Filament Helix Research Articles

Macroscopic and microscopic mechanical behaviors of climbing tendrils

Tendril-bearing climbing plants must recur to the tendril helices with chiral perversion or dual chirality for climbing and to obtain sun exposure. Despite researchers’ prolonged fascination with climbing tendrils since Darwin’s time and even earlier, why the soft and slender tendrils can bear heavy loads such as the self-weight of a plant or additional load caused by rain remains elusive. In this paper, we take towel gourd tendrils as an example and investigate the macroscopic and microscopic mechanical behaviors of tendrils through experiments and simulations. Our study indicates that the tendril filament exhibits rubber-like hyperelastic behaviors and can particularly endure large elongation, which is mainly attributed to the superelasticity of the cellulose fibril helix contained in the cell wall. Combination of the tendril helical structure with dual chirality or chiral perversion at a macroscale and a cellulose filament helix at a subcellular level creates superior elasticity for biological species relying on support and climbing. This study provides deep insight into the structure–property relationship of climbing tendrils, and the relationship is useful for the bioinspired design of composite systems with superior elasticity.

Unusual winding of helices under tension

An unusual overwinding and a subsequently unwinding behavior of helix of filament with twisted microstructures under stretch is analyzed from the nonlinear variation of material chirality (i.e., chirality in material properties), motivated by recent single-molecule experiments for B-DNA. We establish a refined Cosserat rod model, which reproduces the deformation behaviors of filament incorporating its microscopic chirality. The helix of filament with twisted microstructures is found to exhibit a DNA-like nonlinear variation of material chirality under large elongation. The multiscale interactions between microscopic chirality of twisted microstructures and macroscopic chirality of helix determine the nonlinear coupling behaviors of hierarchically chiral structured materials. These results provide a fundamental principle for understanding the functions and mechanisms of chiral materials.

Study of yarn twist angle using numerical simulation

The twist angle is an important parameter of the yarn which determines the degree and shape of the filament’s deformation produced along their length. So, in this paper, a 3D numerical modelling of filaments assemblies has been developed. The twist behaviour of the multifilament was simulated using ABAQUS finite element package by modelling the yarns as 3D continuum elements. The distribution of filament helix angle according to the radial position of the yarn is evaluated. Results show that the helix angle draws near zero in the yarn axis but does not fall to zero in presence of migration, and becomes bigger as the filaments are positioned on the surface. Moreover, the twist angle is calculated for different twist level which proves that twist angle is highly affected by torsion level. The target of the research was to establish a relationship between twist angle, twist value and yarn linear density for a series of models varying in the filament radius, twist value and filament number.

The structural dynamics of α-tropomyosin on F-actin shape the overlap complex between adjacent tropomyosin molecules

Coiled-coil tropomyosin, localized on actin filaments in virtually all eukaryotic cells, serves as a gatekeeper regulating access of the motor protein myosin and other actin-binding proteins onto the thin filament surface. Tropomyosin’s modular pseudo-repeating pattern of approximately 39 amino acid residues is designed to allow binding of the coiled-coil to successive actin subunits along thin filaments. Even though different tropomyosin isoforms contain varying numbers of repeat modules, the pseudo-repeat length, in all cases, matches that of a single actin subunit. Thus, the seven pseudo-repeats of 42nm long muscle tropomyosin bind to seven successive actin subunits along thin filaments, while simultaneously bending into a super-helical conformation that is preshaped to the actin filament helix. In order to form a continuous cable on thin filaments that is free of gaps, adjacent tropomyosin molecules polymerize head-to-tail by means of a short (∼9 residue) overlap. Several laboratories have engineered peptides to mimic the N- and C-terminal tropomyosin association and to characterize the overlap structure. All overlapping domains examined show a compact N-terminal coiled-coil inserting into a partially opened C-terminal partner, where the opposing coiled-coils at the overlap junction face each other at up to ∼90° twist angles. Here, Molecular Dynamics (MD) simulations were carried out to determine constraints on the formation of the tropomyosin overlap complex and to assess the amount of twisting exhibited by full-length tropomyosin when bound to actin. With the exception of the last 20–40 C- and N-terminal residues, we find that the average tropomyosin structure closely resembles a “canonical” model proposed in the classic work of McLachlan and Stewart, displaying perfectly symmetrical supercoil geometry matching the F-actin helix with an integral number of coiled-coil turns, a coiled-coil helical pitch of 137Å, a superhelical pitch of 770Å, and no localized pseudo-rotation. Over the middle 70% of tropomyosin, the average twisting of the coiled-coil deviates only by 10° from the canonical model and the torsional freedom is very small (std. dev. of 7°). This small degree of twisting cannot yield the orthogonal N- and C-termini configuration observed experimentally. In marked contrast, considerable coiled-coil unfolding, splaying and twisting at N- and C-terminal ends is observed, providing the conformational plasticity needed for head-to-tail nexus formation.

