scholarly journals Cross-linking of bovine rhodopsin with sulfosuccinimidyl 4-(N maleimidomethyl)cyclohexane-1-carboxylate affects its functionality

2020 ◽  
Vol 477 (12) ◽  
pp. 2295-2312
Author(s):  
Rafael Medina ◽  
Deisy Perdomo ◽  
Carolina Möller ◽  
José Bubis
Keyword(s):  
Conformational Changes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Three Dimensional ◽  
Binding Activity ◽  
Dimensional Structure ◽  
Cross Linking ◽  
Bovine Rhodopsin ◽  
Rhodopsin Kinase ◽  
Cross Links ◽  
Guanine Nucleotide Binding

Rhodopsin is the photoreceptor protein involved in visual excitation in retinal rods. The functionality of bovine rhodopsin was determined following treatment with sulfosuccinimidyl 4-(N maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC), a bifunctional reagent capable of forming covalent cross-links between suitable placed lysines and cysteines. Denaturing polyacrylamide gel electrophoresis showed that rhodopsin incubated with sulfo-SMCC generated intermolecular dimers, trimers, and higher oligomers, although most of the sulfo-SMCC-treated protein remained as a monomer. Minor alterations on the absorption spectrum of light-activated sulfo-SMCC-treated rhodopsin were observed. However, only ∼2% stimulation of the guanine nucleotide binding activity of transducin was measured in the presence of sulfo-SMCC-cross-linked photolyzed rhodopsin. Moreover, rhodopsin kinase was not able of phosphorylating sulfo-SMCC-cross-linked rhodopsin after illumination. Rhodopsin was purified in the presence of either 0.1% or 1% n-dodecyl β-d-maltoside, to obtain dimeric and monomeric forms of the protein, respectively. Interestingly, no generation of the regular F1 and F2 thermolytic fragments was perceived with sulfo-SMCC-cross-linked rhodopsin either in the dimeric or monomeric state, implying the formation of intramolecular connections in the protein that might thwart the light-induced conformational changes required for interaction with transducin and rhodopsin kinase. Structural analysis of the rhodopsin three-dimensional structure suggested that the following lysine and cysteine pairs: Lys66/Lys67 and Cys316, Cys140 and Lys141, Cys140 and Lys248, Lys311 and Cys316, and/or Cys316 and Lys325 are potential candidates to generate intramolecular cross-links in the protein. Yet, the lack of fragmentation of sulfo-SMCC-treated Rho with thermolysin is consistent with the formation of cross-linking bridges between Lys66/Lys67 and Cys316, and/or Cys140 and Lys248.

scholarly journals The Three-dimensional Structure of Bovine Rhodopsin Determined by Electron Cryomicroscopy

2003 ◽  
Vol 278 (50) ◽  
pp. 50217-50225 ◽  
Author(s):  
Angelika Krebs ◽  
Patricia C. Edwards ◽  
Claudio Villa ◽  
Jade Li ◽  
Gebhard F. X. Schertler
Keyword(s):  
Three Dimensional ◽  
Dimensional Structure ◽  
Electron Cryomicroscopy ◽  
Three Dimensional Structure ◽  
Bovine Rhodopsin

Televisualization: An Aid To Collaborative Research In Molecular Structure Biology

1998 ◽  
Vol 4 (S2) ◽  
pp. 32-33
Author(s):  
M. F. Schmid ◽  
P. Matsudaira ◽  
M. T. Dougherty ◽  
M. B. Sherman ◽  
C. Henn ◽  
...  
Keyword(s):  
Image Processing ◽  
Actin Filaments ◽  
Collaborative Research ◽  
Horseshoe Crab ◽  
Hexagonal Lattice ◽  
Three Dimensional ◽  
Limulus Polyphemus ◽  
Dimensional Structure ◽  
Cross Linking ◽  
Tilt Series

Collaboration between local microscopists and image processing specialists, and their remote biological colleagues, has been hampered by the difficulty of i) transferring the three-dimensional reconstructions of macromolecules resulting from the cryomicroscopy and image processing, ii) viewing the results in a meaningful way, and iii) communicating the results and the interpretations derived therefrom to each other.The acrosomal process is an intracellular quasi-crystalline organelle in the head of the sperm of the horseshoe crab Limulus polyphemus. It consists of 100 - 130 actin-scruin filaments packed together in a pseudo-hexagonal lattice and is up to 60 μm long with a diameter of 0.1 μm. Scruin-scruin interactions are responsible for cross-linking the actin filaments together in the bundle. Our goal was to reveal interfilament interactions in the bundle. We have taken tilt series images in the electron microscope to reconstruct its three-dimensional structure at 45 Å resolution.

