JAST
2012 September;3(2):154-159.
Published online 2012 July 20.
doi:http://dx.doi.org/10.5355/JAST.2012.154
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| Copyright ⓒ 2010 Journal of Analytical Science & Technology |
| NMR spectroscopic study of a Glutaredoxin1 from Clostridium oremlandii |
| Eun Hye Lee1,2, Eun-Hee Kim1, Hwa-Young Kim3, Kwang Yeon Hwang2*, Hye-Yeon Kim1* |
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
3Department of Biochemistry and Molecular Biology, Yeungnam University College of Medicine, Daegu 705-717, Korea |
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Corresponding Author:
Hye-Yeon Kim, Kwang Yeon Hwang ,Tel: 82-43-240-5087, Email: hyeyeon@kbsi.re.kr; chahong@korea.ac.kr |
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ABSTRACT |
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| Grx1 is a thiol-disulfide oxidoreductase from gram positive bacterium Clostridium oremlandii(strain OhILAs), that plays a role in maintaining the cellular redox homeostasis. Here we report the protein purification and NMR spectroscopic study of recombinant Grx1 double mutant U13C/C16S. We collected 3D NMR spectra and assigned all the backbone chemical shifts including C?, C?, CO, HN, and N of Grx1. The secondary structure estimated from chemical shifts shows that Grx1 protein is well folded and consists of three ?-helices and four ?-strands. This NMR result is very useful for further structural and functional study of Grx1 protein. |
| Keywords: Grx1, Clostridium oremlandii, backbone assignment, NMR |
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FIGURES |
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Fig.1
Purification of Grx1. Samples from all purification steps were confirmed by the SDS-PAGE analysis. The expressed Grx1 protein was firstly purified using the HisTrap column (A) and then its eluents were applied to the Superdex 75 gel chromatography column (B). M, protein size marker; T, total fraction; S, soluble fraction; P, pellet; FT, flow-through; E, Elution; C, protein before gel filtration. |
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Fig.2
1H-15N HSQC spectrum of Grxl . All assigned residues are labeled and one crowded regions are magnified (insets). The assigned set of cross peaks from amide side-chains of Asn and Gln residues is indicated using a gray horizontal bar.
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Fig.3
The secondary structure of Grx1 was estimated from the assigned backbone chemical shifts. The probabilities of ?-helix and ?-sheet are represented using a positive and a negative vertical bar, respectively. The positive and negative lines indicated the boundaries of secondary structure for ?-helix and ?-sheet, respectively. The red arrows indicate two residues, H15 and S16, of which 1H-15N HSQC peaks were not identified. |
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TABLES |
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Table.1
Assigned backbone chemical shifts (1HN, 15N, 13CO, 13C? and 13C?) of Grx1 |
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