+publisher:"University of Colorado" +contributor:("Deborah S. Wuttke")

University of Colorado

1. 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)

Subjects/Keywords: Aptamer; NMR; Structure; Vascular endothelial growth factor; VEGF; Biochemistry

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APA (6^th 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 (16^th 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 August 18, 2019. https://scholar.colorado.edu/chem_gradetds/227.

MLA Handbook (7^th Edition):

Hunt, Sabrina Robin. “Structure Determination of Vascular Endothelial Growth Factor Heparin-Binding Domain in Complex with a Dna Aptamer.” 2017. Web. 18 Aug 2019.

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 2019 Aug 18]. 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

2. Dickerson, Sarah Michelle. Interplay Between the Helicase-Primase and the Dna Polymerase-Processivity Factor During Herpes Dna Replication.

Degree: PhD, Chemistry & Biochemistry, 2017, University of Colorado

URL: https://scholar.colorado.edu/chem_gradetds/239

► There are seven proteins essential for Herpes Simplex Virus Type 1 (HSV-1) DNA replication. While some of their roles are known, many of their… (more)

▼ There are seven proteins essential for Herpes Simplex Virus Type 1 (HSV-1) DNA replication. While some of their roles are known, many of their mechanisms remain unclear. Understanding the mechanism by which herpes replicates its viral genome may provide insight into targeting critical pathways to more effectively treat infection. This work identifies new roles and mechanisms of the essential enzymes at the herpes replication fork: the helicase-primase (UL5-UL8-UL52) and polymerase-processivity factor (UL30-UL42). I determined that as with other helicases, herpes helicase-primase displays varied ability to displace different bound proteins. Though the polymerase-processivity factor enhances helicase activity considerably, bound proteins still greatly impede unwinding during coupled DNA replication. From streptavidin displacement studies, I propose a model where the monomeric helicase-primase may largely or completely encircle the lagging strand template much like multimeric helicases which form rings around the DNA. I exploited a variety of minicircle DNA substrates to determine how the herpes helicase-primase and polymerase-processivity factor function together during DNA replication. I determined that herpes replication machinery effectively utilizes noncognate enzymes, i.e. the herpes polymerase-processivity factor utilizes E. coli DnaB helicase to synthesize longer products. However, the cognate enzyme pair is more efficient, suggesting tight coordination. Though UL30-UL42 stimulates DNA unwinding by the helicase-primase, UL30-UL42 does not stimulate the ATPase activity of the helicase-primase. Continuing my investigation of coupling at the replication fork, I found that the leading and lagging strand polymerases are not tightly coupled; i.e., the inhibition of lagging strand DNA synthesis does not impact leading strand synthesis. The processivity factor, UL42, enhances the processivity of the polymerase, UL30, by a factor of 8 in an all-or-nothing manner via a contact 24 nucleotides upstream of the primer 3’-terminus. In the absence of the processivity factor, the polymerase is essentially nonfunctional at the replication fork under conditions where the helicase must unwind the DNA in front of the polymerase. While the polymerase can function with a helicase lacking the UL8 subunit, UL8 increases the length and number of DNA products synthesized during helicase-polymerase coupled DNA replication. I discuss in detail the potential roles that UL42 and UL8 play to achieve efficient herpes replication. Advisors/Committee Members: Robert D. Kuchta, Jennifer F. Kugel, Deborah S. Wuttke, Carlos E. Catalano, Dylan J. Taatjes.

Subjects/Keywords: Herpes Simplex Virus Type 1 (HSV-1) DNA replication; viral genomes; treatment; helicase-primase (UL5-UL8-UL52); polymerase-processivity factor (UL30-UL42); Biochemistry

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APA (6^th Edition):

Dickerson, S. M. (2017). Interplay Between the Helicase-Primase and the Dna Polymerase-Processivity Factor During Herpes Dna Replication. (Doctoral Dissertation). University of Colorado. Retrieved from https://scholar.colorado.edu/chem_gradetds/239

Chicago Manual of Style (16^th Edition):

Dickerson, Sarah Michelle. “Interplay Between the Helicase-Primase and the Dna Polymerase-Processivity Factor During Herpes Dna Replication.” 2017. Doctoral Dissertation, University of Colorado. Accessed August 18, 2019. https://scholar.colorado.edu/chem_gradetds/239.

MLA Handbook (7^th Edition):

Dickerson, Sarah Michelle. “Interplay Between the Helicase-Primase and the Dna Polymerase-Processivity Factor During Herpes Dna Replication.” 2017. Web. 18 Aug 2019.

Vancouver:

Dickerson SM. Interplay Between the Helicase-Primase and the Dna Polymerase-Processivity Factor During Herpes Dna Replication. [Internet] [Doctoral dissertation]. University of Colorado; 2017. [cited 2019 Aug 18]. Available from: https://scholar.colorado.edu/chem_gradetds/239.

Council of Science Editors:

Dickerson SM. Interplay Between the Helicase-Primase and the Dna Polymerase-Processivity Factor During Herpes Dna Replication. [Doctoral Dissertation]. University of Colorado; 2017. Available from: https://scholar.colorado.edu/chem_gradetds/239

University of Colorado

3. Gerhardt, Alana. Synergistic Effects of Interfaces and Agitation on Particle Formation in Therapeutic Protein Formulations in Pre-filled Syringes.

Degree: PhD, 2014, University of Colorado

URL: http://scholar.colorado.edu/chbe_gradetds/60

► Pre-filled syringes are commonly used storage and delivery devices for protein therapeutics because of their convenience and ease of use. However, in a pre-filled… (more)

