Optimized negative-staining protocol for examining lipid-protein interactions by electron microscopy.

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.

Optimized negative-staining electron microscopy for lipoprotein studies.

BACKGROUND: Negative-staining (NS), a rapid, simple and conventional technique of electron microscopy (EM), has been commonly used to initially study the morphology and structure of proteins for half a century. Certain NS protocols however can cause artifacts, especially for structurally flexible or lipid-related proteins, such as lipoproteins. Lipoproteins were often observed in the form of rouleau as lipoprotein particles appeared to be stacked together by conventional NS protocols. The flexible components of lipoproteins, i.e. lipids and amphipathic apolipoproteins, resulted in the lipoprotein structure being sensitive to the NS sample preparation parameters, such as operational procedures, salt concentrations, and the staining reagents. SCOPE OF REVIEW: The most popular NS protocols that have been used to examine lipoprotein morphology and structure were reviewed. MAJOR CONCLUSIONS: The comparisons show that an optimized NS (OpNS) protocol can eliminate the rouleau artifacts of lipoproteins, and that the lipoproteins are similar in size and shape as statistically measured from two EM methods, OpNS and cryo-electron microscopy (cryo-EM). OpNS is a high-throughput, high-contrast and high-resolution (near 1nm, but rarely better than 1nm) method which has been used to discover the mechanics of a small protein, 53kDa cholesterol ester transfer protein (CETP), and the structure of an individual particle of a single protein by individual-particle electron tomography (IPET), i.e. a 14Å-resolution IgG antibody three-dimensional map. GENERAL SIGNIFICANCE: It is suggested that OpNS can be used as a general protocol to study the structure of proteins, especially highly dynamic proteins with equilibrium-fluctuating structures.