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PubMed2003

Structural criteria for regulation of membrane fusion and virion incorporation by the murine leukemia virus TM cytoplasmic domain.

The cytoplasmic domains of viral glycoproteins influence the trafficking and subcellular localization of the glycoproteins and their incorporation into virions. They also promote correct virus morphology and viral budding. The cytoplasmic domains of murine-leukemia-virus envelope-protein TM subunits regulate membrane fusion. During virion maturation the carboxy-terminal 16 amino acid residues of the TM protein are removed by the retroviral protease. Deletion of these residues activates envelope-protein-mediated membrane fusion. Our quantitative analysis of the effects of Moloney murine leukemia virus TM mutations on envelope-protein function support the proposition that a trimeric coiled coil in the TM cytoplasmic domain inhibits fusion. The data demonstrate that cleavage of the TM cytoplasmic domain is not required for viral entry and provide evidence for a model in which fusogenic and nonfusogenic conformations of the envelope protein exists in an equilibrium that is regulated by the cytoplasmic domain. In addition, a conserved tyrosine residue in the TM cytoplasmic domain was shown to play an important role in envelope-protein incorporation into retroviral particles.

3T3 Cells↗
PubMed2006

Important role for the transmembrane domain of severe acute respiratory syndrome coronavirus spike protein during entry.

The spike protein (S) of severe acute respiratory syndrome coronavirus (SARS-CoV) is responsible for receptor binding and membrane fusion. It contains a highly conserved transmembrane domain that consists of three parts: an N-terminal tryptophan-rich domain, a central domain, and a cysteine-rich C-terminal domain. The cytoplasmic tail of S has previously been shown to be required for assembly. Here, the roles of the transmembrane and cytoplasmic domains of S in the infectivity and membrane fusion activity of SARS-CoV have been studied. SARS-CoV S-pseudotyped retrovirus (SARSpp) was used to measure S-mediated infectivity. In addition, the cell-cell fusion activity of S was monitored by a Renilla luciferase-based cell-cell fusion assay. S(VSV-Cyt), an S chimera with a cytoplasmic tail derived from vesicular stomatitis virus G protein (VSV-G), and S(MHV-TMDCyt), an S chimera with the cytoplasmic and transmembrane domains of mouse hepatitis virus, displayed wild-type-like activity in both assays. S(VSV-TMDCyt), a chimera with the cytoplasmic and transmembrane domains of VSV-G, was impaired in the SARSpp and cell-cell fusion assays, showing 3 to 25% activity compared to the wild type, depending on the assay and the cells used. Examination of the oligomeric state of the chimeric S proteins in SARSpp revealed that S(VSV-TMDCyt) trimers were less stable than wild-type S trimers, possibly explaining the lowered fusogenicity and infectivity.

Amino Acid Sequence↗
PubMed2001

The structure of the fusion glycoprotein of Newcastle disease virus suggests a novel paradigm for the molecular mechanism of membrane fusion.

BACKGROUND: Membrane fusion within the Paramyxoviridae family of viruses is mediated by a surface glycoprotein termed the "F", or fusion, protein. Membrane fusion is assumed to involve a series of structural transitions of F from a metastable (prefusion) state to a highly stable (postfusion) state. No detail is available at the atomic level regarding the metastable form of these proteins or regarding the transitions accompanying fusion. RESULTS: The three-dimensional structure of the fusion protein of Newcastle disease virus (NDV-F) has been determined. The trimeric NDV-F molecule is organized into head, neck, and stalk regions. The head is comprised of a highly twisted beta domain and an additional immunoglobulin-like beta domain. The neck is formed by the C-terminal extension of the heptad repeat region HR-A, capped by a four-helical bundle. The C terminus of HR-A is encased by a further helix HR-C and a 4-stranded beta sheet. The stalk is formed by the remaining visible portion of HR-A and by polypeptide immediately N-terminal to the C-terminal heptad repeat region HR-B. An axial channel extends through the head and neck and is fenestrated by three large radial channels located approximately at the head-neck interface. CONCLUSION: We propose that prior to fusion activation, the hydrophobic fusion peptides in NDV-F are sequestered within the radial channels within the head, with the central HR-A coiled coil being only partly formed. Fusion activation then involves, inter alia, the assembly of a complete HR-A coiled coil, with the fusion peptides and transmembrane anchors being brought into close proximity. The structure of NDV-F is fundamentally different than that of influenza virus hemagglutinin, in that the central coiled coil is in the opposite orientation with respect to the viral membrane.

