+publisher:"University of North Carolina" +contributor:("Carter, Charles")

University of North Carolina

1. Chandrasekaran, Srinivas Niranj. Parameter optimization on the convergence surface of PATH simulations.

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

URL: https://cdr.lib.unc.edu/record/uuid:a3c79ded-6255-40ca-8f2d-5da0e7dadb9c

► Computational treatments of protein conformational changes tend to focus on the trajectories themselves, despite the fact that it is the transition state structures that contain… (more)

▼ Computational treatments of protein conformational changes tend to focus on the trajectories themselves, despite the fact that it is the transition state structures that contain information about the barriers that impose multi-state behavior. PATH is an algorithm that computes a transition pathway between two protein crystal structures, along with the transition state structure, by minimizing the Onsager-Machlup action functional. It is rapid but depends on several unknown input parameters whose range of different values can potentially generate different transition-state structures. Transition-state structures arising from different input parameters cannot be uniquely compared with those generated by other methods. I outline modifications that I have made to the PATH algorithm that estimates these input parameters in a manner that circumvents these difficulties, and describe two complementary tests that validate the transition-state structures found by the PATH algorithm. First, I show that although the PATH algorithm and two other approaches to computing transition pathways produce different low-energy structures connecting the initial and final ground-states with the transition state, all three methods agree closely on the configurations of their transition states. Second, I show that the PATH transition states are close to the saddle points of free-energy surfaces connecting initial and final states generated by replica-exchange Discrete Molecular Dynamics simulations. I show that aromatic side-chain rearrangements create similar potential energy barriers in the transition-state structures identified by PATH for a signaling protein, a contractile protein, and an enzyme. Finally, I observed, but cannot account for, the fact that trajectories obtained for all-atom and Cα-only simulations identify transition state structures in which the Cα atoms are in essentially the same positions. The consistency between transition-state structures derived by different algorithms for unrelated protein systems argues that although functionally important protein conformational change trajectories are to a degree stochastic, they nonetheless pass through a well-defined transition state whose detailed structural properties can rapidly be identified using PATH. In the end, I outline the strategies that could enhance the efficiency and applicability of PATH. Advisors/Committee Members: Chandrasekaran, Srinivas Niranj, Carter, Charles, Campbell, Sharon, Dokholyan, Nikolay, Hermans, Jan, Zhang, Qi.

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

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APA (6^th Edition):

Chandrasekaran, S. N. (2016). Parameter optimization on the convergence surface of PATH simulations. (Thesis). University of North Carolina. Retrieved from https://cdr.lib.unc.edu/record/uuid:a3c79ded-6255-40ca-8f2d-5da0e7dadb9c

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):

Chandrasekaran, Srinivas Niranj. “Parameter optimization on the convergence surface of PATH simulations.” 2016. Thesis, University of North Carolina. Accessed January 18, 2021. https://cdr.lib.unc.edu/record/uuid:a3c79ded-6255-40ca-8f2d-5da0e7dadb9c.

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

MLA Handbook (7^th Edition):

Chandrasekaran, Srinivas Niranj. “Parameter optimization on the convergence surface of PATH simulations.” 2016. Web. 18 Jan 2021.

Vancouver:

Chandrasekaran SN. Parameter optimization on the convergence surface of PATH simulations. [Internet] [Thesis]. University of North Carolina; 2016. [cited 2021 Jan 18]. Available from: https://cdr.lib.unc.edu/record/uuid:a3c79ded-6255-40ca-8f2d-5da0e7dadb9c.

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

Council of Science Editors:

Chandrasekaran SN. Parameter optimization on the convergence surface of PATH simulations. [Thesis]. University of North Carolina; 2016. Available from: https://cdr.lib.unc.edu/record/uuid:a3c79ded-6255-40ca-8f2d-5da0e7dadb9c

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 18, 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. 18 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 18]. 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. Garrett, Christopher. Characterization of a Key Mycobacterium tuberculosis Lipase, LipY.

