Figure 4: 2D free energy landscape diagram of Tau_plane , Tau_glyc , Tau_phos , Tau_p356 and Tau_p352 along with the structure corresponding to the local energy minima. The figure shows the wide distribution of structural ensembles in Tau_plane with a large energy barrier, while in Tau_glyc and Tau_phos, we have comparatively narrow distribution and fewer local energy minima.
Figure S4 shows Tau_plane having four funnels each corresponding to a local minimum with an energy barrier separating them suggesting the folding process is rugged and not stable. The 2D contour plot, Figure 5 , indicates the wide distribution of ensembles suggesting many of the confirmations are trapped in high energy states. Tau_glyc shows two local minima, without much energy barrier separating them and the conformational ensembles are not widely distributed as in Tau_plane. This suggests a comparatively stable folding process in Tau_glyc as compared to Tau_plane. Tau_phos system have a single energy minimum, suggesting the protein folds into a single native structure, but we can see that the funnel is broad and have many confirmations trapped in high energy states. Five local energy minima observed in the FEL of Tau_p356 indicate the rugged nature of the folding process. These local minima are separated by a large energy barrier, suggesting the possibility of the protein getting trapped in one local energy minima. Tau_p352 FEL indicates two local minima, one at low energy and another at high energy, separated by a large energy barrier, suggesting the entrapment of protein in high energy states. Tau_plane having four local energy minima metastable states have an energy barrier of about 3-4 KJ/mol separating them, while in tau_glyc we have two low energy metastable states with ~‘2KJ/mol energy barrier. In Tau_phos the we have a single prominent lower energy state separated by the next higher energy state by a transition barrier of ~3KJ/mol. In the case of tau_p356 and tau_p352 the lowest energy metastable states are separated by an energy barrier of ~ 6-7 KJ/mol.
Structural analysis of the lowest energy metastable conformations from each of the systems obtained from FEL were performed. The four local energy minima conformations of Tau_plane (I-IV) was analyzed for secondary structure preference, especially beta sheet formation, and none of the four local energy minimal structures had beta sheet confirmations, while only III had a 5 percent isolated beta bridge. Both local minima confirmations (V & VI) of Tau_glyc had beta sheets with 10 percent beta sheets in each structure. Both V and VI were analyzed for salt bridges and we found that V had two salt bridges Asp345-Lys343 & Glu372- Lys375, while VI also had two salt bridges, Glu380-Lys347 & Glu380-Lys353. The lowest energy conformation of Tau_phos VII had zero percent beta sheets but had around 10 percent residues with beta bridges and no salt bridges. The energy minimal metastable structures of Tau_p356 and Tau_p352 did not have any beta sheets but largely exist in random coils and helix. This suggests that the N-glycosylation induced folding of tau is able to form beta sheet rich regions in the protein, while the unmodified tau is having an unstable folding process with none of the lowest energy metastable structures having beta sheet propensity.