The Journal of Physical Chemistry B ARTICLE Figure 6. Sequence alignment of NrfB with N-terminal sequence of MtrA, MtrA0 (top), and C-terminal sequence of MtrA, MtrA00 (bottom). Identical residues are shaded with blue, and the CXXCH motifs are indicated with asterisks. Using SAXS data, the dummy residue model for MtrA determined by ab initio shape reconstructions is at least two times longer in one dimension than the perpendicular axes (Figure 5b). This aspect ratio of MtrA can be checked by a simple geometric calculation using the Rg and Rc values extra- polated to zero protein concentration. For a prolate ellipsoid of uniform density with a length 2c and cross-sectional semiaxes a and b, Rg2 ¼ ða2 þ b2 þ c2Þ=5 ¼ Rc2 þ c2=5 3 ð Þ Approximating the cross section as circular, that is, a = b, substitution of the experimental Rg and Rc gives semiaxes of 24.5 and 61.6 Å with an axial ratio of 2.5. As this axial ratio is >1, that of a sphere, MtrA must be elongated along one dimension, con- sistent with P(r) and the ab initio shape reconstructions. This axial ratio is also in reasonable agreement with that obtained by sedimentation velocity AUC. Modeling MtrA as a prolate ellipsoid in SedFit yielded an axial ratio of 2.2 from the ﬁtted hydrodynamic frictional ratio. Additionally, the theoretical s20,w of the SAXS model was calculated with SOMO50 for comparison with the value determined experimentally by AUC. Using a partial speciﬁc volume of 0.73 mL/g (the average value for proteins36) and the molecular weight for a monomeric MtrA, the theoretical s20,w was calculated to be 3.1 S, in reasonable agreement with the experimental value of 3.4 S. Together, these results support the overall shapes of the reconstructed models that are up to 104 Å in length and up to 50 Å in the widest cross- sectional dimension. Previously, it has been proposed that MtrA may be similar in shape to an end-to-end repeat of the pentaheme protein NrfB.12 The shape reconstructions presented here suggest that the cross section of MtrA is elliptical rather than spherical, similar to the pentaheme NrfB from E. coli, and does appear somewhat evocative of a repeat of the NrfB fold (Figure 5b), where NrfB has dimensions of 40 Å 30 Å 20 Å in its crystal structure (Figure 5b).28 However, the sequence alignments suggest that the potential relationship between NrfB and MtrA should only be taken so far: whereas the ﬁve CXXCH motifs in the N-terminal domain of MtrA (MtrA0) can be aligned with those in NrfB (NCBI BLAST51), giving an E value of 1 10 15 (Figure 6, top), the two halves of MtrA do not align particularly well with one another (E value is 1 10 4), and the C-terminal domain of MtrA (MtrA00) aligns with NrfB quite poorly, yielding an E value of only 0.007 (Figure 6, bottom). Moreover, whereas one molecule of NrfB will ﬁt within the SAXS-derived surface reconstruction of MtrA, it is not possible to model two end-to- end NrfB monomers such that the pentaheme modules are within electron transfer distance without imposing arbitrary changes to the structural scaﬀold. Therefore, it is unclear if the entirety of MtrA could be reasonably modeled as being com- posed of two NrfB modules, in terms of key details such as the stacking of heme cofactors. We do note that repetition of multiheme motifs is not unprecedented among c-type cyto- chromes because sequence analysis of the dodecaheme cyto- chrome from Geobacter sulfurreducens shows that it consists of four triheme domains that align with the triheme cyto- chrome c7.52,53 Regardless of applicability of NrfB as a structural model for the entirety of MtrA, our data provide the ﬁrst structural evidence that MtrA is indeed elongated like a wire. With this information in hand, we can consider two of the proposed roles for MtrA: receiving electrons from inner mem- brane-bound CymA and directing them across the periplasm to the outer membrane, and shuttling electrons through the outer membrane to MtrC via the β-barrel porin protein MtrB (Figure 1).19 First, we can now build upon our previous result that MtrA can directly accept electrons from the inner-membrane bound quinol oxidoreductase CymA,19,54 and we can estimate that one ∼100 Å long MtrA molecule contacting CymA could span much of the 130 250 Å distance of periplasmic space.26 Without knowing to what degree CymA or MtrB are extended into the periplasm and with such a large uncertainty in the periplasmic distance, we cannot predict how far a single MtrA molecule would need to travel or if it would need to travel at all to engage in redox shuttling. We can say that in the absence of a stabilizing factor, it is unlikely that end-to-end MtrA molecules are involved in this process because our data show that MtrA alone does not oligo- merize up to protein concentrations of 4 mg/mL. The second proposed function for MtrA is that it could pass electrons directly to the outer membrane cytochrome MtrC when both are docked within the MtrB β-barrel. (See Figure 1.) Whereas the structure of MtrB is unknown, it is predicted to be a 28-strand β-barrel protein using the PRED-TMBB prediction program.55,56 The size of a transmembrane β-barrel can be 11212 dx.doi.org/10.1021/jp203603r |J. Phys. Chem. B 2011, 115, 11208–11214