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Efficient and selective isotopic labeling of hemes to facilitate the study of multiheme proteins
 
Bruno M. Fonseca1, Ming Tien2, Mario Rivera3, Liang Shi4, and Ricardo O. Louro1
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Moreover, to confirm that the ΔhemA E. coli LS543 strain is capable of expressing larger and more complex multiheme cytochromes c, an expression vector containing the gene of the decaheme cytochrome MtrA from S. oneidensis MR-1 of approximately 37 kDa, was used. Figure 1B shows that the resulting ΔhemA E. coli LS544 strain is also capable of expressing this protein, opening the door to the detailed characterization of these larger multiheme c-type cytochromes.

The auto-induction method developed by Studier (41) showed the best expression yields compared with the other protein expression methods tested (Figure 1A). This approach has the advantage of allowing the induction to occur gradually. This gradual process is essential for correct incorporation of the hemes, and allows the cultures to reach higher cell densities, thereby increasing the protein yield. A yield of approximately 4 mg pure STC(D2N) per liter of cell culture was obtained. To the best of our knowledge, to present date, this is the highest yield obtained for isotopically labeled multiheme cytochromes (40) and is comparable to other strategies used for overexpressing nonlabeled multiheme cytochromes c (25, 26, 46).

Thus, this expression method allows the efficient production of specifically isotopic labeled hemes and also their correct incorporation into a multiheme cytochrome c (Figures 2 and 3).



Using 1,2-13C-labeled dALA causes the incorporation of 13C at the methyl groups at the periphery of the heme macrocycle and also at the β- and carboxylate carbons of the propionate groups (Figure 2A). Figure 2 shows that adventitious unlabeled carbons in the methyl positions is below the detection limit of NMR experiments as can be confirmed by verifying the lack of residual peaks at the center of each doublet in spectra obtained without 13C decoupling (Figure 2A). In low-spin paramagnetic cytochromes, the heme methyls are reasonably sharp and typically located in a clean spectral region in the 13C dimension (Figure 3). This allows for a simple identification of these NMR signals that facilitates their assignment, since the remainder of the protein contains 13C only at natural abundance (╛1%).

This now opens the possibility to characterize in detail the structure and function of multiheme cytochromes containing a large number of hemes thanks to the greater spectral dispersion obtained in the 13C frequency versus the 1H frequency (Figure 3). The position of the heme methyl signals in low-spin paramagnetic hemes can be used to determine the orientation of the axial ligands and the placement of the magnetic axes system associated with the unpaired electron (47). When a multiheme cytochrome is titrated, the position of the methyl signals changes in ways that can be related with the oxidized fraction allowing for the determination of the relative reduction potentials of the hemes (48). The specific 13C labeling enabled by the method reported here is further suitable for characterizing proteins of large size or containing paramagnetic centers (49), because it allows the use of direct heteronuclear detection experiments such as 13C-13C NOESY, which may be more suitable than 1H based experiments in these cases.

Also, since dALA is a versatile labeling source for hemes, with different labeled carbons in dALA, different kinds of information can be obtained. For instance, considering NMR applications, using 5-13C-labeled dALA, the heme carbons attached to the meso protons can be labeled. Measurements of the residual dipolar coupling (RDC) of these signals provide information on the relative spatial orientation of the hemes (50).

A further general advantage of the method described here when applied to NMR spectroscopy is that the need for a highly concentrated sample, or even a pure sample, may be eliminated. Under aerobic conditions E. coli only expresses the cytochrome of interest, assuring the specific and efficient use of the labeled dALA in the biosynthesis of the hemes for this protein. Therefore, the 13C NMR spectrum is dominated by the signals of the labeled hemes. This advantage may facilitate the future characterization of multiheme c-type cytochromes that are difficult to express and purify, such as those associated to cell membranes, and may also allow the in cell characterization of cytochromes.

In conclusion, a strategy to efficiently produce multiheme cytochromes labeled at selected carbons in the hemes was developed. The simplicity of the method and its ability to produce isotopically labeled multiheme c-type cytochromes with a yield comparable to that obtained from the expression of unlabeled proteins, makes this approach potentially applicable to many different heme proteins. This is true even for those cytochromes that are not of the c-type and therefore dispense the need for covalent attachment of the heme to the polypeptide chain. The methodology will also enable the detailed structural and functional characterization of large multiheme cytochromes. A detailed characterization of these proteins, which mediate microbe-mineral or microbe-electrode contact, is essential to develop rationally designed bioelectrochemical devices and bioengineered systems for bioenergy production and bioremediation of environmental contaminants (13).

Acknowledgments

The plasmid pEC86 used in this work was a gift from Prof. L Thöny-Meyer. B.M.F. is the recipient of a PhD fellowship from Fundação para a Ciência e Tecnologia (FCT; SFRH/BD/41205/2007). L.S. was supported by the Subsurface Biogeochemical Research program/Office of Biological and Environmental Research, U.S. Department of Energy. Research in the author's laboratories was supported by grants PTDC/BIA-PRO 098158/2008, MIT-Pt BS-BB/1014/2008 from FCT awarded to R.O.L. and a grant from the National Science Foundation (MCB-0818488) awarded to M.R. This work was also supported by FCT through grant PEst-OE/EQB/LA0004/2011. The NMR data were collected at The Portuguese National NMR Network (REDE/1517/RMN/2005), supported by “Programa Operacional Ciência e Inovação (POCI) 2010” and FCT.

Competing interests

The authors declare no competing interests.

Correspondence
Address correspondence to Ricardo O. Louro, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República-EAN, 2780-157 Oeiras, Portugal. e-mail: [email protected]">[email protected]

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