▼ Pre-filled syringes are commonly used storage and delivery devices for protein therapeutics because of their convenience and ease of use. However, in a pre-filled syringe, a protein molecule encounters several interfaces which may negatively impact its stability. In addition, protein formulations in pre-filled syringes may also be subject to agitation stresses during transportation. Exposure to interfaces and to agitation has been shown to promote aggregation and particle formation in protein formulations, and these stresses are hypothesized to work synergistically to cause more aggregation and particle generation than either stress alone. In this work, we investigated the synergistic effects of interfaces and agitation on protein formulations in pre-filled syringes, primarily using a monoclonal antibody. First, we characterized the adsorption of this antibody to the silicone oil-water interface and showed that the tertiary structure of the antibody was perturbed after adsorption. This perturbation promoted aggregation of the antibody when it was exposed to both silicone oil microdroplets and agitation. However, when the ionic strength of the formulation was increased, the antibody was not perturbed after adsorption, and less aggregation was observed after exposure to silicone oil microdroplets and agitation. When the antibody formulation was incubated in pre-filled syringes, the highest particle concentrations were observed in formulations that were agitated in siliconized syringes with an air bubble. From these results, we proposed an interfacial mechanism, wherein capillary forces at the three-phase contact line in the syringe pulled gelled protein aggregates and silicone oil droplets from the silicone oil-water interface into the bulk, to describe how air-water interfaces, silicone oil-water interfaces, and agitation worked synergistically to generate particles in pre-filled syringes. Furthermore, we proposed two ways to reduce particle generation in pre-filled syringes. First, silicone oil coatings which are strongly adhered to the glass syringe wall were less able to be removed from the wall and, therefore, resulted in less particles. Second, we observed that concentrations of polysorbate 20 above and below the CMC were able to inhibit gelation of the protein layer adsorbed at the silicone oil-water interface which minimized particle generation in agitated, siliconized syringes with an air bubble. Advisors/Committee Members: Theodore W. Randolph, John F. Carpenter, Joel L. Kaar, Daniel K. Schwartz, Deborah S. Wuttke.

Subjects/Keywords: monoclonal antibody; particle formation; pre-filled syringes; protein aggregation; silicone oil; Biochemical and Biomolecular Engineering; Medicinal Chemistry and Pharmaceutics

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APA (6^th Edition):

Gerhardt, A. (2014). Synergistic Effects of Interfaces and Agitation on Particle Formation in Therapeutic Protein Formulations in Pre-filled Syringes. (Doctoral Dissertation). University of Colorado. Retrieved from http://scholar.colorado.edu/chbe_gradetds/60

Chicago Manual of Style (16^th Edition):

Gerhardt, Alana. “Synergistic Effects of Interfaces and Agitation on Particle Formation in Therapeutic Protein Formulations in Pre-filled Syringes.” 2014. Doctoral Dissertation, University of Colorado. Accessed August 18, 2019. http://scholar.colorado.edu/chbe_gradetds/60.

MLA Handbook (7^th Edition):

Gerhardt, Alana. “Synergistic Effects of Interfaces and Agitation on Particle Formation in Therapeutic Protein Formulations in Pre-filled Syringes.” 2014. Web. 18 Aug 2019.

Vancouver:

Gerhardt A. Synergistic Effects of Interfaces and Agitation on Particle Formation in Therapeutic Protein Formulations in Pre-filled Syringes. [Internet] [Doctoral dissertation]. University of Colorado; 2014. [cited 2019 Aug 18]. Available from: http://scholar.colorado.edu/chbe_gradetds/60.

Council of Science Editors:

Gerhardt A. Synergistic Effects of Interfaces and Agitation on Particle Formation in Therapeutic Protein Formulations in Pre-filled Syringes. [Doctoral Dissertation]. University of Colorado; 2014. Available from: http://scholar.colorado.edu/chbe_gradetds/60

University of Colorado

4. Dickey, Thayne Henderson. Structural Plasticity in the Recognition of ssDNA by the Telomeric Protein Pot1.

Degree: PhD, Chemistry & Biochemistry, 2014, University of Colorado

URL: http://scholar.colorado.edu/chem_gradetds/111

► Telomere dysfunction has been implicated in several diseases including cancer and aging. The two main roles of telomeres are often defined as end-protection and… (more)

▼ Telomere dysfunction has been implicated in several diseases including cancer and aging. The two main roles of telomeres are often defined as end-protection and length regulation and the shelterin complex lies at the heart of both of these functions. Pot1 is the single-stranded DNA-binding component of shelterin and is important for genome stability, telomere length regulation, and C-strand resection, all functions that rely on its ssDNA-binding ability. In the work presented here, we structurally and biochemically characterize the dual OB-fold DNA-binding domain of Pot1 from the model organism S. pombe. This work provides insight into potential mechanisms of telomere length regulation and elucidates novel and broadly applicable features of protein/nucleic acid recognition. X-ray crystal structures of the second OB-fold of Pot1, Pot1pC, bound to various ssDNA sequences reveal a unique plasticity at the DNA-binding interface. Global rearrangements of the entire interface allow the accommodation of complementary base substitutions despite the abundance of apparently base-specific hydrogen bonds. This structural plasticity likely explains the ability of S. pombe Pot1 to accommodate the natural heterogeneity in S. pombe telomeric sequence. Furthermore, these mechanisms of accommodation allow for a high-affinity/low-specificity binding mode that is likely utilized by other sequence nonspecific ssDNA and ssRNA-binding proteins. We also use NMR and biochemical techniques to study how the behavior of the individual OB-folds is modulated in the context of the complete DNA-binding domain. Multiple DNA-binding domains often exist within a single protein or complex in biology, but the functional importance of these tandem arrangements is rarely known. The work described here reveals a malleable interface between domains that allows for multiple DNA-binding modes. These binding modes have differing structural and biochemical features that may be important for telomere length regulation. Advisors/Committee Members: Deborah S. Wuttke, Robert T. Batey, Marcelo C. Sousa, Loren E. Hough, Charles McHenry.

Subjects/Keywords: Plasticity; Pombe; Pot1; protein nucleic acid; shelterin; telomere; Biochemistry; Molecular Genetics

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APA (6^th Edition):

Dickey, T. H. (2014). Structural Plasticity in the Recognition of ssDNA by the Telomeric Protein Pot1. (Doctoral Dissertation). University of Colorado. Retrieved from http://scholar.colorado.edu/chem_gradetds/111

Chicago Manual of Style (16^th Edition):

Dickey, Thayne Henderson. “Structural Plasticity in the Recognition of ssDNA by the Telomeric Protein Pot1.” 2014. Doctoral Dissertation, University of Colorado. Accessed August 18, 2019. http://scholar.colorado.edu/chem_gradetds/111.

MLA Handbook (7^th Edition):

Dickey, Thayne Henderson. “Structural Plasticity in the Recognition of ssDNA by the Telomeric Protein Pot1.” 2014. Web. 18 Aug 2019.

Vancouver:

Dickey TH. Structural Plasticity in the Recognition of ssDNA by the Telomeric Protein Pot1. [Internet] [Doctoral dissertation]. University of Colorado; 2014. [cited 2019 Aug 18]. Available from: http://scholar.colorado.edu/chem_gradetds/111.

