Protein Dynamics Studied by NMR. Kinetics of the Adipocyte Fatty Acid-Binding Protein and Oligomerisation of the Low Molecular Weight Protein Tyrosine Phosphatase.

Tomas Åkerud

Protein Dynamics Studied by NMR. Kinetics of the Adipocyte Fatty Acid-Binding Protein and Oligomerisation of the Low Molecular Weight Protein Tyrosine Phosphatase.

Tomas Åkerud

Biophysical Chemistry

Research output: Thesis › Doctoral Thesis (compilation)

Abstract

Nuclear magnetic resonance spectroscopy (NMR) has been used to determine the kinetics of ligand binding to the adipocyte fatty acid-binding protein (A-FABP), as well as the oligomerisation and inter- and intramolecular dynamics of the low molecular weight-protein tyrosine phosphatase (LMW-PTP) in solution.

NMR spectroscopy is sensitive to dynamic processes on timescales ranging from picoseconds to days. Using 15N isotope-enriched protein samples, dynamic processes have been probed with high spatial resolution, by monitoring each amide group in the protein.

The kinetics of ligand binding to A-FABP was studied by 15N-longitudinal relaxation-exchange experiments. Using competitive binding experiments, it proved feasible to determine the kinetic rate constants of ligands with higher affinity than otherwise possible using protein NMR. The study included the important natural ligand oleate, a fatty acid mimetic denoted BVT.1961 (3-[4-hydroxy-3-isopropylphenyl]-propionic acid) and a fatty acid mixture consisting of myristate, palmitate, palmitoleate, cis-9,10-methylene-hexadecanoate, cis- vaccenoate and stearate. The measured off-rates ranged between 0.8 and 5 s–1 and the dissociation constants between 0.5 and 10 m M, with the lowest values for oleate and the highest for BVT.1961.

The oligomerisation of LMW-PTP was extensively characterised using concentration dependent chemical shifts, as well as concentration dependent longitudinal and transverse relaxation rates. The chemical shift changes were confined to a region of the protein surface that corresponds to the interface between the two molecules in the dimeric crystal structure of of LMW-PTP. The oligomerisation state and dissociation constants for the oligomers were determined by fitting hydrodynamic models to the concentration dependent relaxation rates. The model that fitted the experimental data best was a monomer-dimer-tetramer equilibrium of LMW-PTP.

Original language	English
Qualification	Doctor
Awarding Institution	Biophysical Chemistry
Supervisors/Advisors	[unknown], [unknown], Supervisor, External person
Award date	2004 Apr 16
Publisher	Tomas Åkerud, Dept. of Biophysical Chemistry, Lund University, Box 124, SE-22100 Lund, Sweden,
ISBN (Print)	91-628-6008-9
Publication status	Published - 2004

Bibliographical note

Defence details

Date: 2004-04-16
Time: 10:15
Place: Lecture Hall B, Kemicentrum, Lund Institute of Technology

External reviewer(s)

Name: Homans, Steven W.
Title: Prof
Affiliation: University of Leeds, UK

---

Article: I.Intramolecular Dynamics of Low Molecular Weight Protein Tyrosine Phosphatase in Monomer-Dimer Equilibrium Studied by NMR. A Model for Changes in Dynamics upon Target Binding. Tomas Åkerud, Eva Thulin, Robert L. Van Etten and Mikael Akke, J. Mol. Biol. 322, 137-52 (2002)

Article: II.Combined Use of NMR Relaxation Measurements and Hydrodynamic Calculations toStudy Protein Association. Evidence for Tetramers of Low Molecular Weight Protein Tyrosine Phosphatase in Solution. Pau Bernadó, Tomas Åkerud, José García de la Torre, Mikael Akke and Miquel Pons, J. Am. Chem. Soc. 125, 916-23 (2003)

