scholarly journals Spectral dependence of irreversible light-induced fluorescence quenching: Chlorophyll forms with maximal emission at 700–702 and 705–710 nm as spectroscopic markers of conformational changes in the core complex

2017 ◽  
Vol 1858 (7) ◽  
pp. 529-543 ◽  
Author(s):  
Sherzod Nematov ◽  
Anna Paola Casazza ◽  
William Remelli ◽  
Vakhobjon Khuvondikov ◽  
Stefano Santabarbara
Keyword(s):  
Fluorescence Quenching ◽  
Conformational Changes ◽  
Spectral Dependence ◽  
Core Complex ◽  
The Core ◽  
Chlorophyll Forms

scholarly journals Reconstitution of cargo-induced LC3 lipidation in mammalian selective autophagy

2021 ◽  
Vol 7 (17) ◽  
pp. eabg4922
Author(s):  
Chunmei Chang ◽  
Xiaoshan Shi ◽  
Liv E. Jensen ◽  
Adam L. Yokom ◽  
Dorotea Fracchiolla ◽  
...  
Keyword(s):  
Complex I ◽  
Kinase Activity ◽  
Protein Aggregates ◽  
Class Iii ◽  
Phosphatidylinositol 3 Kinase ◽  
Giant Unilamellar Vesicles ◽  
Core Complex ◽  
Selective Autophagy ◽  
The Core ◽  
Stimulation Of

Selective autophagy of damaged mitochondria, protein aggregates, and other cargoes is essential for health. Cargo initiates phagophore biogenesis, which entails the conjugation of LC3 to phosphatidylethanolamine. Current models suggest that clustered ubiquitin chains on a cargo trigger a cascade from autophagic cargo receptors through the core complexes ULK1 and class III phosphatidylinositol 3-kinase complex I, WIPI2, and the ATG7, ATG3, and ATG12ATG5-ATG16L1 machinery of LC3 lipidation. This was tested using giant unilamellar vesicles (GUVs), GST-Ub4 as a model cargo, the cargo receptors NDP52, TAX1BP1, and OPTN, and the autophagy core complexes. All three cargo receptors potently stimulated LC3 lipidation on GUVs. NDP52- and TAX1BP1-induced LC3 lipidation required all components, but not ULK1 kinase activity. However, OPTN bypassed the ULK1 requirement. Thus, cargo-dependent stimulation of LC3 lipidation is common to multiple autophagic cargo receptors, yet the details of core complex engagement vary between the different receptors.

Structural and geochronological constraints on the role of partial melting during the formation of the Shuswap metamorphic core complex at the latitude of the Thor-Odin dome, British Columbia

1999 ◽  
Vol 36 (6) ◽  
pp. 917-943 ◽  
Author(s):  
Olivier Vanderhaeghe ◽  
Christian Teyssier ◽  
Richard Wysoczanski
Keyword(s):  
Partial Melting ◽  
Metamorphic Core Complex ◽  
High Grade ◽  
Core Complex ◽  
Genetic Link ◽  
The Core ◽  
Lower Unit ◽  
Geochronological Constraints ◽  
Metamorphic Core ◽  
Middle Unit

At the latitude of the Thor-Odin dome, the Shuswap metamorphic core complex exposes a ~15 km thick structural section composed of an upper unit that preserved Mesozoic metamorphism, structures, and cooling ages, separated from the underlying high-grade rocks by low-angle detachment zones. Below the detachments, the core of the complex consists of an amphibolite-facies middle unit overlying a migmatitic lower unit exposed in the core of the Thor-Odin dome. Combined structural and super high resolution ion microprobe (SHRIMP) U-Pb geochronology studies indicate that the pervasive shallowly dipping foliation and east-west lineation developed in the presence of melt during Paleocene time. SHRIMP analyses of complexly zoned zircon grains suggest that the migmatites of the lower unit crystallized at ~56 Ma, and a syntectonic leucogranite at ~60 Ma. We suggest that leucogranite migrated upward from the migmatites through an array of dikes and sills that permeated the middle unit and ponded to form laccoliths spatially related to the detachment zones. The similarity in ages of inherited zircon cores in the two migmatite and the leucogranite samples suggests a genetic link consistent with the structural analysis. Following the crystallization of migmatites, the terrane cooled rapidly, as indicated by argon thermochronology. We propose that exhumation of the core of the Canadian Cordillera during the formation of the Shuswap metamorphic core complex occurred from ~60 to 56 Ma at a time when the crust was significantly partially molten. These structural and temporal relationships suggest a genetic link between mechanical weakening of the crust by partial melting, late-orogenic collapse, and exhumation of high-grade rocks in the hinterland of a thermally mature orogenic belt.

