Disorder profile of nebulin encodes a vernierlike position sensor for the sliding thin and thick filaments of the skeletal muscle sarcomere.

Nebulin is an about 1µm long intrinsically disordered scaffold for the thin filaments of skeletal muscle sarcomere. It is a multifunctional elastic protein that wraps around actin filament, stabilizes thin filaments, and regulates Ca-dependent actomyosin interactions. This study investigates whether the disorder profile of nebulin might encode guidelines for thin and thick filament interactions in the sarcomere of the skeletal muscle. The question was addressed computationally by analyzing the predicted disorder profile of human nebulin (6669 residues, ∼200 actin-binding repeats) by pondr and the periodicity of the A-band stripes (reflecting the locations of myosin-associated proteins) in the electron micrographs of the sarcomere. Using the detrended fluctuation analysis, a scale factor for the A-band stripe image data with respect to the nebulin disorder profile was determined to make the thin and thick filaments aligned to have maximum correlation. The empirical mode decomposition method was then applied to identify hidden periodicities in both the nebulin disorder profile and the rescaled A-band data. The decomposition reveals three characteristic length scales (45 nm, 100 nm, and 200 nm) that are relevant for correlational analysis. The dynamical cross-correlation analyses with moving windows at various sarcomere lengths depict a vernierlike design for both periodicities, thus enabling nebulin to sense position and fine tune sarcomere overlap. This shows that the disorder profile of scaffolding proteins may encode a guideline for cellular architecture.

Engineering of an elastic scaffolding polyprotein based on an SH3-binding intrinsically disordered titin PEVK module.

Titin is a large elastic protein found in muscle that maintains the elasticity and structural integrity of the sarcomere. The PEVK region of titin is intrinsically disordered, highly elastic and serves as a hub to bind signaling proteins. Systematic investigation of the structure and affinity profile of the PEVK region will provide important information about the functions of titin. Since PEVK is highly heterogeneous due to extensive differential splicing from more than one hundred exons, we engineered and expressed polyproteins that consist of a defined number of identical single exon modules. These customized polyproteins reduce heterogeneity, amplify interactions of less dominant modules, and most importantly, provide tags for atomic force microscopy and allow more readily interpretable data from single-molecule techniques. Expression and purification of recombinant polyprotein with repeat regions presented many technical challenges: recombination events in tandem repeats of identical DNA sequences exacerbated by high GC content, toxicity of polymer plasmid and expressed protein to the bacteria; early truncation of proteins expressed with different numbers of modules; and extreme sensitivity to proteolysis. We have investigated a number of in vitro and in vivo bacterial and yeast expression systems, as well as baculoviral systems as potential solutions to these problems. We successfully expressed and purified in gram quantities a polyprotein derived from human titin exon 172 using Pichia pastoris yeast. This study provides valuable insights into the technical challenges regarding the engineering and purification of a tandem repeat sequence of an intrinsically disordered biopolymer.

Extensive and modular intrinsically disordered segments in C. elegans TTN-1 and implications in filament binding, elasticity and oblique striation.

TTN-1, a titin like protein in Caenorhabditis elegans, is encoded by a single gene and consists of multiple Ig and fibronectin 3 domains, a protein kinase domain and several regions containing tandem short repeat sequences. We have characterized TTN-1's sarcomere distribution, protein interaction with key myofibrillar proteins as well as the conformation malleability of representative motifs of five classes of short repeats. We report that two antibodies developed to portions of TTN-1 detect an approximately 2-MDa polypeptide on Western blots. In addition, by immunofluorescence staining, both of these antibodies localize to the I-band and may extend into the outer edge of the A-band in the obliquely striated muscle of the nematode. Six different 300-residue segments of TTN-1 were shown to variously interact with actin and/or myosin in vitro. Conformations of synthetic peptides of representative copies of each of the five classes of repeats--39-mer PEVT, 51-mer CEEEI, 42-mer AAPLE, 32-mer BLUE and 30-mer DispRep--were investigated by circular dichroism at different temperatures, ionic strengths and solvent polarities. The PEVT, CEEEI, DispRep and AAPLE peptides display a combination of a polyproline II helix and an unordered structure in aqueous solution and convert in trifluoroethanol to alpha-helix (PEVT, CEEEI, DispRep) and beta-turn (AAPLE) structures, respectively. The octads in BLUE motifs form unstable alpha-helix-like structures coils in aqueous solution and negligible heptad-based, alpha-helical coiled-coils. The alpha-helical structure, as modeled by threading and molecular dynamics simulations, tends to form helical bundles and crosses based on its 8-4-2-2 hydrophobic helical patterns and charge arrays on its surface. Our finding indicates that APPLE, PEVT, CEEEI and DispRep regions are all intrinsically disordered and highly reminiscent of the conformational malleability and elasticity of vertebrate titin PEVK segments. The proposed presence of long, modular and unstable alpha-helical oligomerization domains in the BLUE region of TTN-1 could bundle TTN-1 and stabilize oblique striation of the sarcomere.