Amino Acid Sequence↗
PubMed2003

Interactions between the transmembrane segments of the alphavirus E1 and E2 proteins play a role in virus budding and fusion.

The alphavirus envelope is built by heterodimers of the membrane proteins E1 and E2. The complex is formed as a p62E1 precursor in the endoplasmic reticulum. During transit to the plasma membrane (PM), it is cleaved into mature E1-E2 heterodimers, which are oligomerized into trimeric complexes, so-called spikes that bind both to each other and, at the PM, also to nucleocapsid (NC) structures under the membrane. These interactions drive the budding of new virus particles from the cell surface. The virus enters new cells by a low-pH-induced membrane fusion event where both inter- and intraheterodimer interactions are reorganized to establish a fusion-active membrane protein complex. There are no intact heterodimers left after fusion activation; instead, an E1 homotrimer remains in the cellular (or viral) membrane. We analyzed whether these transitions depend on interactions in the transmembrane (TM) region of the heterodimer. We observed a pattern of conserved glycines in the TM region of E1 and made two mutants where either the glycines only (SFV/E1(4L)) or the whole segment around the glycines (SFV/E1(11L)) was replaced by leucines. We found that both mutations decreased the stability of the heterodimer and increased the formation of the E1 homotrimer at a suboptimal fusion pH, while the fusion activity was decreased. This suggested that TM interactions play a role in virus assembly and entry and that anomalous or uncoordinated protein reorganizations take place in the mutants. In addition, the SFV/E1(11L) mutant was completely deficient in budding, which may reflect an inability to form multivalent NC interactions at the PM.

Alphavirus↗
PubMed1984

Purification and properties of 100-kd proteins from coated vesicles and their reconstitution with clathrin.

Bullock brain coated vesicles contain a family of at least six 100-kd polypeptides which have the property of promoting clathrin assembly. These proteins have been purified from Triton X-100-extracted coated vesicles by a combination of gel filtration and chromatography on hydroxylapatite and DE-52 cellulose. Three major 100-kd species occur as complexes with a stoichiometric amount of a 50-kd polypeptide. On cross-linking these complexes, the chief products appear to contain two polypeptides of 100 kd and two of 50 kd. These 100-kd/50-kd complexes will polymerise with low concentrations of clathrin to give a relatively homogeneous population of coats predominantly of the 'barrel' size. In contrast, three other polypeptides of 100 kd lack the 50-kd protein but polymerise with clathrin under the same conditions to yield coats of a wide range of sizes including 'barrels', truncated icosahedra and particles of greater than 100 nm diameter. When clathrin cages are reassembled with a saturating amount of 100-kd/50-kd complexes and studied by electron microscopy, the additional proteins appear to follow the underlying geometry of the clathrin polyhedra, partially filling in the polygonal faces of the cage structures. Saturation appears to require approximately 3 molecules of 100-kd polypeptide per clathrin trimer.

Animals↗
PubMed2001

Distinct cysteine sulfhydryl environments detected by analysis of Raman S-hh markers of Cys-->Ser mutant proteins.