scholarly journals Reovirus core proteins λ1 and σ2 promote stability of disassembly intermediates and influence early replication events

2020 ◽  
Author(s):  
Stephanie Gummersheimer ◽  
Pranav Danthi
Keyword(s):  
Conformational Changes ◽  
Genetic Material ◽  
Point Mutations ◽  
Host Cells ◽  
Capsid Proteins ◽  
Endosomal Membrane ◽  
The Core ◽  
Core Proteins ◽  
Early Replication ◽  
Outer Capsid

ABSTRACTThe capsids of mammalian reovirus contain two concentric protein shells, the core and the outer capsid. The outer capsid is comprised of µ1-σ3 heterohexamers which surround the core. The core is comprised of λ1 decamers held in place by σ2. After entry into the endosome, σ3 is proteolytically degraded and µ1 is cleaved and exposed to form ISVPs. ISVPs undergo further conformational changes to form ISVP*s, resulting in the release of µ1 peptides which facilitate the penetration of the endosomal membrane to release transcriptionally active core particles into the cytoplasm. Previous work has identified regions or specific residues within reovirus outer capsid that impact the efficiency of cell entry. We examined the functions of the core proteins λ1 and σ2. We generated a reovirus T3D reassortant that carries strain T1L derived σ2 and λ1 proteins (T3D/T1L L3S2). This virus displays a lower ISVP stability and therefore converts to ISVP*s more readily. To identify the basis for lability of T3D/T1L L3S2, we screened for hyper-stable mutants of T3D/T1L L3S2 and identified three point mutations in µ1 that stabilize ISVPs. Two of these mutations are located in the C-terminal ϕ region of µ1, which has not previously been implicated in controlling ISVP stability. Independent from compromised ISVP stability, we also found that T3D/T1L L3S2 launches replication more efficiently and produces higher yields in infected cells. In addition to identifying a new role for the core proteins in disassembly events, these data highlight that core proteins may influence multiple stages of infection.IMPORTANCEProtein shells of viruses (capsids) have evolved to undergo specific changes to ensure the timely delivery of genetic material to host cells. The 2-layer capsid of reovirus provides a model system to study the interactions between capsid proteins and the changes they undergo during entry. We tested a virus in which the core proteins were derived from a different strain than the outer capsid. We found that this mismatched virus was less stable and completed conformational changes required for entry prematurely. Capsid stability was restored by introduction of specific changes to the outer capsid, indicating that an optimal fit between inner and outer shells maintains capsid function. Separate from this property, mismatch between these protein layers also impacted the capacity of virus to initiate infection and produce progeny. This study reveals new insights into the roles of capsid proteins and their multiple functions during viral replication.

scholarly journals Baculovirus Per Os Infectivity Factor Complex: Components and Assembly

2019 ◽  
Vol 93 (6) ◽  
Author(s):  
Xi Wang ◽  
Yu Shang ◽  
Cheng Chen ◽  
Shurui Liu ◽  
Meng Chang ◽  
...  
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Critical Role ◽  
Previous Knowledge ◽  
Multiprotein Complex ◽  
Core Complex ◽  
Insect Midgut ◽  
Content Type ◽  
The Core ◽  
Liquid Chromatography Tandem Mass ◽  
The Impact