Council of Science Editors:

Dickey TH. Structural Plasticity in the Recognition of ssDNA by the Telomeric Protein Pot1. [Doctoral Dissertation]. University of Colorado; 2014. Available from: http://scholar.colorado.edu/chem_gradetds/111

University of Colorado

5. Altschuler, Sarah E. Characterization of single-stranded DNA binding and small molecule inhibition of S. pombe Pot1.

Degree: PhD, Chemistry & Biochemistry, 2011, University of Colorado

URL: http://scholar.colorado.edu/chem_gradetds/129

► Many critical cellular functions involve the management of single-stranded DNA (ssDNA). For the vast majority of these processes ssDNA is only transiently present. Telomeres,… (more)

▼ Many critical cellular functions involve the management of single-stranded DNA (ssDNA). For the vast majority of these processes ssDNA is only transiently present. Telomeres, the ends of eukaryotic chromosomes, are a special case where ssDNA is a necessary feature. Telomeres consist of a suite of proteins associated with long tracts of repetitive GT-rich DNA terminating in a conserved 3′ ssDNA overhang. The protection and regulation of these overhangs requires the sequence-specific binding activity of the telomere-end protection (TEP) family of proteins. The telomeric DNA of Schizosaccharomyces pombe consists of a core repeat sequence (GGTTAC) interrupted by spacer elements of variable length and sequence. The ssDNA-binding activity of the S. pombe TEP (SpPot1) is conferred by a DNA-binding domain consisting of two subdomains, Pot1pN and Pot1pC. Previous work has shown that Pot1pN binds a single repeat of the core telomere sequence (GGTTAC) with exquisite specificity, while Pot1pC binds an extended sequence representing 1.5 repeats (GGTTACGGT) with only modest specificity. Fulllength SpPot1 binds the composite 15mer, (GGTTAC)2GGT, and a shorter two-repeat 12mer, (GGTTAC)2, with equally high affinity (< 3 pM), but with substantially different kinetic and thermodynamic properties. The binding mode of the SpPot1/15mer complex is more stable than that of the 12mer complex, with a 2-fold longer half-life and increased tolerance to nucleotide and amino acid substitutions. In addition, SpPot1 accommodates significant sequence alterations within the 3′ end of telomeric oligonucleotides. The data suggest that SpPot1 protection of heterogeneous telomeres is mediated through 5′ sequence recognition and the use of alternate binding modes to maintain high affinity interaction with the ssDNA overhang. Disruption of the end-binding activity of SpPot1 provides an avenue for studying telomere uncapping. The rich chemical diversity manifest in SpPot1/ssDNA interaction suggested binding could be perturbed by small molecule inhibition (SMI). Presented here are the successful implementation of a high-throughput screen and the identification of a single inhibitor of SpPot1/ssDNA binding. The compound binds specifically to the ssDNA-binding surface and inhibits via specific trimerization of the protein. These results suggest the utilization of SMI of TEP proteins as a feasible tool for the continued analysis of telomere-end protection. Advisors/Committee Members: Deborah S. Wuttke, Robert T. Batey, Thomas Cech, Robert Kuchta, Norman Pace.

Subjects/Keywords: Pot1; ssDNA; ssDNA binding; Telomere; Biochemistry

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Altschuler, S. E. (2011). Characterization of single-stranded DNA binding and small molecule inhibition of S. pombe Pot1. (Doctoral Dissertation). University of Colorado. Retrieved from http://scholar.colorado.edu/chem_gradetds/129

Chicago Manual of Style (16^th Edition):

Altschuler, Sarah E. “Characterization of single-stranded DNA binding and small molecule inhibition of S. pombe Pot1.” 2011. Doctoral Dissertation, University of Colorado. Accessed August 18, 2019. http://scholar.colorado.edu/chem_gradetds/129.

MLA Handbook (7^th Edition):

Altschuler, Sarah E. “Characterization of single-stranded DNA binding and small molecule inhibition of S. pombe Pot1.” 2011. Web. 18 Aug 2019.

Vancouver:

Altschuler SE. Characterization of single-stranded DNA binding and small molecule inhibition of S. pombe Pot1. [Internet] [Doctoral dissertation]. University of Colorado; 2011. [cited 2019 Aug 18]. Available from: http://scholar.colorado.edu/chem_gradetds/129.

Council of Science Editors:

Altschuler SE. Characterization of single-stranded DNA binding and small molecule inhibition of S. pombe Pot1. [Doctoral Dissertation]. University of Colorado; 2011. Available from: http://scholar.colorado.edu/chem_gradetds/129

University of Colorado

6. Dalby, Andrew Benjamin. The Molecular Interfaces of Telomerase and Telomere Proteins in Yeast and Humans.

Degree: PhD, Chemistry & Biochemistry, 2014, University of Colorado

URL: http://scholar.colorado.edu/chem_gradetds/136

► Telomeres are the nucleoprotein endcaps of linear chromosomes. Telomeres shorten with each cell division, limiting the capacity of telomeres to protect chromosomal DNA. The… (more)

▼ Telomeres are the nucleoprotein endcaps of linear chromosomes. Telomeres shorten with each cell division, limiting the capacity of telomeres to protect chromosomal DNA. The enzyme telomerase counteracts telomere attrition by synthesizing new telomeric DNA. Telomerase is a ribonucleoprotein complex comprised of TElomerase Reverse Transcriptase (TERT) and Telomerase RNA (TR). To maintain a homeostatic telomere length, telomerase must assemble, traffic to the telomere, and interact with a host of protein cofactors. Since the discovery of telomerase, many key components of the telomerase holoenzyme and telomeric protein cap have been identified. This thesis presents detailed investigations of the molecular interactions of telomerase and associated proteins. In Saccharomyces cerevisiae the Ku heterodimer binds directly to a hairpin of TR to promote the nuclear localization of telomerase. Footprinting experiments examined the Ku binding site and chemical probing investigated the hairpin secondary structure. Heterologous mixing experiments and RNA mutagenesis tested which stem-loop elements mediate Ku binding. These experiments suggest that Ku binds to the terminal loop and proximal stems of the hairpin, recognizing a critical bulge motif in a sequence-independent but structure-specific manner. The TEL-patch consists of amino acids on the surface of the human telomere protein TPP1 that are necessary for telomerase recruitment and processivity stimulation, but the TEL-patch contributions are not fully understood. Single-turnover translocation, substrate-competition, and substrate-telomerase dissociation rate assays tested the impact of the TEL-patch on telomerase catalysis. The competition experiments constitute a step in developing an in vitro telomerase recruitment assay. Furthermore, the TEL-patch increases the apparent rate and efficiency of telomerase translocation, slows the rate of substrate dissociation, and contributes to the preferential binding and extension of TPP1-bound substrates by telomerase. The exact component of telomerase that interacts with the TEL-patch of TPP1 has been unknown. Direct telomerase extension assays identified human TERT separation-of-function alleles that disrupt the telomerase-TPP1 interaction. Perturbation of the interaction resulted in compromised telomere maintenance in cells. A deleterious mutation in the TEN-domain of hTERT was rescued by introducing a compensatory charge-swap mutation in the TEL-patch of TPP1 that restored telomerase stimulation in vitro, telomere maintenance in vivo, and suggests that these proteins interact directly. Advisors/Committee Members: Thomas R. Cech, Deborah S. Wuttke, Arthur Pardi, Roy Parker, Norman R. Pace.

