NMR studies of a Glutaredoxin 2 from Clostridium oremlandii

Abstract

Grx2 is a glutaredoxin from gram positive bacterium Clostridium oremlandii (strain OhILAs), which is Cys-homolog of selenoprotein Grx1. Grx2 is a poor reductant of selenoprotein MsrA not like Grx1 while the reducing activity is reversed in two Grxs for Cys version of MsrA.

The wild-type Grx2 and the C15S mutant were overexpressed in E.coli and purified by affinity chromathography and gel filtration. The 3D NMR spectra was collected and assigned all the backbone chemical shifts including C[alpha], C[beta], C[Omicron], HN, and N of Grx2 and C15S mutant. The protein folding of two proteins were evaluated by circular dichroism.

Here we report the protein purification and NMR spectroscopic study of recombinant Grx2 and the C15S mutant. The HSQC spectrum of two proteins show chemical shift difference for residues 8-19, 52-55,66. The circular dichroism result shows that recombinant proteins are well folded.

The conformation of two proteins resembles the oxidized form (wild-type Grx2) and the reduced form (the C15S mutant). The residues showing chemical shift difference will join the conformational change of Grx2 upon a disulfide formation.

Lee et al. Journal of Analytical Science and Technology 2013, 4:2

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RESEARCH Open Access

NMR studies of a Glutaredoxin 2 from Clostridium oremlandii

Eun Hye Lee1,2, Hae-Kap Cheong1 and Hye-Yeon Kim1*

Abstract

Background: Grx2 is a glutaredoxin from gram positive bacterium Clostridium oremlandii (strain OhILAs), which is Cys-homolog of selenoprotein Grx1. Grx2 is a poor reductant of selenoprotein MsrA not like Grx1 while the reducing activity is reversed in two Grxs for Cys version of MsrA.

Methods: The wild-type Grx2 and the C15S mutant were overexpressed in E.coli and purified by affinity chromathography and gel filtration. The 3D NMR spectra was collected and assigned all the backbone chemical shifts including C, C, C, HN, and N of Grx2 and C15S mutant. The protein folding of two proteins were evaluated by circular dichroism.

Results: Here we report the protein purification and NMR spectroscopic study of recombinant Grx2 and the C15S mutant. The HSQC spectrum of two proteins show chemical shift difference for residues 8-19, 52-55,66. The circular dichroism result shows that recombinant proteins are well folded.

Conclusion: The conformation of two proteins resembles the oxidized form (wild-type Grx2) and the reduced form (the C15S mutant). The residues showing chemical shift difference will join the conformational change of Grx2 upon a disulfide formation.

Keywords: Grx2, MsrA, Clostridium oremlandii, Backbone assignment, NMR

Introduction

Glutaredoxins (Grxs) have been studied in decades and described as glutathionine-dependent reductases of the disulfide formed during its catalytic cycle (Holmgren et al. 2005). Grxs are able to restore the growth of E.coli in a mutant lacking thioredoxin (Trx) (Holmgren 1976).Trxs and Grxs share several functions but Grxs are more versatile in choice of substrate and reaction mechanisms (Holmgren 1989). Two groups of Grxs, dithiol and monothiol Grxs, are divided upon catalytic site and functional mechanism (Lillig et al. 2008). Dithiol Grxs contain the characteristic CPYC active site motif and monothiol Grxs lack the C-terminal active site cysteine in the CGFS motif. Both Grxs utilize glutathionine (GSH) as a substrate and share structural elements of binding GSH. GSH is a major biological compound and has a pivotal role in cellular redox homeostasis (Meister 1994). The ratio of GSH and the oxidized form of GSH,

glutathionine disulfide (GSSG), are major determinants of cellular redox state. Grxs could regulate the cellular processes related with the GSH-GSSG redox state. Many organisms contain a unique composition of Grxs. E.coli contains four Grxs, two classical dithiol Grxs (Grx1 and Grx3), one unusual dithiol Grx (Grx2), and one monothiol Grx (Grx4) (Vlamis-Gardikas & Holmgren 2002; Fernandes & Holmgren 2004). The structures of Grxs have been studied by X-ray crystallography and NMR spectroscopy. Grxs belong to the Trx fold family which consists of a four stranded -sheet surrounded by three -helices. In addition to the active site motif, two additional regions are present for binding of GSH; the residues preceding the cis-proline (consensus: TVP) and the residues following the GG-motif (consensus: GGxdD) (Lillig et al. 2008).

