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Mechanical extension of single molecules of the giant protein titin
Miklós S.Z. Kellermayer1, Steven Smith2, Carlos Bustamante2, Henk L. Granzier3
1 Dept. Biophysics, University Medical School of Pécs, Hungary
2 Dept. Physics, University of California, Berkeley, CA, USA
3 Dept. VCAPP, Washington State University, Pullman, WA, USA
Titin (also known as connectin) is a giant filamentous protein that spans the approximately 1 µm distance from the Z-line to the M-line of the vertebrate-muscle sarcomere. Sequence analysis demonstrated that titin is highly modular in construction: it contains a tandem array of ~300 globular (Ig of FN-type) domains interspersed with unique sequences, most notably the PEVK segment (named after its most prominent amino acids). Several earlier studies have indicated that titin plays an important role in maintaining sarcomeric structural integrity and generating passive muscle force due to the elastic nature of the molecule. To explore the elastic properties of titin, we have stretched single molecules by using force-measuring laser tweezers.
Measurements of the force required to stretch a single molecule revealed that titin behaves as a highly non-linear entropic spring whose behavior can be best described with the wormlike chain (WLC) model. The molecule partially unfolds in a high-force transition that begins at 20-30 pN and refolds in a low-force transition at ~ 2.5 pN. Force hysteresis arises from a difference between the unfolding and refolding kinetics of the molecule relative to the stretch and release rates in the experiments, respectively. Scaling the molecular data up to sarcomeric dimensions reproduces many features of the passive force vs. extension curve of muscle fibers.
Stretching titin with high external forces (above 400 pN) results in the complete mechanical unfolding of the molecule, characterized by the disappearance of force-hysteresis at high forces. Titin refolds following complete mechanical denaturation, as the hysteresis at low forces reappears in subsequent stretch-release cycles.
The addition of 4 M guanidium-HCl (GuCl) abolishes force hysteresis across the entire force spectrum. Since the release force curve retraces the stretch force curve, the molecule is in a state of conformation equilibrium. Such a completely unfolded molecule behaves as a WLC with a persistence length (Lp, measure of bending rigidity) of ~13-17Å. Depending on the tissue-specific isoform, Lp of the denatured titin molecule may vary slightly (rat cardiac 12.9Å, rabbit soleus 15.2Å, rabbit cardiac 15.4Å, rabbit longissimus dorsi 16.6Å). Titin refolds upon the removal of GuCl, as the force hysteresis reappears in subsequent stretch-release cycles.
During repeated stretch and release cycles the force hysteresis does not recover completely, and titin becomes progressively longer by the beginning of each consecutive stretch. Thus, repeated mechanical cycles progressively wear out titin, revealing a process that may rightly be called "molecular fatigue." Molecular fatigue takes place in a limited force range (<25 pN), indicating that the process may be restricted to a segment of titin only. Detailed analysis of the force data demonstrate that slow, incomplete domain refolding cannot solely account for the appearance of molecular fatigue. As experimental data and computer simulation suggest, the involved titin segment is permanently unfolded, and it may be shortened by non-covalent bonds that cross-link various sites along its contour. Each cross-link shortens the segment with the length of the enclosed piece of the chain. At first approximation, the part of the chain enclosed by the cross-link may be modelled as a molecular loop. Upon stretching titin, the cross-links rupture at a rate influenced by the external force and the stretch rate. The force-driven rupture of the cross-links lengthens the molecular segment, and hence titin, in a non-discrete manner by adding the loop perimeter to the titin length during each cross-link rupture event. The cross-links re-form only as the chain is allowed to spend sufficient time in the shortened, low-force state so that the bond-forming sites can diffusively encounter each other. The possibility of non-specific titin-bead interaction, which may artificially display a behavior similar to that caused by intrachain cross-links, was excluded by following the non-specific binding of single, fluorescently labelled titin molecules to microscopic beads by using confocal microscopy. We speculate that the segment displaying molecular fatigue may include the PEVK segment and/or other unique sequences in titin. Since titin's molecular fatigue occurs in the physiologically relevant force range of < 25 pN/molecule, it may play an important role in adjusting the length of the effectively elastic region in titin. The kinetic mechanisms involved may dynamically adjust titin's apparent stiffness according to the rate of mechanical perturbations. |
