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W Gibson - One of the best experts on this subject based on the ideXlab platform.
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Cytomegalovirus Capsid Protease: Biological Substrates Are Cleaved More Efficiently by Full-Length Enzyme (pUL80a) than by the Catalytic Domain (Assemblin)
Journal of virology, 2011Co-Authors: Steve M. Fernandes, Edward J. Brignole, W GibsonAbstract:We compared the full-length capsid maturational protease (pPR, pUL80a) of human cytomegalovirus with its proteolytic domain (Assemblin) for the ability to cleave two biological substrates, and we found that pPR is more efficient with both. Affinity-purified, refolded enzymes and substrates were combined under defined reaction conditions, and cleavage was monitored and quantified following staining of the resulting electrophoretically separated fragments. The enzymes were stabilized against self-cleavage by a single point mutation in each cleavage site (ICRMT-pPR and IC-Assemblin). The substrates were pPR itself, inactivated by replacing its catalytic nucleophile (S132A-pPR), and the sequence-related assembly protein precursor (pAP, pUL80.5). Our results showed that (i) ICRMT-pPR is 5- to 10-fold more efficient than Assemblin for all cleavages measured (i.e., the M site of pAP and the M, R, and I sites of S132A-pPR). (ii) Cleavage of substrate S132A-pPR proceeded M>R>I for both enzymes. (iii) Na2SO4 reduced M- and R-site cleavage efficiency by ICRMT-pPR, in contrast to its enhancing effect for both enzymes on I site and small peptide cleavage. (iv) Disrupting oligomerization of either the pPR enzyme or substrate by mutating Leu382 in the amino-conserved domain reduced cleavage efficiency two- to fourfold. (v) Finally, ICRMT-pPR mutants that include the amino-conserved domain, but terminate with Pro481 or Tyr469, retain the enzymatic characteristics that distinguish pPR from Assemblin. These findings show that the scaffolding portion of pPR increases its enzymatic activity on biologically relevant protein substrates and provide an additional link between the structure of this essential viral enzyme and its biological mechanism.
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Enzymatic Activities of Human Cytomegalovirus Maturational Protease Assemblin and Its Precursor (pPR, pUL80a): Maximal Activity of pPR Requires Self-Interaction through Its Scaffolding Domain
Journal of virology, 2007Co-Authors: Edward J. Brignole, W GibsonAbstract:Herpesviruses encode an essential, maturational serine protease whose catalytic domain, Assemblin (28 kDa), is released by self-cleavage from a 74-kDa precursor (pPR, pUL80a). Although there is considerable information about the structure and enzymatic characteristics of Assemblin, a potential pharmacologic target, comparatively little is known about these features of the precursor. To begin studying pPR, we introduced five point mutations that stabilize it against self-cleavage at its internal (I), cryptic (C), release (R), and maturational (M) sites and at a newly discovered “tail” (T) site. The resulting mutants, called ICRM-pPR and ICRMT-pPR, were expressed in bacteria, denatured in urea, purified by immobilized metal affinity chromatography, and renatured by a two-step dialysis procedure and by a new method of sedimentation into glycerol gradients. The enzymatic activities of the pPR mutants were indistinguishable from that of IC-Assemblin prepared in parallel for comparison, as determined by using a fluorogenic peptide cleavage assay, and approximated rates previously reported for purified Assemblin. The percentage of active enzyme in the preparations was also comparable, as determined by using a covalent-binding suicide substrate. An unexpected finding was that, in the absence of the kosmotrope Na2SO4, optimal activity of pPR requires interaction through its scaffolding domain. We conclude that although the enzymatic activities of Assemblin and its precursor are comparable, there may be differences in how their catalytic sites become fully activated.