Visualization of Troponin on Muscle Thin Filaments by Single Particle Analysis

Troponin on the thin filaments of striated muscle couples Ca-concentration changes to movement of tropomyosin. Ca-free troponin is thought to shift tropomyosin to the myosin-blocking position, a constraint that is released after Ca binds. Although the location and regulatory movements of tropomyosin have been defined at near-atomic resolution, the organization of troponin on thin filaments has not been determined definitively, and different models of troponin position on actin are contradictory. Here novel single-particle analysis (SPA) protocols were designed to reconstruct thin filament structure from electron micrographs of negatively-stained cardiac muscle thin filaments at low-Ca. Troponin-tropomyosin has a tendency to dissociate from filaments under EM conditions, and only filaments showing evidence of bound troponin were chosen for analysis. The axial location of troponin densities on actin was identified by newly developed algorithms. Filament polarity was determined according to Narita and Maeda (2007). Filaments were divided computationally into 46.8 nm particles centered on troponin. SPA was carried out on these particles without applying helical averaging, and successively refined reconstructions did not degrade the troponin-tropomyosin signal. Tropomyosin strands extended smoothly and continuously over adjacent actin monomers along the long-pitch filament helix, occupying the myosin blocking position. Tropomyosin atomic models fitted well to corresponding densities close to the binding site on actin described in Li et al. (2011) for troponin-free filaments. The troponin core-domain position and orientation deviated slightly from those proposed by Pirani et al. (2006). A broadened tropomyosin strand density found on the barbed-end side of the troponin core-domain appeared to bifurcate to link tropomyosin and the core-domain. We attribute this density to the tail of troponin-T, whose location corroborates the polarity of troponin on actin proposed by Flicker et al. (1982).

The different muscle-energetics during shortening and stretch.

The helical shape of the thin filaments causes their passive counterclockwise rotation during muscle stretch that increases tensile stress and torque at first by unwinding and then by winding up the four anchoring Z-filaments. This means storage of energy in the series elastic Z-filaments and a considerable decrease of the liberated energy of heat and work to (h—wap), where h is the heat energy and wap the stretch energy induced from outside by an apparatus. The steep thin filament helix with an inclination angle of 70° promotes the passive rotation during stretch, but impedes the smooth sliding of shortening by increased friction and production of frictional heat. The frictional heat may be produced by the contact with the myosin cross-bridges: (1) when they passively snap on drilling thin filaments from cleft to cleft over a distance 2 × 2.7 nm = 5.4 nm between the globular actin monomers in one groove, causing stepwise motion; or (2) when they passively cycle from one helical groove to the next (distance 36 nm). The latter causes more heat and may take place on rotating thin filaments without an effective forward drilling (“idle rotation”), e.g., when they produce “unexplained heat” at the beginning of an isometric tetanus. In an Appendix to this paper the different states of muscle are defined. The function of its most important components is described and rotation model and power-stroke model of muscular contraction is compared.

