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1H, 13C, and 15N Sequence-specific Resonance Assignment and Secondary Structure of Plasmodium falciparum Thioredoxin
Munte, C.E.a, Becker, K.b, Schirmer, R.H.c, and Kalbitzer,
H.R.d
aInstituto de Física de São Carlos, Universidade de São Paulo, 13560-970 São Carlos SP, Brazil; bInterdiziplinäres Forschungszentrum, Universität Giessen, 35392 Giessen, Germany; cBiochemie-Zentrum der Universität, 69120 Heidelberg, Germany; dInstitut für Biophysik und Physikalische Biochemie, Universität Regensburg, 93040 Regensburg, Germany.
The malaria parasite Plasmodium falciparum spends part of its life cycle in human erythrocytes, where it is challenged with enhanced oxidative stress, and where it needs efficient anti-oxidants to maintain its reducing milieu and to protect itself against damage (Rahlfs et al., 2002). Thioredoxin (PfTrx) is one of the major redox-regulatory molecules which, because of its dithiol/disulfide exchange activity, determines the oxidation state of protein cysteines, being therefore a potential target in research towards anti-malarial drugs (Kanzok et al., 2000; Krnajski et al., 2001). We initiated the structure determination of the recombinant 104 amino acid polypeptide, using multidimensional NMR spectroscopy. Sequential backbone resonance assignments were carried out on the basis of HNCA, CBCA(CO)NH, CBCANH, HNCO, 1H-15N-HSQC and 1H-15N-NOESY_HSQC experiments. Side-chain assignments were done using the HCCH-TOCSY experiment with the sample in 2H2O. The chemical shifts for 99% of the backbone 1HN, 13Ca 15NH (without considering the proline residues), for 89% of the 13C', 99% of the 1Ha, and 88% of all side-chain atoms (except the 13C shifts of the aromatic rings) of the PfTrx were assigned. Data were deposited in the BioMagResBank under accession number 6442. The reduced state of the cysteine residues is confirmed by 13Cb chemical shifts at around 28 ppm, compared to 41 ppm typical for cystine residues (Wishart et al., 1995). Using the chemical shift values of the 1Ha, 13Ca, 13Cb and 13C' atoms the Chemical Shift Index was calculated using the computer program csi v. 1.1 (Wishart et al., 1994 – courtesy of Sykes et al., http://www.pence.ualberta.ca/ftp). The predicted secondary elements conserve the same pattern found in other thioredoxins (Friedmann et al., 2003; Wahl et al., 2005), suggesting a similar tertiary structure. References: Rahlfs et al. (2002) Cell. Mol. Life Sci., 59, 1024-1041; Kanzok et al. (2000) J. Biol. Chem., 275, 40181-40186; Krnajski et al. (2001) Mol. Biochem. Parasitol., 112, 219-228; Wishart et al. (1995) J. Biomol. NMR, 5, 67-81; Wishart et al. (1994) J. Biomol. NMR, 4, 171-180; Friedmann et al. (2003) FEBS Lett., 554, 301-305; Wahl et al. (2005) J. Mol. Biol., 345, 1119-1130.
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