Physiology; Statistical, Nonlinear, and Soft Matter Physics
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APA (6th Edition):
Edelmaier, C. (2018). Computational Modeling of Mitosis in Fission Yeast. (Doctoral Dissertation). University of Colorado. Retrieved from https://scholar.colorado.edu/phys_gradetds/237
Chicago Manual of Style (16th Edition):
Edelmaier, Christopher. “Computational Modeling of Mitosis in Fission Yeast.” 2018. Doctoral Dissertation, University of Colorado. Accessed April 14, 2021.
https://scholar.colorado.edu/phys_gradetds/237.
MLA Handbook (7th Edition):
Edelmaier, Christopher. “Computational Modeling of Mitosis in Fission Yeast.” 2018. Web. 14 Apr 2021.
Vancouver:
Edelmaier C. Computational Modeling of Mitosis in Fission Yeast. [Internet] [Doctoral dissertation]. University of Colorado; 2018. [cited 2021 Apr 14].
Available from: https://scholar.colorado.edu/phys_gradetds/237.
Council of Science Editors:
Edelmaier C. Computational Modeling of Mitosis in Fission Yeast. [Doctoral Dissertation]. University of Colorado; 2018. Available from: https://scholar.colorado.edu/phys_gradetds/237
University of Colorado
2.
Fiedler, Brett.
Time-Resolved Fluorescence Techniques for the Development and Characterization of Genetically-Encoded Biosensors.
Degree: PhD, Chemistry & Biochemistry, 2017, University of Colorado
URL: https://scholar.colorado.edu/jila_gradetds/2
► Fluorescent biosensors are important measurement tools for in vivo quantification of pH, concentrations of metal ions and other analytes, and physical parameters such as…
(more)
▼ Fluorescent biosensors are important measurement tools for
in vivo quantification of pH, concentrations of metal ions and other analytes, and physical parameters such as membrane potential. Both the development of these sensors and their implementation in examining cellular heterogeneity requires technology for measuring and sorting cells based on the fluorescence levels before and after chemical or physical perturbations. We developed a droplet microfluidic platform for the screening and separation of cell populations on the basis of the
in vivo response of expressed fluorescence-based biosensors after addition of an exogenous analyte. Screening with this instrument reveals increased heterogeneity in an array of targeted Zn
2+ biosensorsin HeLa cells that helps shed light on the complexities of these sensors in different chemical environments. Subsequently, the instrument is used to screen and assess diversity in a number of HeLa-cell based genetic linker libraries for a family of genetically-encoded Zn
2+ sensors. Progress on sensor characterization is made using time-resolved fluorescence techniques to advance a deeper molecular understanding of these sensors to guide further development.
Advisors/Committee Members: Ralph Jimenez, Amy Palmer, Thomas Perkins, Loren Hough, Robert Parson.
Subjects/Keywords: fluorescence; fret; fluorescent protein; biosensor; microfluidic; cell sorting; Analytical Chemistry; Biophysics; Physical Chemistry
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APA (6th Edition):
Fiedler, B. (2017). Time-Resolved Fluorescence Techniques for the Development and Characterization of Genetically-Encoded Biosensors. (Doctoral Dissertation). University of Colorado. Retrieved from https://scholar.colorado.edu/jila_gradetds/2
Chicago Manual of Style (16th Edition):
Fiedler, Brett. “Time-Resolved Fluorescence Techniques for the Development and Characterization of Genetically-Encoded Biosensors.” 2017. Doctoral Dissertation, University of Colorado. Accessed April 14, 2021.
https://scholar.colorado.edu/jila_gradetds/2.
MLA Handbook (7th Edition):
Fiedler, Brett. “Time-Resolved Fluorescence Techniques for the Development and Characterization of Genetically-Encoded Biosensors.” 2017. Web. 14 Apr 2021.
Vancouver:
Fiedler B. Time-Resolved Fluorescence Techniques for the Development and Characterization of Genetically-Encoded Biosensors. [Internet] [Doctoral dissertation]. University of Colorado; 2017. [cited 2021 Apr 14].