Subjects/Keywords: Ku heterodimer; POT1; Recrutiment; Telomerase; Telomere; TPP1; Biochemistry; Genetics and Genomics

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APA (6^th Edition):

Dalby, A. B. (2014). The Molecular Interfaces of Telomerase and Telomere Proteins in Yeast and Humans. (Doctoral Dissertation). University of Colorado. Retrieved from http://scholar.colorado.edu/chem_gradetds/136

Chicago Manual of Style (16^th Edition):

Dalby, Andrew Benjamin. “The Molecular Interfaces of Telomerase and Telomere Proteins in Yeast and Humans.” 2014. Doctoral Dissertation, University of Colorado. Accessed August 18, 2019. http://scholar.colorado.edu/chem_gradetds/136.

MLA Handbook (7^th Edition):

Dalby, Andrew Benjamin. “The Molecular Interfaces of Telomerase and Telomere Proteins in Yeast and Humans.” 2014. Web. 18 Aug 2019.

Vancouver:

Dalby AB. The Molecular Interfaces of Telomerase and Telomere Proteins in Yeast and Humans. [Internet] [Doctoral dissertation]. University of Colorado; 2014. [cited 2019 Aug 18]. Available from: http://scholar.colorado.edu/chem_gradetds/136.

Council of Science Editors:

Dalby AB. The Molecular Interfaces of Telomerase and Telomere Proteins in Yeast and Humans. [Doctoral Dissertation]. University of Colorado; 2014. Available from: http://scholar.colorado.edu/chem_gradetds/136

University of Colorado

7. Porter, Ely Blanton. The use of Biological Riboswitches and Ribozymes as Scaffolds for Selection and Novel RNA Devices.

Degree: PhD, Chemistry & Biochemistry, 2015, University of Colorado

URL: http://scholar.colorado.edu/chem_gradetds/143

► The utility of RNA as a sensor for in vivo enzyme evolution, metabolomics monitoring, and bacterial control modules is slowly being realized, but is… (more)

▼ The utility of RNA as a sensor for in vivo enzyme evolution, metabolomics monitoring, and bacterial control modules is slowly being realized, but is met with some outstanding challenges. Current work to date on genetically encoded RNA-biosensors has focused on independently developing modular sensors or adaptors. The engineering of adaptor domains has been generally successful in their broad application and modularity, with small molecule fluorescent activators and several strategies for gene regulation now at our disposal. The sensor domains primarily consist of naturally occurring riboswitches and synthetic aptamers generated by in vitro selection. The riboswitch aptamer domains have seen wide application due to their modularity and high affinity, yet their diversity is limited to natural availability. In contrast, hundreds of synthetic aptamers have been created, yet their modularity and application has been met with limited success. Several studies have now shown the faults in assuming that the transition from aptamer to device sensor would be trivial. In this work, a recurrent and model RNA motif is used to introduce robust folding and complexity to a limited library that is selected to bind 5-hydroxytryptophan. The introduced peripheral motif benefits the binding and structure of the RNA, producing an aptamer with higher affinity and specificity than sequences lacking the greater fold. The developed workflow relies on deep sequencing and bioinformatics for the identification of robust elements, reducing the need for tedious validation and allowing the immediate screening of candidate sequences. Additionally, when multiple scaffolds are employed to host limited libraries and parallel selections are carried out, a suite of high affinity aptamers are obtained that are functionally related, yet distant in sequence space. This allows for an aptameric screening strategy, where a desired adaptor platform may be selected and candidate sensors screened for function, an ability not readily afforded by traditional selection techniques. These strategies ease constraints on and expedite the development of high affinity RNA devices, adding yet another robust component to the synthetic biologist's toolbox. Advisors/Committee Members: Robert T. Batey, James A. Goodrich, Deborah S. Wuttke, Roy Parker, Norman R. Pace.

Subjects/Keywords: 5-hydroxytryptophan; Purine; Riboswitch; RNA; SELEX; Serotonin; Biochemistry; Molecular Genetics

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APA (6^th Edition):

Porter, E. B. (2015). The use of Biological Riboswitches and Ribozymes as Scaffolds for Selection and Novel RNA Devices. (Doctoral Dissertation). University of Colorado. Retrieved from http://scholar.colorado.edu/chem_gradetds/143

Chicago Manual of Style (16^th Edition):

Porter, Ely Blanton. “The use of Biological Riboswitches and Ribozymes as Scaffolds for Selection and Novel RNA Devices.” 2015. Doctoral Dissertation, University of Colorado. Accessed August 18, 2019. http://scholar.colorado.edu/chem_gradetds/143.

MLA Handbook (7^th Edition):

Porter, Ely Blanton. “The use of Biological Riboswitches and Ribozymes as Scaffolds for Selection and Novel RNA Devices.” 2015. Web. 18 Aug 2019.

Vancouver:

Porter EB. The use of Biological Riboswitches and Ribozymes as Scaffolds for Selection and Novel RNA Devices. [Internet] [Doctoral dissertation]. University of Colorado; 2015. [cited 2019 Aug 18]. Available from: http://scholar.colorado.edu/chem_gradetds/143.

