Figure 4.

Use of the carrier-guest strategy. (a) Typical force-extension recording of the pFS-2+I27 protein carrying (in this order) the N2B region and unfolding peaks from the “carrier” ubiquitin, three ubiquitin repeats, and the I27. The unfolding peak of the carrier ubiquitin repeat must always precede the unfolding peak of the guest I27 module (23). This carrier ubiquitin shows a larger ΔL_c due to the inclusion in its fold of the MCS and the folded I27. The inset shows a representation of the ubiquitin carrier with the I27 module grafted inside (top; PDB code 1TIT) as visualized by VMD 1.8.6 (48). (b) The ΔL_c analysis of the elements in this construct shows that the folds of the ubiquitin repeats (bottom), ubiquitin carrier (middle), and I27 guest (top) are preserved. (c) F_u histograms of the construct modules (same order). Both histograms show that the carrier-guest strategy does not alter the mechanical properties of the modules involved. All distributions are normalized in b and c. The Gaussian fits to the histograms are shown. (d) Typical force-extension recording of the pFS-2+VAMP2 protein. VAMP2 shows no force peaks in SFMS (i.e., no mechanical stability), thus the unfolding of the ubiquitin carrier is followed by the peakless stretching of the guest VAMP2. The inset shows a representation of the ubiquitin carrier with the cytoplasmic region from VAMP2 (residues 1–94, top) hosted within it, visualized by VMD 1.8.6 (48).