Available from: https://scholar.colorado.edu/jila_gradetds/2.
Council of Science Editors:
Fiedler B. Time-Resolved Fluorescence Techniques for the Development and Characterization of Genetically-Encoded Biosensors. [Doctoral Dissertation]. University of Colorado; 2017. Available from: https://scholar.colorado.edu/jila_gradetds/2
University of Colorado
3.
Hunt, Sabrina Robin.
Structure Determination of Vascular Endothelial Growth Factor Heparin-Binding Domain in Complex with a Dna Aptamer.
Degree: PhD, Chemistry & Biochemistry, 2017, University of Colorado
URL: https://scholar.colorado.edu/chem_gradetds/227
► Vascular endothelial growth factor is a cytokine that is required for the maintenance of healthy vasculature, and it is responsible for a variety of pathogenic…
(more)
▼ Vascular endothelial growth factor is a cytokine that is required for the maintenance of healthy vasculature, and it is responsible for a variety of pathogenic conditions, such as age-related macular degeneration and tumor growth in numerous cancers. As an important therapeutic target, vascular endothelial growth factor has been the focus of many in vitro selection strategies to identify aptamers that bind the protein with high affinity and specificity. One of these is a 25-nucleotide DNA aptamer that targets the heparin-binding domain (HBD) of vascular endothelial growth factor. The work presented here focuses on determining the solution structure of the HBD-aptamer complex using NMR. The structure was determined using molecular dynamics simulations restrained with experimental NMR data. The structure of the complex is displayed as an ensemble of the 10 lowest energy structures with a heavy atom RMSD of 2.0 ± 0.8 Å for the well-defined core of the complex. The HBD has two subdomains that retain a similar backbone conformation between the free and bound states, but the orientation of the two subdomains relative to one another changes. The aptamer forms two Watson-Crick base pairs and two tandem base triples that are proximal to a pyrimidine-rich triloop. The 5’ and 3’ ends of the aptamer base pair and form a tetraloop-like structure. The Watson-Crick base pairs and several unpaired nucleotides form the binding interface that spans both subdomains of the HBD. The structure presented here contributes to the larger goal of better understanding the molecular recognition between aptamers and their targets.
Advisors/Committee Members: Arthur Pardi, Robert T. Batey, Loren Hough, Marcelo C. Sousa, Deborah S. Wuttke.
Subjects/Keywords: Aptamer; NMR; Structure; Vascular endothelial growth factor; VEGF; Biochemistry
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APA (6th Edition):
Hunt, S. R. (2017). Structure Determination of Vascular Endothelial Growth Factor Heparin-Binding Domain in Complex with a Dna Aptamer. (Doctoral Dissertation). University of Colorado. Retrieved from https://scholar.colorado.edu/chem_gradetds/227
Chicago Manual of Style (16th Edition):
Hunt, Sabrina Robin. “Structure Determination of Vascular Endothelial Growth Factor Heparin-Binding Domain in Complex with a Dna Aptamer.” 2017. Doctoral Dissertation, University of Colorado. Accessed April 14, 2021.
https://scholar.colorado.edu/chem_gradetds/227.
MLA Handbook (7th Edition):
Hunt, Sabrina Robin. “Structure Determination of Vascular Endothelial Growth Factor Heparin-Binding Domain in Complex with a Dna Aptamer.” 2017. Web. 14 Apr 2021.
Vancouver:
Hunt SR. Structure Determination of Vascular Endothelial Growth Factor Heparin-Binding Domain in Complex with a Dna Aptamer. [Internet] [Doctoral dissertation]. University of Colorado; 2017. [cited 2021 Apr 14].
Available from: https://scholar.colorado.edu/chem_gradetds/227.
Council of Science Editors:
Hunt SR. Structure Determination of Vascular Endothelial Growth Factor Heparin-Binding Domain in Complex with a Dna Aptamer. [Doctoral Dissertation]. University of Colorado; 2017. Available from: https://scholar.colorado.edu/chem_gradetds/227
University of Colorado
4.
