Skip Residues Research Articles

Cryo-EM structure of tarantula myosin filament: Implications for analysis of myopathy mutations.

The high-resolution structure of myosin thick filaments of invertebrates has aided understanding of key muscle functions and analysis of disease mutations in humans. We previously published a low-resolution tarantula skeletal thick filament cryo-EM structure (Woodhead et al., 2005), which was used to map hypertrophic and dilated cardiomyopathy mutations (Alamo et al., 2017, Nag et al., 2017), present mostly in the head-head and head-tail interaction sites of the interacting-heads motif (IHM). This study suggested that the myosin coiled-coil tails in the filament backbone were arranged in groups of three, forming 40-Å-diameter subfilaments (Wray, 1979), but it lacked the detail required to resolve individual tails and to map the 40% of myopathy mutations that occur in the tail region of the molecule. Here we report a 5.5 Å resolution cryo-EM map of the tarantula thick filament backbone that resolves the densities of the full length of the myosin tails and their packing interactions. The map reveals that the tails do not form subfilaments but are arranged individually in layers forming a “molecular crystal,” in which the myosin coiled-coils gradually move towards the center of the filament, near the paramyosin core, while slowly curving around the filament axis. A similar arrangement was found in insect flight muscle thick filaments (Hu et al., 2016), suggesting it could be a general packing scheme in thick filaments, as originally proposed by Squire (1973). The positions of skip residues predict longer pitch regions and bending of the coiled-coils, as seen in the cryo-EM density. The structure also suggests new interactions of the blocked-head converter region with the backbone of the filament, implying hyperstability of IHMs. Improved resolution, and solution of the vertebrate thick filament structure, should enable precise mapping of tail mutations.

The Myosin II Coiled-Coil Domain Atomic Structure in its Native Environment

The atomic structure of the complete myosin tail within thick filaments isolated from Lethocerus indicus flight muscle is described and compared to crystal structures of recombinant, human cardiac myosin tail segments. Overall, the agreement is good with three exceptions: the proximal S2, in which the filament has heads attached but the crystal structure doesn't, and skip regions 2 and 4. At the head-tail junction, the tail α-helices are asymmetrically structured encompassing well-defined unfolding of 12 residues for one myosin tail, ∼4 residues of the other, and different degrees of α-helix unwinding for both tail α-helices, thereby providing an atomic resolution description of coiled-coil uncoiling at the head-tail junction. Asymmetry is observed in the nonhelical C termini; one C-terminal segment is intercalated between ribbons of myosin tails, the other apparently terminating at Skip 4 of another myosin tail. Between skip residues, crystal and filament structures agree well. Skips 1 and 3 also agree well and show the expected α-helix unwinding and coiled-coil untwisting in response to skip residue insertion. Skips 2 and 4 are different. Skip 2 is accommodated in an unusual manner through an increase in α-helix radius and corresponding reduction in rise/residue. Skip 4 remains helical in one chain, with the other chain unfolded, apparently influenced by the acidic myosin C terminus. The atomic model may shed some light on thick filament mechanosensing and is a step in understanding the complex roles that thick filaments of all species undergo during muscle contraction.

Contiguity and Structural Impacts of a Non-Myosin Protein within the Thick Filament Myosin Layers.

