+publisher:"University of North Carolina" +contributor:("Lee, Andrew")

University of North Carolina

1. Cohen, Rachel. Quinary structure alters protein folding landscapes.

Degree: Chemistry, 2017, University of North Carolina

URL: https://cdr.lib.unc.edu/record/uuid:7b21bc35-13d7-417c-856d-683de8dd6205

► Most knowledge of protein chemistry is derived from experiments performed in dilute, buffered solutions. Although such experiments provide essential information, proteins function in the crowded… (more)

▼ Most knowledge of protein chemistry is derived from experiments performed in dilute, buffered solutions. Although such experiments provide essential information, proteins function in the crowded and complex cellular environment, which imposes an additional level of structure. This quinary structure comprises the transient interactions between macromolecules that provides organization and compartmentalization inside cells. I have used in-cell NMR spectroscopy to characterize quinary structure, and have shown that globular protein stability is affected by quinary interactions involving both the folded state and the unfolded ensemble. Chapter 1 reviews the history of quinary structure in the context of metabolic pathways, and the technological advances that have yielded recent insight into protein behavior in living cells. In Chapter 2, I use the K10H variant of the B domain of protein G (GB1, 6.2 kDa) as a pH reporter in Escherichia coli cells to show that quinary interactions influence the quality of in-cell 15N–1H HSQC NMR spectra. In Chapter 3, I quantify the pH-dependence of GB1 stability in cells. At neutral pH, GB1 stability in cells is comparable to that in buffer. As the pH decreases, the increased number of attractive interactions between E. coli proteins and GB1 destabilizes GB1 relative to buffer alone. I conclude that electrostatic interactions involving surface residues of the folded state contribute to quinary structure. Chapter 4 shows that quinary structure can also affect the unfolded state ensemble. It has been known for many years that the unfolded ensemble of GB1 is stabilized by a non-native hydrophobic staple. Exploiting this idea, I made several mutations that do not change the folded state of GB1, but have a large effect on its stability in buffer. These effects are severely attenuated in cells, demonstrating that the cellular environment can remodel the unfolded ensemble. My work shows that there is more to protein stability than a well-packed hydrophobic core; the key to understanding protein behavior in nature lies in quinary structure. Advisors/Committee Members: Cohen, Rachel, Pielak, Gary J., Brustad, Eric, Redinbo, Matthew R., Riordan, John, Lee, Andrew.

Subjects/Keywords: College of Arts and Sciences; Department of Chemistry

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

APA (6^th Edition):

Cohen, R. (2017). Quinary structure alters protein folding landscapes. (Thesis). University of North Carolina. Retrieved from https://cdr.lib.unc.edu/record/uuid:7b21bc35-13d7-417c-856d-683de8dd6205

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Cohen, Rachel. “Quinary structure alters protein folding landscapes.” 2017. Thesis, University of North Carolina. Accessed January 24, 2021. https://cdr.lib.unc.edu/record/uuid:7b21bc35-13d7-417c-856d-683de8dd6205.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Cohen, Rachel. “Quinary structure alters protein folding landscapes.” 2017. Web. 24 Jan 2021.

Vancouver:

Cohen R. Quinary structure alters protein folding landscapes. [Internet] [Thesis]. University of North Carolina; 2017. [cited 2021 Jan 24]. Available from: https://cdr.lib.unc.edu/record/uuid:7b21bc35-13d7-417c-856d-683de8dd6205.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Cohen R. Quinary structure alters protein folding landscapes. [Thesis]. University of North Carolina; 2017. Available from: https://cdr.lib.unc.edu/record/uuid:7b21bc35-13d7-417c-856d-683de8dd6205

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of North Carolina

2. Falk, Bradley. Probing subunit communication via a singly ligated state of dimeric thymidylate synthase.

Degree: Biochemistry and Biophysics, 2017, University of North Carolina

URL: https://cdr.lib.unc.edu/record/uuid:5277b259-725a-470f-a204-5de0c349ead6

► Enzymes are highly dynamic molecules that undergo motions on a wide variety of timescales that are linked to all aspects of their function from mediating… (more)

▼ Enzymes are highly dynamic molecules that undergo motions on a wide variety of timescales that are linked to all aspects of their function from mediating substrate binding and enabling chemistry to facilitating regulation by effector ligands. NMR studies on a handful of small monomeric enzymes have highlighted the role of dynamics in simple systems, but most enzymes possess added complexity stemming from size, oligomeric structures, multistep and/or multisubstrate mechanisms, and allosteric regulation. In recent years there has been a resurgence of interest in understanding the mechanisms of protein allostery. Canonically, allostery occurs in oligomeric proteins: ligand binding in one subunit alters binding or activity in a symmetry related second subunit. For such systems it is often straightforward to measure cooperative binding thermodynamics, but gaining detailed structural and/or dynamic mechanisms for intersubunit communication has proved to be much more difficult. This work has focused on applying NMR techniques to probe the inner workings of cooperativity and intersubunit communication in a large dimeric enzyme, Escherichia coli thymidylate synthase. A combined ITC and NMR approach showed that binding of substrate dUMP and cofactor mTHF occurs in both binding sites and dUMP binds with equal affinity. However, the two dUMP binding events are characterized by slightly different ΔCp showing that the two binding sites are not formally equivalent and presenting evidence for intersubunit communication. To probe this inequivalence a pair of mixed labeled dimers were created that can only bind substrate in one subunit and can be labeled on either subunit for NMR studies. These mixed labeled dimers enabled an NMR approach to distinguish the step-wise effects of ligand binding to this dimeric enzyme with subunit specificity. Binding studies revealed an asymmetric response to the two dUMP binding events and evidence of an extreme state in the singly bound form. The mixed dimers allow for visualization of singly bound peak positions which are typically elusive for dimeric systems. In thymidylate synthase these singly bound peaks fall in unexpected patterns that yield new insight into intersubunit communication. Advisors/Committee Members: Falk, Bradley, Lee, Andrew, Carter, Charles, Pielak, Gary J., Traut, Thomas, Zhang, Qi.

Subjects/Keywords: School of Medicine; Department of Biochemistry and Biophysics

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

APA (6^th Edition):

Falk, B. (2017). Probing subunit communication via a singly ligated state of dimeric thymidylate synthase. (Thesis). University of North Carolina. Retrieved from https://cdr.lib.unc.edu/record/uuid:5277b259-725a-470f-a204-5de0c349ead6

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Falk, Bradley. “Probing subunit communication via a singly ligated state of dimeric thymidylate synthase.” 2017. Thesis, University of North Carolina. Accessed January 24, 2021. https://cdr.lib.unc.edu/record/uuid:5277b259-725a-470f-a204-5de0c349ead6.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Falk, Bradley. “Probing subunit communication via a singly ligated state of dimeric thymidylate synthase.” 2017. Web. 24 Jan 2021.