Council of Science Editors:

Porter EB. The use of Biological Riboswitches and Ribozymes as Scaffolds for Selection and Novel RNA Devices. [Doctoral Dissertation]. University of Colorado; 2015. Available from: http://scholar.colorado.edu/chem_gradetds/143

University of Colorado

8. Poss, Zachary Copeland. Characterization of a Novel Inhibitor and Substrates of the Mediator Kinases CDK8 and CDK19.

Degree: PhD, Chemistry & Biochemistry, 2015, University of Colorado

URL: http://scholar.colorado.edu/chem_gradetds/167

► (Pol II) transcription that can interact with gene-specific transcription factors and the general transcription machinery. Little is known about the molecular mechanisms by which… (more)

▼ (Pol II) transcription that can interact with gene-specific transcription factors and the general transcription machinery. Little is known about the molecular mechanisms by which Mediator functions. Within this multi-subunit, 1.2MDa assembly there is only one known enzymatic activity, which is carried out by CDK8 and its paralog CDK19. These kinases function in the cell as “modules” consisting of CDK8 or CDK19, Cyclin C, MED12 and MED13. This module can reversibly associate with the Mediator complex to form CDK8/19-Mediator, and can modulate the structure and activity of the assembly upon binding. Few cellular CDK8/19 substrates are known, owing in part to a lack of specific small-molecule inhibitors for these kinases. Here, we assist in the characterization of a new CDK8 substrate, the activation domain of the transcription factor STAT1. We identify the site of phosphorylation using kinase assays and mutagenesis, and show that it is phosphorylated at a single site. We then identify a potent and specific small-molecule inhibitor of CDK8/19 activity. This inhibitor does not inhibit other transcriptional CDKs, and it inhibits CDK8/19 activity with an IC50 in the low nanomolar range. Based upon a co-crystal structure of the small molecule bound to CDK8-CCNC, we identify and test a CDK8/19 mutant that conveys resistance to the inhibitor. To more comprehensively identify substrates of CDK8/19, we used inhibitor treatment coupled with phosphoproteomics to elucidate novel kinase targets from human cells. The biological function of most of these substrates involves Pol II transcription and includes transcription factors, Mediator subunits and chromatin-modifying enzymes. Further use of quantitative mass spectrometry experiments shows that phosphorylation by Mediator kinases is able to regulate protein abundance in select cases for identified substrates. Additionally, targeted inhibition of CDK8/19 alters the expression of a small number of genes, some of which can be linked to phosphoproteomic changes. Collectively, these data represent the first large-scale identification of CDK8/19 kinase substrates to date and point to regulatory functions for CDK8/19 activity. This analysis has helped expand our understanding of the diverse mechanisms by which CDK8/19 control transcription. Advisors/Committee Members: Dylan J. Taatjes, Deborah S. Wuttke, James Goodrich, William Old, Joaquin Espinosa.

Subjects/Keywords: mediator complex; RNA transcription; single site phosphorylation; human cells; targeting; Biochemistry; Molecular Genetics

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APA (6^th Edition):

Poss, Z. C. (2015). Characterization of a Novel Inhibitor and Substrates of the Mediator Kinases CDK8 and CDK19. (Doctoral Dissertation). University of Colorado. Retrieved from http://scholar.colorado.edu/chem_gradetds/167

Chicago Manual of Style (16^th Edition):

Poss, Zachary Copeland. “Characterization of a Novel Inhibitor and Substrates of the Mediator Kinases CDK8 and CDK19.” 2015. Doctoral Dissertation, University of Colorado. Accessed August 18, 2019. http://scholar.colorado.edu/chem_gradetds/167.

MLA Handbook (7^th Edition):

Poss, Zachary Copeland. “Characterization of a Novel Inhibitor and Substrates of the Mediator Kinases CDK8 and CDK19.” 2015. Web. 18 Aug 2019.

Vancouver:

Poss ZC. Characterization of a Novel Inhibitor and Substrates of the Mediator Kinases CDK8 and CDK19. [Internet] [Doctoral dissertation]. University of Colorado; 2015. [cited 2019 Aug 18]. Available from: http://scholar.colorado.edu/chem_gradetds/167.

Council of Science Editors:

Poss ZC. Characterization of a Novel Inhibitor and Substrates of the Mediator Kinases CDK8 and CDK19. [Doctoral Dissertation]. University of Colorado; 2015. Available from: http://scholar.colorado.edu/chem_gradetds/167

University of Colorado

9. Warner, Lisa Rose. Structural Characterization of Proteins from the E. coli β-barrel Assembly Machine using NMR Spectroscopy.

Degree: PhD, Chemistry & Biochemistry, 2011, University of Colorado

URL: http://scholar.colorado.edu/chem_gradetds/128

► The cytosol of Gram-negative bacteria is fortified by an inner membrane and an outer membrane that are separated by ~140 Å of periplasmic space.… (more)

▼ The cytosol of Gram-negative bacteria is fortified by an inner membrane and an outer membrane that are separated by ~140 Å of periplasmic space. Spanning the outer membrane are β-barrel proteins that are integral for basic physiological functions, virulence and multidrug resistance. The E. coli β-barrel assembly machine is a five protein complex responsible for the folding and insertion of β-barrel proteins into the outer membrane. How this molecular machine works in the absence of ATP or a proton gradient has remained elusive. The prevailing theory is that interactions between proteins in the complex, protein chaperones in the periplasm, and outer membrane protein precursors drive this molecular machine. The work presented here contributes to understanding the mechanics of the β-barrel assembly machine by providing a structural description of two of the components: BamC and BamA. NMR is a powerful tool to investigate the structure and dynamics of proteins in solution. Here, a novel approach was used to determine the solutions structure of the 27 kDa BamC by combining a limited NMR dataset of chemical shifts, residual dipolar couplings (RDCs) and nuclear Overhauser effect (NOE) distance restraints with the protein fold prediction program, Rosetta. The structure of BamC was determined to consist of two helix-grip type domains connected by an ∼18-residue flexible linker. The structure was validated with a supplementary NOE NMR dataset including amide-amide ¹H-¹H and isoleucine, leucine and valine methyl methyl ¹H-¹H NOEs. Interestingly, regions of the structural ensemble that did not converge to a unique conformation also showed increased ¹⁵N backbone amide dynamics. The domain orientation and flexibility of the periplasmic POlypeptide TRansport Associated (POTRA) domains of BamA were investigated. Solution domain orientation using RDCs validated the orientation of POTRA4–5 in a spliced crystallographic model of POTRA1–5. The flexibility of POTRA1–5 in solution was assessed with 15N amide backbone dynamic, paramagnetic relaxation enhancement and analysis of RDCs. Previous reports suggested that POTRA1–5 was comprised of two rigid units, POTRA1–2 and POTRA3–5. The results presented here indicate that POTRA1–5 may be more flexible than previously thought. Advisors/Committee Members: Arthur Pardi, Marcelo C. Sousa, Deborah S. Wuttke, Natalie G. Ahn, Elan Eisenmesser.