Lysyl oxidases: from enzyme activity to extracellular matrix cross-links

2019 ◽  
Vol 63 (3) ◽  
pp. 349-364 ◽  
Author(s):  
Sylvain D. Vallet ◽  
Sylvie Ricard-Blum
Keyword(s):  
Extracellular Matrix ◽  
Molecular Mechanisms ◽  
Catalytic Domain ◽  
Lysyl Oxidase ◽  
Dimensional Structure ◽  
Cross Linking ◽  
Copper Binding ◽  
Molecular Features ◽  
Ecm Proteins ◽  
Cross Links

Abstract The lysyl oxidase family comprises five members in mammals, lysyl oxidase (LOX) and four lysyl oxidase like proteins (LOXL1-4). They are copper amine oxidases with a highly conserved catalytic domain, a lysine tyrosylquinone cofactor, and a conserved copper-binding site. They catalyze the first step of the covalent cross-linking of the extracellular matrix (ECM) proteins collagens and elastin, which contribute to ECM stiffness and mechanical properties. The role of LOX and LOXL2 in fibrosis, tumorigenesis, and metastasis, including changes in their expression level and their regulation of cell signaling pathways, have been extensively reviewed, and both enzymes have been identified as therapeutic targets. We review here the molecular features and three-dimensional structure/models of LOX and LOXLs, their role in ECM cross-linking, and the regulation of their cross-linking activity by ECM proteins, proteoglycans, and by inhibitors. We also make an overview of the major ECM cross-links, because they are the ultimate molecular readouts of LOX/LOXL activity in tissues. The recent 3D model of LOX, which recapitulates its known structural and biochemical features, will be useful to decipher the molecular mechanisms of LOX interaction with its various substrates, and to design substrate-specific inhibitors, which are potential antifibrotic and antitumor drugs.

scholarly journals The three-dimensional structure of a class-Pi glutathione S-transferase complexed with glutathione: the active-site hydration provides insights into the reaction mechanism

1998 ◽  
Vol 333 (3) ◽  
pp. 811-816 ◽  
Author(s):  
Antonio PÁRRAGA ◽  
Isabel GARCÍA-SÁEZ ◽  
Sinead B. WALSH ◽  
Timothy J. MANTLE ◽  
Miquel COLL
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Water Molecules ◽  
X Ray Diffraction ◽  
Glutathione S Transferase ◽  
X Ray ◽  
The Right

The structure of mouse liver glutathione S-transferase P1-1 complexed with its substrate glutathione (GSH) has been determined by X-ray diffraction analysis. No conformational changes in the glutathione moiety or in the protein, other than small adjustments of some side chains, are observed when compared with glutathione adduct complexes. Our structure confirms that the role of Tyr-7 is to stabilize the thiolate by hydrogen bonding and to position it in the right orientation. A comparison of the enzyme–GSH structure reported here with previously described structures reveals rearrangements in a well-defined network of water molecules in the active site. One of these water molecules (W0), identified in the unliganded enzyme (carboxymethylated at Cys-47), is displaced by the binding of GSH, and a further water molecule (W4) is displaced following the binding of the electrophilic substrate and the formation of the glutathione conjugate. The possibility that one of these water molecules participates in the proton abstraction from the glutathione thiol is discussed.

scholarly journals Viral Genome Conformations and Contacts across Different Lifecycle Stages

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 109
Author(s):  
Peter G. Stockley ◽  
Nikesh Patel ◽  
Emma L. Wroblewski ◽  
Andrew J. P. Scott ◽  
Carlos P. Mata ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Drug Targets ◽  
Three Dimensional ◽  
Host Cells ◽  
Dimensional Structure ◽  
Viral Genomes ◽  
Starting Point ◽  
B Virus ◽  
Infection Mechanisms ◽  
Conformational Memory

Single-stranded RNA viral genomes (gRNA) are dynamic molecules that permit packaging into virions and their subsequent extrusion during infection. For viruses with such genomes, we discovered a previously unsuspected mechanism that regulates their assembly. This regulation is the result of multiple cognate coat protein (CP)–gRNA contacts distributed across the RNA. Collectively, these interactions make the assembly highly efficient and specific. The regions of the gRNA packaging signals (PSs) driving this assembly are potential drug targets, whilst the manipulation of PS–CP contacts with nonviral RNA cargos is a route towards bespoke virus-like particles. Infectivity depends on the virions being able to transfer their gRNAs into host cells. The starting point for this transfer appears to be an encapsidated RNA with a defined three-dimensional structure, especially around the PSs. A combination of asymmetric cryo-electron microscopy structure determination and X-ray synchrotron footprinting were used to define these contacts and structures in a number of viral examples, including hepatitis B virus and enteroviruses. These tools allow us to look beyond the outer CP layer of the virion shell and to see the functional, asymmetric components that regulate viral infectivity. This revealed yet more unexpected aspects of critical infection mechanisms, such as the RNA conformational changes required for encapsidation, the details of PS–CP contacts regulating the assembly, and the conformational “memory” imposed by encapsidation.