Vancouver:

Falk B. Probing subunit communication via a singly ligated state of dimeric thymidylate synthase. [Internet] [Thesis]. University of North Carolina; 2017. [cited 2021 Jan 24]. Available from: https://cdr.lib.unc.edu/record/uuid:5277b259-725a-470f-a204-5de0c349ead6.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Falk B. Probing subunit communication via a singly ligated state of dimeric thymidylate synthase. [Thesis]. University of North Carolina; 2017. Available from: https://cdr.lib.unc.edu/record/uuid:5277b259-725a-470f-a204-5de0c349ead6

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of North Carolina

3. Kim, Doo Nam. Computer-based Design of β-sheet Containing Proteins.

Degree: Biochemistry and Biophysics, 2016, University of North Carolina

URL: https://cdr.lib.unc.edu/record/uuid:076f3980-6637-481b-bece-d26ad5cdb96a

► Protein design is an excellent test of the minimal determinants of protein structure. Although 70% of naturally occurring proteins contain β-sheets, most previous design efforts… (more)

Subjects/Keywords: School of Medicine; Department of Biochemistry and Biophysics

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

APA (6^th Edition):

Kim, D. N. (2016). Computer-based Design of β-sheet Containing Proteins. (Thesis). University of North Carolina. Retrieved from https://cdr.lib.unc.edu/record/uuid:076f3980-6637-481b-bece-d26ad5cdb96a

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Kim, Doo Nam. “Computer-based Design of β-sheet Containing Proteins.” 2016. Thesis, University of North Carolina. Accessed January 24, 2021. https://cdr.lib.unc.edu/record/uuid:076f3980-6637-481b-bece-d26ad5cdb96a.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Kim, Doo Nam. “Computer-based Design of β-sheet Containing Proteins.” 2016. Web. 24 Jan 2021.

Vancouver:

Kim DN. Computer-based Design of β-sheet Containing Proteins. [Internet] [Thesis]. University of North Carolina; 2016. [cited 2021 Jan 24]. Available from: https://cdr.lib.unc.edu/record/uuid:076f3980-6637-481b-bece-d26ad5cdb96a.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Kim DN. Computer-based Design of β-sheet Containing Proteins. [Thesis]. University of North Carolina; 2016. Available from: https://cdr.lib.unc.edu/record/uuid:076f3980-6637-481b-bece-d26ad5cdb96a

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of North Carolina

4. Page, Stephani. Analysis of Receiver Domain Dephosphorylation Kinetics.

Degree: Biochemistry and Biophysics, 2016, University of North Carolina

URL: https://cdr.lib.unc.edu/record/uuid:c10f28e9-b05d-4513-a60f-56e3289c2734

► Plants and microorganisms use two-component signal transduction systems (TCSs) to mediate responses to stimuli. Canonical TCSs consist of a sensory component, the sensor kinase (SK),… (more)

▼ Plants and microorganisms use two-component signal transduction systems (TCSs) to mediate responses to stimuli. Canonical TCSs consist of a sensory component, the sensor kinase (SK), and a response-mediating component. The phosphorylation state of the SK is regulated by the presence of stimuli. The response-mediating component, the response regulator (RR) accepts the phosphoryl group from the SK. Receiver domains are the conserved domain of RRs, and contain the site of phosphorylation, a conserved Asp. Receiver domain dephosphorylation results in signal termination, and can occur through phosphotransfer to water or histidine. The kinetics of self-catalyzed Asp → water phosphotransfer (autodephosphorylation) of RRs appear to be tuned to the timescales of the biological processes in which RRs participate, and reported rate constants span six orders of magnitude. More complex variations of TCSs, called phosphorelays, contain histidine-containing phosphotransfer domain (Hpt), which contain the phosphoaccepting histidine in Asp → His phosphotransfer. First, we propose a strategy for assessment of functional variation within protein families. We used bioinformatics to guide experimental analysis of non-conserved receiver domain active site residues D+2 and T+2 (two amino acids C-terminal to conserved Asp phosphorylation site and Thr/Ser, respectively). Using bioinformatics, we were able to determine a representative and feasible set of D+2/T+2 pairs to assess for kinetic effects on receiver domain autodephosphorylation. We also report results of additional bioinformatics analysis of receiver domain sequences assigned to RR subfamilies (determined by the presence of specific output domains). Second, this work represents the first reported demonstration of a small molecule analog for Hpts in TCSs. Imidazole is the functional group of His. Here, we characterize the imidazole-mediated dephosphoryation reaction of receiver domains. Additionally, we use imidazole to probe non-conserved active site residues as potential determinants of TCS Asp → His phosphotransfer reactions. Further, we show the structure of a receiver domain in the presence of a phosphomimic and imidazole. This work presents novel approaches to answering fundamental questions regarding receiver domain dephosphorylation reactions in two-component systems, and proposes avenues to further probe phosphotransfer reactions in TCSs and functional variation in protein families in general. Advisors/Committee Members: Page, Stephani, Bourret, Robert R., Campbell, Sharon, Collins, Edward, Cotter, Peggy, Lee, Andrew.

Subjects/Keywords: School of Medicine; Department of Biochemistry and Biophysics

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

APA (6^th Edition):

Page, S. (2016). Analysis of Receiver Domain Dephosphorylation Kinetics. (Thesis). University of North Carolina. Retrieved from https://cdr.lib.unc.edu/record/uuid:c10f28e9-b05d-4513-a60f-56e3289c2734

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Page, Stephani. “Analysis of Receiver Domain Dephosphorylation Kinetics.” 2016. Thesis, University of North Carolina. Accessed January 24, 2021. https://cdr.lib.unc.edu/record/uuid:c10f28e9-b05d-4513-a60f-56e3289c2734.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Page, Stephani. “Analysis of Receiver Domain Dephosphorylation Kinetics.” 2016. Web. 24 Jan 2021.

Vancouver:

Page S. Analysis of Receiver Domain Dephosphorylation Kinetics. [Internet] [Thesis]. University of North Carolina; 2016. [cited 2021 Jan 24]. Available from: https://cdr.lib.unc.edu/record/uuid:c10f28e9-b05d-4513-a60f-56e3289c2734.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Page S. Analysis of Receiver Domain Dephosphorylation Kinetics. [Thesis]. University of North Carolina; 2016. Available from: https://cdr.lib.unc.edu/record/uuid:c10f28e9-b05d-4513-a60f-56e3289c2734

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of North Carolina

5. Fant, Andrew. The Effect of Data Curation on the Accuracy of Quantitative Structure-Activity Relationship Models.

Degree: 2015, University of North Carolina

URL: https://cdr.lib.unc.edu/record/uuid:fc71a688-33be-402d-a99d-3d1c75efe86d

► In the 33 years since the first public release of GenBank, and the 15 years since the publication of the first pilot assembly of the… (more)

▼ In the 33 years since the first public release of GenBank, and the 15 years since the publication of the first pilot assembly of the human genome, drug discovery has been awash in a tsunami of data. But it has only been within the past decade that medicinal chemists and chemical biologists have had access to the same sorts of large-scale, public-access databases as bioinformaticians and molecular biologists have had for so long. The release of this data has sparked a renewed interest in computational methods for rational drug design, but questions have arisen recently about the accuracy and quality of this data. The same question has arisen in other scientific disciplines, but it has a particular urgency to practitioners of Quantitative Structure-Activity Relationship (QSAR) modeling. By its nature QSAR modeling depends on both activity data and chemical structures. While activities are usually expressed as numerical scalar values, a form ubiquitous throughout the sciences, chemical structures (especially that must be interpretable as such by computer software) are stored in a variety of specialized formats which are much less common and mostly ignored outside of cheminformatics and related fields. While previous research has determined that a 5% error rate in data being used for modeling can cause a QSAR model to be non-predictive and useless for its intended purpose, and workflows have been proposed which reduce the effect of inconsistent chemical structure representations on model accuracy, a fundamental question remains: “how accurate are the structure and activity data freely available to researchers?” To this end, we have undertaken two surveys of data quality, one focusing on chemical structure information in Internet resources and a second examining the uncertainty associated with compounds reported in the medicinal chemistry literature as abstracted in ChEMBL. The results of these studies have informed the creation of an improved workflow for the curation of structure-activity data which is intended to identify problematic data points in raw data extracted from databases so that an expert human curator can examine the underlying literature and resolve discrepancies between reported values. This workflow was in turn applied to the creation of two QSAR models that were used to implement a virtual screen seeking molecules capable of binding to both the serotonergic reuptake transporter and the alpha2a adrenergic receptor. While no suitable compounds were identified in the initial screening process, regions of chemical space that may yield truly novel alpha 2a receptor ligands have been identified. These regions can be targeted in future efforts. Basing data curation workflows on manual processes by human curators is not particularly viable, as humans have a tendency to introduce errors by inattention even as they identify and repair other problems. Computers cannot effectively curate data either. While they are highly accurate when programmed properly, they lack human creativity and insight that would allow… Advisors/Committee Members: Fant, Andrew, Tropsha, Alexander, Elston, Timothy, Lee, Andrew, Rusyn, Ivan, Singleton, Scott.