Article: III.Structure-Based Screening As Applied to Human FABP4: A Highly Efficient Alternative to HTS for Hit Generation. Maria J. P. van Dongen, Jonas Uppenberg, Stefan Svensson, Thomas Lundbäck, Tomas Åkerud, Mats Wikström and Johan Schultz,J. Am. Chem. Soc. 124, 11874-80 (2002)

Article: IV.Kinetics of Competitive Ligand Binding Determined by Protein NMR Spectroscopy.Binding of an Organic Molecule to Adipocyte Fatty Acid Binding Protein in the Presence of Natural Ligands. Tomas Åkerud, Jonas Uppenberg, Mats Hamberg, Agneta Tjernberg, Johan Schultz and Mikael AkkeManuscript

Article: V.The Kinetics of Oleate Binding to the Adipocyte Fatty Acid-Binding Protein Studied by NMR. Tomas Åkerud and Mikael AkkeManuscript

Subject classification (UKÄ)

Physical Chemistry (including Surface- and Colloid Chemistry)

Free keywords

Physical chemistry
Fysikalisk kemi

Cite this

@phdthesis{0bce4735957447a4bb96c26189e4474d,

title = "Protein Dynamics Studied by NMR. Kinetics of the Adipocyte Fatty Acid-Binding Protein and Oligomerisation of the Low Molecular Weight Protein Tyrosine Phosphatase.",

abstract = "Nuclear magnetic resonance spectroscopy (NMR) has been used to determine the kinetics of ligand binding to the adipocyte fatty acid-binding protein (A-FABP), as well as the oligomerisation and inter- and intramolecular dynamics of the low molecular weight-protein tyrosine phosphatase (LMW-PTP) in solution. NMR spectroscopy is sensitive to dynamic processes on timescales ranging from picoseconds to days. Using 15N isotope-enriched protein samples, dynamic processes have been probed with high spatial resolution, by monitoring each amide group in the protein. The kinetics of ligand binding to A-FABP was studied by 15N-longitudinal relaxation-exchange experiments. Using competitive binding experiments, it proved feasible to determine the kinetic rate constants of ligands with higher affinity than otherwise possible using protein NMR. The study included the important natural ligand oleate, a fatty acid mimetic denoted BVT.1961 (3-[4-hydroxy-3-isopropylphenyl]-propionic acid) and a fatty acid mixture consisting of myristate, palmitate, palmitoleate, cis-9,10-methylene-hexadecanoate, cis- vaccenoate and stearate. The measured off-rates ranged between 0.8 and 5 s–1 and the dissociation constants between 0.5 and 10 m M, with the lowest values for oleate and the highest for BVT.1961. The oligomerisation of LMW-PTP was extensively characterised using concentration dependent chemical shifts, as well as concentration dependent longitudinal and transverse relaxation rates. The chemical shift changes were confined to a region of the protein surface that corresponds to the interface between the two molecules in the dimeric crystal structure of of LMW-PTP. The oligomerisation state and dissociation constants for the oligomers were determined by fitting hydrodynamic models to the concentration dependent relaxation rates. The model that fitted the experimental data best was a monomer-dimer-tetramer equilibrium of LMW-PTP.",