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Cytomegalovirus Assemblin (pUL80a): Cleavage at Internal Site Not Essential for Virus Growth; Proteinase Absent from Virions
Journal of virology, 2002Co-Authors: Chee Kai Chan, Edward J. Brignole, W GibsonAbstract:The human cytomegalovirus (HCMV) maturational proteinase is synthesized as an enzymatically active 74-kDa precursor that cleaves itself at four sites. Two of these, called the maturational (M) and release (R) sites, are conserved in the homologs of all herpesviruses. The other two, called the internal (I) and cryptic (C) sites, have recognized consensus sequences only among cytomegalovirus (CMV) homologs and are located in the 28-kDa proteolytic portion of the precursor, called Assemblin. I-site cleavage cuts Assemblin in half without detected effect on its enzymatic behavior in vitro. To investigate the requirement for this cleavage during virus infection, we used the CMV-bacterial artificial chromosome system (E. M. Borst, G. Hahn, U. H. Koszinowski, and M. Messerle, J. Virol. 73:8320-8329, 1999) to construct a virus encoding a mutant I site (Ala143 to Val) intended to be blocked for cleavage. Characterizations of the resulting mutant (i) confirmed the presence of the mutation in the viral genome and the inability of the mutant virus to effect I-site cleavage in infected cells; (ii) determined that the mutation has no gross effect on the rate of virus production or on the amounts of extracellular virions, noninfectious enveloped particles (NIEPs), and dense bodies; (iii) established that Assemblin and its cleavage products are present in NIEPs but are absent from CMV virions, an apparent difference from what is found for virions of herpes simplex virus; and (iv) showed that the 23-kDa protein product of C-site cleavage is more abundant in mutant virus-than in wild-type virus-infected cells and NIEPs. We conclude that the production of infectious CMV requires neither I-site cleavage of Assemblin nor the presence of Assemblin in the mature virion.
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Assemblin Homolog of Herpes Simplex Virus Type 1 Retains Proteolytic Activity When Expressed as a Recombinant Two-Chain Enzyme
Virology, 1997Co-Authors: Matthew R T Hall, W GibsonAbstract:The herpes simplex virus type 1 (HSV) maturational proteinase is synthesized as a precursor that undergoes two autoproteolytic cleavages; one at its (M)aturational site, which eliminates its carboxyl ‘‘tail,’’ and a second at its (R)elease site, which separates the amino proteolytic half of the precursor from its nonproteolytic carboxyl half. In cytomegalovirus (CMV) the proteolytic half of the precursor, called Assemblin, undergoes a third cleavage at an (I)nternal site that converts it from a single-chain to a two-chain enzyme that retains activity. The HSV Assemblin homolog has no I site and therefore does not form a counterpart two-chain enzyme. In the work reported here we have cloned and expressed HSV sequences that encode mimics of the An and Ac subunits of two-chain CMV Assemblin. We show that when these HSV sequences are coexpressed in eukaryotic cells, the resulting subunits associate spontaneously to form an active two-chain enzyme. We also show that the two-chain HSV enzyme, like the natural one-chain form, retains its marked preference for HSV over CMV substrates, and that intertypic recombinant two-chain Assemblin (e.g., HSV An/CMV Ac) does not form because the cross-species subunits do not interact. We conclude from these results that (i) there are not intrinsic structural differences in the HSV Assemblin homolog that preclude its functioning as a CMV-like two-chain enzyme, (ii) the substrate selectivity shown by the single-chain HSV enzyme was not noticeably relaxed in the HSV two-chain mimic, and (iii) the interactive domains, through which the An and Ac portions of the single-chain enzymes associate, differ between HSV and CMV. q 1997 Academic Press
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Independently cloned halves of cytomegalovirus Assemblin, An and Ac, can restore proteolytic activity to Assemblin mutants by intermolecular complementation
1997Co-Authors: Matthew R T Hall, W Gibson, Mutants Intermolecular ComplementationAbstract:mutants by intermolecular complementation. restore proteolytic activity to Assemblin cytomegalovirus Assemblin, An and Ac, can Independently cloned halves o
D Wade Gibson - One of the best experts on this subject based on the ideXlab platform.
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Chemical Rescue of I-site Cleavage in Living Cells and in Vitro Discriminates between the Cytomegalovirus Protease, Assemblin, and Its Precursor, pUL80a
The Journal of biological chemistry, 2005Co-Authors: Stephen A. Mccartney, Edward J. Brignole, Keli Kolegraff, Amy N. Loveland, Lashon M. Ussin, D Wade GibsonAbstract:Abstract Chemical rescue is an established approach that offers a directed strategy for designing mutant enzymes in which activity can be restored by supplying an appropriate exogenous compound. This method has been used successfully to study a broad range of enzymes in vitro, but its application to living systems has received less attention. We have investigated the feasibility of using chemical rescue to make a conditional-lethal mutant of the cytomegalovirus (CMV) maturational protease. The 28-kDa CMV serine protease, Assemblin, has a Ser-His-His catalytic triad and an internal (I) cleavage site near its midpoint. We found that imidazole can restore I-site cleavage to mutants inactivated by replacing the critical active site His with Ala or with Gly, which rescued better. Comparable rescue was observed for counterpart mutants of the human and simian CMV Assemblin homologs and occurred in both living cells and in vitro. Cleavage was established to be at the correct site by amino acid sequencing and proceeded at ∼11%/h in bacteria and ∼30%/h in vitro. The same mutations were unresponsive to chemical rescue in the context of the Assemblin precursor, pUL80a. This catalytic difference distinguishes the two forms of the CMV protease.
