It is well known that ubiquitin exhibits a two state folding behavior and hence the characterization of the transition state is important. The transition state is a high energy state posited at the peak of the highest energy barrier. Since the TS is kinetically and thermodynamically unstable, it was suggested that the structure of the protein should undergo rapid changes after passing this state. Figure 4 shows a plot of the radius of gyration of the protein as unfolding proceeds. There seem to be two distinct phases in the plot identified by their different slopes. The bend in the plot occurs GSI-IX Gamma-secretase inhibitor around 2.5�C3.0 ns which is probably the location of the TS since it involves a conformation which is near native except for the breaking of the b1�Cb5 interaction as mentioned above. However, a more definitive method for obtaining the TS was used by Li et al. who identified the TS by analyzing the RMSD between all pairs of conformations generated during a simulated trajectory. The pairwise RMSD values can be projected onto a 2D or 3D Cartesian space where the distance between conformations correspond to their RMSD from each other. In this work, 50 ps snapshots were chosen from the unfolding trajectory resulting in 160 snapshots or frames. Pairwise RMSDs for each pair of conformations were calculated and were projected onto a 3D Cartesian space following the work of Dastidar et. al. The projected plot is shown in Figure 5. The line connecting two conformations XAV939 implies sequential evolution in time. The distance between the conformations is proportional to the RMSD distances between the conformations. Hence, conformations close to each other in the RMSD space will cluster together. However, it must be noted that the coexistence of conformations in a cluster does not imply structural similarity of the conformations but rather that there are no abrupt changes in structure between them. This is at the basis of the so called ����chaining effect���� which is well known in hierarchical cluster analysis and leads to a grouping of very different objects in the same cluster. Since this is based on the similarity of adjacent conformations, completely unrelated structures can exist in the same cluster. In Figure 5, there are two well-defined clusters. The first cluster extends from 0�C2 ns. As mentioned earlier the major structural change takes place at around 2.5 ns and involves the breaking of the b1�Cb5 interaction. Hence the period between the two clusters probably indicates the transition between the native structure and the transient intermediate. This occurs between 2.0 and 2.25 ns and could thus be taken to represent the TS and which also corresponds well with the putative TS inference from the plot of the radius of gyration with unfolding time. In order to observe the topological variation during the unfolding trajectory, each 50 ps snapshot was analyzed using Recurrence Quantification Analysis.