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

URL: https://cdr.lib.unc.edu/record/uuid:80107563-6511-4298-9a76-7185a0292ea6

► The causative agent of tuberculosis, Mycobacterium tuberculosis is estimated to be present in roughly a third of the world’s population. One of the hallmarks of… (more)

Subjects/Keywords: Biochemistry; Biophysics; 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):

Garrett, C. (2015). Characterization of a Key Mycobacterium tuberculosis Lipase, LipY. (Thesis). University of North Carolina. Retrieved from https://cdr.lib.unc.edu/record/uuid:80107563-6511-4298-9a76-7185a0292ea6

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):

Garrett, Christopher. “Characterization of a Key Mycobacterium tuberculosis Lipase, LipY.” 2015. Thesis, University of North Carolina. Accessed January 18, 2021. https://cdr.lib.unc.edu/record/uuid:80107563-6511-4298-9a76-7185a0292ea6.

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

MLA Handbook (7^th Edition):

Garrett, Christopher. “Characterization of a Key Mycobacterium tuberculosis Lipase, LipY.” 2015. Web. 18 Jan 2021.

Vancouver:

Garrett C. Characterization of a Key Mycobacterium tuberculosis Lipase, LipY. [Internet] [Thesis]. University of North Carolina; 2015. [cited 2021 Jan 18]. Available from: https://cdr.lib.unc.edu/record/uuid:80107563-6511-4298-9a76-7185a0292ea6.

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

Council of Science Editors:

Garrett C. Characterization of a Key Mycobacterium tuberculosis Lipase, LipY. [Thesis]. University of North Carolina; 2015. Available from: https://cdr.lib.unc.edu/record/uuid:80107563-6511-4298-9a76-7185a0292ea6

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

University of North Carolina

4. Jacak, Ronald. Explicit Consideration of Solubility and Interaction Specificity in Computational Protein Design.

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

URL: https://cdr.lib.unc.edu/record/uuid:eee31c27-82d0-4e6b-bb39-6456860e0255

► Most successes to date in computational protein design have relied on optimizing sequences to fit well for a single structure. Multistate design represents a new… (more)

▼ Most successes to date in computational protein design have relied on optimizing sequences to fit well for a single structure. Multistate design represents a new approach to designing proteins in which sequences are optimized for multiple contexts usually given by multiple structures (states). In multistate design simulations, sequences that either stabilize the target state or destabilize alternate competing states are selected. This dissertation describes the application of multistate design to two problems in protein design: designing sequences for solubility and increasing binding specificity in protein-protein interface design. Previous studies with the modeling program Rosetta have shown that many designed proteins have patches of hydrophobic surface area that are considerably larger than what is seen in native proteins. These patches can lead to nonspecific association and aggregation. We use a multistate design approach to address protein solubility by disfavoring the aggregated state through the addition of a new solubility term to the Rosetta energy function. The score term explicitly detects and penalizes the formation of hydrophobic patches during design. Designing with this new score term results in proteins with naturally occurring frequencies of hydrophobic amino acids on the surface without large hydrophobic patches. Designing protein-protein interfaces with high affinity and specificity is still a challenge for protein design algorithms. Multistate design is well-suited for addressing the problem of specificity because it can explicitly disfavor off-target interactions. Using a new implementation in Rosetta, multistate design is applied to the orthogonal interface design problem: redesign a protein with many partners to interact with only one of the partners. We use the RalA signaling network as the model system and make our design goal a redesigned RalA that only interacts with the effector RalBP1. Multistate design is able to recover several of the known mutants important for effector binding and predicts many new mutations that alter binding specificity. From in silico predictions, single-state design for Ral/RalBP1 by itself is not sufficient to destabilize RalA's interactions with its other effectors. Only multistate design is able to destabilize both of the negative states and give the desired interaction specificity. Advisors/Committee Members: Jacak, Ronald, Kuhlman, Brian, Campbell, Sharon, Carter, Charles, Der, Channing, Elston, Timothy.

