The Journal of Physical Chemistry B Figure 1. Proposed Fe(III) oxide electron transfer pathway in Shewa- nella oneidensis MR-1. MtrA is shown in two locations due to its potential dual functionality. been shown to be localized to the outermembrane.7,20 22 It has been suggested by Hartshorne and coworkers that the MtrCAB complex may represent a more widely used bioenergetic strategy for the electrical connection of the cellular periplasm with the exterior in a range of microbes capable of diﬀerent bioenergetic pathways as orthologs can be found in Shewanella and other organisms.19 Notably, in these cases, a decaheme protein similar to MtrA would serve as the critical electrical conduit to pass charge to reach the cellular surface. Currently, however, no high- resolution structures exist for the MtrCAB complex or any of its proposed substituents. Therefore, the molecular details of this DMR pathway generally and MtrA in particular remain poorly understood. MtrA may perform a unique role in the electron transfer pathway as an intermediate periplasmic protein that is able to transport electrons from CymA to the outer membrane protein MtrC. With 333 amino acid residues and 10 low-spin heme cofactors proposed to possess bis-histidine coordination,9 MtrA has a notably low amino acid to heme ratio of 33, whereas an average heme-bearing protein has approximately 60 70 amino acids per heme unit.23 Crystal structures of other multiheme cytochromes have shown that the hemes are packed as a wire with edge-to-edge distances between 4 and 8 Å.24,25 A similar packing of cofactors in MtrA would suggest that the protein is ∼100 Å long and thus able to span a substantial portion of the 130 250 Å distance across the periplasm.26 In addition to shuttling electrons across the periplasm, MtrA is also thought to contact the outer membrane protein MtrC directly by docking in the MtrB porin protein. Such a function requires that at least one end of MtrA be narrow enough to ﬁt in the MtrB porin barrel. In the absence of a crystal structure, the overall structure of MtrA has been hypothesized based on a sequence alignment with the structurally characterized NrfB, a pentaheme c-type cytochrome from Escherichia coli that puriﬁes as a 40 kDa decaheme homo- dimer on a size exclusion column.7,27 Sequence alignment of NrfB with the N-terminal half of MtrA shows alignment of the heme-binding motifs, leading to the hypothesis that MtrA forms two pentaheme modules with dimensions of an end-to-end NrfB homodimer: 80 Å in length and 30 Å, on average, in diameter.12,28 These dimensions would presumably allow MtrA to insert partially into the MtrB porin. However, other than the heme-binding motifs of the N-terminal half of MtrA, the sequences of NrfB and MtrA show poor alignment, particularly ARTICLE along the C-terminal portion of the MtrA sequence. Therefore, it remains unknown if MtrA is structurally shaped like an elec- tron “wire” and therefore able to perform both of its proposed functions. Here we present the ﬁrst structural characterization of MtrA from S. oneidensis using two complementary solution-based techniques: SAXS and analytical ultracentrifugation (AUC). Whereas AUC is particularly useful for probing the oligomeriza- tion state of proteins, SAXS is sensitive to electron density distributions with inhomogeneities on the length scales of 10 to 100 Å and thereby provides further structural detail of proteins in solution. Although SAXS does not provide high-resolution structural models, it is particularly useful for investigating pro- teins that are challenging to crystallize and has been successful in the characterization of the overall shape and length of spherical as well as elongated proteins.29 32 Data from both techniques show that MtrA is monomeric over a wide range of protein concentra- tions and possesses a rod-like aspect ratio with a maximum dimension of 104 Å. This corroborates the previously proposed model of MtrA serving as a functional wire.12,19 The data presented in this Article are a step toward understanding the structure of MtrA and how it shuttles electrons across the periplasmic space of Shewanella. ’ EXPERIMENTAL METHODS Protein Expression and Purification. The plasmid contain- ing mtrA, LS246, was provided by Dr. Liang Shi (Pacific North- west National Laboratory). E. coli JM109(DE3) cells were transformed first with the pEC86 plasmid (provided by Linda Th€ony-Meyer of ETH Zurich) containing the ccmABCDEFG genes, then transformed with LS246. MtrA was expressed as previously described.9 Cells were harvested at 8000g for 15 min at 4 C. Lysis was completed through osmotic shock by resus- pending the cell pellet in TES buffer (200 mM Tris-HCl, pH 8.0, 1 mM EDTA, and 500 mM sucrose) containing 1 mg/mL lyso- zyme and incubating on ice for 1 h. The lysed cells were spun at 18 000g for 30 min at 4 C. Purification was carried out similarly to the published protocol,9 with a few modifications. The supernatant was loaded onto DEAE resin, equilibrated with 10 mM Tris-HCl, pH 8.0 and MtrA was eluted using a linear gradient of 0 300 mM NaCl. Fractions containing MtrA were pooled and concentrated to ∼1 mL using a 10 kDa MWCO Amicon centrifugal device. This sample was loaded onto a HiPrep 26/60 Sephacryl S-100 high-resolution gel filtration column and eluted with 20 mM HEPES, pH 8.0, 50 mM NaCl using a GE AKTA FPLC system. Fractions were tested for purity using SDS-PAGE and UV vis absorbance; those displaying a single band and a purity index (A409/A280) greater than 2.0 were pooled and concentrated for a final purity index of 2.8. Analytical Ultracentrifugation. Sedimentation velocity AUC was performed using a Beckman XL-I analytical ultracentrifuge (Beckman Coulter, Brea, CA) at the MIT Biophysical Instru- mentation Facility. Fresh samples of 0.5 mg/mL MtrA were dialyzed against 20 mM HEPES, pH 8.0, 50 mM NaCl. The protein solution and dialysate were loaded into two-sector cells and equilibrated for 10 h to reach a temperature of 20.0 C prior to centrifugation at a rotor speed of 40 000 rpm. The protein concentration across the cell was measured by monitoring the heme absorbance at 420 nm at 2 min intervals. The apparent sedimentation coeﬃcient distribution, g(s*), was generated from 42 scans with a peak broadening limit of 180 kDa 11209 dx.doi.org/10.1021/jp203603r |J. Phys. Chem. B 2011, 115, 11208–11214