Simple SummaryHexapods and crustaceans (Pancrustacea) represent nearly 80% of known living animals. Species within this clade exhibit exquisite muscle types propelling ingenious means of locomotion, likely contributing to their evolutionary success. Flightin, a myosin-binding protein, first identified in the flight muscle of Drosophila, is defined by WYR, a protein domain exclusive to Pancrustacea. In Drosophila, flightin imparts stiffness to the thick filament and is essential for their length determination and structural integrity. Here, we build on results from the three-dimensional reconstruction of the Lethocerus flight muscle thick filament to advance the hypothesis that flightin influences thick filament mechanics, and by extension muscle function, by acting as a cinch in the filament core.Myosin dimers arranged in layers and interspersed with non-myosin densities have been described by cryo-EM 3D reconstruction of the thick filament in Lethocerus at 5.5 Å resolution. One of the non-myosin densities, denoted the ‘red density’, is hypothesized to be flightin, an LMM-binding protein essential to the structure and function of Drosophila indirect flight muscle (IFM). Here, we build upon the 3D reconstruction results specific to the red density and its engagement with the myosin coiled-coil rods that form the backbone of the thick filament. Each independent red density winds its way through the myosin dimers, such that it links four dimers in a layer and one dimer in a neighboring layer. This area in which three distinct interfaces within the myosin rod are contacted at once and the red density extends to the thick filament core is designated the “multiface”. Present within the multiface is a contact area inclusive of E1563 and R1568. Mutations in the corresponding Drosophila residues (E1554K and R1559H) are known to interfere with flightin accumulation and phosphorylation in Drosophila. We further examine the LMM area in direct apposition to the red density and identified potential binding residues spanning up to ten helical turns. We find that the red density is associated within an expanse of the myosin coiled-coil that is unwound by the third skip residue and the coiled-coil is re-oriented while in contact with the red density. These findings suggest a mechanism by which flightin induces ordered assembly of myosin dimers through its contacts with multiple myosin dimers and brings about reinforcement on the level of a single myosin dimer by stabilization of the myosin coiled-coil.

Secondary Structure of the Novel Myosin Binding Domain WYR and Implications within Myosin Structure.

Simple SummaryWYR is a conserved protein domain characteristic of the muscle protein flightin, estimated to have originated in the ancestor to hexapods and crustaceans ~500 MYA. This study characterizes the secondary structure of WYR and shows that it binds the coiled-coil motif of muscle myosin and changes its structural properties. Characterizing WYR and its role in conjunction with myosin provides valuable insight into enduring evolutionary processes driving the success of Insecta. The relationship between WYR and myosin further reveals a means of ultrastructural regulation capable of informing the molecular structure–function relationships essential for the mechanical properties and structural stability of muscle.Structural changes in the myosin II light meromyosin (LMM) that influence thick filament mechanical properties and muscle function are modulated by LMM-binding proteins. Flightin is an LMM-binding protein indispensable for the function of Drosophila indirect flight muscle (IFM). Flightin has a three-domain structure that includes WYR, a novel 52 aa domain conserved throughout Pancrustacea. In this study, we (i) test the hypothesis that WYR binds the LMM, (ii) characterize the secondary structure of WYR, and (iii) examine the structural impact WYR has on the LMM. Circular dichroism at 260–190 nm reveals a structural profile for WYR and supports an interaction between WYR and LMM. A WYR–LMM interaction is supported by co-sedimentation with a stoichiometry of ~2.4:1. The WYR–LMM interaction results in an overall increased coiled-coil content, while curtailing ɑ helical content. WYR is found to be composed of 15% turns, 31% antiparallel β, and 48% ‘other’ content. We propose a structural model of WYR consisting of an antiparallel β hairpin between Q92-K114 centered on an ASX or β turn around N102, with a G1 bulge at G117. The Drosophila LMM segment used, V1346-I1941, encompassing conserved skip residues 2-4, is found to possess a traditional helical profile but is interpreted as having <30% helical content by multiple methods of deconvolution. This low helicity may be affiliated with the dynamic behavior of the structure in solution or the inclusion of a known non-helical region in the C-terminus. Our results support the hypothesis that WYR binds the LMM and that this interaction brings about structural changes in the coiled-coil. These studies implicate flightin, via the WYR domain, for distinct shifts in LMM secondary structure that could influence the structural properties and stabilization of the thick filament, scaling to modulation of whole muscle function.

The myosin II coiled-coil domain atomic structure in its native environment

The atomic structure of the complete myosin tail within thick filaments isolated from Lethocerus indicus flight muscle is described and compared to crystal structures of recombinant, human cardiac myosin tail segments. Overall, the agreement is good with three exceptions: the proximal S2, in which the filament has heads attached but the crystal structure doesn't, and skip regions 2 and 4. At the head-tail junction, the tail α-helices are asymmetrically structured encompassing well-defined unfolding of 12 residues for one myosin tail, ∼4 residues of the other, and different degrees of α-helix unwinding for both tail α-helices, thereby providing an atomic resolution description of coiled-coil "uncoiling" at the head-tail junction. Asymmetry is observed in the nonhelical C termini; one C-terminal segment is intercalated between ribbons of myosin tails, the other apparently terminating at Skip 4 of another myosin tail. Between skip residues, crystal and filament structures agree well. Skips 1 and 3 also agree well and show the expected α-helix unwinding and coiled-coil untwisting in response to skip residue insertion. Skips 2 and 4 are different. Skip 2 is accommodated in an unusual manner through an increase in α-helix radius and corresponding reduction in rise/residue. Skip 4 remains helical in one chain, with the other chain unfolded, apparently influenced by the acidic myosin C terminus. The atomic model may shed some light on thick filament mechanosensing and is a step in understanding the complex roles that thick filaments of all species undergo during muscle contraction.

