Past exam of the biological physics course of the University of Oxford / 2017 / 3

In spectrin unfolding by force spectroscopy, the cantilever measures tension through its calibrated deflection, while displacement of the pulling point increases the separation of the protein attachments. The protein extension is obtained after allowing for cantilever compliance and any linker extension; it is not simply the commanded displacement.

A folded spectrin repeat stores much of its backbone in a compact domain. Between unfolding events, the already released chain is stretched: increasingly straight conformations have fewer accessible configurations, and the resisting entropic force rises nonlinearly. A peak is reached when a still-folded domain crosses an unfolding barrier on the timescale of the imposed loading. Its released backbone increases the available contour length abruptly. At nearly the same end-to-end distance the fractional extension $x/L$ is then smaller, so tension drops. Pulling resumes on the next, longer-chain elastic branch. Repeated release of domains produces the saw-tooth sequence.

The record is therefore evidence for sequential mechanical unfolding of modular structure, not repeated stretching of a fixed-length rod. For the nominal eight-domain construct, successive branches correspond to increasing numbers of unfolded repeats. The order and rupture forces can fluctuate because barrier crossing is thermally activated and depends on loading rate, structural heterogeneity and pulling geometry; equivalent repeats need not rupture at identical force.

The horizontal separation between worm-like-chain fits at the same force estimates the added contour length, whereas the vertical force drop at one extension reflects redistribution of tension. These are distinct measurements. A repeat increment near the specified $31.7\mathrm{nm}$ is consistent with repeated similar structural units. The gain is a net release relative to the folded span and attachment geometry; $31.7/106\mathrm{nm}$ is not automatically the universal contour length of a peptide residue.

During unloading, relaxation and possible refolding can restore compact domains. Hysteresis would indicate dissipative or kinetically delayed processes. Neither the peak force alone nor an arbitrary saw-tooth area directly equals an equilibrium unfolding free energy: one must separate chain elasticity, cantilever storage, irreversible loss and pulling-rate effects. The worm-like-chain branches describe released-chain elasticity, not the molecular transition state responsible for a rupture peak.

Write $u=x/L$ and $A=k_{B}T{L}_p$. The worm-like chain force-extension interpolation isA fit to an elastic rising branch estimates its contour length $L$ and, if not fixed independently, its persistence length $L_p$. Comparing successive fitted contour lengths gives the length released by a domain and helps distinguish whole-repeat unfolding from partial events. $L_p$ estimates the flexibility of the unfolded chain and the bending rigidity $A$; at a stated temperature, $A=k_{B}T{L}_p$.

For small extension, expansion gives $f\simeq [3k_{B}T/(2L_{p}L)]x$, so the effective entropic spring constant is $3k_{B}T/(2L_{p}L)$. Close to full extension, $f\sim k_{B}T/[4L_p(1-x/L{)}^2]$, giving strong strain stiffening. These regimes constrain the fit differently and correlate its parameters if the measured interval is short. The fit does not by itself identify a unique folding barrier or unfolding kinetic rate.

Persistence-length convention. In a three-dimensional isotropic worm-like chain the standard definition is $⟨\mathbf{t}(s)\cdot \mathbf{t}(0)⟩=e^{-s/L_p}$. Thus at $s=L_p$, $\arccos ⟨\cos \theta ⟩=\arccos (e^{-1})\simeq 68.4^{\circ }$. This is not literally the mean angle $⟨\theta ⟩$. For a simple counterexample, equally probable angles $0^{\circ }$ and $90^{\circ }$ have mean angle $45^{\circ }$ but $\arccos ⟨\cos \theta ⟩=60^{\circ }$. The source's angle description is shorthand for the tangent-correlation convention.

After a domain unfolds, its backbone joins the same continuous chain as previously unfolded material. The new branch differs mainly by an added contour length, while the local backbone bending statistics remain similar. It is therefore consistent to fit successive branches with different $L_j$ and one common $L_p\simeq 0.8\mathrm{nm}$. Tension is transmitted through serial portions of the same chain; one should not add their tensile forces as though they were independent parallel springs.

The persistence length is short compared with the contour increment per repeat, so each released domain contributes many flexible bending segments. Stretching suppresses those thermal modes and raises free energy through loss of entropy, which is precisely the worm-like-chain mechanism. The common $L_p$ is a property of the unfolded backbone under the measurement conditions, not the folded domain's axial length or the number of domains already released. With $T=300\mathrm{K}$ only as an illustrative value, $k_{B}T{L}_p\simeq 3.3\mathrm{pN}\mathrm{n}{\mathrm{m}}^2$.

A common persistence-length fit is evidence for approximately repeat-independent chain flexibility over the observed branches; it is not proof that a continuum inextensible model describes every molecular scale. Residual folded structure, heterogeneous linkers, force-induced backbone stretching, and very short-chain effects can cause deviations. The formal force divergence at $x=L$ signals the model's inextensibility assumption, not a physical infinite tensile force.