Protein Dynamics Studied by NMR. Kinetics of the Adipocyte Fatty Acid-Binding Protein and Oligomerisation of the Low Molecular Weight Protein Tyrosine Phosphatase.
(2004)- 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 <sup>15</sup>N 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 <sup>15</sup>N-longitudinal relaxation-exchange... (More) - 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 <sup>15</sup>N 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 <sup>15</sup>N-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, <i>cis</i>-9,10-methylene-hexadecanoate, <i>cis</i>- vaccenoate and stearate. The measured off-rates ranged between 0.8 and 5 s<sup>–1</sup> 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. (Less) - Abstract (Swedish)
- Popular Abstract in Swedish
1: Tomas Åkerud, doktorand vid Lunds Universitet, har utvecklat en metod för att mäta hur snabbt läkemedelsmolekyler och fettsyror (de naturliga bindarna) tar sig in i och lämnar bindingsfickan i ett protein som är viktigt för fettsyrametabolismen i människa. Proteinet, adipocyt-fettsyrabindande protein (A-FABP), har som funktion att transportera fettsyror, som inte är särskilt vattenlösliga, i fettcellernas vattenfyllda inre. Om proteinets fettsyratransporterande egenskaper kunde blockeras med en läkemedelsmolekyl skulle fettcellernas upplagring av fett kunna hämmas. För att förstå bindningsprocessen i detalj har kärnmagnetisk resonansspektroskopi (NMR) använts. Proteinet har anrikats med den... (More) - Popular Abstract in Swedish
1: Tomas Åkerud, doktorand vid Lunds Universitet, har utvecklat en metod för att mäta hur snabbt läkemedelsmolekyler och fettsyror (de naturliga bindarna) tar sig in i och lämnar bindingsfickan i ett protein som är viktigt för fettsyrametabolismen i människa. Proteinet, adipocyt-fettsyrabindande protein (A-FABP), har som funktion att transportera fettsyror, som inte är särskilt vattenlösliga, i fettcellernas vattenfyllda inre. Om proteinets fettsyratransporterande egenskaper kunde blockeras med en läkemedelsmolekyl skulle fettcellernas upplagring av fett kunna hämmas. För att förstå bindningsprocessen i detalj har kärnmagnetisk resonansspektroskopi (NMR) använts. Proteinet har anrikats med den NMR-aktiva, men icke-radioaktiva isotopen kväve-15 (den naturligt förekommande är kväve-14) för att möjliggöra studier av varje enskild aminosyra i proteinet. För att kunna studera en fettsyra, oljesyra, har denna anrikats med kol-13. Den naturligt förekommande isotopen är kol-12, medan den radioaktiva kol-14 isotopen används till datering av arkeologiska prover. Protein och oljesyra blandas sedan och signaler från fritt och bundet protein och fri och bunden oljesyra kan studeras samtidigt. Hur intensiva de fria och bundna signalerna är bestämmer hur starkt oljesyra och protein interagerar. Tricket med metoden som använts är att göra tidsupplösta mätningar, där magnetiseringen från fri och bunden form blandas så att den genomsnittliga tid som oljesyran befinner sig i bindningsfickan kan bestämmas. Det faktum att kväve-15 atomerna är utspridda i proteinet gör det också möjligt att få information om var bindingsprocessen sker i proteinet. Samma metod har använts för att studera proteinets interaktion med en läkemedelskandidat som tagits fram på Biovitrum i Stockholm.
2: Oligomerisering, bindning till sig själv, har studerats med NMR för proteinet lågmolekylärt fosfatas (LMW-PTP). Proteinets funktion är att ta bort fosfatgrupper bundna till protein. Detta är viktigt i mänskliga celler då funktioner som är livsnödvändiga kontrolleras genom att de andra proteinerna har olika fosforyleringsmönster. När LMW-PTP binder till sig själv döljs den del av proteinet som sköter defosforyleringen och proteinet blir inaktivt. Med NMR har vi visat att detta sker i vattenlösning. Tillsammans med en forskargrupp i Barcelona ledd av Miquel Pons har vi visat att det inaktiva proteinet också binder till sig själv och bildar enheter som innehåller fyra proteinmolekyler. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/466824
- author
- Åkerud, Tomas LU
- supervisor
- opponent
-
- Prof Homans, Steven W., University of Leeds, UK
- organization
- publishing date
- 2004
- type
- Thesis
- publication status
- published
- subject
- keywords
- Physical chemistry, Fysikalisk kemi
- pages
- 150 pages
- publisher
- Tomas Åkerud, Dept. of Biophysical Chemistry, Lund University, Box 124, SE-22100 Lund, Sweden,
- defense location
- Lecture Hall B, Kemicentrum, Lund Institute of Technology
- defense date
- 2004-04-16 10:15:00
- ISBN
- 91-628-6008-9
- language
- English
- LU publication?
- yes
- additional info
- 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
- id
- 0bce4735-9574-47a4-bb96-c26189e4474d (old id 466824)
- date added to LUP
- 2016-04-04 10:28:40
- date last changed
- 2018-11-21 20:58:59
@phdthesis{0bce4735-9574-47a4-bb96-c26189e4474d,
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.<br/><br>
<br/><br>
NMR spectroscopy is sensitive to dynamic processes on timescales ranging from picoseconds to days. Using <sup>15</sup>N isotope-enriched protein samples, dynamic processes have been probed with high spatial resolution, by monitoring each amide group in the protein.<br/><br>
<br/><br>
The kinetics of ligand binding to A-FABP was studied by <sup>15</sup>N-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, <i>cis</i>-9,10-methylene-hexadecanoate, <i>cis</i>- vaccenoate and stearate. The measured off-rates ranged between 0.8 and 5 s<sup>–1</sup> and the dissociation constants between 0.5 and 10 m M, with the lowest values for oleate and the highest for BVT.1961.<br/><br>
<br/><br>
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.}},
author = {{Åkerud, Tomas}},
isbn = {{91-628-6008-9}},
keywords = {{Physical chemistry; Fysikalisk kemi}},
language = {{eng}},
publisher = {{Tomas Åkerud, Dept. of Biophysical Chemistry, Lund University, Box 124, SE-22100 Lund, Sweden,}},
school = {{Lund University}},
title = {{Protein Dynamics Studied by NMR. Kinetics of the Adipocyte Fatty Acid-Binding Protein and Oligomerisation of the Low Molecular Weight Protein Tyrosine Phosphatase.}},
year = {{2004}},
}