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Action at the Assemblin dimer interface.
Nature Structural Biology, 2001Co-Authors: D Wade GibsonAbstract:A recent study of cytomegalovirus Assemblin proteinase suggests that dimer-induced stabilization of the oxyanion hole may activate the enzyme.
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Independently cloned halves of cytomegalovirus Assemblin, An and Ac, can restore proteolytic activity to Assemblin mutants by intermolecular complementation.
Journal of virology, 1997Co-Authors: Matthew R T Hall, D Wade GibsonAbstract:Herpesviruses encode an essential serine proteinase called Assemblin that is responsible for cleaving the precursor assembly protein during the process of capsid formation. In cytomegalovirus (CMV), Assemblin undergoes autoproteolysis at an internal (I) site located near the middle of the molecule. I-site cleavage converts the enzyme to an active two-chain form consisting of the subunits An and Ac. We have recently shown that the recombinant An and Ac subunits can spontaneously associate within eukaryotic cells to yield active two-chain proteinase. This finding indicates that the subunits are able to independently assume their correct functional conformations and led us to test whether they are capable of intermolecular complementation. This was done by coexpressing inactive mutant (point, deletion, and insertion) forms of Assemblin together with the wild-type subunit (either An or Ac) corresponding to the domain of Assemblin that was mutated. Results of these experiments showed that both An and Ac are able to rescue the enzymatic activity of Assemblin mutants. I-site cleavage of the mutated Assemblin occurred during complementation but was not absolutely required, as shown by effective complementation of inactive Assemblins with noncleavable I sites. We have also shown that intermolecular complementation can rescue the activity of an inactive mutant full-length proteinase precursor and can occur between different species of CMV (e.g., human CMV subunit can rescue activity of mutant simian CMV Assemblin). These results indicate that Assemblin is able to form active multimeric structures that may be of functional importance.
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Assemblin, an essential herpesvirus proteinase.
Drug design and discovery, 1997Co-Authors: D Wade Gibson, Matthew R T HallAbstract:The herpesvirus maturational proteinase, called Assemblin, is essential for the production of infectious virus and represents a new molecular target for the development of antivirals. A brief summary of the synthesis, structure, and function of this fascinating enzyme is presented here.
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Cytomegalovirus Assemblin: the amino and carboxyl domains of the proteinase form active enzyme when separately cloned and coexpressed in eukaryotic cells.
Journal of virology, 1996Co-Authors: Matthew R T Hall, D Wade GibsonAbstract:The cytomegalovirus (CMV) serine proteinase Assemblin is synthesized as a precursor that undergoes three principal autoproteolytic cleavages. Two of these are common to the Assemblin homologs of all herpes group viruses: one at the maturational site near the carboxyl end of the precursor and another at the release site near the midpoint of the precursor. Release-site cleavage frees the proteolytic amino domain, Assemblin, from the nonproteolytic carboxyl domain of the precursor. In CMV, a third autoproteolytic cleavage at an internal site divides Assemblin into an amino subunit (An) and a carboxyl subunit (Ac) of approximately the same size that remain associated as an active "two-chain" enzyme. We have cloned the sequences encoding An and Ac as separate genes and expressed them by transfecting human cells with recombinant plasmids and by infecting insect cells with recombinant baculoviruses. When An and Ac from either simian CMV or human CMV were coexpressed in human or insect cells, active two-chain Assemblin was formed. This finding demonstrates that An and Ac do not require synthesis as single-chain Assemblin to fold and associate correctly in these eukaryotic systems, and it suggests that they may be structurally, if not functionally, distinct domains. An interaction between the independently expressed An and Ac subunits was demonstrated by coimmunoprecipitation experiments, and efforts to disrupt the complex indicate that the subunit interaction is hydrophobic. Cell-based cleavage assays of the two-chain Assemblin formed from independently expressed An and Ac also indicate that (i) its specificity for both CMV and herpes simplex virus native substrates is similar to that of single-chain Assemblin, (ii) R-site cleavage is not essential for the activity of two-chain recombinant Assemblin, and (iii) the human CMV and simian CMV An and Ac recombinant subunits are functionally interchangeable.