Subjects/Keywords: NMR; Rosetta; structure; Biochemistry

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APA (6^th Edition):

Warner, L. R. (2011). Structural Characterization of Proteins from the E. coli β-barrel Assembly Machine using NMR Spectroscopy. (Doctoral Dissertation). University of Colorado. Retrieved from http://scholar.colorado.edu/chem_gradetds/128

Chicago Manual of Style (16^th Edition):

Warner, Lisa Rose. “Structural Characterization of Proteins from the E. coli β-barrel Assembly Machine using NMR Spectroscopy.” 2011. Doctoral Dissertation, University of Colorado. Accessed August 18, 2019. http://scholar.colorado.edu/chem_gradetds/128.

MLA Handbook (7^th Edition):

Warner, Lisa Rose. “Structural Characterization of Proteins from the E. coli β-barrel Assembly Machine using NMR Spectroscopy.” 2011. Web. 18 Aug 2019.

Vancouver:

Warner LR. Structural Characterization of Proteins from the E. coli β-barrel Assembly Machine using NMR Spectroscopy. [Internet] [Doctoral dissertation]. University of Colorado; 2011. [cited 2019 Aug 18]. Available from: http://scholar.colorado.edu/chem_gradetds/128.

Council of Science Editors:

Warner LR. Structural Characterization of Proteins from the E. coli β-barrel Assembly Machine using NMR Spectroscopy. [Doctoral Dissertation]. University of Colorado; 2011. Available from: http://scholar.colorado.edu/chem_gradetds/128

University of Colorado

10. Rao, Timsi. Structure of the Essential Yeast Telomerase Protein Est3 from S. cerevisiae and Structure-Guided Investigation of Function.

Degree: PhD, 2012, University of Colorado

URL: http://scholar.colorado.edu/mcdb_gradetds/53

► Telomeres are the nucleoprotein complexes at the ends of linear chromosomes that protect the genome from degradation and chromosomal fusions. Telomeres are replicated by… (more)

▼ Telomeres are the nucleoprotein complexes at the ends of linear chromosomes that protect the genome from degradation and chromosomal fusions. Telomeres are replicated by the specialized enzyme telomerase. The telomerase holoenzyme in S. cerevisiae contains an RNA template and three known protein subunits, Est1, Est2 and Est3 (est = ever shorter telomeres for the phenotype observed upon their deletion). The reverse transcriptase Est2 and the RNA template TLC1 constitute the catalytic core of the telomerase holoenzyme. While Est1 and Est3 are not required for catalysis in vitro, they are strictly required for activity in vivo. The function of Est3 has remained elusive, although genetic data suggests that one mode of Est3's function might be carried out via its interaction with Est2. To provide insights into Est3 function, we have solved its high resolution structure. Because Est3 is a difficult protein target for structure elucidation, the structure was solved using a relatively novel strategy of combining minimal NMR experimental data (chemical shifts, RDCs and NOEs) with Rosetta de novo structure prediction. The structure is an OB-fold, with five-stranded β-barrel, capped with an α-helix and has some specialized features that distinguish it from other OB folds. The canonical loop L45 is quite unusual in the case of Est3, in that it is long and highly structured and plugs on top of the OB-fold canonical ligand binding face. Even in the absence of appreciable sequence relationship, the Est3 structure shows remarkable similarity to HsTPP1-OB structure, not only in the core β-barrel, but also in the positioning of the α-helix at the base and placement of C-terminal tail partially covering the canonical OB-fold binding face. Mapping residues involved in telomerase-association onto the structure reveals a novel protein interaction surface at the base of the β-barrel for Est3 and TPP1-OB. In vivo analysis, using structure-guided mutagenesis of Est3 surface also identified several new, functionally relevant, residues of Est3. The structure also served as a validation tool for an in vivo guided in vitro study that showed a direct correlation of in vivo dominant-negative mutants with their structural retention in vitro. Advisors/Committee Members: Deborah S. Wuttke, Mark Winey, Shelley D. Copley, Andreas Hoenger, Thomas Blumenthal.

Subjects/Keywords: telomeres; proteins; Biochemistry, Biophysics, and Structural Biology; Molecular Biology

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Rao, T. (2012). Structure of the Essential Yeast Telomerase Protein Est3 from S. cerevisiae and Structure-Guided Investigation of Function. (Doctoral Dissertation). University of Colorado. Retrieved from http://scholar.colorado.edu/mcdb_gradetds/53

Chicago Manual of Style (16^th Edition):

Rao, Timsi. “Structure of the Essential Yeast Telomerase Protein Est3 from S. cerevisiae and Structure-Guided Investigation of Function.” 2012. Doctoral Dissertation, University of Colorado. Accessed August 18, 2019. http://scholar.colorado.edu/mcdb_gradetds/53.

MLA Handbook (7^th Edition):

Rao, Timsi. “Structure of the Essential Yeast Telomerase Protein Est3 from S. cerevisiae and Structure-Guided Investigation of Function.” 2012. Web. 18 Aug 2019.

Vancouver:

Rao T. Structure of the Essential Yeast Telomerase Protein Est3 from S. cerevisiae and Structure-Guided Investigation of Function. [Internet] [Doctoral dissertation]. University of Colorado; 2012. [cited 2019 Aug 18]. Available from: http://scholar.colorado.edu/mcdb_gradetds/53.

Council of Science Editors:

Rao T. Structure of the Essential Yeast Telomerase Protein Est3 from S. cerevisiae and Structure-Guided Investigation of Function. [Doctoral Dissertation]. University of Colorado; 2012. Available from: http://scholar.colorado.edu/mcdb_gradetds/53

University of Colorado

11. Ozdilek, Bagdeser Akdogan. Characterizing the RNA Binding Properties of the Intrinsically Disordered FUS Protein and RGG/RG Domains.