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):

Jacak, R. (2011). Explicit Consideration of Solubility and Interaction Specificity in Computational Protein Design. (Thesis). University of North Carolina. Retrieved from https://cdr.lib.unc.edu/record/uuid:eee31c27-82d0-4e6b-bb39-6456860e0255

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):

Jacak, Ronald. “Explicit Consideration of Solubility and Interaction Specificity in Computational Protein Design.” 2011. Thesis, University of North Carolina. Accessed January 18, 2021. https://cdr.lib.unc.edu/record/uuid:eee31c27-82d0-4e6b-bb39-6456860e0255.

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

MLA Handbook (7^th Edition):

Jacak, Ronald. “Explicit Consideration of Solubility and Interaction Specificity in Computational Protein Design.” 2011. Web. 18 Jan 2021.

Vancouver:

Jacak R. Explicit Consideration of Solubility and Interaction Specificity in Computational Protein Design. [Internet] [Thesis]. University of North Carolina; 2011. [cited 2021 Jan 18]. Available from: https://cdr.lib.unc.edu/record/uuid:eee31c27-82d0-4e6b-bb39-6456860e0255.

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

Council of Science Editors:

Jacak R. Explicit Consideration of Solubility and Interaction Specificity in Computational Protein Design. [Thesis]. University of North Carolina; 2011. Available from: https://cdr.lib.unc.edu/record/uuid:eee31c27-82d0-4e6b-bb39-6456860e0255

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

University of North Carolina

5. Pham, Yen Bao. Characterization of a Tryptophanyl-tRNA Synthetase Urzyme and its Intramolecular Complementation Establishes a Model to Study the Catalytic Mechanism and Evolution of the Contemporary Class I Aminoacyl-tRNA Synthetases.

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

URL: https://cdr.lib.unc.edu/record/uuid:8953fcbd-d082-4011-a700-6f46cf19201f

► Aminoacyl-tRNA synthetases (AaRSs) comprise a crucial group of enzymes catalyzing a two-step reaction to activate amino acids and acylate their cognate tRNAs. It is widely… (more)

▼ Aminoacyl-tRNA synthetases (AaRSs) comprise a crucial group of enzymes catalyzing a two-step reaction to activate amino acids and acylate their cognate tRNAs. It is widely known that highly conserved sequences and motifs divide the synthetases evenly into two distinct classes of ten members each. However, the questions of how they might have emerged and evolved and what a reasonable candidate for the ancestral enzyme would be like still remain unsolved. Answering these would not only shed light on the aaRSs evolution but could also help unravel the critical point when the extremely high kinetic barrier of amino acid activation was bypassed, allowing the synthesis of primitive peptide chains. The research object of the studies is a tryptophanyl-tRNA synthetase (TrpRS) Urzyme (Ur-: prefix means primitive, original) which would fit the description of a potential candidate for the ancestral class I aaRS. The TrpRS Urzyme is designed to retain class I signature sequences that comprise the ATP-binding Rossmann fold. Interestingly, it has similar affinity for ATP compared to the full length wild type enzyme but surprisingly low affinity for the cognate amino acid substrate, tryptophan. This might be explained by absence of the connecting peptide 1 (CP1), an insertion that wraps around the tryptophan binding site in the contemporary enzyme and occurs at the same location as the editing domain in four other class I aaRSs (MetRS, LeuRS, IleRS, ValRS). An in cis modular complementation experiment might point out a potential role for CP1 in configuring a confirmation that has high affinity for tryptophan, functionally similar to a primitive proofreading function. Furthermore, an Urzyme containing the C-terminal domain (CTD) in cis has been constructed to further explore CTD's role in tryptophan activation and tRNATrp acylation. The active TrpRS Urzyme establishment confirms an essential prediction of, and is therefore, the first experiment to provide evidential support for the unique Rodin-Ohno hypothesis about the earlier existence of a sense-antisense coding system for both aaRS classes. The successful design of the TrpRS Urzyme has also provided a precedent that has led to the construction of class I and class II aaRS Urzymes of comparable lengths. Advisors/Committee Members: Pham, Yen Bao, Carter, Charles.