Nanomechanics of full-length nebulin: an elastic strain gauge in the skeletal muscle sarcomere.

Nebulin, a family of giant modular proteins (MW 700-800 kDa), acts as a F-actin thin filament ruler and calcium-linked regulator of actomyosin interaction. The nanomechanics of full length, native rabbit nebulin was investigated with an atomic force microscope by tethering, bracketing, and stretching full-length molecules via pairs of site-specific antibodies that were attached covalently, one to a protein resistant self-assembled monolayer of oligoethylene glycol and the other to the cantilever. Using this new nanomechanics platform that enables the identification of single molecule events via an unbiased analysis of detachment force and distance of all force curves, we showed that nebulin is elastic and extends to approximately 1 microm by external force up to an antibody detachment force of approximately 300-400 pN. Upon stretching, nebulin unravels and yields force spectra with craggy mountain range profiles with variable numbers and heights of force peaks. The peak spacings, analyzed by the model-independent, empirical Hilbert-Huang transform method, displayed underlying periodicities at approximately 15 and approximately 22 nm that may result from the unfolding of one or more nebulin modules between force peaks. Nebulin may act as an elastic strain gauge that interacts optimally with actin only under appropriate strain and stress. This stretch to match protein ruler may also exert a compressive force that stabilizes thin filaments against stress during contraction. We propose that the elasticity of nebulin is integral and essential in the muscle sarcomere.

Surface initiated actin polymerization from top-down manufactured nanopatterns.

Protocols to fabricate high aspect-ratio biologically-based nanostructures using a top-down fabricated polymer platform and surface-initiated actin polymerization were developed.

What the buzz was all about: superfast song muscles rattle the tymbals of male periodical cicadas.

Male cicadas produce mating calls by oscillating a pair of superfast tymbal muscles in their anterior abdominal cavity that pull on and buckle stiff-ribbed cuticular tymbal membranes located beneath the folded wings. The functional anatomy and rattling of the tymbal organ in 17 yr periodical cicada, Magicicada cassini (Brood X), were revealed by high-resolution microcomputed tomography, magnetic resonance imaging, electron microscopy, and laser vibrometry to understand the mechanism of sound production in these insects. Each 50 Hz muscle contraction yielded five to six stages of rib buckling in the tymbal, and a small release of muscle tension resulted in a rapid recovery due to the spring-loaded nature of the stiff ribs in the resilin-rich tymbal. The tymbal muscle sarcomeres have thick and thin filaments that are 30% shorter than those in flight muscles, with Z-bands that were thicker and configured into novel perforated hexagonal lattices. Caffeine-treated fibers supercontracted by allowing thick filaments to traverse the Z-band through its open lattice. This superfast sonic muscle illustrates design features, especially the matching hexagonal symmetry of the myofilaments and the perforated Z-band that contribute to high-speed contractions, long endurance, and potentially supercontraction needed for producing enduring mating songs and choruses.

Functionalized self-assembled monolayers on ultraflat gold as platforms for single molecule force spectroscopy and imaging.

Single molecule force spectroscopy is a valuable tool for studying unfolding and nanomechanical properties of proteins. The common practice is to stretch proteins from a surface that was dosed to give a reasonable hit rate and to analyze the curves that exhibit the expected characteristics of a single polymer. Whether the surface-bound proteins are indeed single and isolated remains unclear, and the undesirable protein/surface interactions that obscure informative features of the force curves are implicitly assumed to be absent. In this study, mixed self-assembled monolayers (SAMs) consisting of N-hydroxysuccinimide (NHS) and oligoethylene glycol (OEG) terminated thiols on an ultraflat gold surface were used to covalently immobilize proteins via lysine residues. By the optimization of attachment sites via lysine-NHS linkages amidst a protein-resistant layer of the OEG SAM, it was possible to isolate single proteins for study in a controlled fashion. The single protein distribution on the surface is clearly demonstrated by atomic force microscopy (AFM) imaging. The OEG also significantly reduces nonspecific tip-surface interactions between the cantilever and surface. Stretching covalently attached single proteins produces high-quality and reproducible force-extension curves. This experimental strategy is an attractive platform with which to study protein structure, interactions, and nanomechanical properties of single proteins.