Degree: PhD, 2017, University of Colorado

URL: https://scholar.colorado.edu/mcdb_gradetds/77

► Recent developments in the comprehensive identification of the RNA-binding protein (RBP) repertoire has accelerated discovery of new RBPs. According to these studies, 20% of… (more)

▼ Recent developments in the comprehensive identification of the RNA-binding protein (RBP) repertoire has accelerated discovery of new RBPs. According to these studies, 20% of both known and novel RNA-binding proteins are highly disordered. Moreover, analysis of the human mRNA interactome revealed most disease related mutations are found within intrinsically disordered RNA-binding domains (RBDs). For most of these proteins, their RNA-binding properties are poorly characterized. Thus, deciphering intrinsically disordered RBD-RNA interactions on a molecular scale is essential to understanding their impact upon human physiology and diseases. RGG/RG (arginine/glycine) domains are the second most common RNA binding domain in the human genome, yet their RNA-binding properties have not been well understood. Proteins containing RGG/RG domains regulate all aspects of RNA metabolism including transcription, processing, nucleocytoplasmic shuttling and translation. Proteins such as Fused in Sarcoma (FUS), Fragile X mental retardation (FMRP) and hnRNP U, bind a majority of the cellular transcriptome such that their recognition of RNAs has been considered to be non-specific or “promiscuous”. Here, I report a detailed analysis of the RNA-binding characteristics of the RGG/RG domains from FUS, FMRP and hnRNP U. While previous studies of FUS focused on RNA binding by the RRM and zinc finger (ZnF) domains, my analysis showed RNA binding activity is driven by the RGG/RG domains. Further, I observed a strong synergy between the RRM and adjacent RGG/RG domains to achieve RNA binding affinities of the full-length FUS. To better characterize RNA-binding properties of RGG/RG domains, we have analyzed RGG/RG domains of FUS, FMRP and hnRNP U in vitro against a spectrum of different RNAs with well-defined structural and sequence features. These experiments revealed that RGG/RG domains have different degrees of preference for binding to RNAs but share consistent trends in their selectivity towards RNAs with complex secondary structure. Thus, the binding behavior of RGG domains is best described as “degenerate specificity” reflecting that RGG/RG domains interact with a broad spectrum of RNAs that contain frequently observed sequence/structural elements. This mode of specificity is likely further facilitated by the intrinsically disordered nature of RGG/RG domains that enable them to adopt multiple conformations to adaptively bind RNA. Advisors/Committee Members: Robert T. Batey, Ravinder Singh, Deborah S. Wuttke, Shelley Copley, Thomas Perkins.

Subjects/Keywords: RNA-binding protein; RGG/RG domains; Biochemistry; Molecular Biology

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Ozdilek, B. A. (2017). Characterizing the RNA Binding Properties of the Intrinsically Disordered FUS Protein and RGG/RG Domains. (Doctoral Dissertation). University of Colorado. Retrieved from https://scholar.colorado.edu/mcdb_gradetds/77

Chicago Manual of Style (16^th Edition):

Ozdilek, Bagdeser Akdogan. “Characterizing the RNA Binding Properties of the Intrinsically Disordered FUS Protein and RGG/RG Domains.” 2017. Doctoral Dissertation, University of Colorado. Accessed August 18, 2019. https://scholar.colorado.edu/mcdb_gradetds/77.

MLA Handbook (7^th Edition):

Ozdilek, Bagdeser Akdogan. “Characterizing the RNA Binding Properties of the Intrinsically Disordered FUS Protein and RGG/RG Domains.” 2017. Web. 18 Aug 2019.

Vancouver:

Ozdilek BA. Characterizing the RNA Binding Properties of the Intrinsically Disordered FUS Protein and RGG/RG Domains. [Internet] [Doctoral dissertation]. University of Colorado; 2017. [cited 2019 Aug 18]. Available from: https://scholar.colorado.edu/mcdb_gradetds/77.

Council of Science Editors:

Ozdilek BA. Characterizing the RNA Binding Properties of the Intrinsically Disordered FUS Protein and RGG/RG Domains. [Doctoral Dissertation]. University of Colorado; 2017. Available from: https://scholar.colorado.edu/mcdb_gradetds/77

University of Colorado

12. Lloyd, Neil Ryan. Discrimination of ssRNA by Pot1 and Identification of a Novel CypE Aptamer Through an Optimized RNA SELEX Protocol.

Degree: PhD, 2018, University of Colorado

URL: https://scholar.colorado.edu/chem_gradetds/285

► Protein-ligand specificity forms the fundamental basis for many biological mechanisms with properly tuned binding being required for most biological processes. Aberrant interactions can result… (more)

▼ Protein-ligand specificity forms the fundamental basis for many biological mechanisms with properly tuned binding being required for most biological processes. Aberrant interactions can result in consequences ranging from wasted cellular resources to disease pathologies and death. As such, characterizing interaction specificities is a critical step in understanding biological systems. In this thesis I have characterized the RNA-binding properties of the telomere protection protein Pot1, developed an optimized SELEX protocol to characterize RNA-binding by newly identified RNA-binding proteins, and expanded on the RNA-binding specificity of one of those proteins, the epigenetic regulator CypE. High fidelity binding to ssDNA, but not ssRNA, is integral to the function of the essential telomere end protection protein Pot1, In <i>S. pombe</i>, this presents a unique challenge as the C-terminal domain of the DNA-binding domain, Pot1pC, exhibits non-specific ssDNA recognition, achieved through thermodynamically equivalent alternative binding conformations. Given this malleability, how simultaneous specificity for ssDNA over RNA is achieved was unclear. Examination of the ribose-position specificity of Pot1pC shows that ssDNA specificity is additive but not uniformly distributed across the ligand. High-resolution structures of Pot1pC in complex with RNA-DNA chimeric ligands reveal Pot1pC discriminates against RNA by utilizing conserved non-compensatory binding modes that feature significant rearrangement of the binding interface. These alternative conformations, accessed through both ligand and protein flexibility, recover much, but not all, of the binding energy, leading to the observed reduction in affinities suggesting that intermolecular interfaces are remarkably sophisticated in their tuning of specificity towards flexible ligands. Recent discovery of widespread RNA-binding by unexpected RNA binders highlights the need for functional characterization of these non-canonical RNA-binding domains. SELEX, combined with new sequencing technologies, represents an ideal technique to do this. Using CypE, an RNA-binding cyclophilin involved in splicing and chromatin remodeling, I have optimized a selection protocol for other cyclophilins. Selection against CypE, while not identifying an RNA that binds the cyclophilin, reveals an aptamer with 20-fold tighter binding than previously reported with an extended binding interface on the RRM, suggesting RNA as a competitive ligand for CypE and provoking implications for the role of RNA in CypE gene repression. Advisors/Committee Members: Deborah S. Wuttke, Robert T. Batey, Roy Parker, Robert D. Kuchta, Loren E. Hough.