Matthew R T Hall - One of the best experts on this subject based on the ideXlab platform.
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Assemblin Homolog of Herpes Simplex Virus Type 1 Retains Proteolytic Activity When Expressed as a Recombinant Two-Chain Enzyme
Virology, 1997Co-Authors: Matthew R T Hall, W GibsonAbstract:The herpes simplex virus type 1 (HSV) maturational proteinase is synthesized as a precursor that undergoes two autoproteolytic cleavages; one at its (M)aturational site, which eliminates its carboxyl ‘‘tail,’’ and a second at its (R)elease site, which separates the amino proteolytic half of the precursor from its nonproteolytic carboxyl half. In cytomegalovirus (CMV) the proteolytic half of the precursor, called Assemblin, undergoes a third cleavage at an (I)nternal site that converts it from a single-chain to a two-chain enzyme that retains activity. The HSV Assemblin homolog has no I site and therefore does not form a counterpart two-chain enzyme. In the work reported here we have cloned and expressed HSV sequences that encode mimics of the An and Ac subunits of two-chain CMV Assemblin. We show that when these HSV sequences are coexpressed in eukaryotic cells, the resulting subunits associate spontaneously to form an active two-chain enzyme. We also show that the two-chain HSV enzyme, like the natural one-chain form, retains its marked preference for HSV over CMV substrates, and that intertypic recombinant two-chain Assemblin (e.g., HSV An/CMV Ac) does not form because the cross-species subunits do not interact. We conclude from these results that (i) there are not intrinsic structural differences in the HSV Assemblin homolog that preclude its functioning as a CMV-like two-chain enzyme, (ii) the substrate selectivity shown by the single-chain HSV enzyme was not noticeably relaxed in the HSV two-chain mimic, and (iii) the interactive domains, through which the An and Ac portions of the single-chain enzymes associate, differ between HSV and CMV. q 1997 Academic Press
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Independently cloned halves of cytomegalovirus Assemblin, An and Ac, can restore proteolytic activity to Assemblin mutants by intermolecular complementation.
Journal of virology, 1997Co-Authors: Matthew R T Hall, D Wade GibsonAbstract:Herpesviruses encode an essential serine proteinase called Assemblin that is responsible for cleaving the precursor assembly protein during the process of capsid formation. In cytomegalovirus (CMV), Assemblin undergoes autoproteolysis at an internal (I) site located near the middle of the molecule. I-site cleavage converts the enzyme to an active two-chain form consisting of the subunits An and Ac. We have recently shown that the recombinant An and Ac subunits can spontaneously associate within eukaryotic cells to yield active two-chain proteinase. This finding indicates that the subunits are able to independently assume their correct functional conformations and led us to test whether they are capable of intermolecular complementation. This was done by coexpressing inactive mutant (point, deletion, and insertion) forms of Assemblin together with the wild-type subunit (either An or Ac) corresponding to the domain of Assemblin that was mutated. Results of these experiments showed that both An and Ac are able to rescue the enzymatic activity of Assemblin mutants. I-site cleavage of the mutated Assemblin occurred during complementation but was not absolutely required, as shown by effective complementation of inactive Assemblins with noncleavable I sites. We have also shown that intermolecular complementation can rescue the activity of an inactive mutant full-length proteinase precursor and can occur between different species of CMV (e.g., human CMV subunit can rescue activity of mutant simian CMV Assemblin). These results indicate that Assemblin is able to form active multimeric structures that may be of functional importance.
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Independently cloned halves of cytomegalovirus Assemblin, An and Ac, can restore proteolytic activity to Assemblin mutants by intermolecular complementation
1997Co-Authors: Matthew R T Hall, W Gibson, Mutants Intermolecular ComplementationAbstract:mutants by intermolecular complementation. restore proteolytic activity to Assemblin cytomegalovirus Assemblin, An and Ac, can Independently cloned halves o
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Assemblin, an essential herpesvirus proteinase.
