Nanomechanical Manipulation Of Skeletal-muscle Titin With Force-ramp Optical Tweezers

Abstract

Titin is a filamentous protein that spans the half sarcomere and functions as a molecular spring, a sarcomeric template, and possibly as a mechanosensor. The molecule has become a popular experimental model for exploring mechanically-driven protein folding because of its structure as a tandem array of similar beta-barrel domains. The force versus extension curve of titin, recorded in constant-displacement-rate experiments, is characterized by entropic-chain behavior onto which sudden, stepwise contour-length fluctuations caused by domain unfolding are superimposed. Recent force-clamp experiments revealed a complex, multi-stage force response during folding, suggesting that the unfolded chain collapses not solely by entropic mechanisms.To explore the nanomechanical detail in titin's folding and unfolding, here we stretched single molecules of purified skeletal-muscle titin with force-ramp optical tweezers. Titin was extended in cycles of stretch and relaxation, during which the loading rate was kept constant by using a fast (500 Hz) feedback. Loading rates ranged between 1-10 pN/s, and minimal relaxation loads were 2-10 pN. We found that above 50 pN partially unfolded titin molecules often deviated significantly from the pure wormlike-chain behavior and displayed a variable stretch modulus of tens to hundreds of pN. When partially unfolded titin was relaxed to 2 pN and restretched immediately, we did not observe significant domain refolding. Thus, while enthalpic elasticity mechanisms may assist the collapse of the unfolded and extended protein chain, complete domain refolding requires longer periods of time spent in a highly contracted state at low (< 2 pN) forces.