Functional and Conformational Analysis of TmMutS2 Figure 2. Nicking endonuclease activity of TmMutS2. (A) The effects of various cations on the nicking endonuclease activity of TmMutS2. The cations were used at a concentration of 10 mM. The nick, l, and sc indicate the nicked, linearized, and supercoiled open circular DNA forms, respectively. (B) A mixture containing 2 mM TmMutS2/TmMutS2-Smr and 1.5 mM plasmid DNA (linear size, 3 kbp) was incubated at temperatures ranging from 20uC to 80uC in the presence or absence of Mg2+ (upper plot). In the middle of the gel, M indicates the DNA size marker. The lower plot presents the percentage yields of the nicked form. (C) Different activities of DNA digestion in the presence of ATP (lanes 3 to 7), ADP (lanes 8 to 12), and ADPnP (lanes 13 to 17). Lane 1 and 2 are TmMutS2 untreated- and treated-plasmid DNA bands in the absence of theh nucleotide at 60uC. Each nucleotide was utilized at a final concentration of 3 mM. doi:10.1371/journal.pone.0034529.g002 Predictions of Structures using SAXS for TmMutS2 Complexes with Various Types of Nucleotides or DNA For conformational analysis of proteins, SAXS can be directly applied to monitor the solution conformations of proteins [34,35]. In this study, we collected SAXS data on the monodispersed solutions of TmMuS2 and its complexes with nucleotides (ADP and ADPnP) or DNA (linear dsDNA and FWJ-DNA). As shown in Figure 5A, the overlying scattering curves for the various concentrations of TmMutS2 indicate an increase in signal according to the concentration of TmMutS2, including some fluctuations at high S (A˚ ). These signal fluctuations at the high- angle region result from structural features such as the total structure and the organization of domains [36]. Additionally, confirmation of the aggregated state of the protein can be provided by the scattering data, inspecting the linearity at the low S range 2 using a Guinier approximation (lnI(S) vs. S ). The upper inset in Figure 5A presents the apo-TmMutS2, indicating that TmMutS exhibits monodispersity and no aggregation. For complexes of TmMutS2 with nucleotides and DNA, different signals were noted according to the partner molecules in the complex (Figure 5B). The Guinier approximation demonstrated that TmMutS2 complexes also displayed monodispersity and did not aggregate (Figure S7). To obtain information about the structures of proteins from SAXS data, the P(R) spectra, histograms for the existence probabilities of atoms at all distances within scattering particles, were constructed (Figure 5C). From these spectra, the radius of PLoS ONE | www.plosone.org 5 April 2012 | Volume 7 | Issue 4 | e34529