Very little is known about the character or functional relevance of hydrogen-bonded cysteine sulfhydryl (S-H) groups in proteins. The Raman S-H band is a unique and sensitive probe of the local S-H environment. Here, we report the use of Raman spectroscopy combined with site-specific mutagenesis to document the existence of five distinguishable hydrogen-bonded states of buried cysteine sulfhydryl groups in a native protein. The 666 residue subunit of the Salmonella typhimurium bacteriophage P22 tailspike contains eight cysteine residues distributed through the elongated structure. The tailspike cysteine residues display an unusual Raman S-H band complex (2500-2600 cm(-1) interval) indicative of diverse S-H hydrogen-bonding interactions in the native trimeric structure. To resolve specific Cys contributions to the complex Raman band we characterized a set of tailspike proteins with each cysteine replaced by a serine. The mutant proteins, once folded, were structurally and functionally indistinguishable from wild-type tailspikes, except for their Raman S-H signatures. Comparison of the Raman spectra of the mutant and wild-type proteins reveals the following hydrogen-bond classes for cysteine sulfhydryl groups. (i) Cys613 forms the strongest S-H...X bond of the tailspike, stronger than any heretofore observed for a protein. (ii) Cys267, Cys287 and Cys458 form robust S-H...X bonds. (iii) Moderate S-H...X bonding occurs for Cys169 and Cys635. (iv) Cys290 and Cys496 form weak hydrogen bonds. (v) It is remarkable that Cys287 contributes two Raman S-H markers, indicating the population of two distinct hydrogen-bonding states. The sum of the S-H Raman signatures of all eight mutants accurately reproduces the composite Raman band of the wild-type tailspike. The diverse cysteine states may be an outcome of the folding and assembly pathway of the tailspike, which though lacking disulfide bonds in the native state, utilizes transient disulfide bonds in the maturation pathway. This Raman study represents the first detailed assessment of local S-H hydrogen bonding in a native protein and provides information not obtainable directly by other structural probes. The method employed here should be applicable to a wide range of cysteine-containing proteins.

Amino Acid Substitution↗
PubMed1985

The structure of the adenovirus capsid. II. The packing symmetry of hexon and its implications for viral architecture.

The orientation and location of the 240 hexons comprising the outer protein shell of adenovirus have been determined. Electron micrographs of the capsid and its fragments were inspected for the features of hexon known from the X-ray crystallographic model as described in the accompanying paper. A capsid model is proposed with each facet comprising a small p3 net of 12 hexons, arranged as a triangular sextet with three outer hexon pairs. The sextet is centrally placed about the icosahedral threefold axis, with its edges parallel to those of the facet. The outer pairs project over the facet edges on one side of the icosahedral twofold axes at each edge. The model capsid is defined by the underlying icosahedron, of edge 445 A, upon which hexons are arranged. The hexons are thus bounded by icosahedra with insphere radii of 336 A and 452 A. A quartet of hexons forms the asymmetric unit of an icosahedral hexon shell, which can be closed by the addition of pentons at the 12 vertices. Considering the hexon trimer as a complex structure unit, its interactions in the four topologically distinct environments are very similar, with conservation of at least two-thirds of the inter-hexon bonding. The crystal-like construction explains the flat facets and sharp edges characteristic of adenovirus. Larger "adenovirus-like" capsids of any size could be formed using only one additional topologically different environment. The construction of adenovirus illustrates how an impenetrable protein shell can be formed, with highly conserved intermolecular bonding, by using the geometry of an oligomeric structure unit and symmetry additional to that of the icosahedral point group. This contrasts with the manner suggested by Caspar & Klug (1962), in which the polypeptide is the structure unit, and for which the number of possible bonding configurations required of a structure unit tends to infinity as the continuously curved capsid increases in size. The known structures of polyoma and the plant viruses with triangulation number equal to 3 are evaluated in terms of hexamer-pentamer packing, and evidence is presented for the existence of larger subunits than the polypeptide in both cases. It is suggested that spontaneous assembly can occur only when exact icosahedral symmetry relates structure units or sub-assemblies, which would themselves have been formed by self-limiting closed interactions. Without such symmetry, the presence of scaffolding proteins or nucleic acid is necessary to limit aggregation.

Adenoviridae↗
PubMed2003

The tailless icosahedral membrane virus PRD1 localizes the proteins involved in genome packaging and injection at a unique vertex.

The double-stranded DNA (dsDNA) virus PRD1 carries its genome in a membrane surrounded by an icosahedral protein shell. The shell contains 240 copies of the trimeric P3 protein arranged with a pseudo T = 25 triangulation that is reminiscent of the mammalian adenovirus. DNA packaging and infection are believed to occur through the vertices of the particle. We have used immunolabeling to define the distribution of proteins on the virion surface. Antibodies to protein P3 labeled the entire surface of the virus. Most of the 12 vertices labeled with antibodies directed against proteins P5, P2, and P31. These proteins are known to function in virus binding to the cell surface. Proteins P6, P11, and P20 were found on a single vertex per virion. The P6 and P20 proteins are believed to function in DNA packaging. Protein P11 is a pilot protein that is involved in a complex that mediates the early stages of DNA entry to the host cell. Labeling with antibodies to P5 or P2 did not affect the labeling of P6, the unique vertex protein. Labeling with antibodies to the unique vertex protein P6 interfered with the labeling by antibodies to the unique vertex protein P20. We conclude that PRD1 utilizes 11 of its vertices for initial receptor binding. It utilizes a single, unique vertex for both DNA packing during assembly and DNA delivery during infection.

