Author: Sewwandi S. Rathnayake

Publisher:

Published: 2016

Total Pages: 218

ISBN-13:

In the past, lipid droplets were considered as inert cell organelles that store cellular energy in the form of neutral lipids. But today, they are known as cell organelles carrying out a myriad of functions required for the sustenance of life. The structure of intracellular lipid droplets closely resembles that of extracellular particles called lipoproteins. Both structures contain a core of neutral lipids surrounded by a monolayer of phospholipids and proteins. However, their functional roles within living organisms are different, ie, lipoprotein particles transport neutral lipids extracellularly in an organism's circulatory system, whereas lipid droplets store neutral lipids intracellularly. These two structures differ in composition as well. However, literature shows that proteins found on the surface of lipoproteins and lipid droplets share a high degree of sequence and structural similarity. Although structural and functional details of lipoproteins and their components have been widely studied in the past, many structural and functional aspects of lipid droplets and their protein components are still unclear. This work describes the lipid binding properties of two structurally homologous proteins carrying amphipathic alpha helix bundles, apoLp-III and perilipin 3 which are found across two different biological systems, namely, lipoproteins in insects and lipid droplets in humans. Both these proteins are exchangeable in their lipid binding behavior. Studying the lipid interactions of the above proteins is of key importance to understand the basics of neutral lipid metabolism and lipid transport of multicellular organisms. Large quantities of recombinant proteins required for our biophysical characterization studies were expressed in E. coli cells and purified using a combination of chromatography techniques. In order to study the lipid interactions of apoLp-III and perilipin 3, we used Langmuir monolayers composed of various types of phospholipids. Our monolayer work revealed that apoLp-III by itself is a surface active protein and it appears to show a preference for saturated and highly ordered lipids. Consistent with earlier work we find that insertion of apoLp-III into fluid lipid monolayers is highest for diacylglycerol. The effective molecular shape of lipid molecules and the head group charge were also identified as important parameters that affect the lipid binding of ApoLp-III. We also investigated the interaction of full length perilipin 3 and three of its N-terminal truncation mutants with lipid monolayers. We observed that similar to apoLp-III, perilipin 3 is a surface active protein. The C-terminus of perilipin 3 shows strong preference for insertion into saturated, hence more-ordered lipid environments, similar to our previous data on apoLp-III. The C-terminal alpha helix bundle domain of perilipin 3 showed significantly different insertion properties compared to that of the full length protein and the other successively longer N-terminal truncation mutants. Addition of N-terminal sequences to the C-terminal alpha helix bundle domain reduced the lipid insertion ability of perilipin 3. Anionic lipids with negative spontaneous curvature facilitate lipid interaction and insertion of perilipin 3. Lipid monolayers created on the surface of hydrophobic planer chips (HPA) were used to study the kinetics of lipid binding interactions of apoLp-III and perilipin 3 through surface plasmon resonance. Our data show that for both apoLp-III and perilipin 3, lipid association and dissociation occur in a two --step process, suggesting conformational changes of the amphipathic alpha helices upon lipid interaction. These results thus shed important new insight into the protein--lipid interactions of a model exchangeable apolipoprotein with significant implications for its mammalian counterparts.