Single-molecule force spectroscopy has proven extremely beneficial in elucidating folding pathways for membrane proteins. Here, we simulate these measurements, conducting hundreds of unfolding trajectories using our fast Upside algorithm for slow enough speeds to reproduce key experimental features that may be missed using all-atom methods. The speed also enables us to determine the logarithmic dependence of pulling velocities on the rupture levels to better compare to experimental values. For simulations of atomic force microscope measurements in which force is applied vertically to the C-terminus of bacteriorhodopsin, we reproduce the major experimental features including even the back-and-forth unfolding of single helical turns. When pulling laterally on GlpG to mimic the experiment, we observe quite different behavior depending on the stiffness of the spring. With a soft spring, as used in the experimental studies with magnetic tweezers, the force remains nearly constant after the initial unfolding event, and a few pathways and a high degree of cooperativity are observed in both the experiment and simulation. With a stiff spring, however, the force drops to near zero after each major unfolding event, and numerous intermediates are observed along a wide variety of pathways. Hence, the mode of force application significantly alters the perception of the folding landscape, including the number of intermediates and the degree of folding cooperativity, important issues that should be considered when designing experiments and interpreting unfolding data.
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