Papers by Bernard Heymann
Herpes simplex virus type 1 (HSV-1) packages its genome via the portal protein, pUL6, a dodecamer... more Herpes simplex virus type 1 (HSV-1) packages its genome via the portal protein, pUL6, a dodecameric ring at one of the capsid’s twelve vertices. While the portal has been visualized in mature capsids, we aimed to describe it in procapsids to elucidate its role in assembly and maturation. Using cryo-electron tomography, we compared procapsids and empty mature capsids (A-capsids) by subtomogram averaging. The portal is located on the interior surface with its narrower end facing outwards in contact with the capsid shell. The portal is embedded in the underlying scaffold, consistent with assembly initiating on a portal-scaffold complex. Maturation involves angularization of the capsid shell, with an accompanying outward movement of the vertices. In A-capsids, the portal translocates further than the surrounding capsomers, enhancing and presumably strengthening its contacts with the capsid shell. We also found a density overlying and offset from the portal vertex corresponding to the ex...
In vitro channel activity of the C-terminal colicin El channel polypeptide under conditions of va... more In vitro channel activity of the C-terminal colicin El channel polypeptide under conditions of variable electrostatic interaction with synthetic lipid membranes showed distinct maxima with respect to pH and membrane surface potential. The membrane binding energy was determined from fluorescence quenching of the intrinsic tryptophans of the channel polypeptide by liposomes containing N-trinitrophenyl-phosphatidylethanolamine. Maximum in vitro colicin channel activity correlated with an intermediate magnitude of the electrostatic interaction. For conditions associated with maximum activity (40% anionic lipid, / = 0.12 M, pH 4.0), the free energy of binding was AG-9 kcal/mol, with nonelectrostatic and electrostatic components, AGnw-5 kcal/mol and AGel-4 kcal/mol, and an effective binding charge of +7 at pH 4.0. Binding of the channel polypeptide to negative membranes at pH 8 is minimal, whereas initial binding at pH 4 followed by a shift to pH 8 causes only 3-10% reversal of binding, implying that it is kinetically trapped, probably by a hydrophobic interaction. It was inferred that membrane binding and insertion involves an initial electrostatic interaction responsible for concentration and binding to the membrane surface. This is followed by insertion into the bilayer driven by hydrophobic forces, which are countered in the case of excessive electrostatic binding.
In vitro channel activity of the C-terminal colicin El channel polypeptide under conditions of va... more In vitro channel activity of the C-terminal colicin El channel polypeptide under conditions of variable electrostatic interaction with synthetic lipid membranes showed distinct maxima with respect to pH and membrane surface potential. The membrane binding energy was determined from fluorescence quenching of the intrinsic tryptophans of the channel polypeptide by liposomes containing N-trinitrophenyl-phosphatidylethanolamine. Maximum in vitro colicin channel activity correlated with an intermediate magnitude of the electrostatic interaction. For conditions associated with maximum activity (40% anionic lipid, / = 0.12 M, pH 4.0), the free energy of binding was AG-9 kcal/mol, with nonelectrostatic and electrostatic components, AGnw-5 kcal/mol and AGel-4 kcal/mol, and an effective binding charge of +7 at pH 4.0. Binding of the channel polypeptide to negative membranes at pH 8 is minimal, whereas initial binding at pH 4 followed by a shift to pH 8 causes only 3-10% reversal of binding, implying that it is kinetically trapped, probably by a hydrophobic interaction. It was inferred that membrane binding and insertion involves an initial electrostatic interaction responsible for concentration and binding to the membrane surface. This is followed by insertion into the bilayer driven by hydrophobic forces, which are countered in the case of excessive electrostatic binding.
Proceedings of the National Academy of Sciences, Dec 12, 2006
Influenza virus remains a global health threat, with millions of infections annually and the impe... more Influenza virus remains a global health threat, with millions of infections annually and the impending threat that a strain of avian influenza may develop into a human pandemic. Despite its importance as a pathogen, little is known about the virus structure, in part because of its intrinsic structural variability (pleiomorphy): the primary distinction is between spherical and elongated particles, but both vary in size. Pleiomorphy has thwarted structural analysis by image reconstruction of electron micrographs based on averaging ...
Proceedings of the National Academy of Sciences, Dec 12, 2006
Influenza virus remains a global health threat, with millions of infections annually and the impe... more Influenza virus remains a global health threat, with millions of infections annually and the impending threat that a strain of avian influenza may develop into a human pandemic. Despite its importance as a pathogen, little is known about the virus structure, in part because of its intrinsic structural variability (pleiomorphy): the primary distinction is between spherical and elongated particles, but both vary in size. Pleiomorphy has thwarted structural analysis by image reconstruction of electron micrographs based on averaging ...
