Optimized Negative-Staining Protocol for Lipid–Protein Interactions Investigated 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. Insights into the lipid-protein interactions are important in understanding biological processes, and the structure of proteins at the lipid binding stage can help identify their roles and critical functions. Previously, such structural determination was challenging to obtain because the traditional methods, such as X-ray crystallography, are unable to capture the conformational and compositional heterogeneity of protein–lipid complexes. Electron microscopy (EM) is an alternative approach to determining protein structures and visualizing lipid–protein interactions directly, and negative-staining (OpNS), a subset of EM techniques, is a rapid, frequently used qualitative approach. The concern, however, is that current NS protocols often generate artifacts with lipid-related proteins, such as rouleaux formation from lipoproteins. To overcome this artifact formation, Ren and his colleagues have refined early NS protocols, and developed an optimized NS protocol that validated by comparing images of lipoproteins from cryo-electron microscopy (cryo-EM). This optimized NS protocol produces “near native-state” particle images and high contrast images of the protein in its native lipid-binding state, which can be used to create higher-quality three-dimensional (3D) reconstruction by single-particle analysis and electron tomography (e.g. IPET). This optimized protocol is thus a promising hands-on approach for examining the structure of proteins at their lipid-binding status.

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“…Limitations of this study include the fact that, while the cytological findings were suggestive of milk accumulation, special staining such as Sudan Black B or Oil red O stain, which were unavailable at the time of data collection, would have been necessary to definitively confirm the presence of milk in an aspirate sample (Frederiks, 1977 ; Escorcia et al., 2018 ; Liu et al., 2019 ). Additionally, while all samples taken from affected foals cultured Streptococcus equi ssp .…”

The Essence of Lipoproteins in Cardiovascular Health and Diseases Treated by Photodynamic Therapy

“…The last model involves a dimer tunnel, or modified tunnel model (the hydrophobic tunnel is formed by a CETP dimer instead of a monomer) [157]. These models illustrate the basic process of lipid transport, but the exact course of the mechanism is currently unknown, since lipoproteins and CETP-lipoprotein complexes have dynamic properties and additionally have structural flexibility [170][171][172][173].…”

Structure-based mechanism and inhibition of cholesteryl ester transfer protein

“…(ii) the tunnel model: a ternary complex in which CETP bridges HDL and LDL/VLDL and exchanges HDL-CE for mostly VLDL-TG, via a hydrophobic tunnel within CETP [ 30 ]; (iii) the dimer tunnel model: a modified tunnel model, in which the hydrophobic tunnel is formed by a CETP dimer instead of monomer [ 31 ]. Although these models account for the basic lipid transport process [ 32 ], the detailed mechanism is unknown due to the dynamic properties and structural flexibilities of lipoprotein and CETP-lipoprotein complexes [ 33 , 34 ••, 35 – 37 ].…”