F. Peter Ottensmeyer

Three-dimensional Structure of Myelin Basic Protein I. RECONSTRUCTION VIA ANGULAR RECONSTITUTION OF RANDOMLY ORIENTED SINGLE PARTICLES

Myelin basic protein (MBP) plays an integral role in the structure and function of the myelin sheath. In humans and cattle, an 18.5-kDa isoform of MBP predominates and exists as a multitude of charge isomers resulting from extensive and varied post-translational modifications. We have purified the least modified isomer (named C1) of the 18.5-kDa isoform of MBP from fresh bovine brain and imaged this protein as negatively stained single particles adsorbed to a lipid monolayer. Under these conditions, MBP/C1 presented diverse projections whose relative orientations were determined using an iterative quaternion-assisted angular reconstitution scheme. In different buffers, one with a low salt and the other with a high salt concentration, the conformation of the protein was slightly different. In low salt buffer, the three-dimensional reconstruction, solved to a resolution of 4 nm, had an overall “C” shape of outer radius 5.5 nm, inner radius 3 nm, overall circumference 15 nm, and height 4.7 nm. The three-dimensional reconstruction of the protein in high salt buffer, solved to a resolution of 2.8 nm, was essentially the same in terms of overall dimensions but had a somewhat more compact architecture. These results are the first structures achieved directly for this unusual macromolecule, which plays a key role in the development of multiple sclerosis.

Improved preservation of the form and contents of wall vesicles and the golgi apparatus in freeze substituted hyphae ofSaprolegnia

SummarySecretory vesicles involved in cell wall synthesis (wall vesicles) and the Golgi apparatus have been compared in conventionally fixed and freeze substituted hyphae of the oomycete fungusSaprolegnia ferax. Wall vesicles freeze substituted in various fluids range from spherical to tubular and contain an intensely staining, phosphorous rich matrix. In contrast diverse conventional fixations cause artefactual constrictions in most tubular vesicles and loss of their intensely staining contents. These data are interpreted to show the existence of an intravesicular skeletal system, with cellular regulation, to determine vesicle morphology and intravesicular synthesis of a hypothetical phosphorylated glycolipid cell wall precursor. Whilst freeze substitution gives superior preservation of wall vesicle morphology, it does not demonstrate any preferential association between wall vesicles and microtubules thus suggesting that microtubules are only indirectly involved in wall vesicle transport. Freeze substitution is superior to conventional fixation for analysis of the Golgi apparatus because it uniquely reveals both differentiation of a specific single cisterna in each Golgi body and greater differences in membrane thicknesses throughout the endomembrane system.

An electron microscopic and spectroscopic study of murine epiphyseal cartilage: Analysis of fine structure and matrix vesicles preserved by slam freezing and freeze substitution

Newborn mice epiphyseal growth plates were preserved by slam freezing/freeze substitution and examined by conventional electron microscopy, stereopsis, high voltage electron microscopy, and electron spectroscopic imaging (ESI). To illustrate the improved ultrastructure of this cryogenic procedure, conventional, aqueously fixed growth plates were included showing collapsed hypertrophic chondrocytes surrounded by a depleted and condensed extracellular matrix. In contrast, the cryogenically prepared epiphyses contain chondrocytes and extracellular matrix vesicles both in direct contact with proteoglycan filaments retained in an expanded state. ESI is an electron microscopic technique which enables the direct localization of atomic elements superimposed over fine structural details. This technique was used to examine the colocalization of calcium and phosphorus within matrix vesicles and within their associated extracellular environments. Matrix vesicles appeared in three distinct diameter ranges. The integrity of the matrix vesicles was examined at various stages of mineralization and also within the mineralized zone of provisional calcification.

Automatic 3D alignment of projection images of randomly oriented objects

Abstract Reconstruction of the 3D density distribution of macromolecular structures from their 2D projection images or electron micrographs requires knowledge of the relative orientations of the 2D projections. Using a technique that combines the principles of the common axis theorem and quaternion mathematics, the projection directions may be determined a posteriori from the images. This technique is applied to stimulated electron micrographs of the Klenow fragment of DNA polymerase I. These simulated images, with graded signal-to-noise ratios, are subjected to image processing procedures, such as 2D translational and rotational registration and multivariate statistical classification, that would normally be applied to electron micrographs to improve their quality. The alignment procedure is able to determine the relative 3D orientations of the projections correctly even when the images have an initial signal-to-noise ratio of 3.1 and when images within a single class of views are deliverately perturbed in orientation. A relationship is obtained between the parameters used to describe the success of the 3D alignment algorithm and the quality of the 3D reconstruction that might be expected.

HIGH RESOLUTION MICROANALYSIS AND THREE-DIMENSIONAL NUCLEOSOME STRUCTURE ASSOCIATED WITH TRANSCRIBING CHROMATIN

The nucleosome is the ubiquitous and fundamental DNA-protein complex of the eukaryotic chromosome, participating in the packaging of DNA and in the regulation of gene expression. Biophysical studies have implicated changes in nucleosome structure from chromatin that is quiescent to active in transcription. Since DNA within the nucleosome contains a high concentration of phosphorus whereas histone proteins do not, the nucleosome structure is amenable to microanalytical electron energy loss mapping of phosphorus to delineate the DNA within the protein-nucleic acid particle. Nucleosomes associated with transcriptionally active genes were separated from nucleosomes associated with quiescent genes using mercury-affinity chromatography. The three-dimensional image reconstruction methods for the total nucleosome structure and for the 3D DNA-phosphorus distribution combined quaternion-assisted angular reconstitution of sets of single particles at random orientations and electron spectroscopic imaging. The structure of the active nucleosome has the conformation of an open clam-shell, C- or U-shaped in one view, elongated in another, and exhibits a protein asymmetry. A three-dimensional phosphorus map reveals a conformational change in nucleosomal DNA compared to DNA in the canonical nucleosome structure. It indicates an altered superhelicity and is consistent with unfolding of the particle. The results address conformational changes of the nucleosome and provide a direct structural linkage to biochemical and physiological changes which parallel gene expression.

