Mechanical manipulation of single titin molecules with laser tweezers.
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 molecules 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 A (+/- 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.
Kellermayer
M S
MS
Dept. Biophysics, Pécs University Medical School, Hungary.
Smith
S
S
Bustamante
C
C
Granzier
H L
HL
D000818:Animals; D064211:Connectin; D004548:Elasticity; D019277:Entropy; D006321:Heart / Q000502:physiology*; D007834:Lasers; D009119:Muscle Contraction; D009124:Muscle Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000502:physiology*; D018482:Muscle, Skeletal / Q000737:chemistry / Q000502:physiology*; D009206:Myocardium / Q000737:chemistry; D011487:Protein Conformation; D011494:Protein Kinases / Q000737:chemistry* / Q000302:isolation & purification / Q000502:physiology*; D011817:Rabbits; D051381:Rats
D064211:Connectin; D009124:Muscle Proteins; D011494:Protein Kinases