Figure 3. The TmMutL effects on the endonuclease activity of TmMutS2. (A) The 0.8% agarose gel indicates that TmMutL enhances nicking endonuclease activity and the DNA binding affinity of TmMutS2. Lane 1 represents TmMutS2 untreated DNA, and the B at lane 2 indicates EcoRI-treated linearized dsDNA. (B) The addition of 1 mM of nucleotide and 2 mM TmMutL to TmMutS2. doi:10.1371/journal.pone.0034529.g003 gyration (Rg) and the maximum particle dimension (Dmax) were calculated using the GNOM program and summarized in Figure 5D (left table). Rpre and Rcal represent the values estimated from the Guinier approximation and the P(R) spectrum, respectively, and Rcal was selected as the Rg value for further discussion. In the case of TmMutS2-dsDNA and TmMutS2-FWJ- DNA, the Rg and Dmax were similar to those of apo-TmMutS2. However, the distance distributions of TmMutS2-ADP and TmMutS2-ADPnP were narrower than that of apo-TmMutS2 and had a smaller Dmax than apo-TmMutS2. These results indicate that the addition of nucleotides to TmMutS2 induces a smaller protein compared to the apo-TmMutS2 form. Meanwhile, the calculated Rg of apo TmMutS2 was 74.8860.31 A˚, and Dmax was 250 A˚. The hydrodynamic radius (Rh) was also calculated using DLS analysis in a temperature range of 25uC to 55uC. The calculation produced an averaged Rh value of 82.98 (62.05) A˚, indicating that TmMutS2 retains thermostability (Figure 5D, right table). In particular, the Rh at 45uC was 93.26 (60.45) A˚, exhibiting that the ratio of Rh to Rg is approximately 1.21, which is a similar result to the generally observed ratio (,1.29) for known spherical molecules [37]. Thus, TmMutS2 may have a spherical configuration. Based on the scattering data, the structures for the TmMutS2 complexes were re-constructed using the ab initio modeling program DAMMIN (Figure 6). The SAXS models of apo-TmMutS2 and its complexes presented the two-fold symmetry, consistent with the results of SPM, SEC analysis, and the sedimentation velocity measurement that verified the homogeneous dimer conformation of Functional and Conformational Analysis of TmMutS2 TmMutS2 (Figure 1A, 1B, and 1E). In addition, apo-TmMutS2 and its complexed forms seem to have two or four linking domains based on the results of the SPM analysis of the surface-distributed TmMutS2 (size range 30 to 50 nm, Figure 1A). Additionally, the TmMutS2-ADP complex retained the slightly reduced size and small valley in the central region. Unlike apo-TmMutS2 and the TmMutS2-ADP complex, the large conformational changes in TmMutS2-ADPnP were observed in the central region. The compacted TmMutS2-ADPnP complex had the smallest Rg at 62.17 (60.21 A˚) and a Dmax of 180 A˚, as described in above. The compactness of TmMutS2-ADPnP was already proven by the SEC analysis (Figure 1E). The TmMutS2-dsDNA and TmMutS2-FWJ- DNA complexes exhibited lengthened structures compared to the apo-TmMutS2 form. In particular, TmMutS2-FWJ-DNA formed an elongated structure resulting from large conformational changes, indicating that intermolecular interactions between the protein and FWJ-DNA may create a nearly globular structure. These results suggest that TmMutS2 assumes a flexible conformation in the presence of nucleotides and DNA. Superimposition of the ATPase and Smr Models of TmMutS2 to the SAXS Models Although much effort has been expended to define the crystal structure of TmMutS2, no one has done so. Therefore, to confirm the validity of our SAXS models, other structural information about TmMutS2 was required. As shown in Figure S8, the ATPase domain of TmMutS2 was aligned with TaqMutS. The alignment indicates that the secondary structures of the two proteins have similar features. Hence, an ATPase domain model of TmMutS2 computationally predicted from a crystal structure of TaqMutS (Protein Data Bank, PDB No. 1EWQ) was utilized as a template for structural overlapping instead of a whole crystal structure of TmMutS2 (Figure 7A). The solution SAXS models of the apo- and ADP-complexed forms could not be superimposed onto the dimeric ATPase domain of TaqMutS and the monomeric TmMutS2 because they may not form compacted structures. However, the compacted TmMutS2-ADPnP complex model was suitable for superimposition of the ATPase domains to the SAXS model (Figure 7B). In addition, whereas there is variability in the superimposition method using symmetrical differences [38], the normalized spatial discrepancy (NSD) algorithm enables fast superimposition of structural models and provides for quantitative analysis of the similarity between the models. Like error-weighted averaging discrepancy [39], NSD is characterized by fineness parameters that play the role of standard deviations for one- dimensional data sets. As shown in Table S1, the final fineness values for the superimposed SAXS structures were lower than their initial values. These tendencies make it more reliable the SAXS models. In addition to ATPase domain analysis, the Smr domain of TmMutS2 was also characterized using the sequential homology of the Smr domain (683–757) of TmMutS2 with the Smr domain (1691–1770) of human B3bp (Figure S8). Like the preceding modeling, the TmMutS2-Smr domain was theoretically modeled from the high-resolution structure of B3bp-Smr (PDB No. 2D9I), as shown in Figure 8A. Using the TmMutS2-Smr model and the B3bp-Smr structure, the superimposition between TmMutS2- DNA (along with the dsDNA and FWJ-DNA complexes) and SAXS models was carried out (Figure 8B). Although the sizes of the B3bp-Smr structure and SAXS models seem to be different, they are similar in size. The final overlay indicated that the TmMutS2 SAXS models fit well with the respective B3bp-Smr structure and the TmMutS2-Smr model. The final fineness values for the SAXS templates and structures were lowered compared to their initial values (Table S1). In the superimposed models, both PLoS ONE | www.plosone.org 6 April 2012 | Volume 7 | Issue 4 | e34529