Probing the native state of poly-proteins by mechanical force

The folding and unfolding processes of poly-protein has been tremendously studied recently. The poly-protein dynamics under an external force can play an important role in addressing the issue of the mechanics of muscle tissue. In this research, we use a single-molecule technique: magnetic tweezers to observe the dynamics of 8-mer poly-protein L under different loads applied and then in different Tris-buffered salines. Our result shows that more protein domains unfold as the force load becomes larger. At 6, 7 and 8 pN loads, the poly-protein is most likely to stay in state 1, 3 and 6 with 1, 3 and 6 domains unfolded, respectively according to the probability distribution. This can be well explained by our constructed free energy-related model. The fit results give protein L parameters of persistence length of 0.4 nm, contour length of 18.8 nm and the unfolding energy of 6.5 kT, all in reasonable ranges based on previously reported literature.

Besides, we also find the dependency of transition rate on force load and salt. The poly-protein has lower transition rate at high force than at low force due to the free energy tilting effect since high force extremely decreases the possibility of protein unfolding that results in a huge drop in the total number of folding and unfolding events. This inverse proportion effect can also be seen in different TRIS-buffered salines (TRIS-150mM NaCl, TRIS-1M NaCl, and TRIS-1M KCl,). We explore the effect of salt concentration, when the concentration of NaCl is increased, the transition rate increases while the probability distribution remains almost the same, indicating the protein unfolding barrier is lowered without altering the overall energy landscape. We attribute this to, first, the charge shielding effect that more interactions between ions and water molecules occur, causing fewer water molecules available to interact with the charged part of protein than before, and, second, more direct interactions of ions with protein that might affect the electrostatic-related transition rate. Considering the effect of salt type, the two 1M alkali metal-chloride salines are compared. We conclude that ions with larger size have less effect on transition rate because ions with smaller size (Na+) can create stronger bonds with water that increase the interference on the protein interaction with water and can easier penetrate into protein to directly interact with the protein.