Titin as a giant scaffold for integrating stress and Src homology domain 3-mediated signaling pathways: the clustering of novel overlap ligand motifs in the elastic PEVK segment.

The richness of proline sequences in titins qualifies these giant proteins as the largest source of intrinsically disordered structures in nature. An extensive search and analysis for Src homology domain 3 (SH3) ligand motifs revealed a myriad of broadly distributed SH3 ligand motifs, with the highest density in the PEVK segments of human titin. Besides the canonical class I and II motifs with opposite orientations, novel overlapping motifs consisting of one or more of each canonical motif are abundant. Experimentally, the binding affinity and critical residues of these putative titin-based SH3 ligands toward nebulin SH3 and other SH3-containing proteins in muscle and non-muscle cell extracts were validated with peptide array technology and by the sarcomere distribution of SH3-containing proteins. A 28-mer overlapping motif-containing PEVK module binds to nebulin SH3 in and around the canonical cleft, especially to the acidic residues in the loops, as revealed by NMR titration. Molecular dynamics and molecular docking studies indicated that the overlapping motif can bind in opposite orientations with comparable energy and contact areas and predicts correctly orientation-specific contacts in NMR data. We propose that the overlap ligand motifs are a new class of ligands with innate ability to dictate SH3 domain orientation and to facilitate the rate, strength, and stereospecificity of receptor interactions. Proline-rich sequences of titins are candidates as major hubs of SH3-dependent signaling pathways. The interplay of elasticity and dense clustering of mixed receptor orientations in titin PEVK segment have important implications for the mechanical sensing, force sensitivity, and inter-adapter interactions in signaling pathways.

Multimodal imaging of the sonic organ of Porichthys notatus, the singing midshipman fish.

The sonic midshipman fish, Porichthys notatus, is a bottom-dwelling species whose swim bladder has evolved into a highly specialized, sound-producing organ. The males of this species exist in two distinct morphs with different physical characteristics and sexual strategies. The Type I males have a much larger sound organ and are capable of generating a loud approximately 100 Hz tone continuously for over an hour to attract females. This sound is produced by sonic muscle and represents one of the most superfast and super-enduring striated muscles found in nature. Each fiber contains a hollow, tubular contractile apparatus composed of radially arranged myofibrils with extremely broad Z-bands that are supported by a desmin-rich cytoskeleton. We have used micro computed tomography (CT) imaging and magnetic resonance (MR) imaging to visualize the location of the sonic organ in an intact male fish. We have also obtained high-resolution MR images of the excised swim bladders from both male types. The images of the Type I sonic organ are strikingly detailed and high-contrast, revealing both the internal organization of the bladder and the crisscrossing muscle fibers and their mode of attachment to the underlying bladder. The high-contrast variation in these images is due to different T(2) values for fiber bundles and the spaces between the bundles. Direct MR imaging of intact Type I sonic organ in Type I midshipman fish is a powerful approach to understanding the contraction of this superfast muscle and the oscillation of its bladder to produce mating calls, and how placement of the sonic organ in the body of the fish sheds light on its prodigious ability to produce and transmit its loud mating call.

Coiled-coil nanomechanics and uncoiling and unfolding of the superhelix and alpha-helices of myosin.

The nanomechanical properties of the coiled-coils of myosin are fundamentally important in understanding muscle assembly and contraction. Force spectra of single molecules of double-headed myosin, single-headed myosin, and coiled-coil tail fragments were acquired with an atomic force microscope and displayed characteristic triphasic force-distance responses to stretch: a rise phase (R) and a plateau phase (P) and an exponential phase (E). The R and P phases arise mainly from the stretching of the coiled-coils, with the hinge region being the main contributor to the rise phase at low force. Only the E phase was analyzable by the worm-like chain model of polymer elasticity. Restrained molecular mechanics simulations on an existing x-ray structure of scallop S2 yielded force spectra with either two or three phases, depending on the mode of stretch. It revealed that coiled-coil chains separate completely near the end of the P phase and the stretching of the unfolded chains gives rise to the E phase. Extensive conformational searching yielded a P phase force near 40 pN that agreed well with the experimental value. We suggest that the flexible and elastic S2 region, particularly the hinge region, may undergo force-induced unfolding and extend reversibly during actomyosin powerstroke.