Drug design and discovery, 1997Co-Authors: D Wade Gibson, Matthew R T HallAbstract:The herpesvirus maturational proteinase, called Assemblin, is essential for the production of infectious virus and represents a new molecular target for the development of antivirals. A brief summary of the synthesis, structure, and function of this fascinating enzyme is presented here.
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Cytomegalovirus Assemblin: the amino and carboxyl domains of the proteinase form active enzyme when separately cloned and coexpressed in eukaryotic cells.
Journal of virology, 1996Co-Authors: Matthew R T Hall, D Wade GibsonAbstract:The cytomegalovirus (CMV) serine proteinase Assemblin is synthesized as a precursor that undergoes three principal autoproteolytic cleavages. Two of these are common to the Assemblin homologs of all herpes group viruses: one at the maturational site near the carboxyl end of the precursor and another at the release site near the midpoint of the precursor. Release-site cleavage frees the proteolytic amino domain, Assemblin, from the nonproteolytic carboxyl domain of the precursor. In CMV, a third autoproteolytic cleavage at an internal site divides Assemblin into an amino subunit (An) and a carboxyl subunit (Ac) of approximately the same size that remain associated as an active "two-chain" enzyme. We have cloned the sequences encoding An and Ac as separate genes and expressed them by transfecting human cells with recombinant plasmids and by infecting insect cells with recombinant baculoviruses. When An and Ac from either simian CMV or human CMV were coexpressed in human or insect cells, active two-chain Assemblin was formed. This finding demonstrates that An and Ac do not require synthesis as single-chain Assemblin to fold and associate correctly in these eukaryotic systems, and it suggests that they may be structurally, if not functionally, distinct domains. An interaction between the independently expressed An and Ac subunits was demonstrated by coimmunoprecipitation experiments, and efforts to disrupt the complex indicate that the subunit interaction is hydrophobic. Cell-based cleavage assays of the two-chain Assemblin formed from independently expressed An and Ac also indicate that (i) its specificity for both CMV and herpes simplex virus native substrates is similar to that of single-chain Assemblin, (ii) R-site cleavage is not essential for the activity of two-chain recombinant Assemblin, and (iii) the human CMV and simian CMV An and Ac recombinant subunits are functionally interchangeable.
Karl-ferdinand Lechtreck - One of the best experts on this subject based on the ideXlab platform.
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ANALYSIS OF STRIATED FIBER FORMATION BY RECOMBINANT SF-Assemblin IN VITRO
Journal of molecular biology, 1998Co-Authors: Karl-ferdinand LechtreckAbstract:Abstract The basal bodies of green flagellates are often connected to striated microtubule-associated fibers (SMAFs), which are highly ordered bundles of 2 nm filaments. SF-Assemblin (33 kDa) is the principal structural subunit of the SMAFs and consists of a non-helical head domain of approximately 32 residues and an α-helical rod domain that shows a pronounced coiled-coil forming ability. To investigate the functional role of the head domain we expressed N-terminally truncated molecules using a cDNA coding for SF-Assemblin from Chlamydomonas reinhardtii . Recombinant wild-type SF-Assemblin or molecules with an N-terminal deletion of ten residues formed striated fibers with an axial repeat of 28 nm. N-terminal truncations of 19 and 29 residues yielded assembly-incompetent molecules, revealing that the head domain is necessary for the constitution of striated fibers. Further, molecules with an internal deletion of 24 residues or with duplicated segments corresponding to insertions of 29 and 58 residues were constructed. The resulting fibers had altered cross-striation patterns and axial repeats. The observed shifts in the axial repeat corresponded well to the number of inserted or deleted residues, indicating a linear coherence between molecule length and axial repeat. The heptad pattern of the rod domain of SF-Assemblin is regularly interrupted by skip residues. The removal of one or two skip residues had no significant effect on the ultrastructure of the striated fibers. Substitution of skip no. 2 with alanine resulted in a modified, asymmetric cross-striation pattern, indicating a polar architecture of the striated fibers. In summary, various mutations of SF-Assemblin effected the solubility of the molecules, and the axial repeat, cross-striation pattern, or overall appearance of the fibers. Thus, analysis of SF-Assemblin may represent a valuable system to study the interactions involved in the polymerization of fibrous coiled-coil proteins. A model of the SMAFs based on staggered protofilaments consisting of overlapping 36 nm subunits is presented.
