The NMR Study Of ?-Synuclein In Rat Mitochondria

?-Synuclein is intrinsically disordered protein composed of 140 amino acids and its aggregates are the major component of Lewy body,the pathological hallmark of Parkinson's disease.Currently,?-Synuclein has attracted widespread attention because it is closely related to the pathogenesis of Parkinson's disease.Mitochondria are eukaryotic organelles surrounded by two-layer membrane.Mitochondria are known as "Power House" of cell because they are the main engine that generates ATP(adenosine triphosphate)through oxidative phosphorylation within the respiratory chain.Mitochondria are also involved in many cellular processes,such as signal transduction,cell differentiation and apoptosis.Mitochondrial dysfunction can lead to a variety of diseases,such as cancer,aging,inflammation and neurodegenerative diseases including Parkinson's disease.It is estimated that there are about 5 million people worldwide suffering from Parkinson's disease.Numerous scientists are dedicated to investigating the pathogenesis of Parkinson's disease and developing effective therapies.The study of the interaction between ?-Synuclein and mitochondrial membrane as well as the in situ study of ?-Synuclein in mitochondria are the effective way to understand the pathogenesis of Parkinson's disease.In addition,accumulating attention has been attracted in the structure and function of proteins inside cells and even cellular organelles with the deepening development of protein science research.The in situ study of protein in mitochondria probably can provide a novel method for the study of protein in the future.Nuclear magnetic resonance spectroscopy(NMR)which is a non-destructive and non-invasive technique acquiring protein information at atomic level,is a powerful tool for the in-situ study of protein in mitochondria.However,systematic research is lacking on how to import protein into mitochondria and optimal labeling strategies of proteins.Here we used NMR spectroscopy to study the interaction between ?-Synuclein and intact mitochondria isolated from rat liver.We found that ?-synuclein can interact with OMM(outer membrane of mitochondria)and the first 60 residues of N-terminus are crucial for binding to mitochondria,which is different from the previous report,in which ?-synuclein does not interact with mimic OMM(only lipid components of mitochondria are considered in mimic membrane).Some of the possible reasons are that the interaction of ?-synuclein and OMM depends not only on the lipid composition of OMM,but also on other factors such as the curvature of membrane and other components of OMM(such as lipoprotein,sugar,etc.).Our study also indicates that NMR is an effective method for studying the interaction of proteins and intact mitochondriaAdditionally,we develop an in-situ NMR method of protein in mitochondria by combining electroporation technique with NMR spectroscopy.The various isotope-labeled(19F?13C?15N)?-Synuclein proteins were successfully introduced into mitochondria by electroporation,respectively.The normal morphological structure and membrane potential of mitochondria can still be maintained after electroporation,showing that mitochondria are not be damaged by electroporation.We successfully acquired a series of high-resolution 1H-15N HMQC,1H-13C HMQC spectra and 1D 19F spectra of ?-Synuclein in mitochondria,laying a foundation for studying in situ structure and function of proteins in mitochondriaWe further analyzed 1H-15N HMQC spectra of ?-Synuclein and its mutant in mitochondria.We found that the cross-peak signals at residual region near Y39,the N-terminal and C-terminal regions,relative to buffer,disappeared or attenuated dramatically,which are likely to be related to the interactions between ?-Synuclein and components in mitochondria,such as the interactions with IMM(inner membrane of mitochondrial),the molecular chaperones in mitochondria,and the electrostatic interactions with biological macromolecules in mitochondriaIn summary,we established NMR methods of the interaction study between protein and intact mitochondria and of the in-situ study of protein in mitochondria.They provide an effective way for further investigation of the protein study in mitochondria,such as protein structure,dynamics and function.