Actin Filaments Stabilize Locally at Random Sites

After the polymerization of actin monomers into filaments, the actin-bound ATP is hydrolyzed into ADP, a process that was believed to decrease the filament stability. Recent experiments suggest the opposite behavior, however, namely that actin filaments become increasingly stable with time. Several mechanisms for this stabilization have been proposed, ranging from structural transitions of the whole filament helix to pure artifacts arising, e.g., from the capping or surface attachment of the filament ends. We performed novel fluorescence microscopy experiments on single filaments to clarify this controversial issue. We find that filaments do indeed cease to depolymerize in an abrupt manner, and that this transition happens on relatively long time scales that exceed those of both ATP cleavage and phosphate release. We also developed a theory that allows us to distinguish the different possible stabilization mechanisms. A detailed comparison of theory and experiment implies that the sudden truncation of the shrinkage process does neither arise from artifacts nor from a collective transition of the whole filament. Instead, our results provide strong evidence for a local stabilization process occurring at random sites within the filament.

Conformational flexibility of RecA protein filament: Transitions between compressed and stretched states

RecA protein is a central enzyme in homologous DNA recombination, repair and other forms of DNA metabolism in bacteria. It functions as a flexible helix-shaped filament bound on stretched single-stranded or double-stranded DNA in the presence of ATP. In this work, we present an atomic level model for conformational transitions of the RecA filament. The model describes small movements of the RecA N-terminal domain due to coordinated rotation of main chain dihedral angles of two amino acid residues (Psi/Lys23 and Phi/Gly24), while maintaining unchanged the RecA intersubunit interface. The model is able to reproduce a wide range of observed helix pitches in transitions between compressed and stretched conformations of the RecA filament. Predictions of the model are in agreement with Small Angle Neutron Scattering (SANS) measurements of the filament helix pitch in RecA::ADP-AlF(4) complex at various salt concentrations.

Enhanced motility of a Proteus mirabilis strain expressing hybrid FlaAB flagella

Proteus mirabilis has two tandemly arranged flagellin-encoding genes, flaA and flaB. flaA is transcribed from a sigma(28) promoter, while flaB is silent. flaA and flaB can undergo reversible rearrangement to produce a set of hybrid genes referred to as flaAB. Flagellins composed of FlaAB protein have a different amino acid sequence and are antigenically distinct from flagellin composed of FlaA, implicating flagellin gene conversion as a putative virulence mechanism for P. mirabilis. The change in amino acid sequence is also hypothesized to alter the filament helix and, hence, affect the motility of FlaAB-expressing strains. To test this hypothesis, the motility of wild-type P. mirabilis was compared with that of a strain, DF1003, locked into the FlaAB(+) hybrid phase, under conditions of altered ionic strength, pH and viscosity. Cell motion tracking analysis showed that DF1003 has wild-type swimming velocity at physiological conditions, but moves significantly faster and travels further compared to the wild-type at NaCl concentrations greater than 170 mM. DF1003 is also significantly faster than the wild-type at pH 5.2, 5.8 and 8.2, and at 5 and 10 % polyvinylpyrrolidone. Measurements of amplitude and wavelength for isolated flagella subjected to pH 5.8 or 425 mM NaCl showed a loss of helical structure in FlaA flagella compared to FlaAB filaments, a feature that could significantly affect motility under these conditions. These results support a hypothesis that FlaAB flagellin imparts a motile advantage to P. mirabilis in conditions that otherwise may impede bacterial movement. In a broader context, flagellar antigenic variation, commonly thought to serve as means to avoid host defences, may also enhance motility in other bacterial species, thus aiding in the adaptation and survival of the cells.

Local destabilization of the tropomyosin coiled coil gives the molecular flexibility required for actin binding.