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SF-Assemblin, striated fibers, and segmented coiled coil proteins.
Cell motility and the cytoskeleton, 1998Co-Authors: Karl-ferdinand Lechtreck, Michael MelkonianAbstract:The flagellar basal apparatus of many flagellate green algae contains noncontractile striated microtubule-associated fibers (SMAFs). The SMAFs consist of 2-nm protofilaments and are predominantly built from striated fiber (SF)-Assemblin, an acidic 33-kDa protein. In this review we summarize the present knowledge concerning the biochemical properties of SF-Assemblin and the molecular architecture of the SMAFs, provide evidence for homologous proteins and similar filament systems in other eukaryotes, and, finally, discuss possible biological functions of SF-Assemblin.
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SF-Assemblin in Chlamydomonas: sequence conservation and localization during the cell cycle.
Cell motility and the cytoskeleton, 1997Co-Authors: Karl-ferdinand Lechtreck, Carolyn D. SilflowAbstract:Previously, SF-Assemblin has been identified as the filament-forming component of the striated microtubule-associated fibers (SMAFs), which emerge from the basal bodies in several green flagellates. We have sequenced cDNAs coding for SF-Assemblin from Chlalmydomonas reinhardtii and C. eugametos. Comparison of the deduced amino acid sequences with the previously described green algal SF-Assemblins shows identities between 54 and 71%, indicating a strong drift in sequence. Cells of C. reinhardtii were analyzed by double immunofluorescence using polyclonal anti-SF-Assemblin and anti-alpha-tubulin. In interphase cells, SF-Assemblin is associated with all four microtubular flagellar roots. During mitosis the SF-Assemblin-based cytoskeleton is reorganized; it divides in prophase and is reduced to two dot-like structures at each spindle pole in metaphase. During anaphase, the two dots present at each pole are connected again. In telophase we observed an asymmetrical outgrowth of new fibers. These observations suggest a role for SF-Assemblin in reestablishing the microtubular root system characteristic of interphase cells after mitosis.
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Purification of SF-Assemblin.
Methods in cell biology, 1995Co-Authors: Angela Bremerich, Karl-ferdinand Lechtreck, Michael MelkonianAbstract:Publisher Summary Basal apparatuses of many flagellate organisms have with non-contractile cross-striated fibers. In the green algae these fibers have been termed system I fibers, or striated microtubule-associated fibers. System I fibers have been isolated from the biflagellate green alga Spermatozopsis similis . Axonemes and basal body-associated microtubules are removed after homogenization, and basal apparatuses are enriched by differential centrifugation. The isolated basal apparatuses contain two system I fibers that are unequal in length and connected at their proximal ends in such a way as to appear as a single continuous fiber. High-salt extraction and mechanical disintegration of isolated basal apparatuses yield single system I fibers. Dialysis of these extracts against 150m M KCI yields paracrystals that closely resemble the native fibers in filament arrangement and cross-striation pattern. A polyclonal antiserum raised against SF-Assemblin from S. similis labels the system I fibers in S. similis and several other flagellate green algae ( Chlamydomonas reinhardtii, Polytomella parva , and Dunaliella bioculata ) by indirect immunofluorescence. Proteins of the same apparent size as SF-Assemblin are detected by immunoblotting in C. reinhardtii , P. parva , and Nephroselms olivacea , whereas in D.bioculata a 31-kDa protein is immunoreactive. Analysis of the amino acid sequence and the predicted secondary structure of SF-Assemblin shows that the protein has two structural domains: a31-residue-long NH 2 -terminal, nonhelical domain rich in proline, and α-helical rod domain of 253 residues that forms a segmented coiled coil with a 29-residue repeat pattern based on four heptads followed by a skip residue. SF-Assembling belongs to a widespread protein family forming microtubule-associated fibers of 2-nm filaments in eukaryotic cells. SF-Assembling can be enriched after several cycles of disassembly and reassembly, this chapter is an alternative purification procedure using anion-exchange chromatography. There are details on materials, preparation of basal apparatuses, isolation of system I fibers, in vitro reassembly of system I fibers, and anion-exchange chromatography.
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SF-Assemblin, the structural protein of the 2-nm filaments from striated microtubule associated fibers of algal flagellar roots, forms a segmented coiled coil.
