The Journal of Physical Chemistry B estimated by sequence and is dependent on the number of strands, n, and shear number, S,57 a numerical parameter corresponding to the pitch of the β-barrel. Shear numbers range between n and n + 4 with a few exceptions,57 and pore size will increase with larger values of n and S.57 A slightly smaller β-barrel protein, FepA, has 22 strands with a shear number of +24, and its crystal structure gives an elliptical pore size of 30 Å 40 Å.58 Therefore, presuming that MtrB is a subparallel β-barrel as predicted, it is reasonable to think of MtrB with slightly larger pore dimensions than FepA. In this light, the dimensions of MtrA estimated by SAXS (approximately 25 Å 50 Å at the largest cross-section by 104 Å in length) are provocative, suggesting that MtrA would at least be able to insert partially into the β-barrel MtrB, if not span the entire outer membrane. The electron recipient, MtrC, has not been structurally characterized but is 43% similar to OmcA (E value of 7 10 18), which has been estimated by SAXS to have dimensions that are wider and shorter than MtrA (34 Å 65 Å 90 Å for OmcA) but still are of a range that could ﬁt partially into the MtrB barrel structure.13 ’ CONCLUSIONS The combined use of AUC and SAXS has enabled us to characterize the structure of MtrA in solution for the ﬁrst time. We have demonstrated that at all concentrations studied, MtrA exists as an extended monomer with a maximum dimension of 104 Å with an aspect ratio of 2.5 when modeled as a prolate ellipsoid. This is the ﬁrst biophysical evidence that supports the proposed physiological functions of MtrA as a periplasmic elec- tron shuttle. The dimensions and shape indicate that it is possible for MtrA to accept electrons from CymA and then donate them to MtrC via the MtrB β-barrel.19,54 As it has been suggested that decaheme orthologs for MtrA may represent a novel, yet wide- ranging strategy for extracellular redox communication in anae- robic micro-organisms,19 our data represent the latest, key step toward a uniﬁed view of multiheme cytochromes as components of long-distance “wires” in nature. ’ ASSOCIATED CONTENT bS Supporting Information. Kratky analysis of MtrA SAXS data. This material is available free of charge via the Internet at http://pubs.acs.org. ’ AUTHOR INFORMATION Corresponding Author *Tel: 617-358-2816. Fax: 617-353-6466. E-mail: elliott@bu.edu. Author Contributions T)hese authors contributed equally to this publication. ’ ACKNOWLEDGMENT This work was generously supported by the NSF (S.J.E., MCB 0546323) and NIH grant F32GM904862 (N.A.). C.L.D. is a Howard Hughes Medical Institute Investigator. CHESS is sup- ported by the NSF & NIH/NIGMS via NSF award DMR- 0936384. We thank Liang Shi (PNNL) for the donation of the MtrA plasmid, Linda Th€ony-Meyer (ETH Zurich) for the donation of the pEC86 vector, Arthur Woll (CHESS) for assistance with SAXS experimental setup, Debbie Pheasant (MIT BIF) for assistance with AUC data collection, Emre ARTICLE Brookes and Borries Demeler (UT Health Science Center) for assistance with SOMO, Cynthia Kinsland for use of the Cornell Protein Facility, and Rebekah Bjork (MIT) for assistance with SAXS data collection. Molecular graphics images were produced using the UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIH P41 RR001081). ’ REFERENCES (1) Myers, C. R.; Nealson, K. H. Science 1988, 240, 319–321. (2) Myers, C. R.; Nealson, K. H. J. Bacteriol. 