Tropomyosin, a coiled coil protein that binds along the length of actin filaments, contains 40 uninterrupted heptapeptide repeats characteristic of coiled coils. Yet, it is flexible. Regions of tropomyosin that may be important for binding to the filament and for interacting with troponin deviate from canonical coiled coil structure in subtle ways, altering the local conformation or energetics without interrupting the coiled coil. In a region rich in interface alanines (an Ala cluster), the chains pack closer than in canonical coiled coils, and are staggered, resulting in a bend [Brown et al. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 8496-8501]. Brown et al. suggested that bends at alanine clusters allow tropomyosin to wind on the actin filament helix. Another explanation is that local destabilization of the coiled coil, rather than close packing of the chains at Ala clusters per se, allows flexibility. Changing three Ala residues to canonical interface residues, A74L-A78V-A81L, greatly stabilized tropomyosin, measured using circular dichroism and differential scanning calorimetry, and reduced actin affinity >10-fold. Normal actin affinity and stability were restored in a mutant A74Q-A78N-A81Q that mimicked the stability of the Ala cluster but not the close packing of the chains. Analysis and modeling of comparable mutations introduced closer to the N-terminus show that the effects on stability and function depend on context. Models based on tropomyosin crystal structures give insight into possible effects of the mutations on the structure. We conclude that the significance of the Ala clusters in allowing flexibility of tropomyosin is stability-driven.

Three-dimensional structure of thin myocardial filaments in cardiac insufficiency caused by toxic allergic myocarditis

Diffraction patterns of a single myocardial thin filament and its Mg-paracrystals from normal myocardium and in cardiac insufficiency caused by toxic allergic myocarditis were presented, and three-dimensional reconstruction of normal and pathological filaments was carried out. It was shown that in a rigor solution pathological actin protomers are elongated, while normal filaments are kidney-shaped. It is concluded that changes in the thin myocardial filament helix parameters in heart failure caused by toxic allergic myocarditis are due to the loss of conformational mobility of native actin filaments.

Temperature-induced structural changes in the myosin thick filament of skinned rabbit psoas muscle

By using synchrotron radiation and an imaging plate for recording diffraction patterns, we have obtained high-resolution x-ray patterns from relaxed rabbit psoas muscle at temperatures ranging from 1 degree C to 30 degrees C. This allowed us to obtain intensity profiles of the first six myosin layer lines and apply a model-building approach for structural analysis. At temperatures 20 degrees C and higher, the layer lines are sharp with clearly defined maxima. Modeling based on the data obtained at 20 degrees C reveals that the average center of the cross-bridges is at 135 A from the center of the thick filament and both of the myosin heads appear to wrap around the backbone. At 10 degrees C and lower, the layer lines become very weak and diffuse scattering increases considerably. At 4 degrees C, the peak of the first layer line shifts toward the meridian from 0.0047 to 0.0038 A(-1) and decreases in intensity approximately by a factor of four compared to that at 20 degrees C, although the intensities of higher-order layer lines remain approximately 10-15% of the first layer line. Our modeling suggests that as the temperature is lowered from 20 degrees C to 4 degrees C the center of cross-bridges extends radially away from the center of the filament (135 A to 175 A). Furthermore, the fraction of helically ordered cross-bridges decreases at least by a factor of two, while the isotropic disorder (the temperature factor) remains approximately unchanged. Our results on the order/disordering effects of temperature are in general agreement with earlier results of Wray [Wray, J. 1987. Structure of relaxed myosin filaments in relation to nucleotide state in vertebrate skeletal muscle. J. Muscle Res. Cell Motil. 8:62a (Abstr.)] and Lowy et al. (Lowy, J., D. Popp, and A. A. Stewart. 1991. X-ray studies of order-disorder transitions in the myosin heads of skinned rabbit psoas muscles. Biophys. J. 60:812-824). and support Poulsen and Lowy's hypothesis of coexistence of ordered and disordered cross-bridge populations in muscle (Poulsen, F. R., and J. Lowy. 1983. Small angle scattering from myosin heads in relaxed and rigor frog skeletal muscle. Nature (Lond.). 303:146-152.). However, our results added new insights into the disordered population. Present modeling together with data analysis (Xu, S., S. Malinchik, Th. Kraft, B. Brenner, and L. C. Yu. 1997. X-ray diffraction studies of cross-bridges weakly bound to actin in relaxed skinned fibers of rabbit psoas muscle. Biophys. J. 73:000-000) indicate that in a relaxed muscle, cross-bridges are distributed in three populations: those that are ordered on the thick filament helix and those that are disordered; and within the disordered population, some cross-bridges are detached and some are weakly attached to actin. One critical conclusion of the present study is that the apparent order <--> disorder transition as a function of temperature is not due to an increase/decrease in thermal motion (temperature factor) for the entire population, but a redistribution of cross-bridges among the three populations. Changing the temperature leads to a change in the fraction of cross-bridges located on the helix, while changing the ionic strength at a given temperature affects the disordered population leading to a change in the relative fraction of cross-bridges detached from and weakly attached to actin. Since the redistribution is reversible, we suggest that there is an equilibrium among the three populations of cross-bridges.

