Structural Investigation of Lipid Storage Protein by Solid-State NMR

Oklahoma State University Stillwater, Stillwater OK

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Abstract

DESCRIPTION (provided by applicant): The long-term objective of this research project is to understand cellular fat metabolism by investigating the atomic-resolution structure of lipid storage proteins. Abnormal storage of neutral lipids has been implicated in conditions of serious health impacts, including obesity, diabetes, fatty liver, and heart diseases. Neutral lipid triglycerides are a main form of fat and energy reserve in animals. Triglycerides are stored in organelles called lipid droplets, which accumulate in the cytosol of adipocytes. Several proteins found on the surface of the lipid droplets are critical to he regulation of storage and release of triglycerides. However, structures of these proteins and details of how the protein activities are affected by protein-membrane interactions are poorly understood. Lipid storage droplet protein 1 (Lsd1) is known to play a key role in the activation of triglyceride hydrolysis. It is also the first lipid droplet protein that has been successfully purified and reconstituted in lipid droplet-like particles. Structural information is critical to the elucidation of the detailed mechanism of its activity. Solid-state NMR offers the best opportunity to provide important three-dimensional structure and dynamics data for Lsd1 in the functional native-like lipid environment, which poses a significant challenge to conventional structure determination techniques. We will employ novel solid-state NMR techniques with high- field spectrometers, along with circular dichroism and other complementary biophysical and biochemical methods, to obtain important structural and functional data on the Lsd1 lipoprotein complexes. Novel proton-detected, magic-angle spinning NMR methods will be applied for their advantages of high sensitivity and additional proton chemical shift information. We were able to purify Lsd1 as lipoprotein particles in tens of milligrams. Preliminary NMR data demonstrated that high quality multidimensional NMR spectra can be obtained with excellent sensitivity and spectral resolution, and that the lipoprotein samples have shown structural homogeneity and stability after weeks of experiments. This study encompasses these specific aims: (1) To achieve residue-specific resonance assignment for Lsd1, which is an indispensible step toward structure determination using NMR techniques. (2) To calculate three-dimensional structure for Lsd1 using distance and angle restraints measured by NMR. (3) To understand the mechanism of protein-lipid interactions using NMR experiments correlating these two types of molecules. The structural information of Lsd1 accomplished in this study is central to the understanding of its function and to the future progress in the field of fat metabolism, since Lsd1 interacts with the triglyceride lipase and other proteins. PUBLIC HEALTH RELEVANCE: The proposed study aims to improve our understanding of cellular fat metabolism on the basis of three-dimensional structure of lipid storage proteins. Knowledge derived from this study is expected to provide clues for how to influence the balance between fat storage and burning, and to eventually discover new therapeutic strategies to prevent/treat obesity and related health problems such as diabetes, fatty liver, and heart diseases.