Clostridium oremlandii (strain OhILAs) is a selenoprotein-rich organism and contains selenoprotein MsrA and selenoprotein Grx1 (Kim et al. 2006). C.oremlandii has a Cys-homolog protein of selenoprotein Grx1 which is defined as glutaredoxin 2 (Grx2). MsrA catalyzes the

* Correspondence: mailto:[email protected]

Web End [email protected]

1Division of Magnetic Resonance Research, Korea Basic Science Institute, Ochang, Chungbuk, KoreaFull list of author information is available at the end of the article

2013 Lee et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0

Web End =http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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reduction of oxidized methionine residue in cellular proteins. A cysteine residue at the active site of MsrA is oxidized after the catalysis and then recycled by reductases like Trx. Selenoprotein MsrA shows 20-fold higher catalytic activity than its Cys-containing form instead of selenocysteine (Sec). This organism uses Grx proteins, Grx1 or Grx2, for reduction of the oxidized MsrA instead of Trxs. Selenoprotein Grx1 is a strong reductant of selenoprotein MsrA while Grx2 shows poor reducing activity for selenoprotein MsrA (Boschi-Muller et al. 2000). Although Grx1 and Grx2 share sequence homology of 55%, the reducing activity for selenoprotein MsrA is extremely different. Interestingly, the reducing activity of Grxs is reversed between Cys vesion of Grx1 and wild-type Grx2 for Cys version of MsrA. Grx2 shows high reducing activity whereas Cys version Grx1 shows almost no activity in reduction of Cys version of MsrA (Kim et al. 2011). Previously, we reported the backbone assignment result of Cys version Grx1 (Lee et al. 2012). To investigate the structural characteristics of Grx2, we have performed the NMR spectroscopy of Grx2 and its C15S mutant. Grx2 consists of 85 amino acid residues including three cysteine residues in its sequence and contains a conserved CGPC motif of dithiol Grxs. Two cysteine residues are

defined as catalytic and resolving cysteines depending on the role during the cataylsis. Catalytic cysteine reduces the substrate and then the oxidized cysteine is recovered by the resolving cysteine. The resolving cysteine C15 is introduced to obtain the advantages in monitoring the molecular interaction between catalytic cysteines of Grx2 and MsrA. The wild-type and the C15S mutant of Grx2 are subjected to NMR experiments and circular dichroism. Here, we report purification and NMR backbone assignment of recombinant Grx2 proteins.

Methods

Cloning, expression and purification

Grx2 (residues 185) from genomic DNA of Clostridium oremlandii was cloned into the expression vector pET21b (Novagen). The recombinant plasmids were transformed to E.coli BL21(DE3) cells for protein overexpression. The wild-type Grx2 and the C15S mutant of Grx2 (the C15S mutant) were expressed with the C-terminal Histag (LEHHHHHH). The cells were grown in M9 minimal media containing 100 g/ml ampicilin for 13C/15 N double labeling at 37C until OD600 reached0.6. Then protein overexpression was induced by addition of 0.5 mM IPTG at 18C for 20 h. The cells was harvested by centrifugation at 4,500 rpm for 20 min and resuspended in the ice-cold buffer A (20 mM TrisHCl, pH 7.5, 300 mM NaCl, 4 mM MgCl2). Harvested cells were disrupted by sonication and centrifuged at 13,000 rpm for 50 min at 4C. The supernatant was loaded onto HisTrap column (GE Healthcare) equilibrated with buffer A and recombinant protein was eluted by gradient increasing of imidazole concentration. The protein was concentrated to ~2 ml and applied to HiLoad 16/60 Superdex-75 (GE healthcare) equilibrated with 20 mM HEPES, pH 7.0, 100 mM NaCl. The eluted protein was concentrated to 1 mM for NMR study.

