Abstract
Particles containing both lipids and proteins (so-called lipoproteins) are vital to study. They are selfassembling particles that, in the human body, are responsible for the transport of lipids and cholesterol. Due to the increasing problems of obesity and related illnesses in the world, obtaining more knowledge about the cholesterol and lipid metabolism is paramount. As an example, in 2012, cardiovascular disease was still the main cause of death in the U.S. This means that the study of lipoproteins is not only of pure academic interest but vital to current world problems.
Another reason for working with lipoprotein particles are their potential in the study membrane proteins. Membrane proteins are responsible for most of the transport in and out of cells and signaling between cells. As an example G-protein coupled receptors, a class of membrane proteins, are the third largest class of proteins encoded by the human genome. G-protein coupled receptors mediate the majority of hormone and neurotransmitter signals as well as being responsible for perception of light, smell and taste in the human body, and a number of Nobel prizes has been
awarded based on their study. Structural investigations of membrane proteins by traditional X-ray crystallography have proved a difficult challenge, and a surprisingly small amount of membrane proteins has been crystalized so far. This implies that development of lipoproteins as a platform for studying membrane proteins is much needed.
In this thesis, studies have been made on human apolipoprotein A1 (ApoA1) in discoidal complex with phospholipids. The structure and function of the N-terminal domain of ApoA1 was investigated and found to be vital for the accommodation of cholesterol in ApoA1 lipoproteins particles. Furthermore, ApoA1 based proteins were made in deuterated versions in order to study the finer details of their structure in discoidal lipoprotein particles by neutron scattering. This led to a search for finding a
solution to the problem that ApoA1 based discoidal lipoproteins aggregate in heavy water when using currently implemented protocols for changing solvent. The problem was investigated and a workable protocol was developed that does not cause the discs to aggregate when solubilized in heavy water.
Further projects were undertaken to develop and characterize new lipoprotein particles and evaluate their usefulness in handling membrane proteins. A minimalistic approach was tested where the ApoA1 protein was mimicked my small amphipathic helical peptides. The resulting discs were very similar to ApoA1 based discs in size and in their ability to stabilize incorporated membrane proteins.
Furthermore, due to their enhanced dynamical properties, it proved possible to exchange the peptides with ApoA1 based proteins, thereby directly transferring lipids and an incorporated membrane protein from one type of disc directly into another.
An opposite approach for making lipoproteins was also tested. Basically, a protein was designed consisting of 3 N-terminal truncated ApoA1 proteins coupled together with small linkers. The efficiency of stabilizing membrane proteins was tested by incorporating the very large membrane spanning photosystem 1 protein complex into these lipoprotein particles. It was shown that the photosystem 1 could be stabilized in solution without detergent by these newly designed lipoprotein
Another reason for working with lipoprotein particles are their potential in the study membrane proteins. Membrane proteins are responsible for most of the transport in and out of cells and signaling between cells. As an example G-protein coupled receptors, a class of membrane proteins, are the third largest class of proteins encoded by the human genome. G-protein coupled receptors mediate the majority of hormone and neurotransmitter signals as well as being responsible for perception of light, smell and taste in the human body, and a number of Nobel prizes has been
awarded based on their study. Structural investigations of membrane proteins by traditional X-ray crystallography have proved a difficult challenge, and a surprisingly small amount of membrane proteins has been crystalized so far. This implies that development of lipoproteins as a platform for studying membrane proteins is much needed.
In this thesis, studies have been made on human apolipoprotein A1 (ApoA1) in discoidal complex with phospholipids. The structure and function of the N-terminal domain of ApoA1 was investigated and found to be vital for the accommodation of cholesterol in ApoA1 lipoproteins particles. Furthermore, ApoA1 based proteins were made in deuterated versions in order to study the finer details of their structure in discoidal lipoprotein particles by neutron scattering. This led to a search for finding a
solution to the problem that ApoA1 based discoidal lipoproteins aggregate in heavy water when using currently implemented protocols for changing solvent. The problem was investigated and a workable protocol was developed that does not cause the discs to aggregate when solubilized in heavy water.
Further projects were undertaken to develop and characterize new lipoprotein particles and evaluate their usefulness in handling membrane proteins. A minimalistic approach was tested where the ApoA1 protein was mimicked my small amphipathic helical peptides. The resulting discs were very similar to ApoA1 based discs in size and in their ability to stabilize incorporated membrane proteins.
Furthermore, due to their enhanced dynamical properties, it proved possible to exchange the peptides with ApoA1 based proteins, thereby directly transferring lipids and an incorporated membrane protein from one type of disc directly into another.
An opposite approach for making lipoproteins was also tested. Basically, a protein was designed consisting of 3 N-terminal truncated ApoA1 proteins coupled together with small linkers. The efficiency of stabilizing membrane proteins was tested by incorporating the very large membrane spanning photosystem 1 protein complex into these lipoprotein particles. It was shown that the photosystem 1 could be stabilized in solution without detergent by these newly designed lipoprotein
| Originalsprog | Engelsk |
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| Forlag | Department of Chemistry, Faculty of Science, University of Copenhagen |
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| Antal sider | 106 |
| Status | Udgivet - 2013 |