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N Geisler

Publications and source records attributed to N Geisler.

At least 19 recordsLinked to original sources
PubMed1996

Specific recognition of coiled coils by infrared spectroscopy: analysis of the three structural domains of type III intermediate filament proteins.

The central domain of cytoplasmic intermediate filament (IF) proteins from vertebrates contains some 310 residues and forms a double-stranded coiled coil (rod) with a length of about 46 nm. The flanking terminal domains show a high cell type specific variability both in sequence and in length. Using Fourier transform infrared (FTIR) spectroscopy we measured secondary structures of isolated domains of type III and IV IF proteins and of the soluble tetramers and the filaments formed by type III IF proteins. The amide I spectrum of the desmin rod is virtually identical to the spectra of other coiled-coil proteins such as tropomyosin and the myosin rod. All these double-stranded coiled coils reveal spectra distinctly different from classical alpha-helical spectra. The spectrum of coiled coils is a triplet of approximately equally strong bands. One band occurs at normal alpha-helix position, while the other two are found at lower wavenumbers. Theoretical aspects of these findings are discussed in the accompanying paper by W. C. Reisdorf and S. Krimm [(1996) Biochemistry 35, 1383-1386]. The amino-terminal head domain of desmin has a multicomponent spectrum with major fractions of beta-sheet. The carboxy-terminal tail domains of desmin and the neurofilament proteins L and H, the latter in the phosphorylated and in the dephosphorylated forms, have very similar FTIR spectra, indicating mostly random structure. The spectrum of desmin type III protofilaments is very similar to the sum of the spectra of the three isolated domains. Polymerization into filaments seems to induce a small change in secondary structure.

Desmin↗
PubMed1995

Identification of the nicking tyrosine of geminivirus Rep protein.

The replication initiator (Rep) proteins of geminiviruses perform a DNA cleavage and strand transfer reaction at the viral origin of replication. As a reaction intermediate, Rep proteins become covalently linked to the 5' end of the cleaved DNA. We have used tomato yellow leaf curl virus Rep protein for in vivo and in vitro analyses. Isolating a covalent peptide-nucleotide complex, we have identified the amino acid of Rep which mediates cleavage and links the protein to DNA. We show that tyrosine-103, located in a conserved sequence motif, initiates DNA cleavage and is the physical link between geminivirus Rep protein and its origin DNA.

Amino Acid Sequence↗
PubMed1995

Major epiplasmic proteins of ciliates are articulins: cloning, recombinant expression, and structural characterization.

The cytoskeleton of certain protists comprises an extensive membrane skeleton, the epiplasm, which contributes to the cell shape and patterning of the species-specific cortical architecture. The isolated epiplasm of the ciliated protist Pseudomicrothorax dubius consists of two major groups of proteins with molecular masses of 78-80 kD and 11-13 kD, respectively. To characterize the structure of these proteins, peptide sequences of two major polypeptides (78-80 kD) as well as a cDNA representing the entire coding sequence of a minor and hitherto unidentified component (60 kD; p60) of the epiplasm have been determined. All three polypeptides share sequence similarities. They contain repeated valine- and proline-rich motifs of 12 residues with the consensus VPVP--V-V-V-. In p60 the central core domain consists of 24 tandemly repeated VPV motifs. Within the repeat motifs positively and negatively charged residues, when present, show an alternating pattern in register with the V and P positions. Recombinant p60 was purified in 8 M urea and dialyzed against buffer. Infrared spectroscopic measurements indicate 30% beta-sheet. Electron microscopy reveals short filamentous polymers with a rather homogenous diameter (approximately 15-20 nm), but variable lengths. The small polymers form thicker filaments, ribbons, and larger sheets or tubes. A core domain similar to that of P. dubius p60 is also found in the recently described epiplasmic proteins of the flagellate Euglena, the so-called articulins. Our results show that the members of this protein family are not restricted to flagellates, but are also present in the distantly related ciliates where they are major constituents of the epiplasm. Comparison of flagellate and ciliate articulins highlights common features of this novel family of cytoskeletal proteins.

Amino Acid Sequence↗
PubMed1993

Chemical crosslinking with disuccinimidyl tartrate defines the relative positions of the two antiparallel coiled coils of the desmin protofilament unit.

Filaments formed by desmin, the myogenic intermediate filament protein, were crosslinked with the lysine specific crosslinker DST (disuccinimidyl tartrate; 0.64 nm span) and three DST crosslinked peptides were characterized. Two correspond to crosslinks previously obtained with the longer crosslinker EGS (ethylene glycol bis(succinimidylsuccinate), 1.61 nm span) which defined the antiparallel on-stagger relationship of neighbouring coiled coils. The two DST crosslinks now provide the relative positions of the coiled coils within a limit of about 9 alpha-helical residues. The third DST crosslink most likely connecting two helices of a single coiled coil gives a direct measure of the distance spanned in DST crosslinks.

