1985 — 1988 |
Purich, Daniel Lee |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Chemistry of Tubulin Guanine Nucleotide Interactions
In this application, we outline a series of experiments designid to provide a more in-depth understanding of the factors controlling the binding and hydrolytic steps which together constitute the tubulin GTPase reaction and their relationship to microtubule self-assembly: (1) Examination of the medium and intermediate oxygen-18 exchange reactions associated with the assembly-induced GTPase (The goal here is to learn about the reversibility of certain steps in the hydrolytic mechanism and the rate constants which define their rates and fluxes.); (2) determination of the stereochemical course of the hydrolysis using GTP Lambda S labeled with oxygen-16, -17 and -18 stereospecifically in the Lambda-phosphate (This will help discriminate between reactions involving a single in-line attack or two inversions, the latter characteristic of a phosphoryl-protein intermediates); (3) Characterization of the binding mechanism for guanine nucleotides (The objective is to learn more about the tubulin conformational changes preceding or attending ligand binding.); (4) Application of chromium (III) GTP complexes to define the stereochemistry of tubulin binding of metal-neucleotide complexes (The approach taken advantage of the invertness of chromium-nucleotide complexes to ligand exchange to help derive otherwise nearly inaccessible information about the binding of short-lived MgGTP2-binding.); (5) Studies of the time-course of hydrolysis during a single elongation step (This will help to distinguish between prompt and delayed DTP hydrolysis with repsect to tubulin binding to an elongating microtubule.); (6) Use of 5'-guanylyl peroxy-diphosphate to potentially modify the chemical course of GTPase action or to inhibit GTP bining to tubulin (This would represent the first attempt to utilize another hydrolyzable analogue of GTP at the exchangeable nucleotide site); (7) Reexamination of the 32Pi less than greater than GTP and 32Pi less than greater than GDP exchange reactions of porcine microtubule protein (This study promises to resolve the conflicting exchange properties of bovine and porcine brain microtubule proteins); (8) Development of an isothermal polymerization method to eliminate potentially misleading cold-stable forma of microtubule protein (This new approach of rapid isothermal concentration may be helpful in studying the regulation of assembly.). The overall goals of the project will be realized when results of these and other studies integrate into a global mechanism.
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1 |
1991 — 1994 |
Purich, Daniel Lee |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Neuronal Map Interactions With Microtubules
MAP-2a and MAP-2b are high-molecular-weight neuronal microtubule- associated proteins that interact with dendritic microtubules (MTs), and MAP-2c is a corresponding lower-molecular-weight MAP abundant in developing axons. Both proteins share highly conserved N-terminal and C-terminal regions of about 150 and 310 amino acids, respectively, with the latter containing the three 18-amino-acid repeated sequences thought to constitute the MT-binding domain. We propose to further define microtubule binding interactions of the MT-binding fragment, relying on new procedures for preparing the fragment and building on our studies of the three non-identical repeated amino-acid regions. We also plan to characterize m2-peptide displacement of intact MAP-2 from assembled microtubules. To define the amino acid side-chains responsible for binding of peptide-m2 to microtubules and to assess the minimal sequence needed to promote tubule assembly and/or MAP-2 displacement. We plan to study microtubule length redistribution kinetics of microtubules assembled from pure tubulin in the absence or presence of the peptide-m2. We propose to complete the determination of the amino acid sequence of the bovine MT-binding fragment using PCR-derived bovine brain cDNA clones. In a parallel effort, we will investigate phosphorylation of tubule-binding fragment of bovine MAP-2, including: (a) evaluation of the affinity of phosphorylated-tubule-binding fragment for microtubules with the peptide displacement assay; (b) characterization of m2-peptide phosphorylation effects using direct synthesis of m2 analogues containing p-Ser and p-Thr residues; and (c) localization of phosphorylated sites in the MT-binding fragment using enzymatic and chemical cleavage, followed by the diagonal electrophoresis with intervening alkaline phosphatase treatment. Finally, we will carry out microinjection experiments using the MT-binding fragment and synthetic peptides to investigate their efficacy in displacing MAP-2 and tau proteins, and their action in altering cellular distribution of MAP-2 and tau, as well as any changes in cell morphology.