Investigation of the quaternary structure of Neurospora pyruvate kinase by cross-linking with bifunctional reagents: the effect of substrates and allosteric ligands

1977 ◽  
Vol 55 (1) ◽  
pp. 43-49 ◽  
Author(s):  
M. Kapoor ◽  
M. D. O'Brien
Keyword(s):  
Pyruvate Kinase ◽  
Functional Groups ◽  
Conformational Changes ◽  
Quaternary Structure ◽  
Polyacrylamide Gel ◽  
Cross Linking ◽  
Complex Cross ◽  
Dimethyl Suberimidate ◽  
Cross Links

Pyruvate kinase (EC 2.7.1.40) of Neurospora, a tetramer composed of apparently identical subunits, has been shown to be a dimer of dimers by interprotomeric cross-linking experiments in which bifunctional reagents were used. An analysis of the polyacrylamide gel profiles of the enzyme after cross-linking with glutaraldehyde, dimethyl suberimidate, and dimethyl adipimidate shows that the extent of intersubunit cross-linking is influenced markedly by the ligand bound to the enzyme. Bifunctional cross-linking reagents with a shorter distance between the two functional groups form cross-links effectively in the unliganded enzyme. In the FDP – pyruvate kinase complex, cross-linking was observed over longer distances compared with the unliganded enzyme. It is demonstrated that covalent cross-linkers can be used as sensitive indicators of conformational changes induced in pyruvate kinase by substrates and allosteric ligands.

scholarly journals Identification and purification from bovine brain of a guanine-nucleotide-binding protein distinct from Gs, Gi and Go

1987 ◽  
Vol 246 (2) ◽  
pp. 431-439 ◽  
Author(s):  
G L Waldo ◽  
T Evans ◽  
E D Fraser ◽  
J K Northup ◽  
M W Martin ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Binding Activity ◽  
Bovine Brain ◽  
Guanine Nucleotide ◽  
Guanine Nucleotide Binding Protein ◽  
Gtp Binding ◽  
Guanine Nucleotide Binding ◽  
The Brain

A guanine-nucleotide-binding protein (G-protein) was purified from cholate extracts of bovine brain membranes by sequential DEAE-Sephacel, Ultrogel AcA-34, heptylamine-Sepharose and Sephadex G-150 chromatography. Guanosine 5′-[gamma-[35S]thio]triphosphate (GTP[35S])-binding activity copurified with a 25,000 Da peptide and a 35,000-36,000 Da protein doublet. Neither pertussis toxin nor cholera toxin catalysed the ADP-ribosylation of a protein associated with the GTP[35S]-binding activity. Photoaffinity labelling of the purified protein with 8-azido[gamma-32P]GTP indicated that the GTP-binding site resides on the 25,000 Da protein. The 35,000-36,000 Da protein doublet was electrophoretically indistinguishable from the beta-subunits of other GTP-binding proteins, and the 36,000 Da protein was recognized by antiserum to oligomeric Gt. The purified protein specifically bound 17.2 nmol of GTP[35S]/mg of protein. The Kd of the binding site for radioligand was approx. 15 nM. The brain GTP-binding protein co-migrated during SDS/polyacrylamide-gel electrophoresis with a GTP-binding protein, named Gp, purified from human placenta [Evans, Brown, Fraser & Northup (1986) J. Biol. Chem. 261, 7052-7059], and cross-reacted with antiserum raised against the placental protein, but not with antiserum raised to brain Go. SDS/polyacrylamide-gel electrophoresis of the brain and placental GTP-binding proteins in the presence of Staphylococcus aureus V8 protease yielded identical peptide maps.

scholarly journals Structure of Isolated Nucleocapsids from Venezuelan Equine Encephalitis Virus and Implications for Assembly and Disassembly of Enveloped Virus

2003 ◽  
Vol 77 (1) ◽  
pp. 659-664 ◽  
Author(s):  
Angel Paredes ◽  
Kathy Alwell-Warda ◽  
Scott C. Weaver ◽  
Wah Chiu ◽  
Stanley J. Watowich
Keyword(s):  
Conformational Changes ◽  
Rna Virus ◽  
Three Dimensional ◽  
Venezuelan Equine Encephalitis Virus ◽  
Encephalitis Virus ◽  
Dimensional Structure ◽  
Icosahedral Symmetry ◽  
Venezuelan Equine Encephalitis ◽  
Equine Encephalitis Virus ◽  
Enveloped Virus

