Jeffrey G. Forbes

Single molecule measurements of titin elasticity

Titin, with a massive single chain of 3--4MDa and multiple modular motifs, spans the half-sarcomere of skeletal and cardiac muscles and serves important, multifaceted functions. In recent years, titin has become a favored subject of single molecule observations by atomic force microscopy (AFM) and laser optical trap (LOT). Here we review these single titin molecule extension studies with an emphasis on understanding their relevance to titin elasticity in muscle function. Some fundamental aspects of the methods for single titin molecule investigations, including the application of dynamic force, the elasticity models for filamentous titin motifs, the technical foundations and calibrations of AFM and LOT, and titin sample preparations are provided. A chronological review of major publications on recent single titin extension observations is presented. This is followed by summary evaluations of titin domain folding/unfolding results and of elastic properties of filamentous titin motifs. Implications of these single titin measurements for muscle physiology/pathology are discussed and forthcoming advances in single titin studies are anticipated.

The polymerization of actin: Thermodynamics near the polymerization line

Studies of the dependence of actin polymerization on thermodynamic parameters are important for understanding processes in living systems, where actin polymerization and depolymerization are crucial to cell structure and movement. We report measurements of the extent of polymerization, Φ, of rabbit muscle actin as a function of temperature [T=(0–35) °C], initial G-actin concentration [[G0]=(1–3) mg/ml], and initiating salt concentration [[KCl]=(5–15) mmol/l with bound Ca2+], in H2O and D2O buffers and in the presence of adenosine triphosphate (ATP). A preliminary account of the data and analysis for H2O buffers has appeared previously [P. S. Niranjan, J. G. Forbes, S. C. Greer, J. Dudowicz, K. F. Freed, and J. F. Douglas, J. Chem. Phys. 114, 10573 (2001)]. We describe the details of the studies for H2O buffers, together with new data and analysis for D2O buffers. The measurements show a maximum in Φ(T) for H2O buffers and D2O buffers. For H2O buffers, Tp decreases as either [G0] or [KCl] increases. For D2...

Thermodynamic regulation of actin polymerization

A Flory–Huggins-type lattice model of actin polymerization under equilibrium conditions is employed to analyze new spectroscopic measurements for the extent of actin polymerization Φ as a function of temperature T, salt concentration [KCl], and the initial concentration of actin monomers [G0]. The theory subsumes existing mechanisms for actin monomer initiation, dimerization, and chain propagation. The extent of polymerization Φ increases with T to an unanticipated maximum, and the calculations explain this unusual effect as arising from a competition between monomer activation, which diminishes upon heating, and propagating chain growth, which increases upon heating. The actin polymerization is described as a rounded phase transition, and the associated polymerization temperature Tp depends strongly, but nearly linearly on [G0] and [KCl] over the concentration regimes investigated. Our findings support the suggestion that physicochemical changes can complement regulatory proteins in controlling actin pol...

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.

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. —Nahirney, P. C., Forbes, J. G., Morris, H. D., Chock, S. C., Wang, K. What the buzz was all about: Superfast song muscles rattle the tymbals of male periodical cicadas. FASEB J. 20, 2017–2026 (2006)

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.

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-1s 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.

The polymerization of actin: Study by small angle neutron scattering

We report measurements of small angle neutron scattering from solutions of rabbit muscle G-actin at 3.00 mg/mL in D2O buffer solution, with [Ca2+]=0.52 mM and with [KCl]=15, 8.9, and 5.4 mM. We observe the onset of the polymerization of G-actin to F-actin as the temperature is increased. The polymerization takes place on a time scale of 30–45 min for each temperature jump of 2 °C–3 °C. As the temperature is increased further, the average size of the polymers increases, and the characteristic length scale (or correlation length), ξ, of the F-actin in the dilute solution grows: ξ is about 10 A below Tp, and about 70 A a few degrees above Tp. The transition is sharper for lower concentrations of KCl. For the sample with [KCl]=8.9 mM, we observe a peak in ξ at about 2 °C above Tp, which indicates a crossover into the semidilute regime. The transition is essentially reversible, but shows evidence of incomplete depolymerization on cycling. We are unable to apply the available theoretical model for reversible po...

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.