Nowadays it is possible to unravel complex information at all levels of cellular organization by ... more Nowadays it is possible to unravel complex information at all levels of cellular organization by obtaining multi-dimensional image information. At the macromolecular level, three-dimensional (3D) electron microscopy, together with other techniques, is able to reach resolutions at the nanometer or subnanometer level. The information is delivered in the form of 3D volumes containing samples of a given function, for example, the electron density distribution within a given macromolecule. The same situation happens at the cellular level with the new forms of light microscopy, particularly confocal microscopy, all of which produce biological 3D volume information. Furthermore, it is possible to record sequences of images over time (videos), as well as sequences of volumes, bringing key information on the dynamics of living biological systems. It is in this context that work on BioImage started two years ago, and that its first version is now presented here. In essence, BioImage is a database specifically designed to contain multi-dimensional images, perform queries and interactively work with the resulting multi-dimensional information on the World Wide Web, as well as accomplish the required cross-database links. Two sister home pages of BioImage can be accessed at http://www.bioimage.org and http://www-embl.bioimage.org
Nowadays it is possible to unravel complex information at all levels of cellular organization by ... more Nowadays it is possible to unravel complex information at all levels of cellular organization by obtaining multi-dimensional image information. At the macromolecular level, three-dimensional (3D) electron microscopy, together with other techniques, is able to reach resolutions at the nanometer or subnanometer level. The information is delivered in the form of 3D volumes containing samples of a given function, for example, the electron density distribution within a given macromolecule. The same situation happens at the cellular level with the new forms of light microscopy, particularly confocal microscopy, all of which produce biological 3D volume information. Furthermore, it is possible to record sequences of images over time (videos), as well as sequences of volumes, bringing key information on the dynamics of living biological systems. It is in this context that work on BioImage started two years ago, and that its first version is now presented here. In essence, BioImage is a database specifically designed to contain multi-dimensional images, perform queries and interactively work with the resulting multi-dimensional information on the World Wide Web, as well as accomplish the required cross-database links. Two sister home pages of BioImage can be accessed at http://www.bioimage.org and http://www-embl.bioimage.org
Microscopy and Microanalysis
This is an Accepted Manuscript for the Microscopy and Microanalysis 2020 Proceedings. This versio... more This is an Accepted Manuscript for the Microscopy and Microanalysis 2020 Proceedings. This version may be subject to change during the production process.
Microscopy and Microanalysis
Microscopy and Microanalysis, 2016
Bacteriophage φ6 has a tripartite double-stranded RNA genome packaged inside three concentric she... more Bacteriophage φ6 has a tripartite double-stranded RNA genome packaged inside three concentric shells. The three segments code for the various viral proteins, with the large (L) segment containing the genes for producing new procapsids. These are the P1 protein making up the inner shell or capsid, the RNAdependent RNA polymerase, P2, the packaging motor, P4, and the packaging facilitator, P7. The transcription of the L segment is regulated such that it is active at an early stage when the virus infects its host, Pseudomonas syringae, while it is suppressed late in infection. This regulation is associated with a difference in the 5' terminus of the L segment as compared to the medium (M) and small (S) segments (the 5' terminus starts with GU in L and GG in M and S). It was discovered that a host protein, YajQ, is required for the early stage activation of L segment transcription [1]. YajQ binds to the outside of the capsid, while the transcription activity occurs inside the capsid. We are interested in how this communication across the capsid shell occurs.
Journal of Structural Biology, 2010
Structure (London, England : 1993), Jul 31, 2016
HIV-1 Rev protein mediates the nuclear export of viral RNA genomes. To do so, Rev oligomerizes co... more HIV-1 Rev protein mediates the nuclear export of viral RNA genomes. To do so, Rev oligomerizes cooperatively onto an RNA motif, the Rev response element (RRE), forming a complex that engages with the host nuclear export machinery. To better understand Rev oligomerization, we determined four crystal structures of Rev N-terminal domain dimers, which show that they can pivot about their dyad axis, giving crossing angles of 90° to 140°. In parallel, we performed cryoelectron microscopy of helical Rev filaments. Filaments vary from 11 to 15 nm in width, reflecting variations in dimer crossing angle. These structures contain additional density, indicating that C-terminal domains become partially ordered in the context of filaments. This conformational variability may be exploited in the assembly of RRE/Rev complexes. Our data also revealed a third interface between Revs, which offers an explanation for how the arrangement of Rev subunits adapts to the "A"-shaped architecture of ...