Subjects/Keywords: Pharmaceutical chemistry; Chemistry; Pharmacology; Eshelman School of Pharmacy; Division of Chemical Biology and Medicinal Chemistry

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

APA (6^th Edition):

Fant, A. (2015). The Effect of Data Curation on the Accuracy of Quantitative Structure-Activity Relationship Models. (Thesis). University of North Carolina. Retrieved from https://cdr.lib.unc.edu/record/uuid:fc71a688-33be-402d-a99d-3d1c75efe86d

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Fant, Andrew. “The Effect of Data Curation on the Accuracy of Quantitative Structure-Activity Relationship Models.” 2015. Thesis, University of North Carolina. Accessed January 24, 2021. https://cdr.lib.unc.edu/record/uuid:fc71a688-33be-402d-a99d-3d1c75efe86d.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Fant, Andrew. “The Effect of Data Curation on the Accuracy of Quantitative Structure-Activity Relationship Models.” 2015. Web. 24 Jan 2021.

Vancouver:

Fant A. The Effect of Data Curation on the Accuracy of Quantitative Structure-Activity Relationship Models. [Internet] [Thesis]. University of North Carolina; 2015. [cited 2021 Jan 24]. Available from: https://cdr.lib.unc.edu/record/uuid:fc71a688-33be-402d-a99d-3d1c75efe86d.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Fant A. The Effect of Data Curation on the Accuracy of Quantitative Structure-Activity Relationship Models. [Thesis]. University of North Carolina; 2015. Available from: https://cdr.lib.unc.edu/record/uuid:fc71a688-33be-402d-a99d-3d1c75efe86d

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of North Carolina

6. Friedman, Adam. THE SELECTION OF HIGHLY STABLE APTAMERS FROM A 2’-FULLY MODIFIED fGmH RNA LIBRARY.

Degree: 2015, University of North Carolina

URL: https://cdr.lib.unc.edu/record/uuid:c82dc258-3481-4204-a6a3-0f06792bacd7

► When developed as tools for applications within biological systems, RNA aptamers immediately face numerous obstacles, in particular nuclease degradation and post-selection 2’ modification. This study… (more)

Subjects/Keywords: Pharmaceutical chemistry; Biochemistry; Molecular biology; Eshelman School of Pharmacy; Division of Chemical Biology and Medicinal Chemistry

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

APA (6^th Edition):

Friedman, A. (2015). THE SELECTION OF HIGHLY STABLE APTAMERS FROM A 2’-FULLY MODIFIED fGmH RNA LIBRARY. (Thesis). University of North Carolina. Retrieved from https://cdr.lib.unc.edu/record/uuid:c82dc258-3481-4204-a6a3-0f06792bacd7

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Friedman, Adam. “THE SELECTION OF HIGHLY STABLE APTAMERS FROM A 2’-FULLY MODIFIED fGmH RNA LIBRARY.” 2015. Thesis, University of North Carolina. Accessed January 24, 2021. https://cdr.lib.unc.edu/record/uuid:c82dc258-3481-4204-a6a3-0f06792bacd7.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Friedman, Adam. “THE SELECTION OF HIGHLY STABLE APTAMERS FROM A 2’-FULLY MODIFIED fGmH RNA LIBRARY.” 2015. Web. 24 Jan 2021.

Vancouver:

Friedman A. THE SELECTION OF HIGHLY STABLE APTAMERS FROM A 2’-FULLY MODIFIED fGmH RNA LIBRARY. [Internet] [Thesis]. University of North Carolina; 2015. [cited 2021 Jan 24]. Available from: https://cdr.lib.unc.edu/record/uuid:c82dc258-3481-4204-a6a3-0f06792bacd7.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Friedman A. THE SELECTION OF HIGHLY STABLE APTAMERS FROM A 2’-FULLY MODIFIED fGmH RNA LIBRARY. [Thesis]. University of North Carolina; 2015. Available from: https://cdr.lib.unc.edu/record/uuid:c82dc258-3481-4204-a6a3-0f06792bacd7

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of North Carolina

7. Perfetti, Michael. Discovery of Small Molecule and Peptide-based Ligands for the Methyl-Lysine Binding Proteins 53BP1 and PHF1/PHF19.

Degree: 2015, University of North Carolina

URL: https://cdr.lib.unc.edu/record/uuid:10ee9105-3039-4676-88e9-b1619e8893d7

► Improving the understanding of the role of chromatin regulators in the initiation, development, and suppression of cancer and other devastating diseases is critical, as they… (more)

▼ Improving the understanding of the role of chromatin regulators in the initiation, development, and suppression of cancer and other devastating diseases is critical, as they are integral players in the regulation of DNA integrity and gene expression. Developing chemical tools for histone binding proteins that possess cellular activity will allow for further elucidation of the specific function of this class of histone regulating proteins. This research specifically targeted two different classes of Tudor domain containing histone binding proteins that are directly involved in the DNA damage response and modulation of gene transcription activities. The first methyl-lysine binding protein targeted was 53BP1, which is a DNA damage response protein. 53BP1 uses a tandem tudor domain (TTD) to recognize histone H4 dimethylated on lysine 20 (H4K20me2), a post-translational modification (PTM) induced by double-strand DNA breaks. Through a cross-screening approach, two different small molecule ligands were identified for the 53BP1 TTD. Medicinal chemistry and structure-based design techniques were used, in addition to the development of a fragment-based screen, toward the goal of optimizing the potency and selectivity of the identified small molecule ligands. The later small molecule ligand, 61 (UNC2170), was further optimized as a micromolar inhibitor of 53BP1, which demonstrated a 17-fold selectivity for 53BP1 TTD as compared to other screened methyl-lysine (Kme) binding proteins. Structural studies revealed that the N-tert-butyl amine of 61 (UNC2170) anchors the compound in the Kme binding pocket of 53BP1 TTD, making it competitive with endogenous Kme substrates. X-ray crystallography demonstrated that 61 (UNC2170) bound at the interface between two tudor domains of a 53BP1 TTD dimer. Importantly, this compound functions as a 53BP1 antagonist in cellular lysates and shows cellular activity by suppressing class switch recombination, a process that requires a functional 53BP1 tudor domain. These results demonstrate that 61 (UNC2170) is a functionally active, fragment-like ligand for 53BP1 and the first selective small molecule ligand discovered for 53BP1 TTD. Additional research efforts were focused toward development of peptide-based ligands for the histone binding proteins PHF1 and PHF19. These proteins play an active role in the modulation of PRC2 activity that in turn controls various gene expression and repression outcomes. Structure-based design techniques were used to design peptide-based inhibitors for PHF1 and PHF19. Research efforts were focused on conducting an initial Structure Activity Relationship (SAR) study to develop a short, low molecular weight peptide via truncation of the endogenous histone 3 peptide. Structure based design techniques were used to develop a shorter 6- or 7-mer peptide that possessed various amino acid substitutions that specifically bound within the Tudor domain of PHF1 and PHF19 with emphasis on determining a quaternary amine mimic for the tri-methyl lysine 36 residue. These efforts… Advisors/Committee Members: Perfetti, Michael, Frye, Stephen, Lawrence, David, Strahl, Brian, Wang, Greg, Bowers, Albert, Lee, Andrew.