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):

Pham, Y. B. (2010). Characterization of a Tryptophanyl-tRNA Synthetase Urzyme and its Intramolecular Complementation Establishes a Model to Study the Catalytic Mechanism and Evolution of the Contemporary Class I Aminoacyl-tRNA Synthetases. (Thesis). University of North Carolina. Retrieved from https://cdr.lib.unc.edu/record/uuid:8953fcbd-d082-4011-a700-6f46cf19201f

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):

Pham, Yen Bao. “Characterization of a Tryptophanyl-tRNA Synthetase Urzyme and its Intramolecular Complementation Establishes a Model to Study the Catalytic Mechanism and Evolution of the Contemporary Class I Aminoacyl-tRNA Synthetases.” 2010. Thesis, University of North Carolina. Accessed January 18, 2021. https://cdr.lib.unc.edu/record/uuid:8953fcbd-d082-4011-a700-6f46cf19201f.

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

MLA Handbook (7^th Edition):

Pham, Yen Bao. “Characterization of a Tryptophanyl-tRNA Synthetase Urzyme and its Intramolecular Complementation Establishes a Model to Study the Catalytic Mechanism and Evolution of the Contemporary Class I Aminoacyl-tRNA Synthetases.” 2010. Web. 18 Jan 2021.

Vancouver:

Pham YB. Characterization of a Tryptophanyl-tRNA Synthetase Urzyme and its Intramolecular Complementation Establishes a Model to Study the Catalytic Mechanism and Evolution of the Contemporary Class I Aminoacyl-tRNA Synthetases. [Internet] [Thesis]. University of North Carolina; 2010. [cited 2021 Jan 18]. Available from: https://cdr.lib.unc.edu/record/uuid:8953fcbd-d082-4011-a700-6f46cf19201f.

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

Council of Science Editors:

Pham YB. Characterization of a Tryptophanyl-tRNA Synthetase Urzyme and its Intramolecular Complementation Establishes a Model to Study the Catalytic Mechanism and Evolution of the Contemporary Class I Aminoacyl-tRNA Synthetases. [Thesis]. University of North Carolina; 2010. Available from: https://cdr.lib.unc.edu/record/uuid:8953fcbd-d082-4011-a700-6f46cf19201f

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

University of North Carolina

6. Potempa, Marc. Mechanisms Regulating HIV-1 Protease Activity.

Degree: Microbiology and Immunology, 2015, University of North Carolina

URL: https://cdr.lib.unc.edu/record/uuid:0f7ef394-0dc8-43d8-92d7-cd6e230029b7

► The Human Immunodeficiency Virus Type 1 (HIV-1) Protease (PR) has no direct involvement in the early steps of HIV-1 replication. Nonetheless, it is the timely… (more)

▼ The Human Immunodeficiency Virus Type 1 (HIV-1) Protease (PR) has no direct involvement in the early steps of HIV-1 replication. Nonetheless, it is the timely and ordered processing of the viral structural proteins by the HIV-1 PR during virion maturation that facilitates the successful completion of virus entry, reverse transcription, and integration. Though a considerable amount of research has been devoted to deciphering how the enzyme prepares a virus particle for infection, the mechanisms regulating its activities continue to remain incompletely defined. RNA serves as one putative regulatory factor, since efficient processing of the maturation intermediate p15NC requires RNA in vitro. Though previously believed relevant to only p15NC cleavage, I demonstrate that RNA enhances HIV-1 proteolysis reactions in a substrate-independent manner. The increased catalytic activity of the HIV-1 PR results from a direct interaction between RNA and the enzyme, with the magnitude of the effect dependent upon the size of the RNA molecule. Large (>400 base) RNAs accelerated proteolytic processing by over 100-fold under near-physiological conditions. This considerable change stemmed from both improved substrate recognition (Km) and turnover rate (kcat). Variability in amino acid sequence also guides HIV-1 PR activity. However, the absence of any overt patterns across HIV-1 cleavage sites has complicated the delineation of why these differences result in diverse processing efficiencies. To address this question, I generated the largest-to-date dataset of globular proteins cleaved by the HIV-1 PR in near-physiological conditions. From these data, I unravel a number of site-specific processing requirements, and identify potentially important relationships shared between multiple cleavage sites. These results additionally enabled the formation of a preliminary conceptual model for explaining processing site amino acid composition. Advisors/Committee Members: Potempa, Marc, Swanstrom, Ronald, Lemon, Stanley, Margolis, David, De Paris, Kristina, Carter, Charles.