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1 |
1995 — 1997 |
Purich, Daniel Lee |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Tubulin Bound Deoxy Gtp and Ngf Treated Neurons
DESCRIPTION: (adapted from Applicant's Abstract) Tubulin aB- heterodimers bind GTP or GDP at their exchangeable (E-) and nonexchangeable (N-) sites, and E-site GTP hydrolysis is the driving force for dynamic instability and microtubule (MT) cytoskeletal rearrangements in the cell cycle. Rat PC12 pheochromocytoma cells use the MT cytoskeleton to achieve morphologic changes, most particularly outgrowth of branched neurites, in response to nerve growth factor (NGF). Using HPLC analysis for nucleotide content of assembled MTs, the investigators recently discovered that dGTP substitutes for GTP in the tubulin N-site of MTs from PC12 cells grown in the presence of NGF. About 80- 85% of tubulin biosynthesized after NGF treatment contained dGTP, based on a one-to-one binding stoichiometry. This research project will test the following overall hypothesis: "NGF stimulates neurite outgrowth, tubulin biosynthesis, and incorporation of dGTP into tubulin; considering the central role of GTP-regulatory transduction systems in cell proliferation, this hitherto unrecognized link between cell growth conditions and tubulin N-site nucleotide composition may provide a mechanism for adjusting cell GTP levels and sequestering dGTP." The research plan has four specific aims: 1) to determine the time-courses for changes in dGTP pools in NGF-treated PC12 cells in response to NGF; 2) to determine turnover rates of newly synthesized tubulin isotypes relative to the turnover rate of N-site dGTP; 3) to investigate dGTP content of MTs isolated from PC12 cells before and after treatment with a mutant of NGF capable of binding only to the high- affinity NGF receptor; 4) to use video microscopy to establish time- courses of neurite outgrowth in cells treated with NGF and to explore the assembly/disassembly dynamics of MTs isolated from cell bodies and of the dGTP-rich MTs from neurites.
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2004 |
Purich, Daniel Lee |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Actin-Based Motility by Clamped-Filament Motors
DESCRIPTION (provided by applicant): Our recently introduced actoclampin motor model describes how force is generated in actin-based motility through clamped-filament elongation. We proposed a three-step mechanoenzymatic process (called the "Lock, Load & Fire" or "LLF" mechanism): during locking, an affinity-modulated clamp protein (also tethered to the surface of a motile object) binds onto ATP-containing actin monomers situated at a filament's barbed-end; in the loading step, new actin ATP monomers bind onto the barbed end of a clamped-filament, an event that triggers the firing step, whereto ATP hydrolysis on the clamped actin subunit greatly attenuates the clamp's affinity for the filament. ATP hydrolysis initiates clamp translocation and re-locking to the new ATP-containing terminus, starting another LLF cycle. This model explains how surface-tethered filaments can grow while exerting flexural or tensile force on the motile surface, and stochastic simulations reproduce the signature motions of motile Listeria. This elongation motor exploits actin's intrinsic ATPase activity to provide a simple, high fidelity enzymatic reaction cycle for force production that does not require elongating filaments to dissociate from the motile surface. Our research proposal addresses model-based hypotheses designed to test specific features of the LLF mechanism of actin polymerization motors. Specific Aim-1 includes experiments aimed at differentiating the LLF model from Brownian Ratchet-type mechanisms (a) by conducting motility studies near and below the (+)-end critical concentration, and (b) by using covalently cross-linked profilin-actin that cannot release profilin after each LLF cycle. Specific Aim-2 focuses on (a) the role of ATP hydrolysis in motility using slowly hydrolyzing ATP analogues pp(NH)pA and ATPgammaS to identify the likely force-producing step(s) in the LLF mechanism. Specific Aim-3 deals with profilin's potential role in a kinetic proofreading pathway to suppress loading of actin-ADP into clamped-filament motors. In Specific Aim-4, we will apply sedimentation and fluorescence anisotropy measurements to learn if and how ATP hydrolysis modulates the strength of binding interactions of VASP's clamping domain with the ends of actin filaments. Together, these investigations promise to shed new light on actin-based motility by testing fundamental mechanistic properties.