Subjects/Keywords: Pharmaceutical chemistry; Eshelman School of Pharmacy; Division of Chemical Biology and Medicinal Chemistry

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

APA (6^th Edition):

Perfetti, M. (2015). Discovery of Small Molecule and Peptide-based Ligands for the Methyl-Lysine Binding Proteins 53BP1 and PHF1/PHF19. (Thesis). University of North Carolina. Retrieved from https://cdr.lib.unc.edu/record/uuid:10ee9105-3039-4676-88e9-b1619e8893d7

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Perfetti, Michael. “Discovery of Small Molecule and Peptide-based Ligands for the Methyl-Lysine Binding Proteins 53BP1 and PHF1/PHF19.” 2015. Thesis, University of North Carolina. Accessed January 24, 2021. https://cdr.lib.unc.edu/record/uuid:10ee9105-3039-4676-88e9-b1619e8893d7.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Perfetti, Michael. “Discovery of Small Molecule and Peptide-based Ligands for the Methyl-Lysine Binding Proteins 53BP1 and PHF1/PHF19.” 2015. Web. 24 Jan 2021.

Vancouver:

Perfetti M. Discovery of Small Molecule and Peptide-based Ligands for the Methyl-Lysine Binding Proteins 53BP1 and PHF1/PHF19. [Internet] [Thesis]. University of North Carolina; 2015. [cited 2021 Jan 24]. Available from: https://cdr.lib.unc.edu/record/uuid:10ee9105-3039-4676-88e9-b1619e8893d7.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Perfetti M. Discovery of Small Molecule and Peptide-based Ligands for the Methyl-Lysine Binding Proteins 53BP1 and PHF1/PHF19. [Thesis]. University of North Carolina; 2015. Available from: https://cdr.lib.unc.edu/record/uuid:10ee9105-3039-4676-88e9-b1619e8893d7

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of North Carolina

8. Smith, Austin. PROTEIN STRUCTURE, STABILITY AND DYNAMICS IN CELLS AND CELL-LIKE ENVIRONMENTS.

Degree: Chemistry, 2015, University of North Carolina

URL: https://cdr.lib.unc.edu/record/uuid:19a082d1-c0ab-4254-a131-5445a0502aed

► The intracellular milieu is filled with small molecules, nucleic acids, lipids and proteins. Theories have attempted to explain how macromolecules react to this environment for… (more)

Subjects/Keywords: Chemistry; Biophysics; Biochemistry; College of Arts and Sciences; Department of Chemistry

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

APA (6^th Edition):

Smith, A. (2015). PROTEIN STRUCTURE, STABILITY AND DYNAMICS IN CELLS AND CELL-LIKE ENVIRONMENTS. (Thesis). University of North Carolina. Retrieved from https://cdr.lib.unc.edu/record/uuid:19a082d1-c0ab-4254-a131-5445a0502aed

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Smith, Austin. “PROTEIN STRUCTURE, STABILITY AND DYNAMICS IN CELLS AND CELL-LIKE ENVIRONMENTS.” 2015. Thesis, University of North Carolina. Accessed January 24, 2021. https://cdr.lib.unc.edu/record/uuid:19a082d1-c0ab-4254-a131-5445a0502aed.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Smith, Austin. “PROTEIN STRUCTURE, STABILITY AND DYNAMICS IN CELLS AND CELL-LIKE ENVIRONMENTS.” 2015. Web. 24 Jan 2021.

Vancouver:

Smith A. PROTEIN STRUCTURE, STABILITY AND DYNAMICS IN CELLS AND CELL-LIKE ENVIRONMENTS. [Internet] [Thesis]. University of North Carolina; 2015. [cited 2021 Jan 24]. Available from: https://cdr.lib.unc.edu/record/uuid:19a082d1-c0ab-4254-a131-5445a0502aed.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Smith A. PROTEIN STRUCTURE, STABILITY AND DYNAMICS IN CELLS AND CELL-LIKE ENVIRONMENTS. [Thesis]. University of North Carolina; 2015. Available from: https://cdr.lib.unc.edu/record/uuid:19a082d1-c0ab-4254-a131-5445a0502aed

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of North Carolina

9. Thompson, Peter. The structural and functional consequences of the interaction of the vinculin tail domain with F-actin and PIP2.

Degree: Biochemistry and Biophysics, 2015, University of North Carolina

URL: https://cdr.lib.unc.edu/record/uuid:f8dcee81-c727-40cc-94ad-8d3a7736d4a1

► Vinculin is an essential, highly-conserved eukaryotic scaffolding protein. It localizes to focal adhesions and adherens juctions, where it assists in physically linking the actin cytoskeleton… (more)

Subjects/Keywords: Biophysics; Biochemistry; School of Medicine; Department of Biochemistry and Biophysics

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

APA (6^th Edition):

Thompson, P. (2015). The structural and functional consequences of the interaction of the vinculin tail domain with F-actin and PIP2. (Thesis). University of North Carolina. Retrieved from https://cdr.lib.unc.edu/record/uuid:f8dcee81-c727-40cc-94ad-8d3a7736d4a1

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Thompson, Peter. “The structural and functional consequences of the interaction of the vinculin tail domain with F-actin and PIP2.” 2015. Thesis, University of North Carolina. Accessed January 24, 2021. https://cdr.lib.unc.edu/record/uuid:f8dcee81-c727-40cc-94ad-8d3a7736d4a1.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Thompson, Peter. “The structural and functional consequences of the interaction of the vinculin tail domain with F-actin and PIP2.” 2015. Web. 24 Jan 2021.

Vancouver:

Thompson P. The structural and functional consequences of the interaction of the vinculin tail domain with F-actin and PIP2. [Internet] [Thesis]. University of North Carolina; 2015. [cited 2021 Jan 24]. Available from: https://cdr.lib.unc.edu/record/uuid:f8dcee81-c727-40cc-94ad-8d3a7736d4a1.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Thompson P. The structural and functional consequences of the interaction of the vinculin tail domain with F-actin and PIP2. [Thesis]. University of North Carolina; 2015. Available from: https://cdr.lib.unc.edu/record/uuid:f8dcee81-c727-40cc-94ad-8d3a7736d4a1

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of North Carolina

10. Leano, Jonathan. THE CLASP FAMILY REGULATES MICROTUBULE DYNAMICS BY USING AN ARRAY OF TOG-LIKE DOMAINS.

Degree: Biochemistry and Biophysics, 2014, University of North Carolina

URL: https://cdr.lib.unc.edu/record/uuid:d79f2760-7d3a-406f-b147-d36a677c5a21

► CLASP is a key regulator of microtubule (MT) dynamics and bipolar mitotic spindle formation, with mutants displaying chromosome aggregation, aberrant monopolar spindle morphologies, and aneuploidy.… (more)

