MTSlow and MTSFN9–10 were prepared as previously described (14, 15). The high-force MTS (MTShigh) was adapted from MTSlow by replacing the (GPGGA)8 module with another tension-sensitive domain, termed HPst (LSDED FKAVF GMTRS AFANL PLWKQ QALMK EKGLF), derived from the villin headpiece (16). The DNA encoding this construct was assembled by Epoch Life Sciences Inc. (Missouri City, TX) and was cloned into the pJ414 expression vector (DNA 2.0). We used Alexa 546 maleimide (Thermo Fisher Scientific, A10258) as the FRET donor and an Alexa 647 maleimide dye (Thermo Fisher Scientific, A20347) as the FRET acceptor. This modified MTS presents the identical RGD ligand derived from fibronectin as used in MTSlow. The entire MTShigh sequence is presented below:

M G S E I G T G F P F D P H Y V E V L G E R M H Y V D V G P R D G T P V L F L H G N P T S S Y V W R N I I P H V A P T H R S I A P D L I G M G K S D K P D L G Y F F D D H V R F M D A F I E A L G L E E V V L V I H D W G S A L G F H W A K R N P E R V K G I A F M E F I R P I P T W D E W P E F A R E T F Q A F R T T D V G R K L I I D Q N V F I E G T L P M G V V R P L T E V E M D H Y R E P F L N P V D R E P L W R F P N E L P I A G E P A N I V A L V E E Y M D W L H Q S P V P K L L F W G T P G V L I P P A E A A R L A K S L P N A K A V D I G P G L N L L Q E D N P D L I G S E I A R W L S T L E I S G G A G E F K C A G L S D E D F K A V F G M T R S A F A N L P L W K Q Q A L M K E K G L F G K C A G S E N L Y F Q G T V Y A V T G R G D S P A S S A A H H H H H H.

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