Johnson, Laura.
Probing the Dynamics of Red Fluorescent Protein Variants using NMR Spectroscopy: How Directed Development Affected Protein Dynamics.
Degree: PhD, Chemistry & Biochemistry, 2013, University of Colorado
URL: https://scholar.colorado.edu/chem_gradetds/135
► Fluorescent proteins are commonly used genetically encodable tools for probing intracellular events in real time. Red fluorescent proteins (RFPs) are particularly useful because scattering…
(more)
▼ Fluorescent proteins are commonly used genetically encodable tools for probing intracellular events in real time. Red fluorescent proteins (RFPs) are particularly useful because scattering and absorption are reduced in cells excited at longer wavelengths. Development of RFPs with increased quantum yield and wavelength of emission, among other photophysical properties, is an active area of research; but the optimization of one property is often accompanied by other deleterious effects. This work explores how differences in the photophysical properties of various RFPs may arise from changes in protein dynamics as a product of protein directed development. NMR relaxation experiments were used to probe ps-ns dynamics for the backbone amides, as well as the chromophore tyrosine CβH of mCherry, mRaspberry and mRojoB, three closely related monomeric RFPs with different quantum yields. Results indicate restricted dynamics in the backbone amides and the chromophore tyrosine CβH of the three monomeric RFPs tested. The results of NMR relaxation dispersion experiments suggested differences in &mus-ms timescale dynamics in the backbone amides and δ, δ, and γ methyls of isoleucine, leucine and valine residues, respectively, among the same three RFPs. Hydrogen-deuterium exchange experiments were used to show that tdTomato, a dimeric RFP with a high quantum yield, has a higher propensity for local unfolding compared with monomeric mCherry and mRojoB, which have substantially lower quantum yields suggesting a small degree of local unfolding does not abolish fluorescence quantum yield in FPs. Differences in the temperature dependence of fluorescence and secondary structure were observed using fluorescence and CD spectroscopy supporting the idea that directed development affected global stability. How the directed development of RFPs with optimized photophysical properties affects protein dynamics is discussed.
Advisors/Committee Members: Arthur Pardi, Deborah Wuttke, Amy Palmer, Loren Hough, Jim Goodrich.
Subjects/Keywords: Development; Dynamics; NMR Spectroscopy; Quantum Yield; Red Fluorescent Protein; Biochemistry
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APA (6th Edition):
Johnson, L. (2013). Probing the Dynamics of Red Fluorescent Protein Variants using NMR Spectroscopy: How Directed Development Affected Protein Dynamics. (Doctoral Dissertation). University of Colorado. Retrieved from https://scholar.colorado.edu/chem_gradetds/135
Chicago Manual of Style (16th Edition):
Johnson, Laura. “Probing the Dynamics of Red Fluorescent Protein Variants using NMR Spectroscopy: How Directed Development Affected Protein Dynamics.” 2013. Doctoral Dissertation, University of Colorado. Accessed April 14, 2021.
https://scholar.colorado.edu/chem_gradetds/135.
MLA Handbook (7th Edition):
Johnson, Laura. “Probing the Dynamics of Red Fluorescent Protein Variants using NMR Spectroscopy: How Directed Development Affected Protein Dynamics.” 2013. Web. 14 Apr 2021.
Vancouver:
Johnson L. Probing the Dynamics of Red Fluorescent Protein Variants using NMR Spectroscopy: How Directed Development Affected Protein Dynamics. [Internet] [Doctoral dissertation]. University of Colorado; 2013. [cited 2021 Apr 14].
Available from: https://scholar.colorado.edu/chem_gradetds/135.
Council of Science Editors:
Johnson L. Probing the Dynamics of Red Fluorescent Protein Variants using NMR Spectroscopy: How Directed Development Affected Protein Dynamics. [Doctoral Dissertation]. University of Colorado; 2013. Available from: https://scholar.colorado.edu/chem_gradetds/135
University of Colorado
5.