The Atomic Model for Myosin II Coiled-Coil Shows Novel Observations and Insights into Muscle Contraction

The atomic structure of the complete myosin tail of native thick filaments from Lethocerus indicus flight muscle is reported and compared to crystal structures of recombinant, expressed human cardiac myosin tail segments. Overall, the agreement is good with three exceptions, the proximal S2, where the filament has heads attached but the crystal structure does not, Skip 2, and Skip 4. At the head-tail junction the differences in the tail α-helices are asymmetric encompassing unfolding of ∼7 residues of α-helix for one myosin head and a bend in the continuous α-helix of the other. Different degrees of α-helix unwinding is observed for both heads, thereby providing an atomic resolution description of the phenomenon known as coiled-coil “uncoiling” at the head tail junction. Asymmetry is also see in the non-helical C-termini; one C-terminal segment is intercalated between ribbons the other apparently terminates at Skip 4 of one α-helix only. Between skip residues, crystal and filament structures agree well. Skips 1 and 3 also agree well and show the expected α-helix unwinding and coiled coil untwisting response to the skip residue insertion. Skips 2 and 4 are different. Skip 2 is accommodated in an unusual manner through an increase in α-helix radius and corresponding reduction in rise/residue. Skip 4 remains helical in one chain, with the other is unfolded apparently influenced by the acidic C-terminus of a myosin peptide chain. The thick filament structure is a first step in understanding the complex roles that thick filaments of all species undergo during muscle contraction.

Peptidomic Analysis of a Disintegrated Surimi Gel from Deep-Sea Bonefish Pterothrissus gissu.

Surimi gel is a commonly found gelled product in Japan. Disintegration of the surimi gel is mainly caused by proteolytic degradation of the myosin heavy chain (MHC) under an inappropriate heating process. Many studies have reported the decrease in MHC in the disintegrated surimi gel but the mechanistic details of this degradation remain unclear. This study employed peptidomic analysis of disintegrated surimi gels from deep-sea bonefish Pterothrissus gissu to reveal the MHC cleavage causing gel disintegration. More peptides derived from an MHC rod were found in the disintegrated P. gissu surimi gels than in the integrated gel. Most MHC peptides were derived from the Src homology 3 domain or near the skip residues. The results of the terminome analysis suggest that the catalytic type of the proteases is responsible for light meromyosin cleavage activated at ∼35 °C. These results showed the temperature-dependent cleavage of the MHC rod, causing disintegration of the P. gissu surimi gel.

THERMAL STABILITY OF SYNTHETIC PEPTIDES MIMICKING THE SEQUENCE OF THE REGION CONTAINING THE SKIP RESIDUES IN SQUID MYOSIN ROD

Myosin is the major protein in skeletal muscles including those of fish and shellfish. The characteristics of this protein are closely related to the biological function and the quality and physical properties of muscle&lt;br /&gt;food. In the myosin rod (the coiled-coil region of myosin), several amino acid residues, known as skip residues, seem to destabilize the ordered structure (heptad repeat). These residues might be responsible for reducing thermal stability. Attempts were thus made to examine the role of these residues in the rod of squid myosin, based on the thermodynamic properties of synthetic peptides which have been designed to mimic the partial sequence of myosin heavy chain from the squid Todarodes pacificus mantle muscle. Five peptides, namely, with the sequence of Trp1343 -Ala1372 having the skip residue Glu1357 at the center (Peptide WT), without the skip residue (Peptide Δ), with the replacements of the skip residue (Glu) by Ile, Gln and Pro (Peptides E/I, E/Q, and E/P, respectively) to modify the helix forming propensity, were synthesized. The results obtained showed that the stability of the peptides as measured by circular dichroism spectrometry was in the order of Peptide Δ &amp;gt; Peptide WT &amp;gt; Peptide E/Q &amp;gt; Peptide E/P &amp;gt; Peptide E/I. It is suggested that the presence of the skip residues dexterously tunes the stability or flexibility of the coiled-coil structure, thus possibly regulating thick filament formation and further gel formation ability of myosin.