The polymerization of actin: structural changes from small-angle neutron scattering.

We present a new analysis of small-angle neutron-scattering data from rabbit muscle actin in the course of the polymerization from G-actin to F-actin as a function of temperature. The data, from Ivkov et al. [J. Chem. Phys. 108, 5599 (1998)], were taken in D2O buffer with Ca2+ as the divalent cation on the G-actin in the presence of ATP and with KCl as the initiating salt. The new analysis of the data using modeling and the method of generalized indirect fourier transform (O. Glatter, GIFT, University of Graz, Austria, http://physchem.kfunigraz.ac.at/sm/) provide shapes and dimensions of the G-actin monomer and of the growing actin oligomer in solution as a function of temperature and salt concentration. This analysis indicates that the G-actin monomer, under the conditions given above, is a sphere 50-54 A in diameter as opposed to the oblate ellipsoid seen by x-ray crystallography. The F-actin dimensions are consistent with x-ray crystal structure determinations.

The polymerization of actin: extent of polymerization under pressure, volume change of polymerization, and relaxation after temperature jumps.

The protein actin can polymerize from monomeric globular G-actin to polymeric filamentary F-actin, under the regulation of thermodynamic variables such as temperature, pressure, and compositions of G-actin and salts. We present here new measurements of the extent of polymerization (phi) of actin under pressure (P), for rabbit skeletal muscle actin in H2O buffer in the presence of adenosine triposphate and calcium ions and at low (5-15 mM) KCl concentrations. We measured phi using pyrene-labeled actin, as a function of time (t) and temperature (T), for samples of fixed concentrations of initial G-actin and KCl and at fixed pressure. The phi(T,P) measurements at equilibrium have the same form as reported previously at 1 atm: low levels of polymerization at low temperatures, representing dimerization of the actin; an increase in phi at the polymerization temperature (Tp); a maximum in phi(T) above Tp) with a decrease in phi(T) beyond the maximum, indicating a depolymerization at higher T. From phi(T,P) at temperatures below Tp, we estimate the change in volume for the dimerization of actin, DeltaVdim, to be -307+/-10 ml/mol at 279 K. The change of Tp with pressure dTp/dP=(0.3015+/-0.0009) K/MPa=(30.15+/-0.09) mK/atm. The phi(T,P) data at higher T indicate the change in volume on propagation, DeltaVprop, to be +401+/-48 ml/mol at 301 K. The phi(t) measurements yield initial relaxation times rp(T) that reflect the behavior of phi(T) and support the presence of a depolymerization temperature. We also measured the density of polymerizing actin with a vibrating tube density meter, the results of which confirm that the data from this instrument are affected by viscosity changes and can be erroneous.

Titin PEVK segment: charge-driven elasticity of the open and flexible polyampholyte.

The giant protein titin spans half of the sarcomere length and anchors the myosin thick filament to the Z-line of skeletal and cardiac muscles. The passive elasticity of muscle at a physiological range of stretch arises primarily from the extension of the PEVK segment, which is a polyampholyte with dense and alternating-charged clusters. Force spectroscopy studies of a 51 kDa fragment of the human fetal titin PEVK domain (TP1) revealed that when charge interactions were reduced by raising the ionic strength from 35 to 560 mM, its mean persistence length increased from 0.30 +/- 0.04 nm to 0.60 +/- 0.07 nm. In contrast, when the secondary structure of TP1 was altered drastically by the presence of 40 and 80% (v/v) of trifluoroethanol, its force-extension behavior showed no significant shift in the mean persistence length of approximately approximately 0.18 +/- 0.03 nm at the ionic strength of 15 mM. Additionally, the mean persistence length also increased from 0.29 to 0.41 nm with increasing calcium concentration from pCa 5-8 to pCa 3-4. We propose that PEVK is not a simple entropic spring as is commonly assumed, but a highly evolved, gel-like enthalpic spring with its elasticity dominated by the sequence-specific charge interactions. A single polyampholyte chain may be fine-tuned to generate a broad range of molecular elasticity by varying charge pairing schemes and chain configurations.