ABSTRACT Venezuelan equine encephalitis virus (VEEV) is an important human and equine pathogen in the Americas, with widespread reoccurring epidemics extending from South America to the southern United States. Most troubling, VEEV has been made into a weapon by several countries and is currently restricted by the Centers for Disease Control and Prevention as a potential biological warfare and terrorism agent. To facilitate the development of antiviral compounds, the structure of the nucleocapsid isolated from VEEV has been determined by electron cryomicroscopy and image reconstruction and represents the first three-dimensional structure of a nucleocapsid isolated from a single-stranded enveloped RNA virus. The isolated VEEV nucleocapsid undergoes significant reorganization relative to its structure within VEEV. However, the isolated nucleocapsid clearly exhibits T=4 icosahedral symmetry, and its characteristic nucleocapsid hexons and pentons are preserved. The diameter of the isolated nucleocapsid is ∼11.5% larger than that of the nucleocapsid within VEEV, with radial expansion being greatest near the hexons. Significantly, this is the first direct structural evidence showing that a simple enveloped virus undergoes large conformational changes during maturation, suggesting that the lipid bilayer and the transmembrane proteins of simple enveloped viruses provide the energy necessary to reorganize the nucleocapsid during maturation.

scholarly journals Histidine-Rich Defensins from the Solanaceae and Brasicaceae Are Antifungal and Metal Binding Proteins

2020 ◽  
Vol 6 (3) ◽  
pp. 145
Author(s):  
Mark R. Bleackley ◽  
Shaily Vasa ◽  
Peta J. Harvey ◽  
Thomas M. A. Shafee ◽  
Bomai K. Kerenga ◽  
...  
Keyword(s):  
Antifungal Activity ◽  
Metal Binding ◽  
Disulfide Bonds ◽  
Three Dimensional ◽  
Binding Activity ◽  
Dimensional Structure ◽  
Histidine Residues ◽  
Plant Defensins ◽  
Arginine Residues ◽  
Solanaceous Plants

Plant defensins are best known for their antifungal activity and contribution to the plant immune system. The defining feature of plant defensins is their three-dimensional structure known as the cysteine stabilized alpha-beta motif. This protein fold is remarkably tolerant to sequence variation with only the eight cysteines that contribute to the stabilizing disulfide bonds absolutely conserved across the family. Mature defensins are typically 46–50 amino acids in length and are enriched in lysine and/or arginine residues. Examination of a database of approximately 1200 defensin sequences revealed a subset of defensin sequences that were extended in length and were enriched in histidine residues leading to their classification as histidine-rich defensins (HRDs). Using these initial HRD sequences as a query, a search of the available sequence databases identified over 750 HRDs in solanaceous plants and 20 in brassicas. Histidine residues are known to contribute to metal binding functions in proteins leading to the hypothesis that HRDs would have metal binding properties. A selection of the HRD sequences were recombinantly expressed and purified and their antifungal and metal binding activity was characterized. Of the four HRDs that were successfully expressed all displayed some level of metal binding and two of four had antifungal activity. Structural characterization of the other HRDs identified a novel pattern of disulfide linkages in one of the HRDs that is predicted to also occur in HRDs with similar cysteine spacing. Metal binding by HRDs represents a specialization of the plant defensin fold outside of antifungal activity.

scholarly journals Three-dimensional reconstruction of the Z disk of sectioned bee flight muscle.

1989 ◽  
Vol 108 (5) ◽  
pp. 1761-1774 ◽  
Author(s):  
N Q Cheng ◽  
J F Deatherage
Keyword(s):  
Central Region ◽  
Actin Filaments ◽  
Flight Muscle ◽  
Three Dimensional ◽  
Opposite Polarity ◽  
Dimensional Structure ◽  
Three Dimensional Reconstruction ◽  
Thin Sections ◽  
Dimensional Reconstruction ◽  
Cross Links

The three-dimensional structure of the central region of the Z disk of honeybee flight muscle has been determined to a resolution of 70 A by three-dimensional reconstruction from electron micrographs of tilted thin sections. The reconstructions show a complex assembly in which actin filaments terminate and are cross-linked together; a number of structural domains of this network are resolved in quantitative three-dimensional detail. The central region of the Z disk contains two sets of overlapping actin filaments of opposite polarity, which originate in the sarcomeres adjacent to the Z disk, and connections between these filaments. The filaments are deflected by the attachment of cross-links; spacing between filaments change by greater than 100 A during their passage through the Z disk. Each actin filament is linked by connecting structures to four filaments of opposite polarity and two filaments are of the same polarity. Four types of connecting density domain are observed in association with pairs of filaments of opposite polarity: C1, C2, C3, and C5. Two of these, C3 and C5, are associated with the ends of actin filaments. Another connection, C4, is associated with three filaments of the same polarity; C4 is threefold symmetric.

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