Interaction forces between F-actin and titin PEVK domain measured with optical tweezers.
Titin is a giant protein that determines the elasticity of striated muscle and is thought to play important roles in numerous regulatory processes. Previous studies have shown that titin's PEVK domain interacts with F-actin, thereby creating viscous forces of unknown magnitude that may modulate muscle contraction. Here we measured, with optical tweezers, the forces necessary to dissociate F-actin from individual molecules of recombinant PEVK fragments rich either in polyE or PPAK motifs. Rupture forces at a stretch rate of 250 nm/s displayed a wide, nonnormal distribution with a peak at approximately 8 pN in the case of both fragments. Dynamic force spectroscopy experiments revealed low spontaneous off-rates that were increased even by low forces. The loading-rate dependence of rupture force was biphasic for polyE in contrast with the monophasic response observed for PPAK. Analysis of the molecular lengths at which rupture occurred indicated that there are numerous actin-binding regions along the PEVK fragments' contour, suggesting that the PEVK domain is a promiscuous actin-binding partner. The complexity of PEVK-actin interaction points to an adaptable viscoelastic mechanism that safeguards sarcomeric structural integrity in the relaxed state and modulates thixotropic behavior during contraction.
Bianco
Pasquale
P
Department of Biophysics, University of Pécs, Faculty of Medicine, Szigeti út 12, Pécs H-7624, Hungary.
Nagy
Attila
A
Kengyel
András
A
Szatmári
Dávid
D
Mártonfalvi
Zsolt
Z
Huber
Tamás
T
Kellermayer
Miklós S Z
MS
D000199:Actins / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D001696:Biomechanical Phenomena; D055592:Biophysical Phenomena; D001703:Biophysics; D064211:Connectin; D006801:Humans; D066298:In Vitro Techniques; D009119:Muscle Contraction / Q000502:physiology; D009124:Muscle Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D036103:Nanotechnology; D052898:Optical Tweezers; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055672:Static Electricity; D014783:Viscosity
D000199:Actins; D064211:Connectin; D009124:Muscle Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C577285:TTN protein, human; D011494:Protein Kinases