Perkins, Russell James.
Beyond Hydrophobicity: Aqueous Interfaces, Interactions and Reactions.
Degree: PhD, 2018, University of Colorado
URL: https://scholar.colorado.edu/chem_gradetds/273
► Many important chemical reactions from all branches of chemistry occur with water as a solvent. Furthermore, in environmental chemistry, biochemistry, and synthetic chemistry, key…
(more)
▼ Many important chemical reactions from all branches of chemistry occur with water as a solvent. Furthermore, in environmental chemistry, biochemistry, and synthetic chemistry, key reactions occur in heterogeneous aqueous systems, where interfacial effects are particularly important. Despite the importance of aqueous environments and the tremendous amount of work done to study them, there are aspects that require further explanation and remain controversial. I have performed experimental studies to help elucidate the fundamental characteristics of aqueous systems, while examining specific phenomena across several fields. The genetic disorder phenylketonuria (PKU) can result in increased levels of the aromatic amino acid phenylalanine in human serum. Much of my work has focused on the driving forces behind partitioning of aromatic small molecules, including phenylalanine, into air-water or membrane-water interfacial regions, and the consequences of partitioning on interfacial properties. Drastically different behaviors for structurally similar aromatic molecules are observed, differences that cannot be explained by hydrophobic effects. These observations can be explained, however, through the development of a more detailed picture of interactions and partitioning, including the formation of interfacial aggregates. For phenylalanine, this partitioning appears to result in drastic changes in membrane morphology and permeability. This is a likely molecular-level cause for the damage associated with the disease state of PKU. Aqueous systems are further complicated by the reactivity of water. It often serves not only the role of a solvent, but also a reactant, a product, and/or a catalyst. I explore this reactivity using an organic molecule with relevance to environmental chemistry, zymonic acid. Zymonic acid forms spontaneously from pyruvic acid, an important atmospheric species. While zymonic acid exists as a single species in solid form when dissolved in DMSO, once in aqueous solution it quickly reacts with water and equilibrates with at least four other forms. I studied the details and kinetics of these equilibria via time-dependent NMR. Several surprising mechanistic details were uncovered, including a direct enol to geminal diol conversion and base-catalyzed lactone ring formation. The consequences of zymonic acid’s behavior are investigated in the context of environmental and prebiotic chemistry.
Advisors/Committee Members: Veronica Vaida, Joel Eaves, Amy Palmer, Loren Hough, Garret Miyake.
Subjects/Keywords: aqueous interfaces; aromatic aggregation; mechanistic organic chemistry; membrane biophysics; preboitic chemistry; surfactants; Organic Chemistry; Physical Chemistry
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APA ·
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APA (6th Edition):
Perkins, R. J. (2018). Beyond Hydrophobicity: Aqueous Interfaces, Interactions and Reactions. (Doctoral Dissertation). University of Colorado. Retrieved from https://scholar.colorado.edu/chem_gradetds/273
Chicago Manual of Style (16th Edition):
Perkins, Russell James. “Beyond Hydrophobicity: Aqueous Interfaces, Interactions and Reactions.” 2018. Doctoral Dissertation, University of Colorado. Accessed April 14, 2021.
https://scholar.colorado.edu/chem_gradetds/273.
MLA Handbook (7th Edition):
Perkins, Russell James. “Beyond Hydrophobicity: Aqueous Interfaces, Interactions and Reactions.” 2018. Web. 14 Apr 2021.
Vancouver:
Perkins RJ. Beyond Hydrophobicity: Aqueous Interfaces, Interactions and Reactions. [Internet] [Doctoral dissertation]. University of Colorado; 2018. [cited 2021 Apr 14].
Available from: https://scholar.colorado.edu/chem_gradetds/273.
Council of Science Editors:
Perkins RJ. Beyond Hydrophobicity: Aqueous Interfaces, Interactions and Reactions. [Doctoral Dissertation]. University of Colorado; 2018. Available from: https://scholar.colorado.edu/chem_gradetds/273
University of Colorado
6.