The Journal of cell biology, 1993Co-Authors: Klaus Weber, Karl-ferdinand Lechtreck, Angela Bremerich, Norbert Geisler, Uwe Plessmann, Michael MelkonianAbstract:The microtubule associated system I fibers of the basal apparatus of the flagellate green alga Spermatozopsis similis are noncontractile and display a 28-nm periodicity. Paracrystals with similar periodicities are formed in vitro by SF-Assemblin, which is the major protein component of system I fibers. We have determined the amino acid sequence of SF-Assemblin and show that it contains two structural domains. The NH2-terminal 31 residues form a nonhelical domain rich in proline. The rod domain of 253 residues is alpha-helical and seems to form a segmented coiled coil with a 29-residue repeat pattern based on four heptads followed by a skip residue. The distinct cluster of acidic residues at the COOH-terminal end of the motifs (periodicity about 4 nm) may be related to tubulin binding of SF-Assemblin and/or its self assembly. A similar structure has been predicted from cDNA cloning of beta-giardin, a protein of the complex microtubular apparatus of the sucking disc in the protozoan flagellate Giardia lamblia. Although the rod domains of SF-Assemblin and beta-giardin share only 20% sequence identity, they have exactly the same length and display 42% sequence similarity. These results predict that system I fibers and related microtubule associated structures arise from molecules able to form a special segmented coiled coil which can pack into 2-nm filaments. Such molecules seem subject to a strong evolutionary drift in sequence but not in sequence principles and length. This conservation of molecular architecture may have important implications for microtubule binding.
Michael Melkonian - One of the best experts on this subject based on the ideXlab platform.
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SF-Assemblin, striated fibers, and segmented coiled coil proteins.
Cell motility and the cytoskeleton, 1998Co-Authors: Karl-ferdinand Lechtreck, Michael MelkonianAbstract:The flagellar basal apparatus of many flagellate green algae contains noncontractile striated microtubule-associated fibers (SMAFs). The SMAFs consist of 2-nm protofilaments and are predominantly built from striated fiber (SF)-Assemblin, an acidic 33-kDa protein. In this review we summarize the present knowledge concerning the biochemical properties of SF-Assemblin and the molecular architecture of the SMAFs, provide evidence for homologous proteins and similar filament systems in other eukaryotes, and, finally, discuss possible biological functions of SF-Assemblin.
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Purification of SF-Assemblin.
Methods in cell biology, 1995Co-Authors: Angela Bremerich, Karl-ferdinand Lechtreck, Michael MelkonianAbstract:Publisher Summary Basal apparatuses of many flagellate organisms have with non-contractile cross-striated fibers. In the green algae these fibers have been termed system I fibers, or striated microtubule-associated fibers. System I fibers have been isolated from the biflagellate green alga Spermatozopsis similis . Axonemes and basal body-associated microtubules are removed after homogenization, and basal apparatuses are enriched by differential centrifugation. The isolated basal apparatuses contain two system I fibers that are unequal in length and connected at their proximal ends in such a way as to appear as a single continuous fiber. High-salt extraction and mechanical disintegration of isolated basal apparatuses yield single system I fibers. Dialysis of these extracts against 150m M KCI yields paracrystals that closely resemble the native fibers in filament arrangement and cross-striation pattern. A polyclonal antiserum raised against SF-Assemblin from S. similis labels the system I fibers in S. similis and several other flagellate green algae ( Chlamydomonas reinhardtii, Polytomella parva , and Dunaliella bioculata ) by indirect immunofluorescence. Proteins of the same apparent size as SF-Assemblin are detected by immunoblotting in C. reinhardtii , P. parva , and Nephroselms olivacea , whereas in D.bioculata a 31-kDa protein is immunoreactive. Analysis of the amino acid sequence and the predicted secondary structure of SF-Assemblin shows that the protein has two structural domains: a31-residue-long NH 2 -terminal, nonhelical domain rich in proline, and α-helical rod domain of 253 residues that forms a segmented coiled coil with a 29-residue repeat pattern based on four heptads followed by a skip residue. SF-Assembling belongs to a widespread protein family forming microtubule-associated fibers of 2-nm filaments in eukaryotic cells. SF-Assembling can be enriched after several cycles of disassembly and reassembly, this chapter is an alternative purification procedure using anion-exchange chromatography. There are details on materials, preparation of basal apparatuses, isolation of system I fibers, in vitro reassembly of system I fibers, and anion-exchange chromatography.