NMR data acquisition and analysis

NMR experiments were performed at 25C using 1 mM of 13C,15 N-labeled Grx2 and the C15S mutant samples in 20 mM HEPES, pH 7.0, 100 mM NaCl. 10% D2O of total sample volume and 5 mM DTT were added to both samples before experiments. NMR data were collected by Bruker Avance 800-MHz NMR spectrometer (Korea Basic Science Institute, Korea) for three days. The backbone chemical shift were obtained by three-dimensional heteronuclear correlation experiments: HNCO, HN(CA)CO, HNCA, HN(CO)CA, HNCACB, CBCA(CO)NH (Wishart et al. 1995). NMR experiments of Grx2 including three spectra, HSQC, HNCACB and CBCA(CO)NH, were performed at same condition. All NMR data were processed and analyzed by TopSpin (Bruker BioSpin), NMRPipe (Delaglio et al. 1995) and then applied to AutoAssign server (Zimmerman et al. 1997) and further

Figure 1 Protein purification. Samples from all purification steps

were confirmed by the SDS-PAGE analysis. The expressed Grx2

proteins were purified using the HisTrap column and then

applied to the Superdex 75 gel chromatography column. The

purified C15S mutant (A) and wild-type Grx2 (B) proteins

show >98% purity. The migration of the molecular mass markers

is indicated on the left.

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Figure 2 1H-15N HSQC spectra of the C15S mutant and wild-type Grx2. (A) Assigned HSQC spectrum of the C15S mutant. (B) HSQC spectra

overlay of the C15S mutant (black) and wild-type Grx2 (red). Several residues of wild-type Grx2 protein are represented with prime(') marks. All

assigned residues are labeled and one crowded region is magnified (insets). The mutated residue C15S is indicated by red arrow. The assigned

set of cross peaks from amide side-chains of Asn and Gln residues is indicated using a gray horizontal bar. The unassigned peaks are marked by

'*' and the tryptophan side chain is marked by 'sc'.

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Table 1 Assigned backbone chemical shifts (1HN, 15 N, 13CO, 13C and 13C) of the C15S mutantAA HN N C C CO AA HN N C C CO K2 - - 53.11 31.01 172.9 A46 7.573 119.8 51.64 15.38 176.3

N3 9.051 121.6 50.24 36.27 171.9 K47 7.341 116.7 55.65 30.68 175.9 I4 8.905 129.5 58.24 36.53 172.6 T48 8.153 107.8 59.14 68.14 173.8 T5 9.033 124.7 58.69 68.94 169.6 G49 8.49 110.5 42.89 - 171.7 I6 9.087 123.6 55.28 39.13 169.5 W50 8.535 122.5 53.42 27.5 172.8 Y7 8.926 129.2 54.69 36.8 173.5 D51 8.356 118.9 49.69 38.2 173.4 T8 8.784 111.8 57.11 69.57 170.6 T52 7.153 108 57.08 68.83 170.9 K9 6.958 112.2 52.91 34.43 175.1 V53 8.021 110.5 55.83 30.89 170 N10 8.323 119.2 52.42 35.54 172.7 P54 - - 59.2 33.52 173.8 P13 - - 61.69 29.38 176.9 Q55 7.844 116.5 54.31 31 172 Y14 8.659 126.1 58.27 35.19 176.2 V56 8.518 123.9 59.31 30.42 170.6 S15 11.18 130.2 61.41 51.86 172.3 F57 9.482 126.2 53.6 40.89 172.5 K16 7.612 120.4 56.83 29.69 176.5 V58 8.779 118.8 58.27 30.67 173.3 K17 7.694 120.4 56.84 30.24 176.4 D59 9.754 130 54.44 36.77 173.1 A18 8.277 122.1 52.92 16.91 175.6 E60 - - 54.66 26.15 172.9 V19 8.354 118.1 64.72 28.92 175.6 E61 8.533 123.4 53.16 28.25 172.4 S20 8.237 116.1 59.22 59.93 174.3 F62 8.81 127.5 52.56 36.32 172.9 L21 7.68 123.4 55.33 37.92 177.2 L63 8.59 128.3 51.63 40.3 172.3 L22 7.636 118.8 55.57 37.86 176.6 G64 5.136 102.3 41.28 41.28 169.3 S23 8.838 114.5 59.36 60.24 175.3 G65 8.823 108.3 40.98 41.03 171.3 S24 8.109 118.4 58.38 60.28 172.7 C66 8.721 119.9 60.96 35.07 -K25 7.329 119.3 52.69 30.37 174.8 D67 - - 55.04 36.66 176.3 G26 7.779 106 43.67 - 171.7 D68 7.602 118.7 54.92 38.57 176.4 V27 7.012 112 56.67 30.7 172.2 I69 8.009 111.5 62.98 35.16 175.1 D28 8.438 123.8 51.44 38.58 173.1 H70 7.594 120.9 59.86 25.44 175 F29 7.711 118 52.92 39.09 169.9 A71 8.196 124.9 52.91 14.83 178 K30 8.796 122.4 51.96 31.66 171.5 L72 8.091 116.4 54.66 39.86 177.1 E31 8.648 127.8 51.43 28.48 173 D73 7.863 121 54.4 39.05 177 V32 9.056 130.8 58.79 29.63 171.2 R74 8.153 120.6 56.52 27.33 175.8 D33 8.44 126.3 50.56 38.56 176 Q75 7.462 114.5 53.4 27.95 173.6 V34 9.159 120.7 58.59 28.05 173.5 G76 7.854 107.9 42.97 - 172.2 T35 8.458 119.7 64.95 66.01 172.4 I77 7.713 115.7 58.9 36.87 174 H36 8.335 116.7 52.67 28.19 172.2 L78 7.18 123.4 55.31 37.16 175.1 D37 7.393 120.4 49.88 37.87 172.1 D79 8.68 118.1 55.5 37.08 175.5 S38 8.297 118.8 58.21 60.13 174.5 K80 7.165 117.6 56.33 29.42 177.8 K39 8.33 124.3 56.29 28.85 176 K81 7.843 119.8 55.12 29.3 175.3 A40 7.775 120.8 51.76 15.74 178.1 L82 7.896 113.1 52.22 39.01 173.2 F41 7.683 116.6 56.17 36.15 174.1 G83 7.521 104.5 42.96 - 171.8 E42 8.404 120.2 57.2 26.4 176.8 L84 7.835 119.5 52.36 39.76 173.9 D43 7.692 118.8 54.38 37.61 175.6 K85 8.096 120.6 52.74 30.37 173 V44 7.135 121 63.45 28.52 174.6 L86 8.2 123.9 52.29 39.69 174.5 M45 8.028 119.4 56.38 30.65 177 E87 8.413 121.4 53.69 27.68 173.3