Perkins, Russell James.
Beyond Hydrophobicity: Aqueous Interfaces, Interactions and Reactions.
Degree: PhD, 2017, University of Colorado
URL: https://scholar.colorado.edu/chem_gradetds/219
► Many important chemical reactions from all branches of chemistry occur with water as a solvent. Furthermore, in environmental chemistry, biochemistry, and synthetic chemistry, key…
(more)
▼ Many important chemical reactions from all branches of chemistry occur with water as a solvent. Furthermore, in environmental chemistry, biochemistry, and synthetic chemistry, key reactions occur in heterogeneous aqueous systems, where interfacial effects are particularly important. Despite the importance of aqueous environments and the tremendous amount of work done to study them, there are aspects that require further explanation and remain controversial. I have performed experimental studies to help elucidate the fundamental characteristics of aqueous systems, while examining specific phenomena across several fields.
The genetic disorder phenylketonuria (PKU) can result in increased levels of the aromatic amino acid phenylalanine in human serum. Much of my work has focused on the driving forces behind partitioning of aromatic small molecules, including phenylalanine, into air-water or membrane-water interfacial regions, and the consequences of partitioning on interfacial properties. Drastically different behaviors for structurally similar aromatic molecules are observed, differences that cannot be explained by hydrophobic effects. These observations can be explained, however, through the development of a more detailed picture of interactions and partitioning, including the formation of interfacial aggregates. For phenylalanine, this partitioning appears to result in drastic changes in membrane morphology and permeability. This is a likely molecular-level cause for the damage associated with the disease state of PKU.
Aqueous systems are further complicated by the reactivity of water. It often serves not only the role of a solvent, but also a reactant, a product, and/or a catalyst. I explore this reactivity using an organic molecule with relevance to environmental chemistry, zymonic acid. Zymonic acid forms spontaneously from pyruvic acid, an important atmospheric species. While zymonic acid exists as a single species in solid form when dissolved in DMSO, once in aqueous solution it quickly reacts with water and equilibrates with at least four other forms. I studied the details and kinetics of these equilibria via time-dependent NMR. Several surprising mechanistic details were uncovered, including a direct enol to geminal diol conversion and base-catalyzed lactone ring formation. The consequences of zymonic acid’s behavior are investigated in the context of environmental and prebiotic chemistry.
Advisors/Committee Members: Veronica Vaida, Joel Eaves, Amy Palmer, Loren Hough, Garret Miyake.
Subjects/Keywords: aqueous interfaces; aromatic aggregation; mechanistic organic chemistry; membrane biophysics; preboitic chemistry; surfactants; Physical Chemistry
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APA ·
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MLA ·
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CSE |
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APA (6th Edition):
Perkins, R. J. (2017). Beyond Hydrophobicity: Aqueous Interfaces, Interactions and Reactions. (Doctoral Dissertation). University of Colorado. Retrieved from https://scholar.colorado.edu/chem_gradetds/219
Chicago Manual of Style (16th Edition):
Perkins, Russell James. “Beyond Hydrophobicity: Aqueous Interfaces, Interactions and Reactions.” 2017. Doctoral Dissertation, University of Colorado. Accessed April 14, 2021.
https://scholar.colorado.edu/chem_gradetds/219.
MLA Handbook (7th Edition):
Perkins, Russell James. “Beyond Hydrophobicity: Aqueous Interfaces, Interactions and Reactions.” 2017. Web. 14 Apr 2021.
Vancouver:
Perkins RJ. Beyond Hydrophobicity: Aqueous Interfaces, Interactions and Reactions. [Internet] [Doctoral dissertation]. University of Colorado; 2017. [cited 2021 Apr 14].
Available from: https://scholar.colorado.edu/chem_gradetds/219.
Council of Science Editors:
Perkins RJ. Beyond Hydrophobicity: Aqueous Interfaces, Interactions and Reactions. [Doctoral Dissertation]. University of Colorado; 2017. Available from: https://scholar.colorado.edu/chem_gradetds/219
University of Colorado
7.