Amino Acid Sequence↗
PubMed1993

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 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.

Amino Acid Sequence↗
PubMed1992

Chemical cross-linking indicates a staggered and antiparallel protofilament of desmin intermediate filaments and characterizes one higher-level complex between protofilaments.

Tetrameric rods, protofilaments and assembled filaments of desmin, the intermediate filament protein of muscle, have been chemically cross-linked with the lysine specific cross-linkers EGS [ethylene glycol bis(succinimidylsuccinate), 1.61 nm span] and bis(sulfosuccinimidyl) suberate (1.14 nm span). One bis(sulfosuccinimidyl)suberate and two EGS cross-links were isolated from the rod and characterized. They show that the two coiled coils in the rod tetramer are staggered by approximately 15-20 nm and strongly indicate an antiparallel arrangement in which the inner overlapping part of the rod is formed by the amino-terminal helices 1A, 1B and 2A. Both EGS cross-links identified in the rod were also isolated from cross-linked filaments. The isolated rod, therefore, represents a complex also present in identical, or very similar form in protofilaments and in assembled filaments. Cross-linked filaments yielded a third EGS cross-link that must have been formed between neighboring protofilaments. It connects the highly conserved carboxy-terminus of helix 2B of the first protofilament to the overlap region formed by helices 1A and 2A of the second protofilament. The restrictions posed by these cross-links on current filament models are discussed.

Amino Acid Sequence↗
PubMed1990

The two coiled coils in the isolated rod domain of the intermediate filament protein desmin are staggered. A hydrodynamic analysis of tetramers and dimers.

Desmin protofilaments and the proteolytically derived alpha-helical rod domain have been characterized by high-resolution gel permeation chromatography (GPC) using columns calibrated for the determination of viscosity radii. Additional characterization by chemical cross-linking and the determination of sedimentation values allowed the calculation of the molecular dimensions of the molecular species isolated. In dilute buffers GPC separated desmin rod preparations into two complexes: a dimer species (single coiled coil) with a length of 50 +/- 5 nm and a tetramer species (two coiled coils) with a length of 65 +/- 5 nm. Thus the two coiled coils in the tetramer are staggered by approximately 15 nm. The hydrodynamically derived lengths of the rod dimer and tetramer are supported by electron microscopy after metal shadowing. The hydrodynamic properties of desmin protofilaments follow that of the rod tetramer. The data on the hydrodynamic analysis of the rod tetramer of desmin in solution are in full agreement with the structural information recently deduced from paracrystals of the rod of glial fibrillary acid protein [Stewart, M., Quinlan, R.A. & Moir, R.D. (1989) J. Cell Biol. 109, 225-234]. Our results explain the inhomogeneity of molecules encountered in previous electron microscopical analyses.

Animals↗
PubMed1989

Phosphorylation in vitro of vimentin by protein kinases A and C is restricted to the head domain. Identification of the phosphoserine sites and their influence on filament formation.

The in vitro phosphorylation of vimentin, the intermediate filament protein of mesenchymal cells, by kinases A and C is serine-specific and involves only the N-terminal head domain. In oligomeric protofilament units each kinase recognizes five sites, which have been identified by sequence analysis. Kinase C introduces 1.5 mol phosphate/mol vimentin, while kinase A treatment results in 4 mol phosphate/mol. Kinase-A-treated oligomers do not polymerize in standard assays whereas kinase C treatment has no inhibitory effect. Filaments exposed to kinase A remain stable and incorporate only 1.7 mol phosphate/mol vimentin. These phosphates are essentially restricted to two of the five kinase A sites found in protofilament units. Thus the head domain, previously related to in vitro assembly competence and filament stability, changes in accessibility between the oligomeric and polymeric state. We discuss the possibility that in vivo phosphorylation of vimentin filaments by kinase A may not necessarily be accompanied by an extensive depolymerization. It could instead involve a dynamic change of the filament surfaces, which could alter the interaction of the filaments with other cellular structures.

Adenosine Triphosphate↗
PubMed1988

Phosphorylation of desmin in vitro inhibits formation of intermediate filaments; identification of three kinase A sites in the aminoterminal head domain.

