Since force is a vector quantity, the mechanical stability of a protein will depend not only on the magnitude but also on the direction and application point of the force, ttus, a force will couple to a reaction whenever it has a component in the direction of the reaction pathway.
We must first consider the geometrical errors present in SMFS measurements. So far, we have assumed for simplicity an orthogonal pulling geometry, i.e., the polyprotein is pulled at a 90° angle with respect to the substrate plane (Fig. 8.4a). To be more realistic, most of the time the polyprotein would be pulled at a slight angle. However, it has been demonstrated that the range of the geometrical errors introduced in this way is negligible (Carrion-Vazquez et al. 1999b).
Protein domains from muscle (titin), cell adhesion (tenascin, fibronectin), cy-toskeletal (filamin), and surface receptor usually belong to the Ig-like ^-sandwich family of proteins (Fig. 8.8a). ttese are likely to have somewhat similar mechanical topologies at the breakpoint, ttis superfamily of Ig folds, which includes Ig (titin, projectin, Sls-kettin, myomesin), fnlll (tenascin, fibronectin, titin, projectin, myo-mesin), E-set (filamin), and PKD (polycystin-1) types, consists of seven stranded sandwich structures in which the N- and C-terminal strands are parallel to each other and point 180° in different directions (Fig. 8.10a, left), ttey seem to have evolved to withstand forces when connected in series based on a shear mechanical topology of the hydrogen bonds at the breakpoint (i.e., the force vector is orthogonal to the bonds, which are arranged in parallel in the mechanical circuit). Ms arrangement may provide these domains with a considerable resistance to mechanical stretching (Table 8.1, Fig. 8.10a, left). Interestingly, protein L, a protein with no known mechanical function but with a shear mechanical topology at the breakpoint, also has relatively high mechanical stability (Table 8.1, Fig. 8.8b).
On the other hand, the C2 domain, found for instance in the secretory protein synaptotagmin I, is a ^-sandwich domain composed of eight antiparallel strands, with the N-and C-terminal strands pointing in the same direction, and a zipper mechanical topology at the breakpoint, i.e., the force vector is parallel to the bonds which are mechanically arranged in series (Fig. 8.10a, right). As can be seen in Table 8.1, the two proteins with a zipper configuration at the breakpoint studied so far, i.e., C2 domain (Carrion-Vazquez et al. 2000) and E2Lip3 domain (Brockwell et al. 2003), both have low mechanical stability when pulled in the N-C direction (see later).
tte different mechanical resistance between shear (concerted rupture) and zipper (sequential rupture or "peeling") topologies was predicted on the basis of molecular dynamics simulations of proteins (Rohs et al. 1999). ttis concept was experimentally demonstrated for DNA oligomers, choosing sequences where the only difference between the two configurations was the orientation of the tails (the sequence of the DNA was the same, only the topology was different), ttese two configurations were found to be thermodynamically (energetically) and kinetically equivalent when probed with traditional bulk methods (i.e., they have the same binding energy as well as the same thermal on and off rates). However, upon forced dissociation, the complex with the shear geometry has an approximately 3-fold higher mechanical stability and a 15-fold greater unbinding probability than that with the zipper geometry (Albrecht et al. 2003 and references therein).
Recently, it has been possible to experimentally pull proteins from different points of application such that the results of two different pulling geometries can be compared (regular N-C pulling vs. pulling from the terminus and an internal residue; Fig. 8.10b). ttis is possible by taking advantage of the existence of naturally occurring covalent linkages for internal amino acid residues in a couple of proteins (i.e., ubiquitin and E2lip3). ttese experiments show that the pulling direction affects dramatically the mechanical stability of a protein so that the two pulling geometries present very different mechanical stabilities (Table 8.1; Brockwell et al. 2003; Carrion-Vazquez et al. 2003). ttis raises the possibility that evolution may have selected specific mechanisms in unfoldases to take advantage of the "Achilles heels" of their substrate proteins (e.g., the internal ubiquitination of protein substrates for protea-somal degradation maybe one of those), as this would have the advantage of saving energy to the cell.
ttese directional effects on the mechanical stability of a protein are observed because a mechanical force acts locally along a single dimension, instead of globally, ttey illustrate the anisotropy of the mechanical unfolding landscape and certainly could not be found by chemical denaturation.
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