J Mol Biol, 2000
The large number of sequences available for the aquaporin family represents a valuable source of ... more The large number of sequences available for the aquaporin family represents a valuable source of information to incorporate into three-dimensional structure determination. Phylogenetic analysis was used to define type sequences to avoid extreme over-representation of some subfamilies, and as a measure of the quality of multiple sequence alignment. Inspection of the sequence alignment suggested eight conserved segments that define the core architecture of six transmembrane helices and two functional loops, B and E, projecting into the plane of the membrane. The sum of the core segments and the minimum lengths of the interlinking loops constitute the 208 residues necessary to satisfy the aquaporin architecture. Analysis of hydrophobic and conservation periodicity and of correlated mutations across the alignment indicated the likely assignment and orientation of the helices in the bilayer. This assignment is examined with respect to the structure of the erythrocyte aquaporin 1 determined by electron crystallography. The aquaporin 1 tetramer is described as three rings of helices, each ring with a different exposure to the lipid environment. The sequence analysis clearly suggests that two helices are exposed along their whole lengths, two helices are exposed only at their N termini, and two helices are not exposed to lipid. It is further proposed that, besides loops B and E, the highly conserved motifs on helices 1 and 4, ExxxTxxF/L, could line the water channel.
Journal of Biological Chemistry, 2015
Parkinson disease and other progressive neurodegenerative conditions are characterized by the int... more Parkinson disease and other progressive neurodegenerative conditions are characterized by the intracerebral presence of Lewy bodies, containing amyloid fibrils of ␣-synuclein. We used cryo-electron microscopy and scanning transmission electron microscopy (STEM) to study in vitro-assembled fibrils. These fibrils are highly polymorphic. Focusing on twisting fibrils with an inter-crossover spacing of 77 nm, our reconstructions showed them to consist of paired protofibrils. STEM mass per length data gave one subunit per 0.47 nm axial rise per protofibril, consistent with a superpleated -structure. The STEM images show two thread-like densities running along each of these fibrils, which we interpret as ladders of metal ions. These threads confirmed the two-protofibril architecture of the 77-nm twisting fibrils and allowed us to identify this morphotype in STEM micrographs. Some other, but not all, fibril morphotypes also exhibit dense threads, implying that they also present a putative metal binding site. We propose a molecular model for the protofibril and suggest that polymorphic variant fibrils have different numbers of protofibrils that are associated differently.
Advances in experimental medicine and biology, 2012
Herpesviruses, a family of animal viruses with large (125-250 kbp) linear DNA genomes, are highly... more Herpesviruses, a family of animal viruses with large (125-250 kbp) linear DNA genomes, are highly diversified in terms of host range; nevertheless, their virions conform to a common architecture. The genome is confined at high density within a thick-walled icosahedral capsid with the uncommon (among viruses, generally) but unvarying triangulation number T = 16. The envelope is a membrane in which some 11 different viral glycoproteins are implanted. Between the capsid and the envelope is a capacious compartment called the tegument that accommodates ∼20-40 different viral proteins (depending on which virus) destined for delivery into a host cell. A strong body of evidence supports the hypothesis that herpesvirus capsids and those of tailed bacteriophages stem from a distant common ancestor, whereas their radically different infection apparatuses - envelope on one hand and tail on the other - reflect subsequent coevolution with divergent hosts. Here we review the molecular components o...
News in physiological sciences : an international journal of physiology produced jointly by the International Union of Physiological Sciences and the American Physiological Society, 1999
How water permeates cellular membranes and what this means for cell functioning and several disea... more How water permeates cellular membranes and what this means for cell functioning and several diseases are now emerging from the study of the aquaporins (AQPs), the water channel family. A combination of sequence analysis, three-dimensional structure determination, and physiology of the AQP family proteins provides a glimpse into the workings of water channels.
Structural Virology, 2010
Living cells compartmentalize materials and enzymatic reactions to increase metabolic efficiency.... more Living cells compartmentalize materials and enzymatic reactions to increase metabolic efficiency. While eukaryotes use membranebound organelles, bacteria and archaea rely primarily on proteinbound nanocompartments. Encapsulins constitute a class of nanocompartments widespread in bacteria and archaea whose functions have hitherto been unclear. Here, we characterize the encapsulin nanocompartment from Myxococcus xanthus, which consists of a shell protein (EncA, 32.5 kDa) and three internal proteins (EncB, 17 kDa; EncC, 13 kDa; EncD, 11 kDa). Using cryo-electron microscopy, we determined that EncA self-assembles into an icosahedral shell 32 nm in diameter (26 nm internal diameter), built from 180 subunits with the fold first observed in bacteriophage HK97 capsid. The internal proteins, of which EncB and EncC have ferritin-like domains, attach to its inner surface. Native nanocompartments have dense iron-rich cores. Functionally, they resemble ferritins, cage-like iron storage proteins, but with a massively greater capacity (~30,000 iron atoms versus~3,000 in ferritin). Physiological data reveal that few nanocompartments are assembled during vegetative growth, but they increase fivefold upon starvation, protecting cells from oxidative stress through iron sequestration.
Uploads
Papers by Bernard Heymann