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Table 2 Assigned backbone chemical shifts (1HN, 15 N, 13C and 13C) of Grx2

AA HN N C C AA HN N C C K2 - - 53.23 31.02 A46 7.585 119.8 51.66 15.39

N3 9.061 121.6 50.28 36.4 K47 7.343 116.7 55.71 30.68 I4 8.912 129.5 58.31 36.49 T48 8.155 107.8 59.24 68.11 T5 9.047 124.5 63.84 - G49 8.507 110.5 42.94 -I6 9.084 123.5 55.37 39.11 W50 8.558 122.5 53.47 27.49 Y7 8.903 129.2 54.97 36.88 D51 8.375 118.7 49.65 38.21 T8 8.907 111.5 56.84 69.69 T52 7.168 108 57.09 68.89 K9 7.542 114.8 52.83 34.3 V53 8.015 109.4 55.83 30.74 N10 8.344 118.8 52.17 35.64 P54 - - 59.17 33.67 P13 - - 61.83 29.48 Q55 7.721 116.6 54.55 30.74 Y14 9.096 127.1 58.64 34.98 V56 8.494 124.1 59.31 30.37 C15 9.988 128.1 62.55 25.84 F57 9.494 126.2 53.62 40.9 K16 7.625 118.2 56.97 29.52 V58 8.79 118.8 58.31 30.64 K17 7.752 120.3 56.91 30.23 D59 9.758 129.9 54.55 36.8 A18 8.143 122.1 53 16.86 E60 - - 54.74 26.25 V19 8.465 118 64.75 28.91 E61 8.54 123.4 53.27 28.24 S20 8.269 116.1 60 59.79 F62 8.814 127.5 52.62 36.31 L21 7.687 123.4 55.41 37.91 L63 8.601 128.1 51.66 40.33 L22 7.6 118.7 55.69 37.85 G64 5.129 102.3 41.37 -S23 8.838 114.6 59.33 60.05 G65 8.818 108.4 41.08 -S24 8.136 118.3 58.39 60.29 C66 8.751 120 61.06 24.51 K25 7.326 119.3 52.77 30.38 D67 - - 55.07 36.64 G26 7.786 106 43.72 - D68 7.63 118.6 54.99 38.61 V27 6.905 111.3 56.66 30.72 I69 8.015 111.5 62.95 35.16 D28 8.442 123.8 51.56 38.57 H70 7.563 120.8 59.94 25.43 F29 7.726 118 52.98 39.14 A71 8.227 124.9 53.03 14.85 K30 8.791 122.4 51.98 31.75 L72 8.113 116.4 54.73 39.76 E31 8.652 127.8 51.55 28.36 D73 7.856 121 54.46 39.09 V32 9.006 130.8 58.84 29.69 R74 8.169 120.6 56.53 27.34 D33 8.479 126.3 50.68 38.67 Q75 7.475 114.4 53.47 27.94 V34 9.243 120.6 58.56 28.09 G76 7.856 107.9 43.01 -T35 8.424 119.6 64.91 65.99 I77 7.719 115.8 58.92 36.85 H36 8.36 116.7 52.76 28 L78 7.188 123.4 55.39 37.1 D37 7.419 120.4 49.91 37.86 D79 8.676 118.1 55.6 37.11 S38 8.28 118.7 58.31 60.15 K80 7.157 117.6 56.29 29.4 K39 8.338 124.3 56.3 28.85 K81 7.865 119.8 55.23 29.29 A40 7.771 120.8 51.83 15.75 L82 7.9 113.2 52.3 38.96 F41 7.7 116.5 56.24 36.16 G83 7.53 104.6 43.01 -E42 8.425 120.2 57.29 26.38 L84 7.841 119.5 52.41 39.72 D43 7.678 118.8 54.45 37.61 K85 8.116 120.7 52.85 30.36 V44 7.118 121 63.5 28.54 L86 8.2 123.9 52.35 39.69 M45 8.04 119.2 56.4 30.71 E87 8.412 121.5 53.73 27.68

