artifact of coarse-graining. An atomistic simulation using the GBSW protein force field (see Methods and Materials) yields a similar RMSF profile with comparable magnitudes of fluctuation throughout the protein, except in the loop (mapped to residue ID162–165) connecting the second and third strands of the HD domain (also see Fig. 1b). This particular loop has few native contacts with the rest of the protein, and therefore its flexibility is exaggerated in the Go¯ -like model. Nonetheless, it locates on the opposite side of the HD domain from where LNR modules pack. An elevated loop dynamics in this region is not expected to have significant influence on unfolding of either the NRR fold or the HD domain. Therefore, the Go¯ -like model as constructed is capable of realistically describing both the structure and dynamics of human Notch1 NRR. Force-induced unfolding of the NRR Constant velocity SMD simulations were carried out to examine the force-induced unfolding mechanism of the NRR. The external force was applied on the N- and C-termini. This setup was designed to mimic the situation where endocytosis of the Notch- bound ligand could exert mechanical stress on the NRR. Initial simulations with different pulling speeds suggest that there is a dependence of force magnitude on the pulling speed. However, the overall unfolding pathway appears to be insensitive to pulling speeds ranging from 0.1 to 100 A˚/ns, except that faster pulling tends to smear the fine details. Therefore, the final simulations were carried out with a modestly slow pulling speed of 1.0 A˚/ns. This provides a good compromise between resolution and com- putational efficiency. Note that the simulated pulling speed is several orders of magnitude faster than that in either typical single molecule pulling experiments or the putative endocytosis-driven pulling of Notch. However, previous studies have demonstrated that this does not prevent a reliable description of mechanically induced unfolding of proteins [33,36]. Mechanosensing in Notch Fig. 3 plots the average extension force computed from 60 independent SMD runs, together with average fractions of native contacts between various domains and subdomains of the NRR. Representative snapshots along the unfolding pathway are shown in Fig. 4. The average force profile shows several distinct major peaks, designated as Peaks 1–5 in Fig. 3a. Clearly, the NRR responds sensitively to mechanical stretching: increase of the end- to-end distance (distance between N- and C-terminal Ca atoms) from the native value of ,35 A˚ can only be achieved via tertiary unfolding and requires substantial external force. The first major force peak, Peak 1, locates near an end-to-end displacement of 47 A˚. The corresponding average fractions of native contacts between different regions of the NRR, shown in Fig. 3b, demon- strate that Peak 1 arises from unwrapping of LNR-A, which, more precisely, leads to Peak 1*, and the adjacent LNR-AB linker. This linker loop provides a hydrophobic plug that forms extensive contact with the HD domain and helps to protect the S2 site from metalloprotease cleavage (Fig. 1b). With a maximum of ,40 pN, Peak 1 is one of the strongest peaks in the force profile. Once LNR-A (with the adjacent LNR-AB linker) is detached, smaller forces are necessary to lift the LNR-B and LNR-C modules from the HD domain (broad Peak 2). Force-induced unpacking of LNR-B and LNR-C occurs roughly at the same time. Nonetheless, LNR-B appears to be more tightly bound to the HD domain, while LNR-C has a weaker interaction and its mechanical removal is the least cooperative (manifested as a broad transition shown in the blue trace of Fig. 3b). The observation that disengagement of LNR-A, the LNR-AB linker, and, to a lesser extent, LNR-B, provides the strongest resistance to mechanical unfolding is con- sistent with previous cell-based reporter gene assays showing that the removal of all these three segments facilitates ligand-inde- pendent activation of human Notch1 [21]. Importantly, all LNR modules and the HD domain remain largely folded during the initial process of peeling off the LNR Figure 3. Calculated extension force profile and corresponding native fractions of the NRR. a) Average extension force as a function of the end-to-end distance computed from 60 independent coarse-grained SMD pulling simulations. The red trace is the 50-point running average. The major peaks discussed in the main text are marked. b) Average fractions of native contacts formed between different sub-domains of NRR (see Materials and Methods for domain definitions). The LNR-A module only makes a few direct contacts with the HD domain, and the native fraction shown was calculated by including all contacts with the HD domain from the first 40 residues. The HD-b5 trace was computed by considering all native contacts made by residues 1721–1727. doi:10.1371/journal.pone.0022837.g003 PLoS ONE | www.plosone.org 4 July 2011 | Volume 6 | Issue 7 | e22837