The in vitro phosphorylation of chicken desmin by the catalytic subunit of cAMP-dependent protein kinase was analysed. Phosphorylated desmin loses the ability to form intermediate filaments (IFs). Fragmentation at the sole cysteine and mild chymotryptic treatment show a differential phosphorylation of the three structural domains. Only the amino-terminal head domain is the target of the kinase. Peptide analysis shows that serine 29 is fully phosphorylated, while serine 35 and 50 are phosphorylated at least at 22 and 50% respectively. All three sites show the sequence arginine-X-serine with X being a small residue. These results strengthen the view that the nonhelical head domain has a strong influence on filament integrity most likely via a direct influence of some of its arginine residues. Taken together with previous results (Inagaki et al., 1987) on the phosphorylation of vimentin by kinase A, a new view on IFs emerges. Phosphorylation could allow for regulatory processes in assembly and turnover.

Amino Acid Sequence↗
PubMed1987

Location and sequence characterization of the major phosphorylation sites of the high molecular mass neurofilament proteins M and H.

Diagonal fingerprinting allows the specific purification of those tryptic peptides which change electrophoretic mobility due to a dephosphorylation step introduced after the first dimension. Nine tryptic peptides from the tail domain of porcine neurofilament M protein identify a minimum of 6 phosphorylated serines. Unexpectedly, four of the nine peptides characterize a region of degenerate repetitive sequences. Results on neurofilament H tail, although less complete, yield longer sequences of degenerate repetitive character. Here, all serines present appear to be contained in a lysine-serine-proline unit. This motif also occurs in some but not all M peptides. We suggest that degenerate repetitive sequences in neurofilament M and H tails have a high species-specific drift.

Amino Acid Sequence↗
PubMed1987

Binding of two desmin derivatives to the plasma membrane and the nuclear envelope of avian erythrocytes: evidence for a conserved site-specificity in intermediate filament-membrane interactions.

Using solution binding assays, we found that a 45-kDa fragment of desmin, lacking 67 residues from the N terminus, could specifically associate with avian erythrocyte nuclear envelopes but not with plasma membranes from the same cells. It was also observed that a 50-kDa desmin peptide, missing 27 C-terminal residues, retained the ability to bind to both membrane preparations. Displacement experiments with an excess of purified vimentin suggested that the two desmin derivatives were interacting with a previously identified vimentin receptor at the nuclear envelope, the protein lamin B [Georgatos, S. & Blobel, G. (1987) J. Cell Biol. 105, 117-127]. Additional analysis by affinity chromatography confirmed this conclusion. Employing an overlay assay, we demonstrated that the 50-kDa fragment, but not the 45-kDa desmin peptide, was capable of interacting with the plasma membrane polypeptide ankyrin (a known vimentin attachment site), as was intact vimentin. Conversely, the nuclear envelope protein lamin B was recognized by both fragments but not by a chymotryptic peptide composed solely of the helical rod domain of desmin. These data imply that the lamin B-binding site on desmin resides within the 21 residues following its helical rod domain, whereas the ankyrin-associating region is localized within its N-terminal head domain, exactly as in the case of vimentin.

Animals↗
PubMed1986

Are the terminal domains in intermediate filaments organized as octameric complexes? Reevaluation of a recent suggestion.

Recently L. M. Milam and H. Erickson ((1985) J. Ultrastruc. Res. 90, 251-260) reported the isolation of a particle thought to be an octomeric complex of the terminal domains of desmin. This complex was isolated after trypsin treatment of intact filaments. As the existence of such a complex would place important restrictions on the possible packing modes of subunits within the filament we have repeated their procedure and additionally characterized the particle in question by detailed protein chemical data. We find that the particle is not derived from the terminal domains but instead comprises a portion of the carboxy-terminal half of the alpha-helical rod domain. We further show that the terminal domains are very rapidly digested into small peptides during the trypsin treatment of the filaments. No inferences on the structure of intermediate filament are therefore possible from the data in the original report.

Animals↗
PubMed1985

Intermediate filament forming ability of desmin derivatives lacking either the amino-terminal 67 or the carboxy-terminal 27 residues.

Amino acid sequence data and results from limited proteolytic digestion have been used to define the three-domain structure of intermediate filament proteins. A centrally located highly alpha-helical domain of about 310 residues well-conserved in sequence principles and length is flanked by the highly variable sequences of the non-alpha-helical headpiece and tailpiece. A direct involvement in filament formation of one or both terminal domains was previously proposed for desmin since chymotryptic removal of head and tailpiece provided a derivative unable to form filaments. In order to evaluate directly the importance of these regions we have prepared desmin derivatives lacking either the amino-terminal 67 (T-desmin) or carboxy-terminal 27 residues (L-desmin). Whereas the latter derivative is fully polymerization-competent the fragment lacking only the basic and arginine-rich headpiece cannot form filaments on its own and remains in a protofilamentous stage. These structures of T-desmin are not incorporated into filaments when mixed with protofilaments of desmin. If, however, the two proteins are mixed in 7 M-urea subsequent dialysis provides morphologically normal filaments containing T-desmin. The results suggest that at least certain hybrid protofilaments containing less than four headpieces are accepted in the filament. The removal of the 27 carboxy-terminal residues in L-desmin, although not interfering with filament formation, leads to a change in surface since filaments show lateral aggregation at 170 mM but not at 50 mM salt. The results are discussed in relation to current models of intermediate filament structure.