▼ CLASP is a key regulator of microtubule (MT) dynamics and bipolar mitotic spindle formation, with mutants displaying chromosome aggregation, aberrant monopolar spindle morphologies, and aneuploidy. How CLASP binds the microtubule lattice to regulate MT dynamics and facilitate proper spindle assembly remains unknown; however, it has been postulated that cryptic TOG domains underlie CLASP's ability to regulate MT dynamics. In this work, we report the crystal structure of the first cryptic TOG domain (TOG2) from human CLASP1, confirming the presence of a TOG array in CLASP. CLASP1 TOG2 displays a bent architecture at the tubulin-binding surface that contrasts with the flat tubulin-binding surface from XMAP215 family TOG domains. Mutating key tubulin-binding determinants along the tubulin-binding surface of TOG2 abrogated the ability of CLASP to 1) rescue mitotic bipolar spindle formation in Drosophila S2 cells 2) associate CLASP with the MT lattice, and 3) promote in vitro MT polymerization. These findings highlight the mechanistic use of a cryptic TOG domain in CLASP to facilitate bipolar spindle formation and MT polymerization. Determining the crystal structure of TOG1 and the second cryptic TOG-like domain (TOG3) is ongoing. Structural characterization of CLASP's array of TOG domains we shows that differential TOG domain architecture confers distinct functions for each TOG domain including MT lattice association, MT polymerization, and MT stabilization. In addition, CLASP's C-terminal domain (CTD) associates with the coiled-coil regions of various associating factors to recruit CLASP at specific cellular locations and is also a necessary component for CLASP dimerization. To determine the role of CLASP CTD in promoting dimerization and interacting with known CLASP-associating factors, we are structurally and biochemically characterizing the interaction between CLASP CTD and the coiled-coil (CC) domain of CLIP-170, a known CLASP-associating factor. CLASP CTD and CLIP-170 CC form a complex in SEC-MALS and ITC experiments. In addition, CLASP CTD alone exists as a monomer, suggesting that CLASP CTD is necessary, but not sufficient, for dimerization. Further analysis to structurally characterize the interaction between CLASP CTD and CLIP-170 CC is an ongoing goal for this thesis work. Advisors/Committee Members: Leano, Jonathan, Slep, Kevin, Lee, Andrew, Slep, Kevin, Cheney, Richard, Crews, Stephen, Ke, Hengming, Lee, Andrew.

Subjects/Keywords: Biophysics; Biochemistry; School of Medicine; Department of Biochemistry and Biophysics

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

APA (6^th Edition):

Leano, J. (2014). THE CLASP FAMILY REGULATES MICROTUBULE DYNAMICS BY USING AN ARRAY OF TOG-LIKE DOMAINS. (Thesis). University of North Carolina. Retrieved from https://cdr.lib.unc.edu/record/uuid:d79f2760-7d3a-406f-b147-d36a677c5a21

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Leano, Jonathan. “THE CLASP FAMILY REGULATES MICROTUBULE DYNAMICS BY USING AN ARRAY OF TOG-LIKE DOMAINS.” 2014. Thesis, University of North Carolina. Accessed January 24, 2021. https://cdr.lib.unc.edu/record/uuid:d79f2760-7d3a-406f-b147-d36a677c5a21.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Leano, Jonathan. “THE CLASP FAMILY REGULATES MICROTUBULE DYNAMICS BY USING AN ARRAY OF TOG-LIKE DOMAINS.” 2014. Web. 24 Jan 2021.

Vancouver:

Leano J. THE CLASP FAMILY REGULATES MICROTUBULE DYNAMICS BY USING AN ARRAY OF TOG-LIKE DOMAINS. [Internet] [Thesis]. University of North Carolina; 2014. [cited 2021 Jan 24]. Available from: https://cdr.lib.unc.edu/record/uuid:d79f2760-7d3a-406f-b147-d36a677c5a21.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Leano J. THE CLASP FAMILY REGULATES MICROTUBULE DYNAMICS BY USING AN ARRAY OF TOG-LIKE DOMAINS. [Thesis]. University of North Carolina; 2014. Available from: https://cdr.lib.unc.edu/record/uuid:d79f2760-7d3a-406f-b147-d36a677c5a21

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of North Carolina

11. McDonald, Leanna Rose. Towards understanding the mechanisms of allostery: the investigation of the relationship between dynamics, structure and function in the model allosteric protein CheY.

Degree: Biochemistry and Biophysics, 2013, University of North Carolina

URL: https://cdr.lib.unc.edu/record/uuid:b091a6db-f64b-4dda-8728-0eb3ef10e44c

► Allosteric proteins are extremely important in signaling, yet the mechanism(s) of the switch between inactive and active conformations is not clearly understood. It is now… (more)

▼ Allosteric proteins are extremely important in signaling, yet the mechanism(s) of the switch between inactive and active conformations is not clearly understood. It is now widely recognized that dynamics are important to consider for understanding allosteric protein function. Escherichia coli CheY, a response regulator protein from the two-component signal transduction system that regulates bacterial chemotaxis, is an ideal protein for the study of allosteric mechanisms. Here, we report an NMR relaxation study of dynamics over multiple timescales at both backbone and side-chain sites upon an allosteric response to phosphorylation. By utilizing 15N CPMG relaxation dispersion experiments, we monitored the inherent dynamic switching of unphosphorylated CheY. We show that unphosphorylated CheY does not undergo a two-state concerted switch between the inactive and active conformations. Interestingly, partial saturation of Mg2+ enhances the intrinsic allosteric motions. Taken together with chemical shift perturbations, these data indicate that the ms-ms timescale motions underlying CheY allostery are segmental in nature. Upon phosphorylation, CheY allosterically responds with a change in dynamics on both the μs-ms and ps-ns timescale. We observe an apparent decrease and redistribution of μs-ms dynamics upon phosphorylation of CheY. Additionally, methyl groups with the largest changes in ps-ns dynamics localize to the regions of conformational change measured by μs-ms dynamics. The limited spread of changes in ps-ns dynamics suggests a distinct relationship between motions on the μs-ms and ps-ns timescales in CheY. Analysis of an activating mutant A113P yields a similar pattern of side-chain ps-ns dynamic changes as upon phosphorylation. This relationship between an activating mutant and an activating pattern of dynamic changes is further evidence for a distinct relationship between the dynamics on multiple timescales and the function of the protein. The allosteric mechanism utilized by CheY highlights the diversity of roles dynamics play in protein function and the complex mechanisms proteins utilize for allostery. Advisors/Committee Members: McDonald, Leanna Rose, Lee, Andrew.

Subjects/Keywords: Gillings School of Global Public Health; School of Medicine; Department of Biochemistry and Biophysics

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APA (6^th Edition):

McDonald, L. R. (2013). Towards understanding the mechanisms of allostery: the investigation of the relationship between dynamics, structure and function in the model allosteric protein CheY. (Thesis). University of North Carolina. Retrieved from https://cdr.lib.unc.edu/record/uuid:b091a6db-f64b-4dda-8728-0eb3ef10e44c

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

McDonald, Leanna Rose. “Towards understanding the mechanisms of allostery: the investigation of the relationship between dynamics, structure and function in the model allosteric protein CheY.” 2013. Thesis, University of North Carolina. Accessed January 24, 2021. https://cdr.lib.unc.edu/record/uuid:b091a6db-f64b-4dda-8728-0eb3ef10e44c.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

McDonald, Leanna Rose. “Towards understanding the mechanisms of allostery: the investigation of the relationship between dynamics, structure and function in the model allosteric protein CheY.” 2013. Web. 24 Jan 2021.