Low energy loss electron microscopy of chromophores

A novel prism-mirror-prism imaging electron spectrometer with 1 eV energy resolution for a transmission electron microscope permits imaging with spectral energies corresponding to light-optical colour absorptions. The instrument selects the molecular orbital excitations of natural chromophores or of specific dyes normally used in biological light microscopy for delineation and chemical identification, but images them with electron microscopic detail. Heavy atom contrast agents customarily used in electron microscopy are not required. The first results exploit the intrinsic red colour of hematin molecules to demonstrate the potential of the technique and address its spatial resolution. Glycosaminoglycans in cartilage stained with Alcian blue are selectively depicted in situ by means of the electron-induced molecular absorption of this chromophore. Thus, with the use of specific colours the direct or indirect analysis of local chemistry by electron microscopy is possible, and can be carried out with a depiction of spatial detail as small as 16 A, or at least 100-fold finer than observed by light microscopy.

Filaments of surfactant protein A specifically interact with corrugated surfaces of phospholipid membranes

Pulmonary surfactant, a mixture of lipids and surfactant proteins (SPs), plays an important role in respiration and gas exchange. SP-A, the major SP, exists as an octadecamer that can self-associate to form elongated protein filaments in vitro. We have studied here the association of purified bovine SP-A with lipid vesicle bilayers in vitro with negative staining with uranyl acetate and transmission electron microscopy. Native bovine surfactant was also examined by transmission electron microscopy of thinly sectioned embedded material. Lipid vesicles made from dipalmitoylphosphatidylcholine and egg phosphatidylcholine (1:1 wt/wt) generally showed a smooth surface morphology, but some large vesicles showed a corrugated one. On the smooth-surfaced vesicles, SP-As primarily interacted in the form of separate octadecamers or as multidirectional protein networks. On the surfaces of the striated vesicles, SP-As primarily formed regularly spaced unidirectional filaments. The mean spacing between adjacent striations and between adjacent filaments was 49 nm. The striated surfaces were not essential for the formation of filaments but appeared to stabilize them. In native surfactant preparations, SP-A was detected in the dense layers. This latter arrangement of the lipid bilayer-associated SP-As supported the potential relevance of the in vitro structures to the in vivo situation.

Visions in the mist: The zeitgeist of food protein imaging by electron microscopy

The biological activity of a protein is a function of its three-dimensional structure, as well as its interaction with other molecules. This applies equally to foods containing proteins whose functional properties (e.g. solubility, gelation, foaming and emulsification) are dictated primarily by their structures. High-resolution transmission electron microscopy (TEM) is an appropriate tool for determining the structures of biological macromolecules and their complexes, especially when they cannot be studied by X-ray crystallography. Although TEM and appropriate image analysis techniques have been used primarily in molecular structural biology, we have recently adopted this combination into the realm of food science. The principles of TEM, including sample preparation and image analysis techniques, as well as the potential benefits that high-resolution TEM may bring to food protein chemists working in the area of protein structure-function relationships will be discussed in this article.

The reaction of mercuric acetate with histidine and tyrosine

Abstract The reaction of mercuric acetate with polypeptides in an appropriate buffer system has been found to result in the selective binding of two atoms of mercury to each tyrosine and histidine residue. These heavy atom labels are stable to the high chloride concentrations used to displace the excess mercury (II) from other binding sites on the polypeptide. The kinetics and stoichiometry of these reactions have been studied by the binding of radioactive ( 203 Hg) mercuric acetate to synthetic polymers of these amino acids and by ultraviolet-visible spectroscopy. For a polymer containing tyrosine the mercuration kinetics closely match those for the following mechanism: At 60° C , and in a buffer containing 0.05M TRIS-acetate, k 1 was determined to be 9.47 ± 0.27 M −1 min −1 . The best match to the data was for k 1 /k 1 = 5.5 It was discovered that there is an inverse relationship between k 1 and the TRIS buffer concentration. The activation energy of k 1 was determined to be 18.9 ± 0.1 kcal/mole. Chemical analyses of the products obtained from the reaction of mercuric acetate with tyrosine amide, L(-)-histidine and the methyl ester of L(-)-histidine have established that mercuration results in the formation of a Hg-C bond at the C 3 and C 5 sites on the phenolic ring in tyrosine and at the C 4 site in the imidazole ring in histidine. The site of the second mercury retained by histidine in the presence of high chloride concentrations is uncertain but does involve the amine functions of the imidazole ring. The reaction conditions employed also cause the oxidation of methionine and cysteine to methionine sulfoxide and cysteic acid, respectively. Cystine, however, resists oxidation.

Scattered Electrons in Biological Structure Determination

Of the various atomic particles or quanta of electromagnetic radiation available for probing the finestructure of biological objects, electrons have a particularly favourable place. The wavelength of the particle at an energy of 100 keV is about 0.0037 nm, offering the promise of high spatial resolution. Its cross-section of interaction with matter is sufficiently small to penetrate a specimen 100 nm thick with ease, but large enough to interact at least once in that thickness. In addition it can be focused by electrostatic and magnetic fields, permitting the construction of electron microscopes.