ABSTRACT Baculovirus entry into insect midgut cells is dependent on a multiprotein complex of per os infectivity factors (PIFs) on the envelopes of occlusion-derived virions (ODVs). The structure and assembly of the PIF complex are largely unknown. To reveal the complete members of the complex, a combination of blue native polyacrylamide gel electrophoresis, liquid chromatography-tandem mass spectrometry, and Western blotting was conducted on three different baculoviruses. The results showed that the PIF complex has a molecular mass of ∼500 kDa and consists of nine PIFs, including a newly discovered member (PIF9). To decipher the assembly process, each pif gene was knocked out from the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) genome individually by use of synthetic baculovirus technology, and the impact on PIF complex formation was investigated. Deletion of pif8 resulted in the formation of an ∼400-kDa subcomplex. Deletion of pif0, -4, -6, -7, or -9 resulted in a subcomplex of ∼230 kDa, but deletion of pif1, -2, or -3 abolished formation of any complex. Taken together, our data identified a core complex of ∼230 kDa, consisting of PIF1, -2, and -3. This revised the previous knowledge that the core complex was about 170 kDa and contained PIF1 to -4. Analysis of the PIF complex in cellular fractions suggested that it is assembled in the cytoplasm before being transported to the nucleus and subsequently incorporated into the envelopes of ODVs. Only the full complex, not the subcomplex, is resistant to proteolytic attack, indicating the essentiality of correct complex assembly for oral infection. IMPORTANCE Entry of baculovirus into host insects is mediated by a per os infectivity factor (PIF) complex on the envelopes of occlusion-derived viruses (ODVs). Knowledge of the composition and structure of the PIF complex is fundamental to understanding its mode of action. By using multiple approaches, we determined the complete list of proteins (nine) in the PIF complex. In contrast to previous knowledge in the field, the core complex is revised to ∼230 kDa and consists of PIF1 to -3 but not PIF4. Interestingly, our results suggest that the PIF complex is formed in the cytoplasm prior to its transport to the nucleus and subsequent incorporation into ODVs. Only the full complex is resistant to proteolytic degradation in the insect midgut, implying the critical role of the entire complex. These findings provide the baseline for future studies on the ODV entry mechanism mediated by the multiprotein complex.

scholarly journals An Intermolecular π-Stacking Interaction Drives Conformational Changes Necessary to β-Barrel Formation in a Pore-Forming Toxin

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Joshua R. Burns ◽  
Craig J. Morton ◽  
Michael W. Parker ◽  
Rodney K. Tweten
Keyword(s):  
Conformational Changes ◽  
Membrane Attack Complex ◽  
Immune Defense ◽  
Stacking Interaction ◽  
Helical Bundles ◽  
The Core ◽  
Π Stacking ◽  
Π Stacking Interaction ◽  
Membrane Spanning ◽  
Β Sheet

ABSTRACT The crystal structures of the soluble monomers of the pore-forming cholesterol-dependent cytolysins (CDCs) contain two α-helical bundles that flank a twisted core β-sheet. This protein fold is the hallmark of the CDCs, as well as of the membrane attack complex/perforin immune defense proteins and the stonefish toxins. To form the β-barrel pore, a core β-sheet is flattened to align the membrane-spanning β-hairpins. Concomitantly with this conformational change, the two α-helical bundles that flank the core β-sheet break their restraining contacts and refold into two membrane-spanning β-hairpins of the β-barrel pore. The studies herein show that in the monomer structure of the archetype CDC perfringolysin O (PFO), a conserved Met-Met-Phe triad simultaneously contributes to maintaining the twist in this core β-sheet, as well as restricting the α-helical–to–β-strand transition necessary to form one of two membrane-spanning β-hairpins. A previously identified intermolecular π-stacking interaction is now shown to disrupt the interactions mediated by this conserved triad. This is required to establish the subsequent intermolecular electrostatic interaction, which has previously been shown to drive the final conformational changes necessary to form the β-barrel pore. Hence, these studies show that the intermolecular π-stacking and electrostatic interactions work in tandem to flatten the core β-sheet and initiate the α-helical–to–β-strand transitions to form the β-barrel pore. IMPORTANCE A unique feature of the CDC/MACPF/SNTX (cholesterol-dependent cytolysin/membrane attack complex perforin/stonefish toxin) superfamily of pore-forming toxins is that the β-strands that comprise the β-barrel pore are derived from a pair of α-helical bundles. These studies reveal the molecular basis by which the formation of intermolecular interactions within the prepore complex drive the disruption of intramolecular interactions within each monomer of the prepore to trigger the α-helical–to–β-strand transition and formation of the β-barrel pore.

