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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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NMR sample preparation and experiments

Protein for NMR experiments was lyophilized twice using 2H2O (99.9 atom %). The protein was dissolved in approximately 500 µL 2H2O to a final concentration of approximately 0.5 mM in 10 mM phosphate buffer at pH 7.0. The pH value reported is a direct reading without correction for the isotope effect. NMR spectra obtained before and after the lyophilization were identical, showing that the protein structure was not affected by this procedure. The NMR experiments were performed with Bruker Avance and Avance II spectrometers operating at 500 MHz (Bruker BioSpin, Wissembourg, France). 2D-1H-13C heteronuclear multiple quantum correlation (HMQC) spectra were obtained with 2048 points covering a spectral width of 39.7 kHz in the 1H dimension and 256 increments with time-proportional phase incrementation (TPPI) to give a spectral width of 34 kHz in the 13C dimension, using a Δ delay fixed at 3.2 ms, with 16 scans, at a temperature of 25°C. 1D-1H spectra were always performed before and after each 2-D spectrum, in order to verify that no changes had occurred in the protein sample. Partially reduced samples were prepared by flushing out the oxygen from the NMR tube with nitrogen gas and subsequently adding controlled amounts of a freshly prepared 10 mM sodium dithionite solution using a gas-tight syringe. The 1H spectra were calibrated using the water signal as an internal reference. The Bruker TopSpin program (Bruker BioSpin, Wissembourg, France) was used to visualize and analyze the NMR spectra.

Results and discussion

Isotopic labeling of hemes using labeled dALA was originally reported by Druyan et al. (44), which labeled hemes of cytochromes belonging to the rat liver. Wachenfeldt et al. (45) and Rivera et al. (32) used isotopic labeled dALA to label hemes of monoheme cytochromes from bacteria. This was achieved by expressing the protein in a minimal medium supplemented with isotopically labeled dALA. This proved the concept for this type of strategy, although expression in minimal media has a much lower yield relative to expression in complex media, such as LB. However, using this method in a complex medium would lead to expression of proteins containing hemes produced from unlabeled sources available in the medium, decreasing the efficiency of isotopic labeling. A method for isotopic labeling of multiheme cytochromes c using complex medium was proposed by Fernandes et al. (40) that uses two steps of growth. Cells are initially grown in rich medium, harvested, washed, and resuspended in minimal medium supplemented with dALA. However, this procedure is experimentally cumbersome and not very efficient, since the overexpression step also occurs in minimal medium, which limits greatly the expression yields.

Although Woodward et al. (30) and Bryson et al. (33) reported strategies to isotopically label hemes, using mutants of E. coli incapable of synthesizing either the heme or dALA, respectively, these methods are limited with respect to the type of cytochromes that can be expressed. In particular, c-type cytochromes could not be obtained. Nonetheless, these developments opened the possibility of using rich media, such as LB, supplemented with heme or dALA under aerobic conditions to overexpress cytochromes.

Taking advantage of this progress, an E. coli mutant unable to synthesize dALA was created. The commercially available strain JM109(DE3), which had already proven its value for correctly overexpressing large multiheme cytochromes c efficiently, was used as source organism (39). This modified strain was then transformed with the vector pEC86 containing the ccmABCDEFGH (cytochrome c maturation) genes, that endows E. coli with the ability to correctly incorporate hemes in multiheme cytochromes c, when growing aerobically (23). This increases the versatility of this novel ΔhemA E. coli strain with respect to the type of cytochromes it can express efficiently, versus previous methods (30, 32, 33).

Deletion of the hemA gene does not affect the capability of the E. coli strains to express multiheme cytochromes efficiently, provided that the expression medium is supplemented with dALA. This was verified by comparing the multiheme cytochrome expression in the wt E. coli JM109(DE3) strain containing pEC86 with the expression in the ΔhemA E. coli strain LS543 (Figure 1A). For this, an expression vector containing a gene for the 12-kDa small tetraheme cytochrome (STC) from Shewanella oneidensis MR-1, mutated in a surface aspartate to an asparagine (D2N) was inserted in both strains. This modification does not affect the structure of the mutant versus native form, as determined by the pattern of the paramagnetic shifts in the NMR spectra collected in oxidized state (see Figure 2, B and C) but is expected to affect the interaction with physiological partners due to changes in surface electrostatics (38).

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