The SH3 domain of UNC-89 (obscurin) interacts with paramyosin, a coiled-coil protein, in Caenorhabditis elegans muscle.

UNC-89 is a giant polypeptide located at the sarcomeric M-line of Caenorhabditis elegans muscle. The human homologue is obscurin. To understand how UNC-89 is localized and functions, we have been identifying its binding partners. Screening a yeast two-hybrid library revealed that UNC-89 interacts with paramyosin. Paramyosin is an invertebrate-specific coiled-coil dimer protein that is homologous to the rod portion of myosin heavy chains and resides in thick filament cores. Minimally, this interaction requires UNC-89's SH3 domain and residues 294-376 of paramyosin and has a KD of ∼1.1 μM. In unc-89 loss-of-function mutants that lack the SH3 domain, paramyosin is found in accumulations. When the SH3 domain is overexpressed, paramyosin is mislocalized. SH3 domains usually interact with a proline-rich consensus sequence, but the region of paramyosin that interacts with UNC-89's SH3 is α-helical and lacks prolines. Homology modeling of UNC-89's SH3 suggests structural features that might be responsible for this interaction. The SH3-binding region of paramyosin contains a "skip residue," which is likely to locally unwind the coiled-coil and perhaps contributes to the binding specificity.

The Structure of the Relaxed Thick Filaments from Lethocerus Asynchronous Flight Muscle

The structure of relaxed muscle thick filaments is a key element for understanding muscle physiology and the effect of mutations on muscle function. Previous thick filament structures by 3D-EM resolved individual myosin heads and revealed an interacting head motif (IHM) first identified in smooth muscle myosin. A later model based on X-ray fiber diffraction of Lethocerus flight muscle showed a different, intermolecular, interaction between myosin heads. Almost nothing is known about the structure of the long α-helical coiled-coil myosin rod in thick filaments. Here we show the relaxed structure of Lethocerus thick filaments by cryoEM at sufficient resolution to reveal the relative placement of individual myosin heads at ∼20 A resolution, as well as the myosin rod α-helices which can be traced from the head-rod junction all the way to their C-termini at a resolution of 5.5A. In contrast to the model suggested by X-ray diffraction, we find an IHM somewhat different from IHMs of other relaxed thick filaments. The myosin rod follows a tortuous path from the outside of the thick filament to the inside across a region we call the myosin rod annulus. The pitch of the rod coiled-coil is variable with long pitch regions in the approximate location of skip residues. Surprisingly, threading their way among the forest of myosin rods are four copies, matching the 4-fold symmetry of the filament, of four polypeptide chains. Their presence in this location could modulate the mechanical properties of the myosin backbone. Our results demonstrate the ubiquity of the IHM in relaxed myosin filaments but with a variation that might shed light on the mechanism of stretch activation and shortening deactivation. Supported by NIH and AHA.

Skip residues modulate the structural properties of the myosin rod and guide thick filament assembly

The rod of sarcomeric myosins directs thick filament assembly and is characterized by the insertion of four skip residues that introduce discontinuities in the coiled-coil heptad repeats. We report here that the regions surrounding the first three skip residues share high structural similarity despite their low sequence homology. Near each of these skip residues, the coiled-coil transitions to a nonclose-packed structure inducing local relaxation of the superhelical pitch. Moreover, molecular dynamics suggest that these distorted regions can assume different conformationally stable states. In contrast, the last skip residue region constitutes a true molecular hinge, providing C-terminal rod flexibility. Assembly of myosin with mutated skip residues in cardiomyocytes shows that the functional importance of each skip residue is associated with rod position and reveals the unique role of the molecular hinge in promoting myosin antiparallel packing. By defining the biophysical properties of the rod, the structures and molecular dynamic calculations presented here provide insight into thick filament formation, and highlight the structural differences occurring between the coiled-coils of myosin and the stereotypical tropomyosin. In addition to extending our knowledge into the conformational and biological properties of coiled-coil discontinuities, the molecular characterization of the four myosin skip residues also provides a guide to modeling the effects of rod mutations causing cardiac and skeletal myopathies.