Subjects/Keywords: Virology; Molecular biology; Biochemistry; School of Medicine; Department of Microbiology and Immunology

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APA (6^th Edition):

Potempa, M. (2015). Mechanisms Regulating HIV-1 Protease Activity. (Thesis). University of North Carolina. Retrieved from https://cdr.lib.unc.edu/record/uuid:0f7ef394-0dc8-43d8-92d7-cd6e230029b7

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):

Potempa, Marc. “Mechanisms Regulating HIV-1 Protease Activity.” 2015. Thesis, University of North Carolina. Accessed January 18, 2021. https://cdr.lib.unc.edu/record/uuid:0f7ef394-0dc8-43d8-92d7-cd6e230029b7.

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

MLA Handbook (7^th Edition):

Potempa, Marc. “Mechanisms Regulating HIV-1 Protease Activity.” 2015. Web. 18 Jan 2021.

Vancouver:

Potempa M. Mechanisms Regulating HIV-1 Protease Activity. [Internet] [Thesis]. University of North Carolina; 2015. [cited 2021 Jan 18]. Available from: https://cdr.lib.unc.edu/record/uuid:0f7ef394-0dc8-43d8-92d7-cd6e230029b7.

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

Council of Science Editors:

Potempa M. Mechanisms Regulating HIV-1 Protease Activity. [Thesis]. University of North Carolina; 2015. Available from: https://cdr.lib.unc.edu/record/uuid:0f7ef394-0dc8-43d8-92d7-cd6e230029b7

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

University of North Carolina

7. Williams, Tishan. MECHANISM-BASED INHIBITION OF BACTERIAL AND MITOCHONDRIAL TRYPTOPHANYL-TRNA SYNTHESASES IS POTENTIATED BY MG2+•ATP.

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

URL: https://cdr.lib.unc.edu/record/uuid:a44867d0-97df-4434-89c5-d5a4fcb040be

► Eukaryotes have distinct nuclear genes for tryptophanyl-tRNA synthetase (TrpRS) enzymes targeted by N-terminal sequence variations to the cytoplasm (Hc) and mitochondria (Hmt) that share only… (more)

