Biophysical characterization of reactions associated with reverse cholesterol transport

This thesis aimed at improving our understanding of reactions relevant in the reverse cholesterol transport (RCT). RCT facilitates cholesterol homeostasis and is the most important pathway involved in cardiovascular disease. For this purpose three different projects were chosen. Thermodynamics of protein self-association and unfolding was characterized in detail at the example of Apolipoprotein A-1 (Apo A-1). Lipid binding was characterized by means of small peptides that mimic Apo A-1 function. The third project gained insight about the molecular mechanisms of ABCA1`s allocrite flopping. Apo A-1 is the main protein constituent of high density lipoprotein (HDL) and is together with ABCA1 a key player of the RCT.
Apolipoprotein A-1
Protein self-association and unfolding are two processes whose understanding is of utmost importance for the development of biological phamaceuticals as oligomerisation may alter functional properties of proteins. Apo A-1 is a perfect candidate for these investigations as it undergoes a concentration dependent self-association process and has high physiological relevance. Even though Apo A-1 is a highly investigated macromolecule, self-association was not investigated in such a comprehensive approach. Additionally, we used highly purified recombinant human Apo A-1, which was generously provided by H.-J. Schönfeld. For analyzing thermodynamics of self-association and thermal unfolding we introduced new theoretical and experimental methods
Self-association data was obtained by a combination of high sensitivity micro calorimetry and analytical ultracentrifugation. The dissociation reaction of highly concentrated and thus oligomeric Apo A-1 was followed by injection into buffer in an isothermal titration calorimeter (ITC). Dilution of the sample moved the chemical equilibrium towards monomers. Complementary, this equilibrium was analyzed by data obtained from analytical ultracentrifugation in a sedimentation equilibrium mode. If any, self-association was described in former studies as equilibrium between distinct species, for example between monomers and dimers. We introduced a cooperative self-association model that describes the equilibrium of the protein between each possible oligomer in a concentration dependent manner. Furthermore, we introduced a “binding partition function” that represents the sum of all concentrations found in the system. Together with a dissociation degree of the protein we found a link between thermodynamic data and theory of self-association. The binding partition function describes the statistical properties of the system in thermodynamic equilibrium. Hence, it is independent of the theoretical model that is utilized to describe the reaction.
Thermal unfolding of Apo A-1 was followed by circular dichroism spectroscopy (CD) and differential scanning calorimetry (DSC). We found that melting of Apo A-1 caused a transition of α–helix to β–sheet and random coiled secondary structure and appeared to be highly reversibly up to 75 °C. Thermal unfolding of Apo A-1 and in general of proteins is analyzed almost exclusively with an all-or-none model. As a powerful alternative for higly α–helical proteins such as Apo A 1, we introduced the cooperative Zimm-Bragg theory. Zimm-Bragg theory is commonly used for thermal unfolding of peptides, but fits well to our data and to data of other proteins obtained from literature.
Apo A-1 mimetic peptides
Apo A-1 was proposed as drug against cardiovascular disease. However, Apo A-1 mimetic peptides are more promising as they have to be administered in much lower dosage and are produced more easily. Understanding their lipid binding properties is essential for the estimation of in vivo effects as well as for formulation and dosage of possible drugs with these peptides. Apo A-1 structure is featured by several amphiphatic class A motif α-helices. Even though it is the main protein component of HDL, thermodynamic characterization of its lipid binding has not been achieved in detail. As a model of Apo A-1 we used two peptides (4F and P), which are featured by class A amphipathic α-helical sequences. 4F showed Apo A-1 mimetic properties in animal models and clinical studies. We used isothermal titration calorimetry to determine thermodynamic parameters of binding to POPC lipid vesicles. In order to understand this reaction several other experimental methods were used. Static and dynamic light scattering illustrated the ability of the peptides to rupture unilamellar vesicles and form micellar-like particles. In contrast, many other peptides such as cell penetrating peptides (CPPs) only partition into the membrane. This finding is in agreement with a 1:1 lipid-to-peptide stochiometry yielded from ITC data analyzed with a model of n identical binding sites. This behavior might have high physiological relevance as possible rupture of cell membranes is unwanted. Circular dichroism experiments yielded insight into structural transitions as part of the driving force of lipid binding. Associated with lipid binding is a transition of the peptide from β–sheet and random coiled to α-helical secondary structure. Tryptophan fluorescence measurements complemented the studies indicating binding to lipids as well. Thermodynamic calculation proved the structural transition of β–sheet and random coiled to α-helix as well as hydrophobic interactions as driving forces of the reaction.
Further, we studied binding of the peptide 4F to cholesterol by means of ITC. Our results suggested affinity of 4F towards cholesterol but with lower affinity compared to POPC. This might explain the formation of HDL like particles, mainly consisting of phosphocholine lipids. These particles, in turn, could bind to cholesterol with high affinity.
ABCA1
ABCA1 is an ATP binding cassette transporter that flops excess lipids of a cell to the outer membrane leaflet, where it can be picked up by Apo A-1 or HDL particles. Research in the field of ABCA1 is mainly focused on studies in cell culture and in animal models and is therefore rather indirect. Cholesterol efflux by ABCA1 was assumed to be controlled by the copy number of the transporter. The possibility of a direct modulation of the transporter activity by allocrites like in P-glycoprotein (Pgp) as well as the proposed allocrite specificity was rarely investigated in previous studies. Here, we measured the ATPase activity of inside-out vesicles prepared from ABCA1 transfected Human Embryonic Kidney 293 cells by means of a spectroscopic phosphate release assay. Aluminum fluorides were found as strong inhibitor of the nucleotide binding sites (NBD) of ABCA1 in contrast to vanadate. Furthermore, a screening for putative allocrites interacting with the transmembrane domains (TMDs) was performed with numerous compounds. Therewith we found that all compounds with a pegylated chain, a heterocyclic group and a hydrocarbon tail indicated activation of the ABCA1 ATPase.