backbone assignment was performed by Sparky (Goddard & Kneller 2004) software packages.

CD analysis

CD spectra (190250 nm) were measured at 25C on a Jasco J-715 apparatus, using a 1.0 mm path length quartz cell. Recombinant proteins were diluted 20 times with water at a protein concentration of 50 M. The buffer contained 1 mM HEPES, pH 7.0, 5 mM NaCl. The averaged blank spectra were subtracted.

Results and discussion

Sample preparation

The C-terminal Histag fused Grx2 and the C15S mutant proteins were overexpressed in E.coli BL21(DE3). The recombinant proteins were purified by nickel affinity chromatography (HisTrap column) and then applied to size-exclusion column (HiLoad 16/60 Superdex-75 column). The purified protein contained the C-terminal histag which was not removed by further treatment. Through gel filtration, Grx2 protein was eluted at a protein size of 10 kD and it means that Grx2 present as a monomer in solution. The eluted protein showed >98% purity at SDS-PAGE and concentrated to 1 mM for NMR measurements. The final purified proteins are shown in Figure 1.

Backbone assignment

The HSQC spectrum of the C15S mutant shows doublet peaks generated by intermolecular disulfide bond in oxidative condition. The doublet peaks disappear after addtion of DTT to the sample at concentration of 5 mM. We have assigned 92% of the expected backbone

1H-15 N correlations (77 out of 83; Grx2 contains 2 pro-line residues) and 96% of all 13CO, 13C and 13C (239 out of 249; Figure 2). The six residues, M1, K2, Y11, C12, E60 and D67, are not visible in HSQC spectrum. The two residues of C-terminal histag (86LEHHHHHH93) were assigned the backbone chemical shifts (Figure 2A). In HSQC spectrum, three 1H-15 N correlations are unassigned which lost their conectivity between assigned residues. The assigned chemical shifts (C, C, CO, HN, and N) of the C15S mutant were summarized in Table 1. NH2 group of Asn and Gln side-chains generally produce two split HSQC cross peaks that were identified in the measured HSQC spectrum. All possible 4 set of amide side-chains peaks were identified in the HSQC spectrum. Residues N3, N10, Q55 and Q75 made two split HSQC cross peaks which are indicated by gray line between two peaks. There is one tryptophan residue in Grx2 protein and the side chain NH resonance of W50 residue was assigned in HSQC spectrum. The missing residues in HSQC spectrum are expected to be partially solvent-exposed or have possible conformational

