 Solution

A rising force branch means that an already exposed polypeptide segment is being stretched while the remaining structure resists further extraction. At a force peak a structural barrier is crossed; protein denaturation or release of additional chain increases the available polymer contour length, so the force falls at almost the same imposed separation. Stretching then loads the next remaining barrier. The sawtooth pattern therefore reports a sequence of mechanically resistant intermediates, not a regular spring oscillation. The cantilever deflection gives force, while its own deformation must be removed from the imposed separation to infer molecular extension.

A membrane protein also requires work to disrupt membrane anchoring, helix packing and contacts with lipids or neighboring molecules. The retinal-containing interior contributes a bulky, specifically packed scaffold; exposing chain must overcome these steric hindrance as well as hydrogen bond and electrostatic interactions. A force peak need not correspond one-to-one to one complete helix, and its height depends on loading rate and pathway, not just an equilibrium folding Gibbs free energy.

Salt changes electrostatic screening and specific ion binding. Screening can reduce either repulsion or attraction, so “more salt always weakens the protein” is false. The primary study underlying these kinds of traces identifies relatively salt-insensitive steric hindrance around the retinal and additional salt-sensitive barriers. Its interpretation associates some added barriers with screening of destabilizing electrostatic repulsion. The supplied traces likewise show a changed set of intermediate peaks, rather than a uniform rescaling of one curve.^[ref]

Exact assignment of every arrow to a residue requires structural and statistical information beyond one representative trace. No such assignment is inferred here. The printed reference to titin in the following paragraph is inconsistent with the stated specimen and the membrane-protein traces; the physical interpretation here concerns bacteriorhodopsin, while the worm-like-chain expression is a generic polymer approximation.