▼ Eukaryotes have distinct nuclear genes for tryptophanyl-tRNA synthetase (TrpRS) enzymes targeted by N-terminal sequence variations to the cytoplasm (Hc) and mitochondria (Hmt) that share only 14% sequence identity. Indolmycin, a natural tryptophan analog, competes with tryptophan for binding to tryptophanyl-tRNA synthetase (TrpRS) enzymes. Although bacterial and eukaryotic cytosolic TrpRSs have comparable affinities for tryptophan, KM ~2 μM, eukaryotic cytosolic TrpRS enzymes are able to evade inhibition by indolmycin. Tryptophan binding to Bacillus stearothermophilus (Bs) TrpRS is largely promoted by hydrophobic interactions and recognition of the indole nitrogen by the side chain of Asp 132. By contrast, HcTrpRS complements non polar interactions for tryptophan binding with electrostatic and hydrogen bonding interactions, which we show by modelling are inconsistent with indolmycin binding. Our crystallographic and inhibition kinetics data show the non-reactive analog indolmycin can recruit unique polar interactions to form an active-site metal coordination that lies off the normal mechanistic path, enhancing affinity to BsTrpRS and other prokaryotic TrpRS enzymes by 1500-fold over its tryptophan substrate. The Mg2+ ion in the inhibited complex forms significantly closer contacts with triphosphate oxygen atoms of ATP and three water molecules than occur in the catalytically-competent pre-transition state (preTS). Indolmycin binding also leads to weakened interactions between ATP and active-site lysine side-chains. Confirmation of our interpretation of structural consequences of indolmycin binding comes from a 1.82 Å crystal structure of an indolmycin-inhibited HmtTrpRS complex. This structure unequivocally demonstrates the use of similar determinants by mitochondrial and bacterial TrpRS enzymes to bind both ATP and indolmycin, with the mitochondrial enzyme forming similar ATP-enzyme, ATP-metal and indolmycin-enzyme interactions. Indolmycin binds HmtTrpRS ~700-times tighter than tryptophan and Mg2+•ATP leads to an ~80-fold enhancement in indolmycin binding affinity. The oxazolinone- Mg2+•ATP interaction contributes ~-2.2 kcal/mol to the Gibbs free energy of the fully-liganded indolmycin inhibited HmtTrpRS complex. Together, our complementary structural, kinetic and thermodynamic characterization of BsTrpRS and HmtTrpRS establish a shared mechanism for indolmycin inhibition of mitochondrial and prokaryotic TrpRS enzymes, which bind indolmycin 105-106-fold tighter than eukaryotic cytosolic homologs. Advisors/Committee Members: Williams, Tishan, Carter, Charles, Ke, Hengming, Beese, Lorena, Fried, Howard.

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

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APA (6^th Edition):

Williams, T. (2016). MECHANISM-BASED INHIBITION OF BACTERIAL AND MITOCHONDRIAL TRYPTOPHANYL-TRNA SYNTHESASES IS POTENTIATED BY MG2+•ATP. (Thesis). University of North Carolina. Retrieved from https://cdr.lib.unc.edu/record/uuid:a44867d0-97df-4434-89c5-d5a4fcb040be

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):

Williams, Tishan. “MECHANISM-BASED INHIBITION OF BACTERIAL AND MITOCHONDRIAL TRYPTOPHANYL-TRNA SYNTHESASES IS POTENTIATED BY MG2+•ATP.” 2016. Thesis, University of North Carolina. Accessed January 18, 2021. https://cdr.lib.unc.edu/record/uuid:a44867d0-97df-4434-89c5-d5a4fcb040be.

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

MLA Handbook (7^th Edition):

Williams, Tishan. “MECHANISM-BASED INHIBITION OF BACTERIAL AND MITOCHONDRIAL TRYPTOPHANYL-TRNA SYNTHESASES IS POTENTIATED BY MG2+•ATP.” 2016. Web. 18 Jan 2021.

Vancouver:

Williams T. MECHANISM-BASED INHIBITION OF BACTERIAL AND MITOCHONDRIAL TRYPTOPHANYL-TRNA SYNTHESASES IS POTENTIATED BY MG2+•ATP. [Internet] [Thesis]. University of North Carolina; 2016. [cited 2021 Jan 18]. Available from: https://cdr.lib.unc.edu/record/uuid:a44867d0-97df-4434-89c5-d5a4fcb040be.

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

Council of Science Editors:

Williams T. MECHANISM-BASED INHIBITION OF BACTERIAL AND MITOCHONDRIAL TRYPTOPHANYL-TRNA SYNTHESASES IS POTENTIATED BY MG2+•ATP. [Thesis]. University of North Carolina; 2016. Available from: https://cdr.lib.unc.edu/record/uuid:a44867d0-97df-4434-89c5-d5a4fcb040be

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