Steered molecular dynamics (SMD) simulation

In this study, two types of steered MD simulations (constant-velocity (CV) and constant-force (CF)) were used. The calculation of stress-strain, cross-sectional area, helix length Young’s modulus, E, determined from the spring stiffness and k in our current SMD simulations are identical to those reported previously [31].

In the constant-velocity SMD simulations, the two alpha carbons of the first two residues (e.g., N15 and V16 in the case of TM1 helix of EcMscL) on each helix were restrained using a strong harmonic restrain constant of 12 kcal/mol/Ų (i.e., 840 pN/Å). The last C_α at the opposite end of the helix called the SMD atom, is attached to another ‘dummy’ atom by a harmonic spring constant of 1 kcal/mol/Ų (e.g., L48 in the TM1 helix of EcMscL) [31] All CV simulations were performed by applying the pulling rate of 0.1 Å/ps.

In the constant-force method, a constant-force vector is applied onto selected atoms on one end of the helix, while the other end is restrained. The alpha carbons of the first two residues N15 and V16 of the TM1 helix of EcMscL were restrained, whereas the force was applied on the alpha carbon of the last residue L48 in the TM1 helix of EcMscL [31]. In the case of the TM1 helix, for example, a constant-force in the range of 0.1–1.5 kcal/mol/Å (~7 to 104 pN) was applied to the C_α atoms of the last residue in the direction defined by a vector. The time step in all our SMD simulations was 1 fs. The pressure and temperature were controlled similar to all atom MD simulation described above. The helices were equilibrated by first holding them fixed for the first 1 ns where the atomic positions were restrained followed by their backbones being held fixed while the rest of the system was allowed to relax for the following 1 ns. Finally, the whole system was equilibrated in an NPT ensemble for 10 ns without any restraint [31]. For the SMD simulations in water, the ‘SOLVATE’ module, with the TIP3P water molecule [30] was applied in a water to a box of 40 × 40 × 90 Å.