The elasticity of single titin molecules using a two-bead optical tweezers assay.
Titin is responsible for the passive elasticity of the muscle sarcomere. The mechanical properties of skeletal and cardiac muscle titin were characterized in single molecules using a novel dual optical tweezers assay. Antibody pairs were attached to beads and used to select the whole molecule, I-band, A-band, a tandem-immunoglobulin (Ig) segment, and the PEVK region. A construct from the PEVK region expressing >25% of the full-length skeletal muscle isoform was chemically conjugated to beads and similarly characterized. By elucidating the elasticity of the different regions, we showed directly for the first time, to our knowledge, that two entropic components act in series in the skeletal muscle titin I-band (confirming previous speculations), one associated with tandem-immunoglobulin domains and the other with the PEVK region, with persistence lengths of 2.9 nm and 0.76 nm, respectively (150 mM ionic strength, 22 degrees C). Novel findings were: the persistence length of the PEVK component rose (0.4-2.7 nm) with an increase in ionic strength (15-300 mM) and fell (3.0-0.3 nm) with a temperature increase (10-60 degrees C); stress-relaxation in 10-12-nm steps was observed in the PEVK construct and hysteresis in the native PEVK region. The region may not be a pure random coil, as previously thought, but contains structured elements, possibly with hydrophobic interactions.
Medical Research Council Muscle and Cell Motility Unit, King's College London, London SE1 1UL, United Kingdom.
Mark C
MC
Leake
David
D
Wilson
Mathias
M
Gautel
Robert M
RM
Simmons
D000818:Animals; D064211:Connectin; D004548:Elasticity; D006863:Hydrogen-Ion Concentration; D008846:Micromanipulation / Q000379:methods*; D020409:Molecular Motor Proteins / Q000737:chemistry*; D009124:Muscle Proteins / Q000032:analysis / Q000737:chemistry*; D018482:Muscle, Skeletal / Q000737:chemistry*; D009206:Myocardium / Q000737:chemistry*; D055095:Optics and Photonics; D010812:Physical Stimulation / Q000379:methods; D011494:Protein Kinases / Q000032:analysis / Q000737:chemistry*; D011817:Rabbits; D012518:Sarcomeres / Q000737:chemistry*; D013314:Stress, Mechanical; D013696:Temperature
D064211:Connectin; D020409:Molecular Motor Proteins; D009124:Muscle Proteins; D011494:Protein Kinases