sequence-dependent, but native-structure independent, pseudo- torsional potentials. Such sequence flavoring has been shown to enhance the realism of simple Go¯ -like models and provides the ability to recapitulate subtle differences in folding mechanisms of topologically analogous proteins [42,55–57]. The original model generated by the MMTSB Go¯-Model Builder was then modified to include the bound calcium ions and disulfide bonds. Human Notch1 NRR contains a total of ten disulfide bonds (see Fig. 1), three in each LNR module and one in the HD domain. These disulfide bonds are modeled using harmo- nic distance restraint potentials with a force constant of 1.0 kcal/ mol/ A˚2. These harmonic restraints are sufficient to maintain the bond linkages, while providing sufficient flexibility for conforma- tional sampling. The bound calcium ions are coordinated by six to seven carbonyl groups from the peptide backbone and aspartic acid side chains. Here, each calcium ion is represented as a neutral atom with a radius of 2 A˚. Coordination with the carbonyl groups is treated as additional native interactions. The strength of calcium ions with carbonyl groups was chosen to be 2.0 kcal/mol. This parameter was chosen empirically by examining the stability of calcium ion coordination during equilibrium simulations. In addi- tion, to prevent occasional dissociation and loss of calcium ions (such as during high-temperature and/or SMD pulling simula- tions), weak harmonic distance restraints were imposed between the ions and one of the coordinating carbonyl groups to contain the ions within the proximity of their native binding sites. Since there is no evidence that tertiary unfolding of the LNR modules is implicated in Notch activation, the precise approach of maintain- ing disulfide bonding and calcium coordination is not expected to affect the force-induced unfolding simulations. By construction, the protein has a melting temperature of about 350 K and is stably folded at 300 K. Finally, to directly observe the Notch heterodimer disassociation, the bond between residues 1622 and 1670 was deleted during SMD pulling simulations. All the other simulations were carried out with a single continuous peptide chain as used in the X-ray structure determination unless other- wise noted. Equilibrium, SMD and REX-MD simulations using the Go¯ -like model Equilibrium and SMD simulations were performed using CHARMM [58,59]. Langevin dynamics was performed with a time step of 10 fs and a friction coefficient of 0.1 ps21. Lengths of all virtual bonds were kept fixed using SHAKE [60], and the cutoff for non-bonded interactions was 25 A˚. Constant velocity SMD was performed using the AFM module [61] in CHARMM. Multiple pulling speeds ranging from 0.1 to 100 A˚/ns were tested before a final speed of 1.0 A˚/ns was chosen to provide a com- promise between efficiency and resolution. A total of 60 inde- pendent SMD runs of 500 ns each were carried out at 300 K (see Movie S1 for a representative pulling simulation). REX-MD was used to enhance conformational sampling and allow converged folding free energy surfaces to be calculated. REX is an advanced sampling technique that has proven ex- tremely powerful in protein simulations [62]. The basic idea is to simulate multiple independent replicas of the system at different temperatures. Periodically, replicas attempt to exchange simula- tion temperatures according to a Metropolis criterion that preserves the detailed balance and ensures proper canonical ensembles at all temperatures. The resulting random walk in the temperature space helps the system to avoid being kinetically trapped in states of local energy minima. The REX-MD simulation was performed using the MMTSB toolset [63] and CHARMM. Eight replicas were dis- tributed exponentially from 300 to 400 K. Exchange of simulation Mechanosensing in Notch temperatures between neighboring replicas was attempted after every 10 ps, and the total simulation length was 1.0 ms (100,000 REX cycles). Atomistic implicit solvent simulations All atomistic simulations were carried using a previously optimized GBSW implicit solvent protein force field [46,64] based on the CHARMM22/CMAP all-atom force field [65–68]. Implicit solvent is essentially a coarse-grained approach where only the protein is represented at atomic level, and the mean influence of solvent is captured by the free energy cost of solvating the protein (thus solvent is implicit) [69]. The system size is reduced about 10 fold compared to traditional explicit solvent, and thus further facilitate facile sampling of accessible conformation space [70]. Importantly, this particular GBSW force field has been extensively optimized to reproduce the experimental structures and stabilities of a range of helical peptides, b-hairpins, and mini- proteins [46]. The same force field has also been successfully applied to describe the conformational equilibria of several proteins under stable and unstable conditions [71–75]. Therefore, it allows computationally efficient yet reliable simulations of human Notch1 NRR. Both the full NRR and HD domain were simulated at 300 K for 20 ns after proper energy minimization and short MD equilibration. The termini were blocked with acetyl (Ace) and amine (NH2) groups. Default GBSW parameters were used [46]. SHAKE [60] was applied to fix lengths of all hydrogen- involving bonds to allow a dynamics time step of 2 fs. Both protein constructs stabilize rapidly in a few ns, and all structural and dy- namic properties examined in this work appear to converge well. Final analysis was done using the last 16 ns trajectories. Data Analysis All the analysis was carried out using CHARMM, the MMTSB toolset, and additional in-house scripts. For coarse-grained modeling, a given native contact is considered formed if the inter-Ca distance is no more than 1 A˚ greater than the native distance. Folding free energy surfaces were calculated by combining informational from all temperatures from the REX-MD simulation using WHAM [43– 45]. All structural visualizations presented in this work were prepared with the VMD program [76]. Supporting Information Figure S1 Structural properties of the isolated HD domain with and without a chain break in the S1 loop during 20-ns atomistic simulations. a) The backbone RMSD as a function of the simulation time. b) The number of residues in a or b secondary structures as a function of time. c) The final snapshot and d) the average structure from the last 16 ns simulation of the HD domain with a break (cyan), in comparison with the X-ray structure (PDB: 3eto) (purple). The backbone RMSD from the X-ray structure is 2.2 A˚ for the average structure and 3.6 A˚ for the final snapshot. The location of the break in the S1 loop is marked. These results suggest that the break does not de-stabilize the isolated HD domain within the simulation time frame. (TIF) Figure S2 Residue-residue map of correlated motions for the human Notch1 NRR. The maps were computed as the Ca-Ca covariance matrices, extracted from the last 16 ns of a 20- ns equilibrium GBSW simulation. It shows that all LNR modules are tightly coupled with the HD domain, particularly with regions that are direct contacts. This highlights the strength of the inter- domain interactions between the LNR modules and the HD PLoS ONE | www.plosone.org 9 July 2011 | Volume 6 | Issue 7 | e22837