Vancouver:

McDonald LR. Towards understanding the mechanisms of allostery: the investigation of the relationship between dynamics, structure and function in the model allosteric protein CheY. [Internet] [Thesis]. University of North Carolina; 2013. [cited 2021 Jan 24]. Available from: https://cdr.lib.unc.edu/record/uuid:b091a6db-f64b-4dda-8728-0eb3ef10e44c.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

McDonald LR. Towards understanding the mechanisms of allostery: the investigation of the relationship between dynamics, structure and function in the model allosteric protein CheY. [Thesis]. University of North Carolina; 2013. Available from: https://cdr.lib.unc.edu/record/uuid:b091a6db-f64b-4dda-8728-0eb3ef10e44c

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of North Carolina

12. Zhang, Jun. Structure and dynamics of the MAGUK core of PSD-95.

Degree: Biochemistry and Biophysics, 2011, University of North Carolina

URL: https://cdr.lib.unc.edu/record/uuid:f039ae75-0716-4a0b-a379-1691bbc4b994

► Protein allostery plays central roles in regulation of enzyme catalysis, signaling conduction and cellular metabolism. In this research, the interdomain allostery of postsynaptic density protein… (more)

▼ Protein allostery plays central roles in regulation of enzyme catalysis, signaling conduction and cellular metabolism. In this research, the interdomain allostery of postsynaptic density protein 95 (PSD-95), a key component of the postsynapse, was studied using NMR and other biophysical methods. Previous research has identified numerous PSD-95 interaction partners and revealed many PSD-95 mediated biological functions. Interestingly, interdomain allostery within PSD-95 has been found between PDZ3 and the following SH3/GK, and these allosteric events are regulated by phosphorylation. However, the structural mechanism of interdomain allostery and phosphorylation regulation is not addressed by in vivo studies on biological functions or in vitro studies on excised domains. In this dissertation, we studied the structural and dynamic effects of phosphorylation at the PDZ3/SH3 linker (Y397, S415 and S418). Upon phosphorylation, we found that the C-extension α-helix of PDZ3 is unfolded and undocked. We further examined the PDZ3-SH3 construct and showed that phosphorylation interrupts the domain interaction between PDZ3 and SH3. Using chemical shift perturbation and paramagnetic relaxation enhancement, we identified the PDZ3-SH3 interface. These experiments also suggested that CRIPT binding moves PDZ3 away from SH3. To understand interdomain allostery, we modeled the PDZ3-SH3-GK structure using SAXS. Consistent with PRE results, we found PDZ3 is mainly docked to the Hook domain region of the SH3 domain, and CRIPT binding reshuffles the domain packing between PDZ3 and SH3-GK. To obtain a high resolution structural model of PDZ3-SH3-GK, we carried out Rosetta simulations in the presence of PRE and chemical shift perturbation constraints. We found that the PDZ3 domain uses its peptide binding groove to interact with the PDZ3-SH3 linker. This interaction brings PDZ3 close to SH3, whereas it can be disrupted by CRIPT binding. This Rosetta model also reveals that the positively charged face of the Hook domain, which is the binding interface for calmodulin, is masked by the PDZ3 domain. Therefore the model provides a basis for understanding interdomain allostery between SH3 and PDZ3. In this research we also discussed the possible mechanism by which PDZ3 and GK allostery is transferred. Advisors/Committee Members: Zhang, Jun, Lee, Andrew, University of North Carolina at Chapel Hill.

Subjects/Keywords: School of Medicine; Department of Biochemistry and Biophysics

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

APA (6^th Edition):

Zhang, J. (2011). Structure and dynamics of the MAGUK core of PSD-95. (Thesis). University of North Carolina. Retrieved from https://cdr.lib.unc.edu/record/uuid:f039ae75-0716-4a0b-a379-1691bbc4b994

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Zhang, Jun. “Structure and dynamics of the MAGUK core of PSD-95.” 2011. Thesis, University of North Carolina. Accessed January 24, 2021. https://cdr.lib.unc.edu/record/uuid:f039ae75-0716-4a0b-a379-1691bbc4b994.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Zhang, Jun. “Structure and dynamics of the MAGUK core of PSD-95.” 2011. Web. 24 Jan 2021.

Vancouver:

Zhang J. Structure and dynamics of the MAGUK core of PSD-95. [Internet] [Thesis]. University of North Carolina; 2011. [cited 2021 Jan 24]. Available from: https://cdr.lib.unc.edu/record/uuid:f039ae75-0716-4a0b-a379-1691bbc4b994.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Zhang J. Structure and dynamics of the MAGUK core of PSD-95. [Thesis]. University of North Carolina; 2011. Available from: https://cdr.lib.unc.edu/record/uuid:f039ae75-0716-4a0b-a379-1691bbc4b994

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of North Carolina

13. Carroll, Mary J. Probing the dynamics of dihydrofolate reductase with small molecule inhibitors.

Degree: 2011, University of North Carolina

URL: https://cdr.lib.unc.edu/record/uuid:5e109623-f912-4cee-bba0-9fd295b85ec0

► High-resolution protein crystal structures have served as the template from which to devise small molecule modulators of protein function through structure-based drug design for many… (more)

▼ High-resolution protein crystal structures have served as the template from which to devise small molecule modulators of protein function through structure-based drug design for many decades. However, protein function, such as ligand binding, dissociation and catalysis, necessitates structural flexibility of the macromolecule. Protein dynamics are often ignored in structure-based design, largely due to limited information on protein flexibility in the presence of bound ligands (e.g., peptides or small molecules). To gain insight into how protein dynamics are modulated by the binding of small molecule inhibitors, nuclear magnetic resonance spectroscopy has been used herein to characterize both fast and slow timescale motions in the pharmaceutical target protein Escherichia coli dihydrofolate reductase. A series of inhibitors of the enzyme (antifolates) that cover a broad range of binding affinity have been studied while bound to the holoenzyme complex. In the presence of two high-affinity inhibitors, dynamics appear to be essentially identical on both the fast and slow timescales. Specifically, these two inhibitors decouple functional loop switching motions on the slow timescale. One medium-affinity antifolate from the series is shown to be conformationally flexible while bound to the enzyme, eliciting a functional switching motion in a loop distal to its binding. In considering the entire panel of eight antifolates (two of high affinity, one of medium affinity, and a homologous series five of reduced-affinity antifolates), the rates of conformational switching of the enzyme on the slow timescale correlate well with ligand binding affinity and the kinetic rate of ligand dissociation. Within the sites exhibiting slow motions, a consensus set of residues is identified to be sampling a novel and structurally identical excited state that differs from physiological complexes bound to folate-derived ligands. This group of antifolate consensus residues is posited to serve as a mechanical initiator of ligand dissociation, an observation that may be quite prevalent in receptor-ligand interactions. Fast timescale motions across the series of antifolate complexes studied also suggest a correlation of flexibility with ligand binding affinity and a possible linkage between motions on the fast and slow timescales. Advisors/Committee Members: Carroll, Mary J., Lee, Andrew, University of North Carolina at Chapel Hill.

Subjects/Keywords: Eshelman School of Pharmacy; Division of Chemical Biology and Medicinal Chemistry

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

APA (6^th Edition):

Carroll, M. J. (2011). Probing the dynamics of dihydrofolate reductase with small molecule inhibitors. (Thesis). University of North Carolina. Retrieved from https://cdr.lib.unc.edu/record/uuid:5e109623-f912-4cee-bba0-9fd295b85ec0

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Carroll, Mary J. “Probing the dynamics of dihydrofolate reductase with small molecule inhibitors.” 2011. Thesis, University of North Carolina. Accessed January 24, 2021. https://cdr.lib.unc.edu/record/uuid:5e109623-f912-4cee-bba0-9fd295b85ec0.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Carroll, Mary J. “Probing the dynamics of dihydrofolate reductase with small molecule inhibitors.” 2011. Web. 24 Jan 2021.

