Conformational changes of the flavivirus E glycoprotein

Dengue virus, a member of the Flaviviridae family, has a surface composed of 180 copies each of the envelope (E) glycoprotein and the membrane (M) protein. The crystal structure of an N-terminal fragment of E has been determined and compared with a previously described structure. The primary difference between these structures is a 10 degrees rotation about a hinge relating the fusion domain DII to domains DI and DIII. These two rigid body components were used for independent fitting of E into the cryo-electron microscopy maps of both immature and mature dengue viruses. The fitted E structures in these two particles showed a difference of 27 degrees between the two components. Comparison of the E structure in its postfusion state with that in the immature and mature virions shows a rotation approximately around the same hinge. Flexibility of E is apparently a functional requirement for assembly and infection of flaviviruses.     

Fine structural distinction between ganglia of the outer and inner submucosal plexus in porcine small intestine

Ultrastructural differences between ganglia of the plexus submucosus internus (Meissner; PSI) and plexus submucosus externus (Schabadasch; PSE) are described. Comparison revealed a different glia index (ratio glia per neuron) between the PSE (3:1) and the PSI (1:1), the arrangement of PSI neurons in compartments and the appearance of broad membrane-to-membrane appositions inside the compartments of the PSI. Structural and immunohistochemical differences between the two plexuses are discussed. In general, PSE neurons show a wider variety in size and shape than most of the PSI neurons.     

Proteins of Newcastle disease virus envelope: interaction between the outer hemagglutinin-neuraminidase glycoprotein and the inner non-glycosylated matrix protein

Using linear sucrose-density ultracentrifugation analysis of Triton-solubilized Newcastle Disease Virus envelopes, we have evidenced, for the first time, the existence of interactions between the outer hemagglutinin-neuraminidase transmembrane glycoprotein and the inner non-glycosylated peripheral matrix protein. Such interactions seem to be electrostatic. These conclusions are based on the behavior of both proteins at different ionic strengths. When in low ionic strength buffer, hemagglutinin-neuraminidase and matrix proteins band together in the sucrose gradient, whereas at high ionic strength both proteins band at different rates in the gradient. The behavior of the inner matrix protein in our conditions was the expected one for a peripheral protein. The results of these 'in vitro' studies are also discussed in terms of the possible 'in vivo' role of such interactions.     

Amidination of the outer and inner surfaces of the human erythrocyte membrane

We have synthesized a novel imidoester, isethionyl acetimidate, which is unable to penetrate the membrane of the human erythrocyte. It has the same specificity for amino groups as ethyl acetimidate, which penetrates the membrane. Either reagent can be labeled with ³H or ¹⁴C and, thus, be used to convert amines to radioactive amidines. An erythrocyte membrane saturated with either compound functions nearly normally. Therefore, the membrane can be double labeled if the amino groups on the outer surface of a cell are saturated with isethionyl acetimidate (e.g. labeled with ¹⁴C) and the remaining active sites are saturated with ethyl acetimidate (labeled with ³H). Alternatively, the membrane can be isolated after saturation with [¹⁴C]isethionyl acetimidate and treated with [³H]isethionyl acetimidate. From quantitative experiments of this kind we conclude that there are more than ten times as many reactive amino groups in protein on the inner surface than on the outer surface of the membrane. Nearly all of the reactive amino groups in lipid are on the inner surface. The localization of individual polypeptides confirms and extends assignments made previously by other techniques; as many as four major components may span the membrane. The proteins and lipids react to the same extent with ethyl acetimidate in the intact cell as they do in isolated membranes; this implies that the isolation does not load to major structural rearrangements.     

The maturation-dependent conformational change of phage T4 capsid involves the translocation of specific epitopes between the inner and the outer capsid surfaces

After polymerization of the phage T4 prohead is complete, its capsid expands by approximately 16%, is greatly stabilized, and acquires the capacity to bind accessory proteins. These effects are manifestations of a large-scale, irreversible, conformational change undergone by the major capsid protein, gp23 (521 residues) which is cleaved to gp23* (residues 66-521) during this maturation process. In order to explore its structural basis, we have performed immunoelectron microscopy with antibodies raised against synthetic peptides that correspond to precisely defined segments of the amino acid sequence of gp23. These antibodies were used to label purified polyheads (tubular polymorphic variants of the normal icosahedral capsid), in experiments designed to impose constraints on the possible foldings of the gp23/gp23* polypeptide chains in their successive conformational states. Peptide 1 (residues 48-57), part of the gp23-delta domain that is excised when gp23 is converted to gp23*, resides on the inner surface of the precursor surface lattice, but--if not proteolyzed--is found on the outer surface of the mature surface lattice. Peptide 2 (residues 65-73), immediately distal to the cleavage site, is located on the inside of the precursor surface lattice, and remains there subsequent to expansion. Peptide 3 (residues 139-146) is translocated in the opposite direction from peptide 1, i.e., from the outer to the inner surface upon expansion; moreover, expansion greatly increases the polyheads' affinity for these antibodies. Peptide 5 (residues 301-308) is located on the inside in both the precursor and the mature states. Taking into account data from other sources, these observations imply that the conformational change that underlies capsid expansion involves a radical reorganization of the proteins' structure, in which at least three distinct epitopes, situated in widely differing parts of the polypeptide chain, are translocated from one side to the other. Moreover, the amino-terminal portion of gp23/gp23*, around the cleavage site, is particularly affected.     

The importance of being dimeric

Why are there so many dimeric proteins and enzymes? While for heterodimers a functional explanation seems quite reasonable, the case of homodimers is more puzzling. The number of homodimers found in all living organisms is rapidly increasing. A thorough inspection of the structural data from the available literature and stability (measured from denaturation-renaturation experiments) allows one to suggest that homodimers can be divided into three main types according to their mass and the presence of a (relatively) stable monomeric intermediate in the folding-unfolding pathway. Among other explanations, we propose that an essential advantage for a protein being dimeric may be the proper and rapid assembly in the cellular milieu.     

The importance of being K-Ras

The ras genes give rise to a family of related proteins that have strong transforming potential. Typical in vitro studies fail to discriminate between the transforming activity of the Ras proteins. Although activating mutations in ras genes are commonly found in human disease, they are not evenly distributed between the different ras members. Instead, they are concentrated in k-ras. With the absence of evidence to suggest that k-ras DNA is more prone to mutation than h-ras DNA, this imbalance in mutational frequency suggests a special biological role for the K-Ras protein in vivo.