title: Optimized negative-staining protocol for examining lipid-protein interactions by electron microscopy.
abstract: A large number of proteins are capable of inserting themselves into lipids, and interacting with membranes, such as transmembrane proteins and apolipoproteins. Protein-lipid interactions have been identified as one of the keys in understanding biological processes, while the structure of proteins at the lipid-binding stage can provide evidence to help identify their roles and critical functions. However, structure determination of proteins at the lipid-binding stage is rather difficult, because conformational and compositional heterogeneities of the protein-lipid complexes are major barriers to unravel their structures using traditional methods, such as X-ray crystallography. Electron microscopy (EM) is an alternative approach to determine protein structure and has demonstrated a capability in visualizing lipid-protein interactions directly. Among various EM techniques, negative-staining (NS) is an easy, rapid, qualitative approach that is a well-established technique, frequently used in research laboratories. Conventional NS protocols, unfortunately, often generate artifacts with lipid-related proteins, such as the rouleau formation of lipoproteins. To overcome this artifact formation, Ren and his colleagues recently developed an optimized NS protocol that was validated by comparing images of lipoproteins from cryo-electron microscopy (cryo-EM). The optimized NS protocol could produce "near native-state" particle images and high contrast images of the protein in its lipid-binding state that is favorable for three-dimensional (3D) reconstruction by single-particle analysis and individual-particle electron tomography (IPET), suggesting this optimized protocol can be used widely to examine the structure of proteins at lipid-binding stage.
authors: Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.
authors: Mark
authors: M
authors: Garewal
authors: Lei
authors: L
authors: Zhang
authors: Gang
authors: G
authors: Ren
mesh_terms: D000818:Animals; D006801:Humans; D050356:Lipid Metabolism; D008854:Microscopy, Electron / Q000379:methods*; D016624:Negative Staining / Q000379:methods*; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism* / Q000648:ultrastructure*
chemical_list: D011506:Proteins
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