Vancouver:

Carroll MJ. Probing the dynamics of dihydrofolate reductase with small molecule inhibitors. [Internet] [Thesis]. University of North Carolina; 2011. [cited 2021 Jan 24]. Available from: https://cdr.lib.unc.edu/record/uuid:5e109623-f912-4cee-bba0-9fd295b85ec0.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Carroll MJ. Probing the dynamics of dihydrofolate reductase with small molecule inhibitors. [Thesis]. University of North Carolina; 2011. Available from: https://cdr.lib.unc.edu/record/uuid:5e109623-f912-4cee-bba0-9fd295b85ec0

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of North Carolina

14. Jones, Courtney. Segregation of the Biological Functions of Heparan Sulfate.

Degree: 2011, University of North Carolina

URL: https://cdr.lib.unc.edu/record/uuid:baf89370-64fc-4d1e-ba92-41762a55d22f

► Heparan sulfate (HS) represents a major class of glycans that perform central physiological functions. Heparin, a special class of HS polysaccharides, is an anticoagulant that… (more)

▼ Heparan sulfate (HS) represents a major class of glycans that perform central physiological functions. Heparin, a special class of HS polysaccharides, is an anticoagulant that inhibitss factors Xa and IIa via its ability to modulate the antithrombin (AT) mechanism. However, medicinal administration of exogenous heparin has been shown to elicit heparin-induced thrombocytopenia (HIT) in 2-5% of patients. This response results from a Type II hypersensitivity reaction to heparin-platelet factor 4 (PF4) complexes. Studies have shown that the interaction of heparin and PF4 may, in part, be mediated by charge. Emerging HS and glycosaminoglycan microarray techniques are used to interrogate HS's structure and function relationship to develop novel therapeutic agents. However, availability of HS with specific sulfation patterns has been a limiting factor and impedes the accuracy of HS glycomics studies. Although organic synthesis provides oligosaccharides, these may not fully represent the biological functions of polysaccharides. Here, we present a study for developing an enzyme-based approach to synthesize a polysaccharide library with different sulfation patterns. Using different combinations of biosynthetic enzymes, we synthesized eight unique polysaccharides. We discovered that polysaccharides without the iduronic acid residue displayed strong binding affinity to antithrombin and high anti-Xa and anti-IIa activities. We also used this biosynthetic method to regulate the overall charge of these glycans by enzymatically modulating the degree of sulfation in order to reduce the binding of these polysaccharides to PF4. By employing a biosynthetic scheme, we generated different species of polysaccharides encompassing varying sulfation patterns. We discovered that modulation of the amount of N-sulfation at the beginning of synthesis is the most important motif for decreasing the polysaccharide's propensity for PF4. The anticoagulant activity of these polysaccharide species was maintained although their affinity for PF4 was decreased. This enzyme-based synthetic approach could become a general method for discovering new HS structures with unique biological functions. Advisors/Committee Members: Jones, Courtney, Liu, Jian, Jarstfer, Michael, Lee, Andrew, Lawrence, David, Pedersen, Lee.

Subjects/Keywords: Eshelman School of Pharmacy; Division of Chemical Biology and Medicinal Chemistry

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

APA (6^th Edition):

Jones, C. (2011). Segregation of the Biological Functions of Heparan Sulfate. (Thesis). University of North Carolina. Retrieved from https://cdr.lib.unc.edu/record/uuid:baf89370-64fc-4d1e-ba92-41762a55d22f

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Jones, Courtney. “Segregation of the Biological Functions of Heparan Sulfate.” 2011. Thesis, University of North Carolina. Accessed January 24, 2021. https://cdr.lib.unc.edu/record/uuid:baf89370-64fc-4d1e-ba92-41762a55d22f.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Jones, Courtney. “Segregation of the Biological Functions of Heparan Sulfate.” 2011. Web. 24 Jan 2021.

Vancouver:

Jones C. Segregation of the Biological Functions of Heparan Sulfate. [Internet] [Thesis]. University of North Carolina; 2011. [cited 2021 Jan 24]. Available from: https://cdr.lib.unc.edu/record/uuid:baf89370-64fc-4d1e-ba92-41762a55d22f.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Jones C. Segregation of the Biological Functions of Heparan Sulfate. [Thesis]. University of North Carolina; 2011. Available from: https://cdr.lib.unc.edu/record/uuid:baf89370-64fc-4d1e-ba92-41762a55d22f

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of North Carolina

15. Whitley, Matthew James. A Nuclear Magnetic Resonance Investigation of the Role of Structure and Dynamics in the Function of Chymotrypsin Inhibitor 2.

Degree: Biochemistry and Biophysics, 2010, University of North Carolina

URL: https://cdr.lib.unc.edu/record/uuid:8e3a1e7e-76ac-4f81-8dae-57f9074500cc

► The concept of proteins as static folded three-dimensional objects has yielded to the more sophisticated idea that proteins are better understood as dynamic entities constantly… (more)

▼ The concept of proteins as static folded three-dimensional objects has yielded to the more sophisticated idea that proteins are better understood as dynamic entities constantly fluctuating about an average conformation rather than being restricted to any single conformation. With the knowledge that proteins sample many different conformational substates comes the question of whether the intrinsic conformational dynamics plays a role in protein function. The work presented in this dissertation begins to explore this question through the study of two model systems. The effects of subtle hydrophobic core mutations on the structure and internal dynamics of the nonallosteric protein chymotrypsin inhibitor 2 (CI2) were characterized using advanced nuclear magnetic resonance methodologies. These experiments revealed that the dynamics of methyl-bearing side chains respond globally and uniformly to hydrophobic core mutations without apparent regard for the position or chemical nature of the mutation; this is a clear example of long range intraprotein communication, a general property that forms the basis of the more complex phenomenon of allostery. Furthermore, the global increase in side-chain flexibility upon mutation takes place without concomitant structural perturbations. Functional assays of these mutants, however, revealed only subtle changes in inhibitory ability, and thus it appears that CI2 is not evolutionarily optimized to harness internal dynamics to modulate function despite its ability to sense perturbations through altered dynamics. The second model system studied is chemotaxis protein Y (CheY), the response regulator domain of the bacterial chemotaxis pathway. CheY is an allosteric protein, and this work reports a characterization of the dynamics involved in phosphorylation-based allosteric activation. The initial results indicate that the classic framework for understanding CheY allostery is too rigid in its definition of distinct inactive and active conformational states. Many of the key residues used to delineate the allosteric state of CheY are dynamic on multiple time scales, thus indicating that they sample numerous conformations and precluding the definition of a sole inactive or active conformation. The results of this work will be useful as a framework for analysis of larger, more complex proteins of biomedical relevance as our theoretical understanding of and experimental capabilities for studying protein dynamics continue to increase. Advisors/Committee Members: Whitley, Matthew James, Lee, Andrew.