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Figure 3 The strip plot of S15 residue in the C15S mutant using spectra of HNCACB and CBCACONH. The sequential connectivity is

observed in neighboring residues Y14, S15 and K16 except 13C of S15 showing low intensity. The peaks are colored by black (positive peak) and

red (negative peak).

exchange within NMR time scale. The unassigned three peaks may originated from the remained hexahistidine tag. The 1H-15N correlations of Grx2 are assigned on HSQC spectrum based on the C15S mutant assignments and HSQC spectra of two proteins are superposed (Figure 2B). Some ambigouos peaks are assigned by additional experiments of HNCACB, CBCA(CO)NH using 13C,15 N-labeled Grx2. The assigned chemical shifts

(C, C, HN, and N) of Grx2 were summarized in Table 2. The 1H-15 N correlations of Grx2 are assigned execept two correlations which are remained in unassigned in the C15S mutant spectrum. In HSQC spectrum of Grx2,

1H-15 N correlations of 77 residues are shown and they are common residues in the C15S mutant. Most residues are represented at the identical position of HSQC spectrum while some residues show large chemical shift

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Figure 4 The circular dichroism results for recombinant

proteins. The CD spectra at 50 M Grx2 proteins were obtained in

1 mM HEPES at pH 7.0 and 5 mM NaCl at 25C. The values are

expressed as mean residue molar ellipticity () in deg cm2 dmol-1.

change between wild-type Grx2 and the C15S mutant. The chemical shift of S15 residue has extremely high 1H

chemical shift of 11.1 ppm than 9.8 ppm of C15 residue. The 11.1 ppm can be observed in serine residue which has 1H chemical shift range of 3.76 ppm 12.33 ppm according to Biological Magnetic Resonance data Bank. The magnitude of the chemical shift depends upon the type of nucleus and the details of the electron motion in the nearby atoms and molecules (Hobbie 1998). The >1 ppm chemical shift difference may caused by extensive alteration of circumstance near proton in amino group of S15 residue. The strip plot of S15 residue with adjacent residues are represented in Figure 3. The residues K9, Y14 and C15 have chemical shift difference over 0.5 ppm and T8, K16, A18, V19, V53, Q55 and C66 residues have over0.1 ppm. These residues could be grouped to three regions, residues 819 including C15 residue, residues 52TVPQ55, and residue C66. The substitution of resolving cysteine to serine may induce the conformational change near active site that is related to oxidation state of Grx2. However, there is a possibility that the chemical shift difference is occurred by the simple change of chemical environments near C15 or S15 residue without no structural change. In addition, two Grx2 proteins have well-folded structure which are validated by circular dichroism (Figure 4).

Conclusions

The substitution of resolving cysteine to serine occured conformational change and these residues may be related to oxidation state of Grx2. Two proteins show different HSQC spectrum even in the reduced condition made by DTT addition. The addition of 5 mM DTT was not enough to break the intramolecular disulfide bond but the intermolecular disulfide bond. The resolviong C15 residue makes intramolecular disulfide bond with

catalytic C12 residue in wild-type Grx2. Wild-type Grx2 keeps two cysteine residues which can form a disulfide bond while the C15S mutant keeps one cysteine residue and is not able to form it. The conformation of two proteins resembles the oxidized form (wild-type Grx2) and the reduced form (the C15S mutant). The residues showing chemical shift difference will join the conformational change of Grx2 upon a disulfide formation. These results will be useful to the structural study of oxidized and reduced Grx2 and the interaction study with MsrA

Competing interestsThe authors declare that they have no competing interests.

Authors contributionsEHL carried out the smaple preparation, NMR studies and circular dichroism analysis. EHL, HKJ. and H-YK. drafted the manuscript. All authors read and approved the final manuscript.

AcknowledgementsThis work was supported by the NMR research program of Korea Basic Science Institute to H.-Y.K. We thank Kim Hwa-Young (Yeungnam University) for providing the Grx2 constructs.

Author details

1Division of Magnetic Resonance Research, Korea Basic Science Institute, Ochang, Chungbuk, Korea. 2Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul, Korea.

Received: 13 March 2013 Accepted: 13 March 2013 Published: 18 April 2013

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doi:10.1186/2093-3371-4-2Cite this article as: Lee et al.: NMR studies of a Glutaredoxin 2 from Clostridium oremlandii. Journal of Analytical Science and Technology 2013 4:2.

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