keywords = "Physical chemistry, Fysikalisk kemi",

author = "Tomas {\AA}kerud",

note = "Defence details Date: 2004-04-16 Time: 10:15 Place: Lecture Hall B, Kemicentrum, Lund Institute of Technology External reviewer(s) Name: Homans, Steven W. Title: Prof Affiliation: University of Leeds, UK --- Article: I.Intramolecular Dynamics of Low Molecular Weight Protein Tyrosine Phosphatase in Monomer-Dimer Equilibrium Studied by NMR. A Model for Changes in Dynamics upon Target Binding. Tomas {\AA}kerud, Eva Thulin, Robert L. Van Etten and Mikael Akke, J. Mol. Biol. 322, 137-52 (2002) Article: II.Combined Use of NMR Relaxation Measurements and Hydrodynamic Calculations toStudy Protein Association. Evidence for Tetramers of Low Molecular Weight Protein Tyrosine Phosphatase in Solution. Pau Bernad{\'o}, Tomas {\AA}kerud, Jos{\'e} Garc{\'i}a de la Torre, Mikael Akke and Miquel Pons, J. Am. Chem. Soc. 125, 916-23 (2003) Article: III.Structure-Based Screening As Applied to Human FABP4: A Highly Efficient Alternative to HTS for Hit Generation. Maria J. P. van Dongen, Jonas Uppenberg, Stefan Svensson, Thomas Lundb{\"a}ck, Tomas {\AA}kerud, Mats Wikstr{\"o}m and Johan Schultz,J. Am. Chem. Soc. 124, 11874-80 (2002) Article: IV.Kinetics of Competitive Ligand Binding Determined by Protein NMR Spectroscopy.Binding of an Organic Molecule to Adipocyte Fatty Acid Binding Protein in the Presence of Natural Ligands. Tomas {\AA}kerud, Jonas Uppenberg, Mats Hamberg, Agneta Tjernberg, Johan Schultz and Mikael AkkeManuscript Article: V.The Kinetics of Oleate Binding to the Adipocyte Fatty Acid-Binding Protein Studied by NMR. Tomas {\AA}kerud and Mikael AkkeManuscript",

year = "2004",

language = "English",

isbn = "91-628-6008-9",

publisher = "Tomas {\AA}kerud, Dept. of Biophysical Chemistry, Lund University, Box 124, SE-22100 Lund, Sweden,",

type = "Doctoral Thesis (compilation)",

school = "Biophysical Chemistry",

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TY - THES

T1 - Protein Dynamics Studied by NMR. Kinetics of the Adipocyte Fatty Acid-Binding Protein and Oligomerisation of the Low Molecular Weight Protein Tyrosine Phosphatase.

AU - Åkerud, Tomas

N1 - Defence details Date: 2004-04-16 Time: 10:15 Place: Lecture Hall B, Kemicentrum, Lund Institute of Technology External reviewer(s) Name: Homans, Steven W. Title: Prof Affiliation: University of Leeds, UK --- Article: I.Intramolecular Dynamics of Low Molecular Weight Protein Tyrosine Phosphatase in Monomer-Dimer Equilibrium Studied by NMR. A Model for Changes in Dynamics upon Target Binding. Tomas Åkerud, Eva Thulin, Robert L. Van Etten and Mikael Akke, J. Mol. Biol. 322, 137-52 (2002) Article: II.Combined Use of NMR Relaxation Measurements and Hydrodynamic Calculations toStudy Protein Association. Evidence for Tetramers of Low Molecular Weight Protein Tyrosine Phosphatase in Solution. Pau Bernadó, Tomas Åkerud, José García de la Torre, Mikael Akke and Miquel Pons, J. Am. Chem. Soc. 125, 916-23 (2003) Article: III.Structure-Based Screening As Applied to Human FABP4: A Highly Efficient Alternative to HTS for Hit Generation. Maria J. P. van Dongen, Jonas Uppenberg, Stefan Svensson, Thomas Lundbäck, Tomas Åkerud, Mats Wikström and Johan Schultz,J. Am. Chem. Soc. 124, 11874-80 (2002) Article: IV.Kinetics of Competitive Ligand Binding Determined by Protein NMR Spectroscopy.Binding of an Organic Molecule to Adipocyte Fatty Acid Binding Protein in the Presence of Natural Ligands. Tomas Åkerud, Jonas Uppenberg, Mats Hamberg, Agneta Tjernberg, Johan Schultz and Mikael AkkeManuscript Article: V.The Kinetics of Oleate Binding to the Adipocyte Fatty Acid-Binding Protein Studied by NMR. Tomas Åkerud and Mikael AkkeManuscript