Characterization of Three Full-length Human Nonmuscle Myosin II Paralogs

Nonmuscle myosin IIs (NM IIs) are a group of molecular motors involved in a wide variety of cellular processes including cytokinesis, migration, and control of cell morphology. There are three paralogs of the NM II heavy chain in humans (IIA, IIB, and IIC), each encoded by a separate gene. These paralogs are expressed at different levels according to cell type and have different roles and intracellular distributions in vivo. Most previous studies on NM II used tissue-purified protein or expressed fragments of the molecule, which presents potential drawbacks for characterizing individual paralogs of the intact protein in vitro. To circumvent current limitations and approach their native properties, we have successfully expressed and purified the three full-length human NM II proteins with their light chains, using the baculovirus/Sf9 system. The enzymatic and structural properties of the three paralogs were characterized. Although each NM II is capable of forming bipolar filaments, those formed by IIC tend to contain fewer constituent molecules than those of IIA and IIB. All paralogs adopt the compact conformation in the presence of ATP. Phosphorylation of the regulatory light chain leads to assembly into filaments, which bind to actin in the presence of ATP. The nature of interactions with actin filaments is shown with different paralogs exhibiting different actin binding behaviors under equivalent conditions. The data show that although NM IIA and IIB form filaments with similar properties, NM IIC forms filaments that are less well suited to roles such as tension maintenance within the cell.

Characterization of Subfragment-2 Regions of Myosins from Invertebrate and Vertebrate Striated Muscles

Type II myosin, the major contractile component of muscle, is a hexamer composed of two heavy chains and four light chains (two essential and two regulatory light chains). The N terminal globular head portion of heavy chain subunit (called subfragment-1, S1) is connected to rod portion through subfragment-2 (S2) region, which causes the bends in the rod portion of myosin and might make S1 lift off the thick filament to facilitate its reaction with actin. In the present study, S2 regions from various striated muscle myosins were compared including recently sequenced ones from cephalopods to shed light on the uniqueness of these stretches. The sequences of myosins from eleven species were aligned, and the α-helical and coiled-coil formations of S2 regions were estimated. As a result, all the S2 regions were considered to have reduced propensity of coiled-coil structure, especially at around the hinge regions, suggesting these regions are structurally flexible. One skip residues was found for all the species, though glutamine was replaced by threonine for fly and vertebrate counterparts. Prediction of secondary structure revealed uniqueness of cephalopod sequence in the N terminal region of S2. This was also confirmed by coiled-coil propensity prediction. Phylogenetic tree drawn based on the amino acid sequence showed that both of scallop and cephalopod S2s formed clear clusters, and were clearly distant from the other invertebrate (fly, worm) and vertebrate counterparts. From the results obtained, it was suggested that S2 regions of muscle myosins are ingeniously tuned so as to adapt to the contraction speed of each myosin or contractile performance of each muscle.

1P-071 ミオシン・ロッドにおけるスキップ残基の役割 : 合成ペプチドを用いた検討(蛋白質・物性(1),第46回日本生物物理学会年会)

1P-071 ミオシン・ロッドにおけるスキップ残基の役割 : 合成ペプチドを用いた検討(蛋白質・物性(1),第46回日本生物物理学会年会)

Skip Residues and Charge Interactions in Myosin II Coiled-coils: Implications for Molecular Packing

Molecular packing of myosin II coiled-coil rods into myosin filaments and the role of skip residues in the heptad sequence have been investigated. Sequence comparison of rods from skeletal, smooth and non-muscle myosin II shows that different myosin II subtypes have significantly different charge distributions. Analysis of the ionic interactions between adjacent rods with changing molecular overlap relates the different patterns of charge to the different structures of skeletal and smooth muscle myosin II filaments. It is shown in the case of skeletal muscle myosin II that the skip residues have a critical role in keeping these unique patterns of charge in perfect phase. Only one of the previously suggested packing models for myosin II filaments, with a slight modification, is supported, since it satisfies all the sequence-predicted axial shifts between adjacent rods. Such analysis significantly advances understanding of myosin filament assembly properties and will help to provide a basis for the proper understanding of myosin-associated diseases.

