Daniel R. Beniac

Three-dimensional structure of myelin basic protein. II. Molecular modeling and considerations of predicted structures in multiple sclerosis.

A computational model of myelin basic protein (MBP) has been constructed based on the premise of a phylogenetically conserved β-sheet backbone and on electron microscopical three-dimensional reconstructions. Many residues subject to post-translational modification (phosphorylation, methylation, or conversion of arginines to citrullines) were located in loop regions and thus accessible to modifying enzymes. The triproline segment (residues 99-101) is fully exposed on the back surface of the protein in a long crossover connection between two parallel β-strands. The proximity of this region to the underlying β-sheet suggests that post-translational modifications here might have potential synergistic effects on the entire structure. Post-translational modifications that lead to a reduced surface charge could result first in a weakened attachment to the myelin membrane rather than in a gross conformational change of the protein itself. Such mechanisms could be operative in demyelinating diseases such as multiple sclerosis.

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.

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.

Visualisation of E. coli ribosomal RNA in situ by electron spectroscopic imaging and image analysis

Abstract A quantitative analysis of large populations of images of the large and small E. coli ribosomal subunits has been performed to construct two-dimensional maps of the phosphorus distribution within these complexes lying in their characteristic crown and right-lateral orientations, respectively. These elemental maps were interpreted to represent primarily the backbone of the nucleic acid component, and showed congruence to low resolution with current models of ribosomal RNA structure based on biochemical data. Considerations of computerised analysis of electron spectroscopic images of isolated particles suggest a need for new alignment algorithms not based solely on correlation functions.

STRUCTURES OF SMALL SUBUNIT RIBOSOMAL RNAS IN SITU FROM ESCHERICHIA COLI AND THERMOMYCES LANUGINOSUS

Small ribosomal subunits from the prokaryoteEscherichia coli and the eukaryoteThermomyces lanuginosus were imaged electron spectroscopically, and single particle analysis used to yield three-dimensional reconstructions of the net phosphorus distribution representing the nucleic acid (RNA) backbone. This direct approach showed both ribosomal RNAs to have a three domain structure and other characteristic morphological features. The eukaryotic small ribosomal subunit had a prominent bill present in the head domain, while the prokaryotic subunit had a small vestigial bill. Both ribosomal subunits contaied a thick ‘collar’ central domain which correlates to the site of the evolutionarily conserved ribosomal RNA core, and the location of the majority of ribosomal RNA bases that have been implicated in translation. The reconstruction of the prokaryotic subunit had a prominent protrusion extending from the collar, forming a channel approximately 1.5 nm wide and potentially representing a ‘bridge’ to the large subunit in the intact monosome. The basal domain of the prokaryotic ribosomal subunit was protein free. In this region of the eukaryotic subunit, there were two basal lobes composed of ribosomal RNA, consistent with previous hypotheses that this is a site for the ‘non-conserved core” ribosomal RNA.

The in situ architecture of Escherichia coli ribosomal RNA derived by electron spectroscopic imaging and three‐dimensional reconstruction

The structures of the large and small ribosomal subunits of Escherichia coli were reconstructed using spectroscopic electron microscopy and quaternion‐assisted angular reconstitution to resolutions of better than 4 nm. In addition, the distributions of phosphorus within these complexes were reconstructed. The three‐dimensional reconstruction of the distribution of this atomic element is an extension of microanalysis (in two dimensions) for phosphorus identification and mapping, as a signature of the arrangement of the phosphate backbones of the constituent ribosomal RNAs. The results on both the phosphorus reconstructions and the total reconstructions (protein and ribosomal RNA) reveal several passageways through both subunits. The structures correspond favourably with other independent reconstructions of the whole E. coli ribosome from cryoelectron micrographs and their accompanying models of translation (Frank et al., Nature, 376, 441–444, 1995; Stark et al., Structure, 3, 815–821, 1995). The overall reconstructions in conjunction with the phosphorus (rRNA) distributions are the first to be achieved synchronously for this nucleoprotein complex.

Ribosomal proteins ofThermomyces lanuginosus — characterisation by two-dimensional gel electrophoresis and differential disassembly

One- and two-dimensional gel electrophoresis were employed to characterise the proteins derived from the ribosomes of the thermophilic fungusThermomyces lanuginosus. Approximately 32 (29 basic and 3 acidic) and 45 (43 basic and 2 acidic) protein spots were resolved fromTh. lanuginosus small and large ribosomal subunits, respectively. The molecular weight of the small subunit proteins ranged from 9,800–36,000 Da with a number average molecular weight of 20,300 Da. The molecular weight range for the large subunit proteins was 12,000–48,500 Da with a number average molecular weight of 25,900 Da. Most proteins appeared to be present in unimolar amounts. These data are comparable with but not identical to those from other eukaryotic ribosomes. The sensitivities of the ribosomal proteins to increasing concentrations of NH4Cl were also evaluated by two-dimensional gel electrophoresis. Most ribosomal proteins were gradually released over a wide range of salt concentrations but some were preferentially enriched in one or two salt conditions.

Three-dimensional architecture of Thermomyces lanuginosus small subunit ribosomal RNA

Abstract The structure of the small ribosomal subunit of the mildly thermophilic fungus Thermomyces lanuginosus was reconstructed using spectroscopic electron microscopy and quaternion-assisted angular reconstitution to a resolution of roughly 3.3 nm. The distribution of phosphorus within this complex was also reconstructed and represents the arrangement of the phosphate backbone of the constituent ribosomal RNA. The results indicate regions where the extra rRNA of eukaryotes (compared with prokaryotes) is likely to be, specifically in the eukaryotic beak and basal lobes. The overall reconstruction in conjunction with the phosphorus (rRNA) distribution is the first to be achieved synchronously for this eukaryotic nucleoprotein complex.