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1 |
2005 — 2006 |
Purich, Daniel Lee |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Active-Based Motility by Clamped-Filament Motors
DESCRIPTION (provided by applicant): Our recently introduced actoclampin motor model describes how force is generated in actin-based motility through clamped-filament elongation. We proposed a three-step mechanoenzymatic process (called the "Lock, Load & Fire" or "LLF" mechanism): during locking, an affinity-modulated clamp protein (also tethered to the surface of a motile object) binds onto ATP-containing actin monomers situated at a filament's barbed-end; in the loading step, new actin ATP monomers bind onto the barbed end of a clamped-filament, an event that triggers the firing step, whereto ATP hydrolysis on the clamped actin subunit greatly attenuates the clamp's affinity for the filament. ATP hydrolysis initiates clamp translocation and re-locking to the new ATP-containing terminus, starting another LLF cycle. This model explains how surface-tethered filaments can grow while exerting flexural or tensile force on the motile surface, and stochastic simulations reproduce the signature motions of motile Listeria. This elongation motor exploits actin's intrinsic ATPase activity to provide a simple, high fidelity enzymatic reaction cycle for force production that does not require elongating filaments to dissociate from the motile surface. Our research proposal addresses model-based hypotheses designed to test specific features of the LLF mechanism of actin polymerization motors. Specific Aim-1 includes experiments aimed at differentiating the LLF model from Brownian Ratchet-type mechanisms (a) by conducting motility studies near and below the (+)-end critical concentration, and (b) by using covalently cross-linked profilin-actin that cannot release profilin after each LLF cycle. Specific Aim-2 focuses on (a) the role of ATP hydrolysis in motility using slowly hydrolyzing ATP analogues pp(NH)pA and ATPgammaS to identify the likely force-producing step(s) in the LLF mechanism. Specific Aim-3 deals with profilin's potential role in a kinetic proofreading pathway to suppress loading of actin-ADP into clamped-filament motors. In Specific Aim-4, we will apply sedimentation and fluorescence anisotropy measurements to learn if and how ATP hydrolysis modulates the strength of binding interactions of VASP's clamping domain with the ends of actin filaments. Together, these investigations promise to shed new light on actin-based motility by testing fundamental mechanistic properties.
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1 |
2005 — 2010 |
Purich, Daniel Dickinson, Richard (co-PI) [⬀] Weitz, David Ladd, Anthony [⬀] Butler, Jason (co-PI) [⬀] |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Multi-Scale Modeling of Chemical-to-Mechanical Energy Conversion in Actin-Based Motility
PROPOSAL NO.: 0505929 PRINCIPAL INVESTIGATOR: A. Ladd INSTITUTION NAME: University of Florida
MULTI-SCALE MODELING OF CHEMICAL-TO-MECHANICAL ENERGY CONVERSION IN ACTIN-BASED MOTILITY
This grant is to develop and validate a biologically relevant, multi-scale model of force generation by polymerization of the biopolymer, actin. Monomeric actin polymerizes into stiff filaments from surface-bound components, which crosslink and propel the surface forward. How the chemical energy involved in monomer addition is converted into mechanical work is critical in understanding cell motility, as well as for exploiting actin-based motility for micro-/nanoscale sensors and actuators. The extension of the wormlike model of the actin filaments to incorporate bending and torsion, as well as the incorporation of the hydration and gel will be studied. Intellectual merits include: incorporation of molecular-level kinetics and energetics into a mesoscale model of polymerizing and cross-linking filaments; the design of a computational framework for modeling the mechanical properties of solutions of stiff biopolymers such as actin, accounting for its resistance to bending and torsion, position and orientation-dependent chemical functionalization along the molecular backbone; and the coupling of the polymer dynamics to the surrounding solvent. Time-dependent variations in concentration of critical components of the polymerization process will also be studied. The broader impacts of the work include establishment of new collaborations between biochemists, physicists, and bioengineers studying actin networks, and chemical engineers developing numerical methods to simulate polymer solutions. New understanding of the coupling between filament elongation and force generation will be valuable in designing technological applications, such as microscale sensors and actuators using linear molecular motors based on actin motility. A small symposium will be organized to promote an objective discussion of the merits of various computational approaches to polymer simulations. The research will contribute to the education and training of graduate students in a collaborative, multidisciplinary environment and undergraduate students will participate in making specific calculations for software development and applications.
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0.915 |