Morphology of sodium deoxycholate-solubilized apolipoprotein B-100 using negative stain and vitreous ice electron microscopy

The primary and secondary structures of apolipoprotein B-100 (apoB-100) are well established. Previous morphological studies have suggested that apoB is a long, flexible, threadlike molecule that encircles the low density lip oprotein (LDL) particle. Several large domain regions of the protein have b een observed in frozen hydrated LDL and may be involved iu anchoring of the protein to the lipid surface of LDL. Calorimetric studies of sodium deoxyc holate (NaDC)-solubilized apoB indicated a similar number of independently melting domains. We therefore undertook a morphological study of NaDC-solub ilized apoB-100 using negative stain and vitreous ice cryoelectron microsco py, a nonperturbing preservation technique. Negative staining experiments w ere performed in two ways: 1) grids were pulled through NaDC-containing buf fer surfaces on which monolayers of apoB had been promoted, or 2) apoB mole cules were allowed to diffuse onto carbon surfaces of grids that were float ed on sample droplets. Vitrified molecules of apoB were obtained by plungin g a thin fluid layer of protein adhered to a holey carbon-coated grid into supercooled ethane and by preserving the molecules in liquid nitrogen. The majority of molecules prepared in negative stain and vitreous ice were curv ed or arced and had alternating thin and thick regions. In negative stain, the apoB molecules lay on the grid perpendicular to the electron beam and h ad a mean length of 650 Angstrom. It In vitreous ice the molecules were ran domly oriented and their images ranged from 160 to 650 Angstrom in length. Vitrified molecules provided visualization of one or two beaded regions. Si milar regions were observed in negative stain but the overall thickness was two to three times greater. Some vitrified molecules contained ribbon-like portions. Our study supports previously obtained data on molecule length b ut suggests that negative staining overestimates molecule width. These firs t images of vitrified NaDC-solubilized apoB-100 confirm the long, flexible, beaded thread morphology of the molecule and support the unique potential of this technique when coupled with proper molecule orientation and antibod y labeling to correlate the tertiary structure of apoB seen in the intact p article with that of the isolated molecule.-Gantz, D. L., M. T. Walsh, and D. M. Small. Morphology of sodium deoxycholate-solubilized apolipoprotein B -100 using negative stain and vitreous ice electron microscopy.