Impact of pressure on the molecular dynamics of low density lipoprotein

Abstract LDLPRESS is a project of fundamental research and aims to clarify the effects of high hydrostatic pressure on the molecular dynamics and the lipid phase transition behavior of human low density lipoproteins (LDL) and modifications thereof. Pressure is a thermodynamic variable that enables to investigate physical parameter of macromolecules and to explore structural and dynamical properties of supermolecular assemblies. Recent developments of high pressure cells especially adapted to be used in combination with scattering techniques have opened new opportunities to study even more complex biological systems. Accordingly, the focus of this project is on native and biochemically modified LDL nanoparticles, which are macromolecular assemblies of phospholipids, cholesterol and fat stabilized by a protein moiety. LDLPRESS aims at combining and integrating data obtained by two complementary high-pressure scattering techniques: small angle X-ray and neutron scattering techniques. In this way we attempt to determine characteristic pressure-temperature diagrams of LDL probes to correlate them with the dynamical behavior of LDL. The conception of a dynamic landscape similar to natural membranes seems to be reasonable for lipoprotein species, which we assume to be highly sensitive to pressure. However, effects of high pressure on lipoproteins have never been explored before, mainly due to technical reasons. Thus, we are also expecting some technological improvements for the application of advanced scattering techniques for biological systems. In the LDLPRESS project we intend to combine our current knowledge on the mobility and dynamics of lipoprotein species with new data upon pressurization and existing information from high pressure studies on proteins and membranes, thereby taking advantage of and combining the complementary expertise of the project partner in both the field of high-pressure technology and lipoproteins. The project is an interdisciplinary approach divided into three interrelated tasks including all aspects of preparation and modification of LDL samples as well as the biochemical and biophysical characterization of LDL, neutron and X-ray scattering experiments. The main objectives of the project are: (i) To investigate the pressure-dependent dynamic behavior of the core lipids as function of temperature and chemical composition. (ii) To study the effects of composition and physical state of the core lipids on the phospholipid surface shell and the dynamics of the protein moiety. (iii) To study the impact of specific modifications of LDL on the dynamics, organization and interplay of lipids and protein under high pressure conditions. By combining all data we expect to obtain a more comprehensive picture of the structure and dynamics of LDL to be discussed in relation to its biological role. We hope to open new ways for a better understanding of LDL-associated diseases i.e. atherosclerosis and cardiovascular diseases - and to pave the way for new LDL-based solutions in view of medical applications.

In the project LDLPRESS we have studied how the structure, morphology and molecular dynamics of human low density lipoprotein (LDL) particles are affected by mechanical stress using high hydrostatic pressure. Recent technological developments of high pressure cells, in particular in combination with cutting-edge scattering techniques have opened up new opportunities to investigate complex biological systems, like LDL, at high pressure. In the focus of this project were defined LDL nanoparticles, which are high molecular assemblies of phospholipids, cholesterol, triglycerides and a single protein termed apolipoprotein B100. In particular, we have compared native human LDL particles to naturally occurring triglyceride rich and oxidatively modified LDL particles, which are more atherogenic harbouring an increased risk for patients to develop cardiovascular heart diseases. To gain a comprehensive picture of the structure-dynamics interplay of native and atherogenic LDL particles under high pressure we have combined data from Synchrotron X-ray diffraction and neutron scattering. Indeed, we could derive specific information on molecular motions, intrinsic flexibility and conformational stability in terms of structural remodelling of LDL. A refined 3D-model analysis based on a superellipsoid was performed and the overall particle shape and the internal layer structure were reconstructed from small angle X-ray scattering (SAXS) data and correlated with data derived from cryo e.m. 3D-models. We found that both, the structural and dynamical data on LDL nanoparticles revealed distinct differences as function of pressure and temperature, depending on the particles chemical composition. In parallel to the hydrostatic pressure experiments we have investigated whether LDL particles can be stabilized by the use of self-assembling peptides. The aim of this approach was to see whether LDL particles can be selectively modified by peptide motifs to be used as molecular scaffolds in nanomedicine. In summary, we could definitively show that LDL particles depending on their lipid composition show different morphologies and distinct molecular motions. We found that LDL nanoparticles, in particular native LDL, are highly resistant to mechanical stress. This finding could be of outmost importance for lipoprotein based medical applications.