Protter, David Stephen Warren.
Contributions of Intrinsically Disordered Regions of Proteins to the Assembly of Ribonucleoprotein Granules.
Degree: PhD, Chemistry & Biochemistry, 2017, University of Colorado
URL: https://scholar.colorado.edu/chem_gradetds/242
► Cells assemble large, non-membrane bound granules of protein and RNA, termed Ri- bonucleoprotein granules (RNP granules), often in response to a wide variety of…
(more)
▼ Cells assemble large, non-membrane bound granules of protein and RNA, termed Ri- bonucleoprotein granules (RNP granules), often in response to a wide variety of cellular stresses. This behavior is conserved from yeast to mammals. Some RNP granules ap- pear important in the stress response, while others are important for proper organismal development, and still others for control of RNA degradation and transport. Curiously, proteins found within granules are disproportionately likey to contain Intrinsically Dis- ordered Regions. Here, I show that those disordered regions can often drive higher order assembly
in vitro and contribute to granule assembly
in vivo. I found that these domains can make it easier for proteins to undergo a process known as Liquid-Liquid Phase Separa- tion in response to changes in ionic strength, wherein the protein of interest self-partitions into a concentrated liquid phase. The droplets that form mimic many of the behaviors of RNP granules in cells, such as recruitment of other IDR-containing proteins, assembly in response to RNA, and rapid exchange of contents with the surrounding medium. I also found that proteins that form these droplets tend to aggregate over time, turning the dynamic droplets into static structures.
Further, I identified several limitations to my
in vitro model, most importantly the impairment of IDR-based phase separation in the presence of other proteins or cellular lysates. However, I also helped uncover the synergistic relationship between IDRs and the more well studied protein-protein and protein-RNA interactions that are important for granule assembly. I therefore propose an inclusive model of granule assembly which asserts that a wide variety of types of interactions are important, and that it is the sum-total of these interactions that determines whether or not a granule assembles.
Advisors/Committee Members: Roy Parker, Christopher Link, Loren Hough, Marcelo Sousa, Amy Palmer.
Subjects/Keywords: intrinsically disordered region; phase separation; RNP granule; stress granule; Biochemistry
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APA (6th Edition):
Protter, D. S. W. (2017). Contributions of Intrinsically Disordered Regions of Proteins to the Assembly of Ribonucleoprotein Granules. (Doctoral Dissertation). University of Colorado. Retrieved from https://scholar.colorado.edu/chem_gradetds/242
Chicago Manual of Style (16th Edition):
Protter, David Stephen Warren. “Contributions of Intrinsically Disordered Regions of Proteins to the Assembly of Ribonucleoprotein Granules.” 2017. Doctoral Dissertation, University of Colorado. Accessed April 14, 2021.
https://scholar.colorado.edu/chem_gradetds/242.
MLA Handbook (7th Edition):
Protter, David Stephen Warren. “Contributions of Intrinsically Disordered Regions of Proteins to the Assembly of Ribonucleoprotein Granules.” 2017. Web. 14 Apr 2021.
Vancouver:
Protter DSW. Contributions of Intrinsically Disordered Regions of Proteins to the Assembly of Ribonucleoprotein Granules. [Internet] [Doctoral dissertation]. University of Colorado; 2017. [cited 2021 Apr 14].
Available from: https://scholar.colorado.edu/chem_gradetds/242.
Council of Science Editors:
Protter DSW. Contributions of Intrinsically Disordered Regions of Proteins to the Assembly of Ribonucleoprotein Granules. [Doctoral Dissertation]. University of Colorado; 2017. Available from: https://scholar.colorado.edu/chem_gradetds/242
University of Colorado
8.
Xiao, Yao.
Conformational Dynamics in the Regulation of MAP Kinase, ERK2.