Subjects/Keywords: cyclophilin; cyp33; cype; pot1; selex; telomeres; Biochemistry; Molecular Biology

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Lloyd, N. R. (2018). Discrimination of ssRNA by Pot1 and Identification of a Novel CypE Aptamer Through an Optimized RNA SELEX Protocol. (Doctoral Dissertation). University of Colorado. Retrieved from https://scholar.colorado.edu/chem_gradetds/285

Chicago Manual of Style (16^th Edition):

Lloyd, Neil Ryan. “Discrimination of ssRNA by Pot1 and Identification of a Novel CypE Aptamer Through an Optimized RNA SELEX Protocol.” 2018. Doctoral Dissertation, University of Colorado. Accessed August 18, 2019. https://scholar.colorado.edu/chem_gradetds/285.

MLA Handbook (7^th Edition):

Lloyd, Neil Ryan. “Discrimination of ssRNA by Pot1 and Identification of a Novel CypE Aptamer Through an Optimized RNA SELEX Protocol.” 2018. Web. 18 Aug 2019.

Vancouver:

Lloyd NR. Discrimination of ssRNA by Pot1 and Identification of a Novel CypE Aptamer Through an Optimized RNA SELEX Protocol. [Internet] [Doctoral dissertation]. University of Colorado; 2018. [cited 2019 Aug 18]. Available from: https://scholar.colorado.edu/chem_gradetds/285.

Council of Science Editors:

Lloyd NR. Discrimination of ssRNA by Pot1 and Identification of a Novel CypE Aptamer Through an Optimized RNA SELEX Protocol. [Doctoral Dissertation]. University of Colorado; 2018. Available from: https://scholar.colorado.edu/chem_gradetds/285

University of Colorado

13. Hunt, Sabrina Robin. Structure Determination of Vascular Endothelial Growth Factor Heparin-Binding Domain in Complex with a DNA Aptamer.

Degree: PhD, 2017, University of Colorado

URL: https://scholar.colorado.edu/chem_gradetds/270

► 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)

Subjects/Keywords: aptamer; nmr; structure; vascular endothelial growth factor; vegf; Biochemistry; Molecular Genetics

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th 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/270

Chicago Manual of Style (16^th 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 August 18, 2019. https://scholar.colorado.edu/chem_gradetds/270.

MLA Handbook (7^th Edition):

Hunt, Sabrina Robin. “Structure Determination of Vascular Endothelial Growth Factor Heparin-Binding Domain in Complex with a DNA Aptamer.” 2017. Web. 18 Aug 2019.

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 2019 Aug 18]. Available from: https://scholar.colorado.edu/chem_gradetds/270.

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/270

University of Colorado

14. McKercher, Marissa A. Multimodal Recognition of Diverse Peptides by the SH2 Domains of PLCγ1 and SH2B1.

Degree: PhD, 2018, University of Colorado

URL: https://scholar.colorado.edu/chem_gradetds/272

► SH2 domains recognize phosphotyrosine (pY)-containing peptide ligands and regulate a wide array of signaling events within receptor tyrosine kinase pathways. SH2 domains have individualized specificity… (more)

▼ SH2 domains recognize phosphotyrosine (pY)-containing peptide ligands and regulate a wide array of signaling events within receptor tyrosine kinase pathways. SH2 domains have individualized specificity for peptides, encoded in the amino acids neighboring the pY of the ligand. In contrast to this simple view of signaling specificity, high-throughput array studies have identified several SH2 domains capable of recognizing peptides containing chemically distinct amino acids at the positions neighboring the pY. For example, the C-terminal SH2 domain (PLCC) of phospholipase C-γ1 (PLCγ1) typically binds peptides containing small and hydrophobic amino acids adjacent to the pY, but can also recognize unexpected peptides containing amino acids with polar or bulky side chains that deviate from this pattern. Similarly, the SH2 domain of Src homology 2 B adaptor protein 1 (SH2B1) can recognize peptides containing either hydrophobic or acidic amino acids at the +3 position C-terminal to the pY. This multimodal specificity may enable these proteins to participate in diverse, previously unrecognized, signaling pathways in response to binding chemically dissimilar partners and facilitate their ability to act as signaling hubs. To better understand this multimodal specificity, we have used thermodynamic and structural approaches, including isothermal titration calorimetry (ITC), nuclear magnetic resonance (NMR), and X-ray crystallography, to elucidate the mechanisms of diverse peptide binding to PLCC and SH2B1. We have identified hydrophobic and charged residues that play distinct roles in peptide binding to each SH2 domain. High resolution crystal structures of PLCC and SH2B1 have also identified conformational plasticity within the peptide ligands of PLCC and within several loops of SH2B1, which appears to contribute to the ability of these domains to recognize diverse ligands. A better understanding of the adaptability of PLCC and SH2B1 will expand the ability of researchers to identify biological ligands of SH2 domains, and will be necessary for the rational development of small molecule therapeutics to target, and selectively inhibit, a desired SH2 domain/ligand interface. Advisors/Committee Members: Deborah S. Wuttke, Joseph J. Falke, Marcelo C. Sousa, Michael R. Shirts, Loren E. Hough.

Subjects/Keywords: phosphotyrosine; src homology 2 domain; peptides; hydrophobic; amino acids; Biochemistry; Molecular Biology

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

McKercher, M. A. (2018). Multimodal Recognition of Diverse Peptides by the SH2 Domains of PLCγ1 and SH2B1. (Doctoral Dissertation). University of Colorado. Retrieved from https://scholar.colorado.edu/chem_gradetds/272

Chicago Manual of Style (16^th Edition):

McKercher, Marissa A. “Multimodal Recognition of Diverse Peptides by the SH2 Domains of PLCγ1 and SH2B1.” 2018. Doctoral Dissertation, University of Colorado. Accessed August 18, 2019. https://scholar.colorado.edu/chem_gradetds/272.

MLA Handbook (7^th Edition):

McKercher, Marissa A. “Multimodal Recognition of Diverse Peptides by the SH2 Domains of PLCγ1 and SH2B1.” 2018. Web. 18 Aug 2019.

Vancouver:

McKercher MA. Multimodal Recognition of Diverse Peptides by the SH2 Domains of PLCγ1 and SH2B1. [Internet] [Doctoral dissertation]. University of Colorado; 2018. [cited 2019 Aug 18]. Available from: https://scholar.colorado.edu/chem_gradetds/272.

Council of Science Editors:

McKercher MA. Multimodal Recognition of Diverse Peptides by the SH2 Domains of PLCγ1 and SH2B1. [Doctoral Dissertation]. University of Colorado; 2018. Available from: https://scholar.colorado.edu/chem_gradetds/272

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