PY - 2004

Y1 - 2004

N2 - Nuclear magnetic resonance spectroscopy (NMR) has been used to determine the kinetics of ligand binding to the adipocyte fatty acid-binding protein (A-FABP), as well as the oligomerisation and inter- and intramolecular dynamics of the low molecular weight-protein tyrosine phosphatase (LMW-PTP) in solution. NMR spectroscopy is sensitive to dynamic processes on timescales ranging from picoseconds to days. Using 15N isotope-enriched protein samples, dynamic processes have been probed with high spatial resolution, by monitoring each amide group in the protein. The kinetics of ligand binding to A-FABP was studied by 15N-longitudinal relaxation-exchange experiments. Using competitive binding experiments, it proved feasible to determine the kinetic rate constants of ligands with higher affinity than otherwise possible using protein NMR. The study included the important natural ligand oleate, a fatty acid mimetic denoted BVT.1961 (3-[4-hydroxy-3-isopropylphenyl]-propionic acid) and a fatty acid mixture consisting of myristate, palmitate, palmitoleate, cis-9,10-methylene-hexadecanoate, cis- vaccenoate and stearate. The measured off-rates ranged between 0.8 and 5 s–1 and the dissociation constants between 0.5 and 10 m M, with the lowest values for oleate and the highest for BVT.1961. The oligomerisation of LMW-PTP was extensively characterised using concentration dependent chemical shifts, as well as concentration dependent longitudinal and transverse relaxation rates. The chemical shift changes were confined to a region of the protein surface that corresponds to the interface between the two molecules in the dimeric crystal structure of of LMW-PTP. The oligomerisation state and dissociation constants for the oligomers were determined by fitting hydrodynamic models to the concentration dependent relaxation rates. The model that fitted the experimental data best was a monomer-dimer-tetramer equilibrium of LMW-PTP.

AB - Nuclear magnetic resonance spectroscopy (NMR) has been used to determine the kinetics of ligand binding to the adipocyte fatty acid-binding protein (A-FABP), as well as the oligomerisation and inter- and intramolecular dynamics of the low molecular weight-protein tyrosine phosphatase (LMW-PTP) in solution. NMR spectroscopy is sensitive to dynamic processes on timescales ranging from picoseconds to days. Using 15N isotope-enriched protein samples, dynamic processes have been probed with high spatial resolution, by monitoring each amide group in the protein. The kinetics of ligand binding to A-FABP was studied by 15N-longitudinal relaxation-exchange experiments. Using competitive binding experiments, it proved feasible to determine the kinetic rate constants of ligands with higher affinity than otherwise possible using protein NMR. The study included the important natural ligand oleate, a fatty acid mimetic denoted BVT.1961 (3-[4-hydroxy-3-isopropylphenyl]-propionic acid) and a fatty acid mixture consisting of myristate, palmitate, palmitoleate, cis-9,10-methylene-hexadecanoate, cis- vaccenoate and stearate. The measured off-rates ranged between 0.8 and 5 s–1 and the dissociation constants between 0.5 and 10 m M, with the lowest values for oleate and the highest for BVT.1961. The oligomerisation of LMW-PTP was extensively characterised using concentration dependent chemical shifts, as well as concentration dependent longitudinal and transverse relaxation rates. The chemical shift changes were confined to a region of the protein surface that corresponds to the interface between the two molecules in the dimeric crystal structure of of LMW-PTP. The oligomerisation state and dissociation constants for the oligomers were determined by fitting hydrodynamic models to the concentration dependent relaxation rates. The model that fitted the experimental data best was a monomer-dimer-tetramer equilibrium of LMW-PTP.

KW - Physical chemistry

KW - Fysikalisk kemi

M3 - Doctoral Thesis (compilation)

SN - 91-628-6008-9

PB - Tomas Åkerud, Dept. of Biophysical Chemistry, Lund University, Box 124, SE-22100 Lund, Sweden,

ER -

Protein Dynamics Studied by NMR. Kinetics of the Adipocyte Fatty Acid-Binding Protein and Oligomerisation of the Low Molecular Weight Protein Tyrosine Phosphatase.

Abstract

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Subject classification (UKÄ)

Free keywords

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