Structure and periodicities of cross-bridges in relaxation, in rigor, and during contractions initiated by photolysis of caged Ca2+

Ultra-rapid freezing and electron microscopy were used to directly observe structural details of frog muscle fibers in rigor, in relaxation, and during force development initiated by laser photolysis of DM-nitrophen (a caged Ca2+). Longitudinal sections from relaxed fibers show helical tracks of the myosin heads on the surface of the thick filaments. Fibers frozen at approximately 13, approximately 34, and approximately 220 ms after activation from the relaxed state by photorelease of Ca2+ all show surprisingly similar cross-bridge dispositions. In sections along the 1,1 lattice plane of activated fibers, individual cross-bridge densities have a wide range of shapes and angles, perpendicular to the fiber axis or pointing toward or away from the Z line. This highly variable distribution is established very early during development of contraction. Cross-bridge density across the interfilament space is more uniform than in rigor, wherein the cross-bridges are more dense near the thin filaments. Optical diffraction (OD) patterns and computed power density spectra of the electron micrographs were used to analyze periodicities of structures within the overlap regions of the sarcomeres. Most aspects of these patterns are consistent with time resolved x-ray diffraction data from the corresponding states of intact muscle, but some features are different, presumably reflecting different origins of contrast between the two methods and possible alterations in the structure of the electron microscopy samples during processing. In relaxed fibers, OD patterns show strong meridional spots and layer lines up to the sixth order of the 43-nm myosin repeat, indicating preservation and resolution of periodic structures smaller than 10 nm. In rigor, layer lines at 18, 24, and 36 nm indicate cross-bridge attachment along the thin filament helix. After activation by photorelease of Ca2+, the 14.3-nm meridional spot is present, but the second-order meridional spot (22 nm) disappears. The myosin 43-nm layer line becomes less intense, and higher orders of 43-nm layer lines disappear. A 36-nm layer line is apparent by 13 ms and becomes progressively stronger while moving laterally away from the meridian of the pattern at later times, indicating cross-bridges labeling the actin helix at decreasing radius.

Actin-actin contact: chemical cross-linking between actin and the 2.6-kDa peptide from subdomain 4 of actin.

Previously, we demonstrated that the 2.6-kDa peptide extending from Arg177 to Tyr198 in subdomain 4 of rabbit skeletal actin bound to actin itself, inhibited the elongation of actin filament, and severed F-actin. The corresponding segment in actin, therefore, is thought to contain the most critical actin-actin contact [Hori, K. and Morita, F. (1992) J. Biochem. 112, 401-408; Hori, K., Itoh, T., Takahashi, K., and Morita, F. (1994) Biochim. Biophys. Acta 1186, 35-42]. In this paper, we report on the binding site in actin for the 2.6-kDa peptide studied by using a zero-length cross-linker, 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC). We conducted limited digestion of actin cross-linked with the 125I-labeled 2.6-kDa peptide with various proteases, and developed peptide maps. The cross-linked region of the 2.6-kDa peptide was found to be within the region of Ala114 to Glu167 in actin by identifying the radioactive peptide fragments. The region was further restricted by isolation of radioactive peptide from alpha-chymotryptic digest of the cross-linked actin. The binding site of the 2.6-kDa peptide was finally assigned to be within the 24 amino acid segment from Ala144 to Glu167, which lies in subdomain 3 of actin. Using computer graphics, actin-actin contact provided by the two segments was suggested to be along the left-handed genetic helix of actin filament.

