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

Muscle Proteomics and Nanotechnology Section, Laboratory of Muscle Biology, NIAMS, NIH, DHHS, Bethesda, MD 20892-8024, USA.
Journal of Muscle Research and Cell Motility (Impact Factor: 2.09). 02/2005; 26(6-8):291-301. DOI: 10.1007/s10974-005-9035-4
Source: PubMed


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 ∼
∼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.

Download full-text


Available from: Albert J Jin, Oct 06, 2015
  • Source
    • "One conclusion drawn from these experiments is that the unique sequences in titin generate elastic forces mainly according to an entropic-spring mechanism, although evidence abounds suggesting that additional factors determine the elasticity of the PEVK domain [27, 58, 72, 76, 92, 132, 137]. In any case, these and related studies [27, 58, 62, 66, 67, 92, 135] clearly demonstrated that the PEVK domain and the N2B us extend at forces at which the Ig domain unfolding probability is still very low. If one compares the WLC parameters for the distinct molecular-spring elements in titin, it becomes immediately obvious that these elements have different mechanical stabilities . "
    [Show abstract] [Hide abstract]
    ABSTRACT: Perturbation of a protein away from its native state by mechanical stress is a physiological process immanent to many cells. The mechanical stability and conformational diversity of proteins under force therefore are important parameters in nature. Molecular-level investigations of "mechanical proteins" have enjoyed major breakthroughs over the last decade, a development to which atomic force microscopy (AFM) force spectroscopy has been instrumental. The giant muscle protein titin continues to be a paradigm model in this field. In this paper, we review how single-molecule mechanical measurements of titin using AFM have served to elucidate key aspects of protein unfolding-refolding and mechanisms by which biomolecular elasticity is attained. We outline recent work combining protein engineering and AFM force spectroscopy to establish the mechanical behavior of titin domains using molecular "fingerprinting." Furthermore, we summarize AFM force-extension data demonstrating different mechanical stabilities of distinct molecular-spring elements in titin, compare AFM force-extension to novel force-ramp/force-clamp studies, and elaborate on exciting new results showing that AFM force clamp captures the unfolding and refolding trajectory of single mechanical proteins. Along the way, we discuss the physiological implications of the findings, not least with respect to muscle mechanics. These studies help us understand how proteins respond to forces in cells and how mechanosensing and mechanosignaling events may proceed in vivo.
    Full-text · Article · May 2008 · Pflügers Archiv - European Journal of Physiology
  • Source
    • "There are two distinct types of PEVK domains encoded by individual differentiallyspliced exons: Group P exons encode neutral or basic PPAK domains and Group E exons encode acidic polyglutamate-rich domains. The number, specific charge characteristics, and interactions of the domains are important in the overall elasticity of the PEVK segment [Forbes et al., 2005]. Additionally, certain PEVK segments contain SH 3 -binding motifs that may be important for SH 3 -based signaling in the muscle sarcomere [Ma et al., 2006]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: We previously discovered a large titin-like protein-c-titin-in chicken epithelial brush border and human blood platelet extracts that binds alpha-actinin and organizes arrays of myosin II bipolar filaments in vitro. RT-PCR analysis of total RNA from human megakaryoblastic (CHRF-288-11) and mouse fibroblast (3T3) nonmuscle cells reveal sequences identical to known titin gene exon sequences that encode parts of the Z-line, I-band, PEVK domain, A-band, and M-line regions of striated muscle titins. In the nonmuscle cells, these sequences are differentially spliced in patterns not reported for any striated muscle titin isoform. Rabbit polyclonal antibodies raised against expressed protein fragments encoded by the Z-repeat and kinase domain regions react with the c-titin band in Western blot analysis of platelet extracts and immunoprecipitate c-titin in whole platelet extracts. Immunofluorescent localization demonstrates that the majority of the c-titin colocalizes with alpha-actinin and actin in 3T3 and Indian Muntjac deer skin fibroblast stress fibers. Our results suggest that differential expression of titin gene exons in nonmuscle cells yields multiple novel isoforms of the protein c-titin that are associated with the actin stress fiber structures.
    Full-text · Article · Jun 2007 · Cell Motility and the Cytoskeleton
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: 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.
    Full-text · Article · May 2006 · Biophysical Journal
Show more