TY - BOOK

T1 - Modelling small-angle scattering data from complex protein-lipid systems

AU - Kynde, Søren Andreas Røssell

PY - 2013

Y1 - 2013

N2 - This thesis consists of two parts.The rst part is divided into five chapters.Chapter 1 gives a general introduction to the bio-molecular systems that havebeen studied. These are membrane proteins and their lipid environments in the form of phospholipid nanodiscs. Membrane proteins are particularly interesting to study because they are common targets for pharmaceutical drugs. At the same time they are unfortunately unstable in solution which make them challenging to study. Phospholipid nanodiscs are small patches of lipid membrane stabilised by a belt of amphipathic helices. They can act as carriers of membrane proteins. Together they form monodisperse soluble aggregates of about 10 nm in size.Chapter 2 introduces the method of small-angle scattering. Small-angle X-ray and neutron scattering are well suited for studying particles in solution on length scales from 1 to 100 nm. This makes the techniques very well suited for the study of the nanodisc system.Chapter 3 explains two different modelling approaches that can be used in the analysis of small-angle scattering data from lipid-protein complexes. These are the continuous approach where the system of interest is modelled as a few regular geometric objects and the discrete approach were models are build from a large number of points. It is the basic hypothesis of this thesis, that analysis of smallangle scattering data can be approached in a way that combines the continuous and the discrete modelling methods, and that such an approach can combine the bene ts of each of the methods and give unique structural information about relevant bio-molecular complexes in solution. Chapter 4 describes the work behind a proposal of a small-angle neutron scattering instrument for the European Spallation Source under construction in Lund. The instrument is speci cally optimised for bio-molecular samples such as the ones studied in the rest of this thesis. Chapter 5 summarises the results of the thesis. In particular claims of the hypothesis are evaluated. The second part of the thesis consists of four papers in manuscript form. The first three papers each describes a protein system that has successfully been measured with small-angle scattering methods and subsequently analysed using the hybrid approach.Paper I governs the transmembrane protein bacteriorhodopsin embedded into a phospholipid nanodisc. The modelling is based on a crystal structure of bacteriorhodopsin and a continuous model of the nanodisc. The position and orientation of the membrane protein relative to the nanodisc is determined as well as the structural changes of the nanodisc.Paper II describes the use of the same approach to determine the relative position of a nanodisc and the membrane anchored enzyme cytochrome P450 3a4. In addition it is shown that the shape of the protein can be reconstructed while attached to the nanodisc without the need for a crystal structure. Paper III governs the partially unfolded protein PLZF protein, which is related to a certain type bone marrow cancer. It is demonstrated that a discrete model candescribe the folded parts of the protein while the unfolded linker chains are betterdescribed by a continuous model for a gaussian-chain.The last Paper IV summarises the ndings and recommendations of the workgroup behind the bio-SANS instrument proposal for the ESS.

AB - This thesis consists of two parts.The rst part is divided into five chapters.Chapter 1 gives a general introduction to the bio-molecular systems that havebeen studied. These are membrane proteins and their lipid environments in the form of phospholipid nanodiscs. Membrane proteins are particularly interesting to study because they are common targets for pharmaceutical drugs. At the same time they are unfortunately unstable in solution which make them challenging to study. Phospholipid nanodiscs are small patches of lipid membrane stabilised by a belt of amphipathic helices. They can act as carriers of membrane proteins. Together they form monodisperse soluble aggregates of about 10 nm in size.Chapter 2 introduces the method of small-angle scattering. Small-angle X-ray and neutron scattering are well suited for studying particles in solution on length scales from 1 to 100 nm. This makes the techniques very well suited for the study of the nanodisc system.Chapter 3 explains two different modelling approaches that can be used in the analysis of small-angle scattering data from lipid-protein complexes. These are the continuous approach where the system of interest is modelled as a few regular geometric objects and the discrete approach were models are build from a large number of points. It is the basic hypothesis of this thesis, that analysis of smallangle scattering data can be approached in a way that combines the continuous and the discrete modelling methods, and that such an approach can combine the bene ts of each of the methods and give unique structural information about relevant bio-molecular complexes in solution. Chapter 4 describes the work behind a proposal of a small-angle neutron scattering instrument for the European Spallation Source under construction in Lund. The instrument is speci cally optimised for bio-molecular samples such as the ones studied in the rest of this thesis. Chapter 5 summarises the results of the thesis. In particular claims of the hypothesis are evaluated. The second part of the thesis consists of four papers in manuscript form. The first three papers each describes a protein system that has successfully been measured with small-angle scattering methods and subsequently analysed using the hybrid approach.Paper I governs the transmembrane protein bacteriorhodopsin embedded into a phospholipid nanodisc. The modelling is based on a crystal structure of bacteriorhodopsin and a continuous model of the nanodisc. The position and orientation of the membrane protein relative to the nanodisc is determined as well as the structural changes of the nanodisc.Paper II describes the use of the same approach to determine the relative position of a nanodisc and the membrane anchored enzyme cytochrome P450 3a4. In addition it is shown that the shape of the protein can be reconstructed while attached to the nanodisc without the need for a crystal structure. Paper III governs the partially unfolded protein PLZF protein, which is related to a certain type bone marrow cancer. It is demonstrated that a discrete model candescribe the folded parts of the protein while the unfolded linker chains are betterdescribed by a continuous model for a gaussian-chain.The last Paper IV summarises the ndings and recommendations of the workgroup behind the bio-SANS instrument proposal for the ESS.

UR - https://soeg.kb.dk/permalink/45KBDK_KGL/fbp0ps/alma99122341708005763

M3 - Ph.D. thesis

BT - Modelling small-angle scattering data from complex protein-lipid systems

PB - The Niels Bohr Institute, Faculty of Science, University of Copenhagen

ER -