Comparison of 13Å helical and 7Å crystallographic projection maps of frozen, hydrated acrosomal bundle from limulus sperm

Actin associates with crosslinking proteins to form bundles and networks. These assemblies can provide a scaffold to anchor organelles, a support for the cell membrane or can attach the cell to the extracellular matrix or to other cells. Each crosslinker must have at least two actin binding sites, one for each actin filament. Scruin, in the Limulus acrosomal bundle, shows no homology with other actin cross-linking proteins. However, it has two homologous domains also found in the sequence encoded by kelch, a gene in Drosophila that is important in nutrient transport into the oocyte, and in MIPP, a mouse placental protein.We have determined a 13Å helical 3-dimensional structure of the actin-scruin complex, which is the basic repeat unit of the acrosomal bundle in Limulus sperm and have fitted the F-actin filament of Holmes. Each scruin binds to two adjacent actin molecules along a single F-actin filament helix. In the present investigation, we used our high resolution (7Å) spot scan electron images of ice-embedded acrosomal bundles for further analysis of the actin atomic map and scruin-scruin contacts. Spot-scan images were obtained from the frozen, hydrated acrosomal bundles in a 400 kV electron cryomicroscope.

Three-dimensional structure of a single filament in the Limulus acrosomal bundle: scruin binds to homologous helix-loop-beta motifs in actin.

Frozen, hydrated acrosomal bundles from Limulus sperm were imaged with a 400 kV electron cryomicroscope. Segments of this long bundle can be studied as a P1 crystal with a unit cell containing an acrosomal filament with 28 actin and 28 scruin molecules in 13 helical turns. A novel computational procedure was developed to extract single columns of superimposed acrosomal filaments from the distinctive crystallographic view. Helical reconstruction was used to generate a three-dimensional structure of this computationally isolated acrosomal filament. The scruin molecule is organized into two domains which contact two actin subunits in different strands of the same actin filament. A correlation of Holmes' actin filament model to the density in our acrosomal filament map shows that actin subdomains 1, 2, and 3 match the model density closely. However, actin subdomain 4 matches rather poorly, suggesting that interactions with scruin may have altered actin conformation. Scruin makes extensive interactions with helix-loop-beta motifs in subdomain 3 of one actin subunit and in subdomain 1 of a consecutive actin subunit along the genetic filament helix. These two actin subdomains are structurally homologous and are closely spaced along the actin filament. Our model suggests that scruin, which is derived from a tandemly duplicated gene, has evolved to bind structurally homologous but non-identical positions across two consecutive actin subunits.

X-ray scattering by myosin S-1: implications for the steric blocking model of muscle control.

Steric blocking and unblocking1–3 of the myosin interaction sites in muscle thin filaments by tropomyosin provides a simple yet elegant method of regulating contraction in skeletal muscle. Recently, this model was challenged4 by the finding that tropomyosin is located on the opposite side of the thin filament helix axis from where Moore et al. reported that the myosin cross-bridge, S-1, bound5. However, new three-dimensional image reconstructions of electron micrographs of thin filaments decorated with S-1 were obtained by Taylor and Amos6, who proposed that the prior assignment of the S-1 binding site of Moore et al.5 was incorrect, and that consequently tropomyosin and S-1 are located on the same side of the actin helix. These past difficulties and the current lack of agreement7 on the location of the S-1 binding site suggested that another technique would be useful. We report here that the shape of S-1, derived from reconstructions using the Taylor and Amos actomyosin interpretation, gives a better fit to the X-ray scattering pattern observed from S-1 in solution than alternative interpretations.