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SF-Assemblin, the structural protein of the 2-nm filaments from striated microtubule associated fibers of algal flagellar roots, forms a segmented coiled coil.
The Journal of cell biology, 1993Co-Authors: Klaus Weber, Karl-ferdinand Lechtreck, Angela Bremerich, Norbert Geisler, Uwe Plessmann, Michael MelkonianAbstract:The microtubule associated system I fibers of the basal apparatus of the flagellate green alga Spermatozopsis similis are noncontractile and display a 28-nm periodicity. Paracrystals with similar periodicities are formed in vitro by SF-Assemblin, which is the major protein component of system I fibers. We have determined the amino acid sequence of SF-Assemblin and show that it contains two structural domains. The NH2-terminal 31 residues form a nonhelical domain rich in proline. The rod domain of 253 residues is alpha-helical and seems to form a segmented coiled coil with a 29-residue repeat pattern based on four heptads followed by a skip residue. The distinct cluster of acidic residues at the COOH-terminal end of the motifs (periodicity about 4 nm) may be related to tubulin binding of SF-Assemblin and/or its self assembly. A similar structure has been predicted from cDNA cloning of beta-giardin, a protein of the complex microtubular apparatus of the sucking disc in the protozoan flagellate Giardia lamblia. Although the rod domains of SF-Assemblin and beta-giardin share only 20% sequence identity, they have exactly the same length and display 42% sequence similarity. These results predict that system I fibers and related microtubule associated structures arise from molecules able to form a special segmented coiled coil which can pack into 2-nm filaments. Such molecules seem subject to a strong evolutionary drift in sequence but not in sequence principles and length. This conservation of molecular architecture may have important implications for microtubule binding.
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SF-Assemblin, the Structural Protein of the 2-nm Filaments from Striated Microtubule Associated Fibers of Algal Flagellar Roots, Forms a Segmented Coiled Coil
1993Co-Authors: Klaus Weber, Angela Bremerich, Norbert Geisler, Uwe Plessmann, Michael MelkonianAbstract:Abstract. The microtubule associated system I fibers of the basal apparatus of the flagellate green alga Spermatozopsis similis are noncontractile and display a 28-rim periodicity. Paracrystals with similar periodicities are formed in vitro by SF-Assemblin, which is the major protein component of system I fibers. We have determined the amino acid sequence of SF-Assemblin and show that it contains two structural domains. The NH2-terminal 31 residues form a nonhelical domain rich in proline. The rod domain of 253 residues is a-helical and seems to form a segmented coiled coil with a 29-residue repeat pattern based on four heptads followed by a skip residue. The distinct cluster of acidic residues at the COOH-terminal end of the motifs (periodicity about 4 rim) may be related to tubuli
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Striated microtubule-associated fibers: identification of Assemblin, a novel 34-kD protein that forms paracrystals of 2-nm filaments in vitro.
The Journal of cell biology, 1991Co-Authors: Karl-ferdinand Lechtreck, Michael MelkonianAbstract:Microtubule-associated fibers from the basal apparatus of the green flagellate alga Spermatozopsis similis exhibit a complex cross-striation pattern with 28-nm periodicity and consist of 2-nm filaments arranged in several layers. Fibers enriched by mechanical disintegration and high salt extraction (2 M NaCl) of isolated basal apparatuses are soluble in 2 M urea. Dialysis of solubilized fibers against 150 mM KCl yields paracrystals which closely resemble the native fibers in filament arrangement and striation pattern. Paracrystals purified through several cycles of disassembly and reassembly are greatly enriched (greater than 90%) in a single protein of 34 kD (Assemblin) as shown by SDS-PAGE. A rabbit polyclonal antibody raised against Assemblin labels the striated fibers as shown by indirect immunofluorescence of isolated cytoskeletons or methanol permeabilized cells and immunogold EM. Two-dimensional electrophoresis (isoelectric focusing and SDS-PAGE) resolves Assemblin into at least four isoforms (a-d) with pI's of 5.45, 5.55, 5.75, and 5.85. The two more acidic isoforms are phosphoproteins as shown by in vivo 32PO4-labeling and autoradiography. Amino acid analysis of Assemblin shows a high content of helix-forming residues (leucine) and a relatively low content of glycine. We conclude that Assemblin may be representative of a class of proteins that form fine filaments alongside microtubules.