The Elasticity of Individual Titin PEVK Exons Measured by Single Molecule Atomic Force Microscopy

Sarkar, Atom; Caamano, Sofia; Fernández, Julio M.

doi:10.1074/jbc.c400573200

Cited by 58 publications

(49 citation statements)

References 25 publications

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“…Although Ottenheijm et al demonstrated that the content of titin in COPD diaphragm was not different to non-COPD diaphragm, they found that there was an upregulation of gene expression in the titin molecule and postulated that this might be due to alternative splicing of the titin gene, resulting in a longer molecule. This ability of the titin molecule to lengthen, specifically through alternative splicing in the proline, glutamate, valine, and lysine (PEVK) region of the gene, has been previously demonstrated by Sarkar et al (30). The results of the present study, therefore, provide an important independent confirmation of Ottenheijm et al's (25) findings specifically that, when stretched, the reduction in passive tension generated in COPD diaphragm probably occurs through an elongation of the titin molecule.…”

Section: Discussionsupporting

confidence: 81%

Passive properties of the diaphragm in COPD

Moore

Stubbings²,

Swallow³

et al. 2006

Journal of Applied Physiology

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tural adaptations that occur in the diaphragm muscle of patients with chronic obstructive pulmonary disease (COPD), namely an increase in type I fibers and a decrease in type II fibers, have been explored in terms of the active contractile properties of the diaphragm. The aim of this study was to test the passive properties of the diaphragm by measuring the force response of relaxed diaphragm muscle fibers to stretching to determine the effect of COPD on these properties. Costal diaphragm biopsies were taken from patients with COPD and from controls with normal pulmonary function. From these biopsies, titin expression was assessed in diaphragm homogenates by gel electrophoresis, and the restoring force was measured by incremental stretching of single fibers in the relaxed state and measuring the force response to stretching. A quadratic model was used to illustrate the relationship between restoring force and muscle fiber length, and it revealed that COPD fibers generate significantly lower restoring forces than control fibers as judged by the area under the force-length curve. Furthermore, this finding applies to both type I and type II fibers. Gel electrophoresis revealed different titin isoforms in COPD and controls, consistent with the conclusion that COPD results not only in a change in muscle fiber-type distribution but in a structural change in the titin molecule in all muscle fiber types within the diaphragm. This may assist the muscle with the energetic changes in the length of the diaphragm required during breathing in COPD.titin; passive tension; chronic obstructive pulmonary disease CHRONIC OBSTRUCTIVE PULMONARY disease (COPD) is one of the leading causes of death and disability in the developed world (17,20) and is characterized by progressive airflow limitation, which is largely irreversible (20). Because of the increased resistance to airflow, pulmonary hyperinflation can develop (18), the consequence of which is a flattening of the diaphragm resulting in a loss of its natural, curved, anatomic shape. This decreases the zone of apposition of the diaphragm to the chest wall (2, 13), which is essential for development of transdiaphragmatic pressure, and so the capacity of the diaphragm to generate negative intrathoracic pressures decreases in pulmonary hyperinflation (13,27).It is has been documented that in COPD the diaphragm undergoes an adaptation to compensate for the mechanical stresses that pulmonary hyperinflation places on it. These changes involve an alteration in the myosin heavy chain (MHC) isoform expression (14), with an increase in the proportion of type I fibers, and there is evidence of structural change also occurring within the diaphragm fibers at a subcellular level. Studies have shown that in chronic hyperinflation the resting sarcomere length in human diaphragm muscle fibers decreases (23). In emphysematous rodent models, it has been proposed that the total number of sarcomeres available in the shortened muscle fiber may be fewer than in normal controls, due to a loss in their n...