Bacteriophage PRD1↗
PubMed1994

Assembly of synthetic laminin peptides into a triple-stranded coiled-coil structure.

Laminin, a large multidomain glycoprotein specific to basement membranes, is a heterotrimer with alpha, beta, and gamma chains held together in an alpha-helical coiled-coil structure. Synthetic peptides comprising two 51-mers (B1 and B2) from the beta 1 and gamma 1 subunits and a 55-mer (M) from alpha 2 were used to study the molecular mechanisms in laminin chain assembly. Using the synthetic peptides in various mixing experiments, the heterotrimer (B1-B2-M) was preferentially produced. The thermal stability of the heterotrimer increased dramatically (by approximately 20 degrees C) over that of the B1-B2 heterodimer as measured by circular dichroism (CD) spectroscopy. The B1-B1 homodimer (Tm = 60 degrees C) showed higher thermal stability when compared to B1-B2 and B2-B2 dimers. However, the B1 + B2 mixture produced principally the B1-B2 heterodimer. These results suggested that the preferential formations of heterodimer was regulated by kinetic interactions between each chain. The B2 and M peptides have many hydrophobic isoleucine residues which were replaced by leucines. These substitutions were predicted to favor an alpha-helical conformation and a higher propensity for zipper formation. B2L and ML, in which all isoleucine residues were replaced by leucine, showed significantly increased alpha-helicities. While B2L was able to form heterodimers and heterotrimers similar to B2, ML was not able to participate in heterotrimer formation as efficiently as the M peptide. The thermal stability of B1-B2L was comparable to that of B1-B2, but B2L and/or ML containing trimers showed lower thermal stability than B1-B2-M. These results suggest that the isoleucine residues in the alpha 2 and gamma 1 chains are critical for stabilizing the heteromeric triple-stranded coiled-coil structure.

Amino Acid Sequence↗
PubMed1997

Collagen-based structures containing the peptoid residue N-isobutylglycine (Nleu): synthesis and biophysical studies of Gly-Nleu-Pro sequences by circular dichroism and optical rotation.

Single-chain peptide-peptoid structures, Ac-(Gly-Nleu-Pro)n-NH2 (n = 3, 6, and 10) and (Gly-Nleu-Pro)n-NH2 (n = 1 and 9), and template-assembled collagen analogs, KTA-[Gly-(Gly-Nleu-Pro)n-NH2]3 (n = 3 and 6; KTA represents cis,cis-1,3,5-trimethylcyclohexane-1,3, 5-tricarboxylic acid, also known as the Kemp triacid; Nleu denotes N-isobutylglycine), were prepared by solid-phase peptide synthesis methods. Biophysical studies using circular dichroism (CD) and optical rotation measurements show that these collagen analogs form triple-helical conformations when the chain is longer than a critical length. Unlike collagen-based structures composed of Gly-Pro-Hyp and Gly-Pro-Nleu sequences, results reveal that the presence of a positive CD peak between 220 and 225 nm is indicative of triple-helical conformations for these collagen-based structures composed of Gly-Nleu-Pro sequences. Results also indicate that the Gly-Nleu-Pro sequence possesses a higher triple-helical propensity than the Gly-Pro-Nleu sequence as demonstrated by the higher melting temperatures, the faster triple-helix folding, and the lower minimum concentration necessary to detect triple-helicity for the single-chain structures. Therefore, we conclude that the Nleu residue in the second position of the trimeric repeat is more effective in inducing triple-helix formation than Pro in the same position.

Amino Acid Sequence↗
PubMed1999

Electrospray ionization mass spectrometric determination of the molecular mass of the approximately 200-kDa globin dodecamer subassemblies in hexagonal bilayer hemoglobins.