1990, 172, 6232–6238. (3) Lovley, D. R. Microbiol. Rev. 1991, 55, 259–287. (4) Lovley, D. R.; Phillips, E. J. P.; Gorby, Y. A.; Landa, E. R. Nature 1991, 350, 413–416. (5) Myers, C. R.; Carstens, B. P.; Antholine, W. E.; Myers, J. M. J. Appl. Microbiol. 2000, 88, 98–106. (6) Lovley, D. R. Annu. Rev. Microbiol. 1993, 47, 263–290. (7) Beliaev, A. S.; Saﬀarini, D. J. Bacteriol. 1998, 180, 6292–6297. (8) Beliaev, A. S.; Saﬀarini, D.; McLaughlin, J. L.; Hunnicutt, D. Mol. Microbiol. 2001, 39, 722–730. (9) Pitts, K. E.; Dobbin, P. S.; Reyes-Ramirez, F.; Thomson, A. J.; Richardson, D. J.; Seward, H. E. J. Biol. Chem. 2003, 278, 27758–27765. (10) Xiong, Y.; Shi, L.; Baowei, C.; Mayer, M. U.; Lower, B. H.; Londer, Y. Y.; Bose, S.; Hochella, M. F.; Fredrickson, J. K.; Squier, T. C. J. Am. Chem. Soc. 2006, 128, 13978–13979. (11) Shi, L.; Squire, T. C.; Zachara, J.; Fredrickson, J. Mol. Microbiol. 2007, 65, 12–20. (12) Clarke, T. A.; Holley, T.; Hartshorne, R. S.; Fredrickson, J. K.; Zachara, J. M.; Shi, L.; Richardson, D. J. Biochem. Soc. Trans. 2008, 36, 1005–1010. (13) Johs, A.; Shi, L.; Droubay, T.; Ankner, J. F.; Liang, L. Biophys. J. 2010, 98, 3035–3043. (14) Firer-Sherwood, M. A; Pulcu, G. S.; Elliott, S. J. J. Biol. Inorg. Chem. 2008, 13, 849–854. (15) Hartshorne, R. S.; Jepson, B. N.; Clarke, T. A.; Field, S. J.; Fredrickson, J.; Zachara, J.; Shi, L.; Butt, J. N.; Richardson, D. J. J. Biol. Inorg. Chem. 2007, 12, 1083–1094. (16) Lower, B. H; Shi, L.; Yongsunthon, R.; Droubay, T. C.; McCready, D. E.; Lower, S. K. J. Bacteriol. 2007, 189, 4944–4952. (17) Myers, C. R.; Myers, J. M. J. Bacteriol. 1997, 179, 1143–1152. (18) Schwalb, C.; Chapman, S. K.; Reid, G. A. Biochem. Soc. Trans. 2002, 30, 658–662. (19) Hartshorne, R. S.; Reardon, C. L.; Ross, D.; Nuester, J.; Clarke, T. A.; Gates, A. J.; Mills, P. C.; Fredrickson, J. K.; Zarchara, J. M.; Shi, L.; Beliaev, A. S.; Marshall, M. J.; Tien, M.; Brantley, S.; Butt, J. N.; Richardson, D. J. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 22169– 22174. (20) Lower, B. H.; Yongsunthon, R.; Shi, L.; Wildling, L.; Gruber, H. J.; Wigginton, N. S.; Reardon, C. L.; Pinchuk, G. E.; Droubay, T. C.; Boily, J.-F.; Lower, S. K. Appl. Environ. Microbiol. 2009, 75, 2931–2935. (21) Donald, J. W.; Hicks, M. G.; Richardson, D. J.; Palmer, T. J. Bacteriol. 2008, 190, 5127–5131. (22) Shi, L.; Chen, B.; Wang, Z.; Elias, D. A.; Mayer, M. U.; Gorby, Y. A. J. Bacteriol. 2006, 188, 4705–4714. (23) Sharma, S.; Cavallaro, G.; Rosato, A. J. Biol. Inorg. Chem. 2010, 15, 559–571. (24) Taylor, P.; Pealing, S. L.; Reid, G. A.; Chapman, S. K.; Walkinshaw, M. D. Nat. Struct. Biol. 1999, 6, 1108–1112. (25) Igarashi, N.; Moriyama, H.; Fujiwara, T.; Fukumori, Y.; Tanaka, N. Nat. Struct. Biol. 1997, 4, 276–284. (26) Seltmann, G., Holst, O. Periplasmic Space and Rigid Layer. Periplasmic Space and Rigid Layer. In The Bacterial Cell Wall; Springer: New York, 2002; pp 103 128. (27) Clarke, T. A.; Dennison, V.; Seward, H. E.; Burlat, B.; Cole, J. A.; Hemmings, A. M.; Richardson, D. J. J. Biol. Chem. 2004, 279, 41333–41339. 11213 dx.doi.org/10.1021/jp203603r |J. Phys. Chem. B 2011, 115, 11208–11214