CDNA cloning of myosin rod and the complete primary structure of myosin heavy chain of walleye pollack fast skeletal muscle

SUMMARY: The amino acid sequences of myosin rod containing subfragment-2 (S2) and light meromyosin (LMM) were determined by cDNA cloning for walleye pollack fast skeletal myosin heavy chain. While S2 and LMM were composed of 442 and 656 amino acid residues, a total of 1937 amino acid residues accounted for the whole myosin heavy chain molecule with previously determined sequence for the subfragment-1 heavy chain region of this fish. Both regions for S2 and LMM showed a seven-residue repeat pattern characteristic to fibrous proteins with a coiled-coil structure of two α-helices, displaying a, b, c, d, e, f, and g where positions a and d were frequently occupied by hydrophobic amino acids and c and g often contained charged residues. The occurrence of a 28-residue unit with repetitive sequence was also strongly suggested, when one and three skip residues were adopted into S2 and LMM, respectively. Thus, walleye pollack S2 and LMM consisted of 17 and 24 zones with a 28-residue repeat rearrangement. There were several amino acid substitutions which might account for a low thermal stability of walleye pollack myosin heavy chain in comparison with the sequences of higher vertebrate counterparts. However, it seemed difficult to interpret such low stability only from the comparison in the 28-residue repeat arrangement at the primary structure.

Analysis of striated fiber formation by recombinant SF-assemblin in Vitro

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.

Cryo-atomic force microscopy of smooth muscle myosin

The motor and regulatory domains of the head and the 14-nm pitch of the alpha-helical coiled-coil of the tail of extended (6S) smooth-muscle myosin molecules were imaged with cryo atomic force microscopy at 80-85 K, and the effects of thiophosphorylation of the regulatory light chain were examined. The tail was 4 nm shorter in thiophosphorylated than in nonphosphorylated myosin. The first major bend was invariant, at approximately 51 nm from the head-tail junction (H-T), coincident with low probability in the paircoil score. The second major bend was 100 nm from the H-T junction in nonphosphorylated and closer to a skip residue than the bend (at 95 nm) in thiophosphorylated molecules. The shorter tail and distance between the two major bends induced by thiophosphorylation are interpreted to result from melting of the coiled-coil. An additional bend not previously reported occurred, with a lower frequency, approximately 24 nm from the H-T. The range of separation between the two heads was greater in thiophosphorylated molecules. Occasional high-resolution images showed slight unwinding of the coiled-coil of the base of the heads. We suggest that phosphorylation of MLC20 can affect the structure of extended, 6S myosin.

The cruciated microtubule-associated fibers of the green alga Dunaliella bioculata consist of a 31 kDa SF-assemblin

Cytoskeletons of Dunaliella bioculata, the biflagellate wallless green alga, were isolated and analyzed using a monoclonal and a polyclonal antibody raised against SF-assemblin, the major protein of the two striated microtubule-associated fibers of the alga Spermatozopsis similis. Indirect immunofluorescence showed antigenic structures associated with the four microtubular flagellar roots. SDS-PAGE followed by immunoblot analysis revealed a cross-reacting polypeptide of 31 kDa. This protein of D. bioculata was isolated using gel filtration chromatography in 8 M urea and in vitro reassembly of striated fibers. Microsequencing of the purified protein yielded various peptides, which could be aligned along the sequence of SF-assemblin from S. similis. A complete sequence of the Dunaliella protein was obtained by cDNA cloning. It documents the non helical head domain followed by a helical rod domain with a 29 residue repeat pattern based on four heptads followed by a skip residue. Compared to SF-assemblin of S. similis the SF-assemblin of Dunaliella has a shorter head and a slightly longer rod domain. The two algal SF-assemblins share only 57% sequence identity. We conclude that SF-assemblin and related proteins in various protists are representatives of a new class of alpha-helical proteins characterized by the ability to form a special segmented coiled coil and to assemble into striated fibers of 2 nm protofilaments in vivo and in vitro.

Chapter 44 Purification of SF-Assemblin

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.