Mechanical Manipulation of Single Titin Molecules with Laser Tweezers
- 194 Downloads
Titin (also known as connectin) is a giant filamentous polypeptide of multi-domain construction spanning between the Z- and M-lines of the vertebrate muscle sarcomere. The molecule is significant in maintaining sarcomeric structural integrity and generating passive muscle force via its elastic properties. Here we summarize our efforts to characterize titin’s elastic properties by manipulating single molecules with force-measuring laser tweezers. The titin molecule can be described as an entropic spring in which domain unfolding occurs at high forces during stretch and refolding at low forces during release. Statistical analysis of a large number (>500) of stretch-release experiments and comparison of experimental data with the predictions of the wormlike chain theory permit the estimation of unfolded titin’s mean persistence length as 16.86 Å (±0.11 SD). The slow rates of unfolding and refolding compared with the rates of stretch and release, respectively, result in a state of non-equilibrium and the display of force hysteresis. Folding kinetics as the source of non-equilibrium is directly demonstrated here by the abolishment of force hysteresis in the presence of chemical denaturant. Experimental observations were well simulated by superimposing a simple domain folding kinetics model on the wormlike chain behavior of titin and considering the characteristics of the compliant laser trap. The original video presentation of this paper may be viewed on the web at http://www.pote.hu/ mm/prezentacio/mkpres/mkpres. htm.URL
KeywordsPersistence Length Partial Release Contour Length Thick Filament Passive Force
Unable to display preview. Download preview PDF.
- Fürst DO, Osborn M, Nave R, Weber K. The organization of titin filaments in the half-sarcomere revealed by monoclonal antibodies in immunoelectron microscopy: a map often nonrepetitive epitopes starting at the Z line extends close to the M line. J Cell Biol 1988;106:1563–72.PubMedCrossRefGoogle Scholar
- Itoh Y, Suzuki T, Kimura S, Ohashi K, Higuchi H, Sawada H, et al., Extensible and less-extensible domains of connectin filaments in stretched vertebrate skeletal muscle sarcomeres as detected by immunofluorescence and immunoelectron microscopy using monoclonal antibodies. J Biochem 1988;104:504–508.PubMedGoogle Scholar