Hexagonal bilayer hemoglobins (Hbs) are approximately 3.6-MDa complexes of approximately 17-kDa globin chains and 24-32-kDa, nonglobin linker chains in a approximately 2:1 mass ratio found in annelids and related species. Studies of the dissociation and reassembly of Lumbricus terrestris Hb have provided ample evidence for the presence of a approximately 200-kDa linker-free subassembly consisting of monomer (M) and disulfide-bonded trimer (T) subunits. Electrospray ionization mass spectrometry (ESI-MS) of the subassemblies obtained by gel filtration of partially dissociated L. terrestris and Arenicola marina Hbs showed the presence of noncovalent complexes of M and T subunits with masses in the 213. 3-215.4 and 204.6-205.6 kDa ranges, respectively. The observed mass of the L. terrestris subassembly decreased linearly with an increase in de-clustering voltage from approximately 215,400 Da at 60 V to approximately 213,300 Da at 200 V. In contrast, the mass of the A. marina complex decreased linearly from 60 to 120 V and reached an asymptote at approximately 204,600 Da (180-200 V). The decrease in mass was probably due to the progressive removal of complexed water and alkali metal cations. ESI-MS at an acidic pH showed both subassemblies to consist of only M and T subunits, and the experimental masses demonstrated them to have the composition M(3)T(3). Because there are three isoforms of M and four isoforms of T in Lumbricus and two isoforms of M and 5 isoforms of T in Arenicola, the masses of the M(3)T(3) subassemblies are not unique. A random assembly model was used to calculate the mass distributions of the subassemblies, using the known ESI-MS masses and relative intensities of the M and T subunit isforms. The expected mass of randomly assembled subassemblies was 213,436 Da for Lumbricus Hb and 204,342 Da for Arenicola Hb, in good agreement with the experimental values.

Animals↗
PubMed2005

External scaffold of spherical immature poxvirus particles is made of protein trimers, forming a honeycomb lattice.

During morphogenesis, poxviruses undergo a remarkable transition from spherical immature forms to brick-shaped infectious particles lacking helical or icosahedral symmetry. In this study, we show that the transitory honeycomb lattice coating the lipoprotein membrane of immature vaccinia virus particles is formed from trimers of a 62-kD protein encoded by the viral D13L gene. Deep-etch electron microscopy demonstrated that anti-D13 antibodies bound to the external protein coat and that lattice fragments were in affinity-purified D13 preparations. Soluble D13 appeared mostly trimeric by gel electrophoresis and ultracentrifugation, which is consistent with structural requirements for a honeycomb. In the presence or absence of other virion proteins, a mutated D13 with one amino acid substitution formed stacks of membrane-unassociated flat sheets that closely resembled the curved honeycombs of immature virions except for the absence of pentagonal facets. A homologous domain that is present in D13 and capsid proteins of certain other lipid-containing viruses support the idea that the developmental stages of poxviruses reflect their evolution from an icosahedral ancestor.

Capsid Proteins↗
PubMed2004

Assembly and kinetic folding pathways of a tetrameric beta-sheet complex: molecular dynamics simulations on simplified off-lattice protein models.

We have performed discontinuous molecular dynamic simulations of the assembly and folding kinetics of a tetrameric beta-sheet complex that contains four identical four-stranded antiparallel beta-sheet peptides. The potential used in the simulation is a hybrid Go-type potential characterized by the bias gap parameter g, an artificial measure of a model protein's preference for its native state, and the intermolecular contact parameter eta, which measures the ratio of intermolecular to intramolecular native attractions. The formation of the beta-sheet complex and its equilibrium properties strongly depend on the size of the intermolecular contact parameter eta. The ordered beta-sheet complex in the folded state and nonaligned beta-sheets or tangled chains in the misfolded state are distinguished by measuring the squared radius of gyration Rg2 and the fraction of native contacts Q. The folding yield for the folded state is high at intermediate values of eta, but is low at both small and large values of eta. The folded state at small eta is liquid-like, but is solid-like at both intermediate and large eta. The misfolded state at small eta contains nonaligned beta-sheets and tangled chains with poor secondary structure at large eta. Various folding pathways via dimeric and trimeric intermediates are observed, depending on eta. Comparison with experimental results on protein aggregation indicates that intermediate eta values are most appropriate for modeling fibril formation and small eta values are most appropriate for modeling the formation of amorphous aggregates.