Amino Acid Sequence↗
PubMed1985

The complete amino acid sequence of the major mammalian neurofilament protein (NF-L).

The first complete amino acid sequence of a neurofilament protein has been established. Porcine NF-L contains 548 residues corresponding to a molecular mass of approximately 62 kDa. This value is noticeably smaller than the 68-72 kDa estimates from gel electrophoresis. Sequence comparison among the 6 non-epithelial intermediate filament (IF) proteins of warm-blooded vertebrates shows that the three NF proteins are the most remote members. Additionally and unexpectedly they reveal among each other lower sequence identity than the three non-neuronal IF proteins GFAP, desmin, and vimentin where the last two are particularly closely related. Certain schemes of IF protein evolution are discussed.

Amino Acid Sequence↗
PubMed1985

Antiparallel orientation of the two double-stranded coiled-coils in the tetrameric protofilament unit of intermediate filaments.

The chymotryptically excised middle domain of desmin slightly exceeds in length the structurally conserved alpha-helical middle region documented in all intermediate filament proteins by amino acid sequence data. This rod domain is a protofilament derivative with a tetrameric organization, thus indicating the presence of two double-stranded coiled-coil units. We now show by immunoelectron microscopy that Fab fragments of a desmin-specific monoclonal antibody mixed with the rod lead to dumb-bell-shaped structures. The tagging of both ends together with the length of the rod (48 nm) argues for an antiparallel orientation of the two coiled-coils without a major stagger. This information combined with the lateral 21 nm periodicity of the intermediate filament observed by us and others leads to a structural hypothesis similar to those entertained from X-ray data on wool alpha-keratins, although here an antiparallel tetrameric unit of some 60 to 66 nm is invoked, which has never been isolated. The structure that we discuss allows for the existence of both the particles, and the antibody experiment strongly supports the antiparallel orientation postulated in both approaches. The tube-like filament structure proposed for the intermediate filament agrees with recent mass per unit length measurements and allows for two minor classes of intermediate filaments with different values in this property as also found experimentally.

Antibodies, Monoclonal↗
PubMed1984

SDS-PAGE strongly overestimates the molecular masses of the neurofilament proteins.

Direct molecular mass determination of the three porcine neurofilament proteins (H, M and L) was performed in 6 M guanidine-HCl using analytical gel filtration and sedimentation equilibrium centrifugation. The results show that SDS-PAGE strongly overestimates the values of the 'higher molecular mass' components H and M. This discrepancy stems from the carboxyterminal extensions known to have unusual amino acid composition.

Animals↗
PubMed1984

Hybrid character of a large neurofilament protein (NF-M): intermediate filament type sequence followed by a long and acidic carboxy-terminal extension.

The sequence of the amino-terminal 436 residues of porcine neurofilament component NF-M (apparent mol. wt. in gel electrophoresis 160 kd), one of the two high mol. wt. components of mammalian neurofilaments, reveals the typical structural organization of an intermediate filament (IF) protein of the non-epithelial type. A non-alpha-helical arginine-rich headpiece with multiple beta-turns (residues 1-98) precedes a highly alpha-helical rod domain able to form double-stranded coiled-coils (residues 99-412) and a non-alpha-helical tailpiece array starting at residue 413. All extra mass of NF-M forms, as a carboxy-terminal tailpiece extension of approximately 500 residues, an autonomous domain of unique composition. Limited sequence data in the amino-terminal region of this domain document a lysine- and particularly glutamic acid-rich array somewhat reminiscent of the much shorter tailpiece extension of NF-L (apparent mol. wt. 68 kd), the major neurofilament protein. NF-M is therefore a true intermediate filament protein co-polymerized with NF-L via presumptive coiled-coil type interactions and not a peripherally bound associated protein of a filament backbone built exclusively from NF-L. Along the structurally conserved coiled-coil domains the two neurofilament proteins show only approximately 65% sequence identity, a value similar to that seen when NF-L and NF-M are compared with mesenchymal vimentin. The highly charged and acidic tailpiece extensions of all triplet proteins particularly rich in glutamic acid seem unique to the neurofilament type of IFs. They could form extra-filamentous scaffolds suitable for interactions with other neuronal components.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