Subjects/Keywords: School of Medicine; Department of Biochemistry and Biophysics

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

APA (6^th Edition):

Whitley, M. J. (2010). A Nuclear Magnetic Resonance Investigation of the Role of Structure and Dynamics in the Function of Chymotrypsin Inhibitor 2. (Thesis). University of North Carolina. Retrieved from https://cdr.lib.unc.edu/record/uuid:8e3a1e7e-76ac-4f81-8dae-57f9074500cc

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Whitley, Matthew James. “A Nuclear Magnetic Resonance Investigation of the Role of Structure and Dynamics in the Function of Chymotrypsin Inhibitor 2.” 2010. Thesis, University of North Carolina. Accessed January 24, 2021. https://cdr.lib.unc.edu/record/uuid:8e3a1e7e-76ac-4f81-8dae-57f9074500cc.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Whitley, Matthew James. “A Nuclear Magnetic Resonance Investigation of the Role of Structure and Dynamics in the Function of Chymotrypsin Inhibitor 2.” 2010. Web. 24 Jan 2021.

Vancouver:

Whitley MJ. A Nuclear Magnetic Resonance Investigation of the Role of Structure and Dynamics in the Function of Chymotrypsin Inhibitor 2. [Internet] [Thesis]. University of North Carolina; 2010. [cited 2021 Jan 24]. Available from: https://cdr.lib.unc.edu/record/uuid:8e3a1e7e-76ac-4f81-8dae-57f9074500cc.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Whitley MJ. A Nuclear Magnetic Resonance Investigation of the Role of Structure and Dynamics in the Function of Chymotrypsin Inhibitor 2. [Thesis]. University of North Carolina; 2010. Available from: https://cdr.lib.unc.edu/record/uuid:8e3a1e7e-76ac-4f81-8dae-57f9074500cc

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of North Carolina

16. Boyer, Joshua Alan. Dialing-Up Distal Sites in Proteins: The Search for the Mechanical Consequences of Thermodynamic Coupling in eglin C.

Degree: Biochemistry and Biophysics, 2009, University of North Carolina

URL: https://cdr.lib.unc.edu/record/uuid:961e7408-a3c0-461b-a588-1c65039f49d4

► Proteins have evolved to exploit long-range dynamic and structural effects as a means of regulating function. Understanding this communication between distal sites in proteins is… (more)

Subjects/Keywords: School of Medicine; Department of Biochemistry and Biophysics

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

APA (6^th Edition):

Boyer, J. A. (2009). Dialing-Up Distal Sites in Proteins: The Search for the Mechanical Consequences of Thermodynamic Coupling in eglin C. (Thesis). University of North Carolina. Retrieved from https://cdr.lib.unc.edu/record/uuid:961e7408-a3c0-461b-a588-1c65039f49d4

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Boyer, Joshua Alan. “Dialing-Up Distal Sites in Proteins: The Search for the Mechanical Consequences of Thermodynamic Coupling in eglin C.” 2009. Thesis, University of North Carolina. Accessed January 24, 2021. https://cdr.lib.unc.edu/record/uuid:961e7408-a3c0-461b-a588-1c65039f49d4.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Boyer, Joshua Alan. “Dialing-Up Distal Sites in Proteins: The Search for the Mechanical Consequences of Thermodynamic Coupling in eglin C.” 2009. Web. 24 Jan 2021.

Vancouver:

Boyer JA. Dialing-Up Distal Sites in Proteins: The Search for the Mechanical Consequences of Thermodynamic Coupling in eglin C. [Internet] [Thesis]. University of North Carolina; 2009. [cited 2021 Jan 24]. Available from: https://cdr.lib.unc.edu/record/uuid:961e7408-a3c0-461b-a588-1c65039f49d4.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Boyer JA. Dialing-Up Distal Sites in Proteins: The Search for the Mechanical Consequences of Thermodynamic Coupling in eglin C. [Thesis]. University of North Carolina; 2009. Available from: https://cdr.lib.unc.edu/record/uuid:961e7408-a3c0-461b-a588-1c65039f49d4

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of North Carolina

17. Mauldin, Randall Vernon. Multi-timescale dynamic effects of antifolate binding and mutations in dihydrofolate reductase.

Degree: Biochemistry and Biophysics, 2009, University of North Carolina

URL: https://cdr.lib.unc.edu/record/uuid:7329276e-8d3e-41a0-b33c-7b303a3150f5

► Long-range amino acid communication is crucial for biological processes such as protein signaling, enzyme catalysis and allosteric regulation. In this work, the underlying dynamic networks… (more)

Subjects/Keywords: School of Medicine; Department of Biochemistry and Biophysics

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

APA (6^th Edition):

Mauldin, R. V. (2009). Multi-timescale dynamic effects of antifolate binding and mutations in dihydrofolate reductase. (Thesis). University of North Carolina. Retrieved from https://cdr.lib.unc.edu/record/uuid:7329276e-8d3e-41a0-b33c-7b303a3150f5

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Mauldin, Randall Vernon. “Multi-timescale dynamic effects of antifolate binding and mutations in dihydrofolate reductase.” 2009. Thesis, University of North Carolina. Accessed January 24, 2021. https://cdr.lib.unc.edu/record/uuid:7329276e-8d3e-41a0-b33c-7b303a3150f5.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Mauldin, Randall Vernon. “Multi-timescale dynamic effects of antifolate binding and mutations in dihydrofolate reductase.” 2009. Web. 24 Jan 2021.

Vancouver:

Mauldin RV. Multi-timescale dynamic effects of antifolate binding and mutations in dihydrofolate reductase. [Internet] [Thesis]. University of North Carolina; 2009. [cited 2021 Jan 24]. Available from: https://cdr.lib.unc.edu/record/uuid:7329276e-8d3e-41a0-b33c-7b303a3150f5.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Mauldin RV. Multi-timescale dynamic effects of antifolate binding and mutations in dihydrofolate reductase. [Thesis]. University of North Carolina; 2009. Available from: https://cdr.lib.unc.edu/record/uuid:7329276e-8d3e-41a0-b33c-7b303a3150f5

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of North Carolina

18. Clarkson, Michael. Dynamic Effects of Mutations in Eglin c.

Degree: Biochemistry and Biophysics, 2006, University of North Carolina

URL: https://cdr.lib.unc.edu/record/uuid:5a070fe7-5aff-4908-9e6c-782a95fe1a25

► In order to investigate the mechanism of dynamic propagation in proteins and the role these responses play in energetic communication, a series of novel dynamic… (more)

Subjects/Keywords: School of Medicine; Department of Biochemistry and Biophysics

Record Details Similar Records Cite « Share

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

APA (6^th Edition):

Clarkson, M. (2006). Dynamic Effects of Mutations in Eglin c. (Thesis). University of North Carolina. Retrieved from https://cdr.lib.unc.edu/record/uuid:5a070fe7-5aff-4908-9e6c-782a95fe1a25

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Clarkson, Michael. “Dynamic Effects of Mutations in Eglin c.” 2006. Thesis, University of North Carolina. Accessed January 24, 2021. https://cdr.lib.unc.edu/record/uuid:5a070fe7-5aff-4908-9e6c-782a95fe1a25.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Clarkson, Michael. “Dynamic Effects of Mutations in Eglin c.” 2006. Web. 24 Jan 2021.

Vancouver:

Clarkson M. Dynamic Effects of Mutations in Eglin c. [Internet] [Thesis]. University of North Carolina; 2006. [cited 2021 Jan 24]. Available from: https://cdr.lib.unc.edu/record/uuid:5a070fe7-5aff-4908-9e6c-782a95fe1a25.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Clarkson M. Dynamic Effects of Mutations in Eglin c. [Thesis]. University of North Carolina; 2006. Available from: https://cdr.lib.unc.edu/record/uuid:5a070fe7-5aff-4908-9e6c-782a95fe1a25

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

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Open Access Theses and Dissertations