BPS2026 – The poly-E motif of titin exhibits greater mechanical compliance at acidic pH

The intrinsically disordered PEVK region in the I-band region of titin functions as an entropic spring to produce elasticity within the muscle. Our lab has previously shown that the poly-E motif becomes more collapsed and flexible under acidic conditions. These studies were conducted in the absence of force, and we have now extended these studies using magnetic tweezers-based force-extension experiments at different pH values. An isolated region of one of the longest poly-E segments of PEVK was tested at pH 7.2 and pH 6.0. Force was applied between 15 pN to 55 pN and resulting extension was analyzed. Poly-E displayed more extension at pH 6.0 compared to pH 7.2. These results are consistent with our previous results and align with predictions from the worm-like chain (WLC) model which expects greater compliance at lower persistence length. We are currently testing constructs of the PPAK motif and combinations of poly-E and PPAK to further assess the pH dependence of the force. To investigate whether our single-molecule results may apply to titin’s PEVK when it is in its in-situ microenvironment, inside the sarcomere, we performed mechanical testing on isolated myofibrils. Myofibrils were immunolabeled at the N2A and distal PEVK (F146.9) regions permitting real-time tracking of titin’s PEVK length during stretch. Forces were measured using optical force nano-levers. Titin’s PEVK segment demonstrated a significantly decreased elastic modulus at physiological lengths at pH ≤ 6.5; at pH = 6.0 the elastic modulus of the PEVK segment was reduced by ∼58%. Thus, in isolated myofibrils, titin’s PEVK segment also shows increased compliance at low, yet physiological pH levels. These results support our model that the poly-E regions act as pH sensors to tune the stiffness of the muscle by modulating the compliance of the PEVK region.