The Journal of Physical Chemistry B ARTICLE Figure 2. Overall and cross-sectional size of 0.5, 1, 2, 3, and 4 mg/mL MtrA studied by SAXS. (a) Guinier plots show good linearity at low q, indicating a lack of nonspeciﬁc aggregation. The radius of gyration, Rg, and normalized zero-angle scattering intensity I(0)/c were determined from linear ﬁts (black) to the data (blue), where the Guinier approxima- tion (eq 1) holds. (b) Rg and I(0)/c are concentration-independent, suggesting that the oligomerization state does not change under these conditions. (c) Cross-sectional Guinier plots display linear regions, indicative of elongated shape of MtrA. The cross-sectional radius of gyration Rc was determined from linear ﬁts (black) to the data (blue), where eq 2 holds. (d) Rc is also concentration-independent. Protein concentration increases from bottom to top in (a) and (c). using the dc/dt method implemented in the program DCDT+.33 The diﬀusion-deconvoluted sedimentation coeﬃcient distribu- tion, c(s), was generated from 280 scans in the program SedFit.34 The sedimentation coeﬃcients were converted to standard values (s20,w) using a solvent viscosity and density of 1.0221 10 2 poise and 1.00185 g/mL (calculated in Sednterp35), respectively. The average partial speciﬁc volume of proteins, 0.73 mL/g,36 was used for this conversion, as the heme contribution to this quantity cannot be easily determined. The molecular weight of monomeric MtrA with 10 hemes was calculated to be 42.8 kDa. Small-Angle X-ray Scattering. SAXS was performed at the Cornell High Energy Synchrotron Source G1 station (CHESS, Cornell University, Ithaca, NY) using a 10.5 keV 250 μm square X-ray beam with a flux of several 1012 photons/s. Data were collected at room temperature on a 1024 1024 pixel CCD detector similar to that described in ref 37 at a sample-to-detector distance of 1 m; the transmission intensity was measured with a PIN diode beamstop. Protein solution scattering was measured from fresh samples of 0.5, 1, 2, 3, and 4 mg/mL MtrA in 20 mM HEPES, pH 8.0, 50 mM NaCl contained in acrylic laminate static cells with 7.5 μm thick polyimide ﬁlm windows.38 Background scattering was measured with buﬀer in the same cells. Detailed data acquisition and reduction procedures are described by Ando et al.38 Trans- mission-normalized background subtraction yielded the scatter- ing proﬁles, scattering intensity (I) versus momentum transfer (q); here q = 4π/λ sin θ, where λ is the X-ray wavelength and 2θ is the scattering angle with respect to the X-ray beam. The foldedness of MtrA under the investigated conditions was veriﬁed by Kratky analysis; a representative plot is shown in Figure S1 of the Supporting Information. Figure 3. Molecular weight determination of 0.5 mg/mL MtrA by sedimentation velocity AUC. The apparent sedimentation coeﬃcient distributions g(s*) ﬁt with single Gaussian, black and magenta curves, respectively, and the diﬀusion-deconvoluted sedimentation coeﬃcient distribution c(s), blue curve, indicate that MtrA is monomeric. The zero-angle scattering intensity I(0) and overall radius of gyration Rg were obtained from a Guinier approximation to the low-q region of the scattering proﬁles satisfying the condition, q*Rg < 1.3.39 IðqÞ≈Ið0Þ expð R2q2=3Þ g 1 ð Þ In the case of elongated proteins, a cross-sectional radius of ginyreaqtio1n.3,9Rc, can be determined using a similar approximation as R2q2=2Þ IðqÞ q≈Ið0Þ expð 2 ð Þ c Rg and I(0) are determined from linear ﬁts to Guinier plots, 2 ln(I(q)) versus q . Likewise, Rc is determined from cross- sectional Guinier plots, ln(I(q)*q) versus q2. ’ RESULTS The size and concentration dependence of MtrA were in- vestigated by SAXS over a concentration range of 0.5 4 mg/mL. Guinier analysis was used to determined the overall radius of gyration, Rg, and the concentration-normalized forward scatter- ing intensity, I(0)/c, which are functions of spatial size and molecular mass, respectively.40 The Guinier plots are linear at the low q-range displayed in Figure 2a at all concentrations, indicat- ing that MtrA has a well-deﬁned size under the solution condi- tions studied and that radiation-induced aggregation is not an issue. Rg and I(0)/c, determined from linear ﬁts to the data over the q range satisfying the q*Rg < 1.3 condition (q = 0.0146 to 0.0416 Å 1), display very little concentration dependence (Figure 2b), indicating that no change in oligomerization state occurs over the investigated concentration range. The oligomerization state of MtrA was determined by sedi- mentation velocity AUC at one of the protein concentrations investigated by SAXS under identical solution conditions. A single peak is observed in g(s*) that is well-described by a single Gaussian over the range s20,w = 2 7 S, indicative of a single dominant species under nearly ideal conditions (black and magenta curves in Figure 3).41 The slight peak broadening in g(s*) at s20,w < 2 S is consistent with the observation of a small peak in c(s) (blue curve in Figure 3) and can be attributed to proteolysis (unpublished data). The molecular weight and s20,w of the dominant species were determined to be 35.6 kDa and 3.4 S by the Svedberg relation in DCDT+. Similar values of 36.9 kDa and 3.4 S were determined from c(s) in SedFit, most consistent with that of a 42.8 kDa monomeric state. The shape of the MtrA monomer was analyzed by SAXS. Using the indirect Fourier transform method implemented in the 11210 dx.doi.org/10.1021/jp203603r |J. Phys. Chem. B 2011, 115, 11208–11214