Degree: PhD, Chemistry & Biochemistry, 2015, University of Colorado
URL: https://scholar.colorado.edu/chem_gradetds/168
► The MAP kinase, extracellular signal-regulated kinase 2 (ERK2), is a key regulator of cell signaling. Aberrant up-regulation of ERK2 has been correlated with various…
(more)
▼ The MAP kinase, extracellular signal-regulated kinase 2 (ERK2), is a key regulator of cell signaling. Aberrant up-regulation of ERK2 has been correlated with various diseases. ERK2 can be activated by MAP kinase kinases through dual phosphorylation at the activation loop. It remains a challenging question how changes in conformational dynamics contribute to kinase activation. NMR relaxation dispersion experiments were used to monitor changes in Ile, Leu, and Val (ILV) methyl motions in microsecond-millisecond timescale upon activation of ERK2. A structure-based procedure was developed to assign 13C1H3-labeled methyls, by comparing NMR distance constraints with the X-ray structure. This procedure yielded 60% of the methyl assignments in inactive and active forms of ILV 13C1H3-methyl labeled ERK2. In inactive ERK2, localized conformational dynamics was observed among methyls. Upon activation, the dynamics of assigned methyls in ERK2 were altered throughout the kinase core, including many residues in the catalytic pocket. The majority of methyls in active ERK2 fit to a single conformational exchange process, suggesting global domain motions involving interconversion between two states. A mutant of ERK2, engineered to enhance flexibility at the hinge region linking the N- and C-terminal domains, induced two-state conformational exchange throughout the kinase core. A mono-phospho-mimetic form of this mutant showed 25% of the dual-phosphorylated ERK2 activity. Thus, activation of ERK2 leads to a dramatic shift in conformational exchange, from a “tense” (T) state to a “relaxed” (R) state, likely through release of constraints at the hinge. To understand the effects on the conformational dynamics of ERK2 during catalysis and upon inhibitor binding, complexes of ERK2 with various ligands were formed. The binding of nucleotides and/or peptide substrates showed no significant perturbation to the T/R conformational equilibrium, with small enhancement of the T state population in active ERK2. In addition, differential conformational stabilization effects, which were not previously reported for ERK2, were observed upon the binding of different tight-binding inhibitors of ERK2. This thesis reports that ERK2 activation enhances microsecond-millisecond interconversion between conformers underlying different enzyme intermediates, thus linking protein dynamics to the catalytic cycle. The perturbations of conformational equilibrium by inhibitors reflect a novel allosteric mechanism in ERK2.
Advisors/Committee Members: Natalie G. Ahn, Arthur Pardi, Marcelo Sousa, Johannes Rudolph, Loren Hough.
Subjects/Keywords: ERK2; MAP kinase; aberrant up-regulation; interconversion; conformation exchange; Biochemistry; Cell Biology
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APA ·
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MLA ·
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CSE |
Export
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Manager
APA (6th Edition):
Xiao, Y. (2015). Conformational Dynamics in the Regulation of MAP Kinase, ERK2. (Doctoral Dissertation). University of Colorado. Retrieved from https://scholar.colorado.edu/chem_gradetds/168
Chicago Manual of Style (16th Edition):
Xiao, Yao. “Conformational Dynamics in the Regulation of MAP Kinase, ERK2.” 2015. Doctoral Dissertation, University of Colorado. Accessed April 14, 2021.
https://scholar.colorado.edu/chem_gradetds/168.
MLA Handbook (7th Edition):
Xiao, Yao. “Conformational Dynamics in the Regulation of MAP Kinase, ERK2.” 2015. Web. 14 Apr 2021.
Vancouver:
Xiao Y. Conformational Dynamics in the Regulation of MAP Kinase, ERK2. [Internet] [Doctoral dissertation]. University of Colorado; 2015. [cited 2021 Apr 14].
Available from: https://scholar.colorado.edu/chem_gradetds/168.
Council of Science Editors:
Xiao Y. Conformational Dynamics in the Regulation of MAP Kinase, ERK2. [Doctoral Dissertation]. University of Colorado; 2015. Available from: https://scholar.colorado.edu/chem_gradetds/168
. 
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