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Section: Discussionsupporting

confidence: 81%

Passive properties of the diaphragm in COPD

Moore

Stubbings²,

Swallow³

et al. 2006

Journal of Applied Physiology

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“…High-persistence-length values were also recently observed in chemically denatured proteins using FRET techniques (37). Such high-persistence lengths have also been observed in PEVK proteins (38)(39)(40) and considered to be an indication that, in addition to entropy, intramolecular interactions also play a role in the force-extension behavior of this natively disordered muscle protein (41,42). Thus, it is now clear that the apparent persistence length of Ϸ0.4 nm, resulting from WLC fits to protein unfolding data from atomic force microscopy experiments (43), reflected a phenomenological stiffness, comprising effects due to both chain entropy and hydrophobic collapse.…”

Section: Resultsmentioning

confidence: 52%

“…Polyproteins of ubiquitin, I27 and the PEVK-I27 chimera were cloned and expressed as described elsewhere (20,39,45). The PEVK-I27 construct is based on the human titin I27 and PEVK (exon 161) sequences.…”

Section: Methodsmentioning

confidence: 99%

Signatures of hydrophobic collapse in extended proteins captured with force spectroscopy

Walther¹,

Gräter

Dougan³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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We unfold and extend single proteins at a high force and then linearly relax the force to probe their collapse mechanisms. We observe a large variability in the extent of their recoil. Although chain entropy makes a small contribution, we show that the observed variability results from hydrophobic interactions with randomly varying magnitude from protein to protein. This collapse mechanism is common to highly extended proteins, including nonfolding elastomeric proteins like PEVK from titin. Our observations explain the puzzling differences between the folding behavior of highly extended proteins, from those folding after chemical or thermal denaturation. Probing the collapse of highly extended proteins with force spectroscopy allows separation of the different driving forces in protein folding.atomic force microscopy ͉ molecular dynamics ͉ protein folding ͉ single molecule P roteins can reversibly fold from a random coil conformation into a well defined native state, a process constituting a major research area in biology. Traditionally, experiments involved varying the ambient environment, such as changing the temperature or pressure, or using denaturing chemicals. Protein folding probed under these conditions has revealed two-state folding for many small proteins (1-3). Based on such experiments, Wolynes and colleagues proposed that the energy landscape of a collapsing polypeptide is funnel-shaped under folding conditions (4-6). In this scenario, the protein's energy decreases as it forms favorable interactions, thus driving it toward the native state. In the classic folding experiments, as well as in the theoretical models, proteins start in the denatured state from collapsed random coil conformations that are only a few Ångström larger than their native state (7,8). In these conformations, the side chains of the collapsed polypeptide are in close proximity to each other. It is widely accepted that, under such conditions, protein folding is driven mostly by hydrophobic interactions that are finely balanced by entropy (9-11). However, given that the denatured state in these experiments is not well defined, it has proved difficult to separate the hydrophobic, electrostatic, and entropic contributions to protein collapse and folding. We use single-molecule force-clamp spectroscopy to bring proteins to an extended conformation of Ͼ80% of their contour length, where the side chains are separated and exposed to the solvent, and native contact formation is rare (12-14). Thus, proteins are driven to the outer regions of the folding landscape, which have not been explored so far. Studying the collapse of such extended proteins greatly simplifies the folding dynamics, permitting a more direct identification of the major driving forces (11).Highly extended proteins have been routinely described as entropic chains using models of polymer elasticity such as the worm-like chain (WLC) model (15) or the freely rotating chain model (16). In this simplified picture, the collapse of a protein from an extended state is driven by...

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“…Those studies of intact titin and the Ig/Fn3 domains have provided a deeper understanding of how the elasticity of titin is adjusted by the sequential unfolding of Ig/Fn3 domains and how its elasticity recovers after domain refolding (Kellermayer et al, 1997;Rief et al, 1997;Carrion-Vazquez et al, 2000). More recently, the mechanical properties of expressed PEVK fragments based on cardiac and soleus PEVK exons were reported Linke et al, 2002;Watanabe et al, 2002;Labeit et al, 2003;Sarkar et al, 2005). Each study observed an unexpectedly broad distribution in the measured elastic persistence length of the PEVK segment.…”

Section: Introductionmentioning

confidence: 99%

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

Forbes¹,

Jin²,

Ma³

et al. 2006

J Muscle Res Cell Motil

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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.

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The Elasticity of Individual Titin PEVK Exons Measured by Single Molecule Atomic Force Microscopy

Cited by 58 publications

References 25 publications

Passive properties of the diaphragm in COPD

Passive properties of the diaphragm in COPD

Signatures of hydrophobic collapse in extended proteins captured with force spectroscopy

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

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