Binding Sites↗
PubMed2003

New high-dimensional networks based on polyoxometalate and crown ether building blocks.

Three novel supramolecular assemblies constructed from polyoxometalate and crown ether building blocks, [(DB18C6)Na(H(2)O)(1.5)](2)Mo(6)O(19).CH(3)CN, 1, and [(Na(DB18C6)(H(2)O)(2))(3)(H(2)O)(2)]XMo(12)O(40).6DMF.CH(3)CN (X = P, 2, and As, 3; DB18C6 = dibenzo-18-crown-6; DMF = N,N-dimethylfomamide), have been synthesized and characterized by elemental analyses, IR, UV-vis, EPR, TG, and single crystal X-ray diffraction. Compound 1 crystallizes in the tetragonal space group P4/mbm with a = 16.9701(6) A, c = 14.2676(4) A, and Z = 2. Compound 2 crystallizes in the hexagonal space group P6(3)/m with a = 15.7435(17) A, c = 30.042(7) A, gamma = 120 degrees, and Z = 2. Compound 3 crystallizes in the hexagonal space group P6(3)/m with a = 15.6882(5) A, c = 29.9778(18) A, gamma = 120 degrees, and Z = 2. Compound 1 exhibits an unusual three-dimensional network with one-dimensional sandglasslike channels based on the extensive weak forces between the oxygen atoms on the [Mo(6)O(19)](2)(-) polyoxoanions and the CH(2) groups of crown ether molecules. Compounds 2 and 3 are isostructural, and both contain a novel semiopen cagelike trimeric cation [(Na(DB18C6)(H(2)O)(2))(3)(H(2)O)(2)](3+). In their packing arrangement, an interesting 2-D "honeycomblike" "host" network is formed, in which the [XMo(12)O(40)](3)(-) (X = As and P) polyoxoanion "guests" resided.

Journal Article↗
PubMed2007

Coupled ATP and DNA binding of adeno-associated virus Rep40 helicase.

Adeno-associated virus 2 Rep40 helicase is involved in packaging single-stranded genomic DNA into virions. ATPase activity was stimulated 5-10-fold by DNA, depending upon assay conditions. The concentration dependence of Rep40 ATPase activity in the absence and presence of DNA indicates that the monomer is inactive and that the active enzyme is at least a dimer. Binding to oligonucleotides, examined by fluorescence anisotropy, was positively cooperative and required ATP or ATPgammaS; ADP and AMPPCP did not promote binding. The cooperativity and the nucleotide requirement were also demonstrated by surface plasmon resonance. Although the Rep40 behaves as a monomer in solution, it binds to DNA as an oligomer. The requirement of a nucleotide for DNA binding and the stimulation of ATPase activity by DNA indicate that the two processes are linked. Glutaraldehyde cross-linking generated a species that migrates as a trimer on sodium dodecyl sulfate (SDS) gel electrophoresis; ATPS promoted the formation of this species and higher order oligomers. The predominant cross-linked species was a trimer in the absence of ATPgammaS, regardless of whether duplex or single-stranded DNA was present. In the presence of duplex or single-stranded DNA and ATPgammaS, glutaraldehyde cross-linking generated a species that behaved as a dimer on SDS gel elctrophoresis. Sucrose-gradient velocity sedimentation of Rep40 gave an S20,w of 3 in the absence of ligands or in the presence of a 26 bp duplex DNA. The S20,w was 3.5 in the presence of ATPgammaS and 7 and 7.6 in the presence of DNA and ATPgammaS.

Adenosine Triphosphate↗
PubMed2001

Influence of adenoviral fiber mutations on viral encapsidation, infectivity and in vivo tropism.