scholarly journals Characterization of the translocon of the outer envelope of chloroplasts

2003 ◽  
Vol 160 (4) ◽  
pp. 541-551 ◽  
Author(s):  
Enrico Schleiff ◽  
Jürgen Soll ◽  
Michael Küchler ◽  
Werner Kühlbrandt ◽  
Roswitha Harrer
Keyword(s):  
Three Dimensional ◽  
Dependent Manner ◽  
Interior Structure ◽  
Two Dimensional ◽  
Outer Envelope ◽  
Core Complex ◽  
The Core ◽  
Precursor Proteins ◽  
Stable Core

The protein translocon of the outer envelope of chloroplasts (Toc) consists of the core subunits Toc159, Toc75, and Toc34. To investigate the molecular structure, the core complex was purified. This core complex has an apparent molecular mass of ∼500 kD and a molecular stoichiometry of 1:4:4–5 between Toc159, Toc75, and Toc34. The isolated translocon recognizes both transit sequences and precursor proteins in a GTP-dependent manner, suggesting its functional integrity. The complex is embedded by the lipids phosphatidylcholine and digalactosyldiacylglyceride. Two-dimensional structural analysis by EM revealed roughly circular particles consistent with the formation of a stable core complex. The particles show a diameter of ∼130 Å with a solid ring and a less dense interior structure. A three-dimensional map obtained by random conical tilt reconstruction of electron micrographs suggests that a “finger”-like central region separates four curved translocation channels within one complex.

scholarly journals The Structure of an Injectisome Export Gate Demonstrates Conservation of Architecture in the Core Export Gate between Flagellar and Virulence Type III Secretion Systems

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Steven Johnson ◽  
Lucas Kuhlen ◽  
Justin C. Deme ◽  
Patrizia Abrusci ◽  
Susan M. Lea
Keyword(s):  
Type Iii Secretion ◽  
Complex Analysis ◽  
Secretion Systems ◽  
Core Complex ◽  
Type Iii ◽  
The Core ◽  
Cryo Electron Microscopy ◽  
Type Iii Secretion Systems ◽  
Equivalent Complex ◽  
Resolution Structure

ABSTRACT Export of proteins through type III secretion systems (T3SS) is critical for motility and virulence of many major bacterial pathogens. Proteins are exported through a genetically defined export gate complex consisting of three proteins. We have recently shown at 4.2 Å that the flagellar complex of these three putative membrane proteins (FliPQR in flagellar systems, SctRST in virulence systems) assembles into an extramembrane helical assembly that likely seeds correct assembly of the rod. Here we present the structure of an equivalent complex from the Shigella virulence system at 3.5 Å by cryo-electron microscopy. This higher-resolution structure yields a more precise description of the structure and confirms the prediction of structural conservation in this core complex. Analysis of particle heterogeneity also suggests how the SctS/FliQ subunits sequentially assemble in the complex. IMPORTANCE Although predicted on the basis of sequence conservation, the work presented here formally demonstrates that all classes of type III secretion systems, flagellar or virulence, share the same architecture at the level of the core structures. This absolute conservation of the unusual extramembrane structure of the core export gate complex now allows work to move to focusing on both mechanistic studies of type III but also on fundamental studies of how such a complex is assembled.

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