Targeting of adenovirus (Ad)-encoded therapeutic genes to specific cell types has become a major goal in gene therapy. Redirecting the specificity of infection requires the abrogation of the natural interaction between the viral fiber and its cellular receptors (CAR) and the simultaneous introduction of a new binding specificity into the viral capsid. To abrogate the natural affinity of the fiber, we have mutated residues presumed to be directly or indirectly involved in CAR-binding in the knob domain of the fiber protein. These residues are located in the AB loop (Ser408) and in the DG loop (Tyr491, Ala494, Ala503). The mutations Ser408Glu, Tyr491Asp, Ala494Asp and Ala503Asp did not prevent the incorporation of trimeric fibers in the viral capsid but led to loss of CAR binding in vitro. Infectivity of the mutant viruses could be restored in vitro by introducing a ligand at the C-terminal end of the knob, confirming that the reduced infectivity of the fiber-modified virus was due to an impaired interaction of the viral particle with the CAR receptor. However, after systemic delivery, the in vivo biodistribution of impaired CAR-binding viruses without addition of a specific ligand was not altered when compared with wild-type Ad.

Adenoviruses, Human↗
PubMed2005

Maturation of papillomavirus capsids.

The papillomavirus capsid is a nonenveloped icosahedral shell formed by the viral major structural protein, L1. It is known that disulfide bonds between neighboring L1 molecules help to stabilize the capsid. However, the kinetics of inter-L1 disulfide bond formation during particle morphogenesis have not previously been examined. We have recently described a system for producing high-titer papillomavirus-based gene transfer vectors (also known as pseudoviruses) in mammalian cells. Here we show that papillomavirus capsids produced using this system undergo a maturation process in which the formation of inter-L1 disulfide bonds drives condensation and stabilization of the capsid. Fully mature capsids exhibit improved regularity and resistance to proteolytic digestion. Although capsid maturation for other virus types has been reported to occur in seconds or minutes, papillomavirus capsid maturation requires overnight incubation. Maturation of the capsids of human papillomavirus types 16 and 18 proceeds through an ordered accumulation of dimeric and trimeric L1 species, whereas the capsid of bovine papillomavirus type 1 matures into more extensively cross-linked forms. The presence of encapsidated DNA or the minor capsid protein, L2, did not have major effects on the kinetics or extent of capsid maturation. Immature capsids and capsids formed from L1 mutants with impaired disulfide bond formation are infectious but physically fragile. Consequently, capsid maturation is essential for efficient purification of papillomavirus-based gene transfer vectors. Despite their obvious morphological differences, mature and immature capsids are similarly neutralizable by various L1- and L2-specific antibodies.

Animals↗
PubMed2025

Retention and Rearrangement of Membrane Protein Complexes' Higher Order Structure by Collisionally Activated Dissociation- and Electron Capture Dissociation-Mass Spectrometry.

Membrane proteins (MPs) challenge biophysical and structural biology methods. Native mass spectrometry (MS) has emerged as a powerful tool to study MP structures and their modulation by lipids. We examine the pathways by which three MPs, tetrameric Aquaporin Z (AqpZ), trimeric ammonium transporter (AmtB), and pentameric mechanosensitive channel of large conductance (MscL) decompose following collisionally activated dissociation (CAD) or electron capture dissociation (ECD) in native top-down MS. MPs subjected to CAD typically decompose along well-characterized pathways releasing highly charged monomers and low-charge state complementary subunits, but a lesser pathway accessed by low pressure collisions cleaves the backbone into fragments covering much of the sequence. Collisions also rearrange structures, e.g., AmtB subunits rearrange to form novel interactions and/or salt bridges that surprisingly retain a formerly surface-exposed segment despite ejecting the binding interface. MscL dissociation pathways depend on precursor charge-state, a behavior observed in a few soluble complexes, yet unaddressed mechanistically. Salt bridges in low charge state complexes stabilize subunits from ejection while facilitating only smaller, local rearrangements that release covalently cleaved products from transmembrane regions. With fewer opposite charges, on average, higher charge state molecules can rearrange intersubunit salt bridges on the experimental time scale to partition charge asymmetrically and free a subunit. ECD with supplemental activation can retain higher order structures of proteins and inform about the strongly interacting regions that preclude product ion release. With extensive regions lacking ionizable residues, MPs enable key interactions that guide the structure and dynamics of gas phase protein assemblies to be probed.

Mass Spectrometry↗