Plasmodium falciparum Apicoplast Transit Peptides are Unstructured in vitro and During Apicoplast Import
Version of Record online: 7 JUL 2011
© 2011 John Wiley & Sons A/S
Volume 12, Issue 9, pages 1124–1138, September 2011
How to Cite
Gallagher, J. R., Matthews, K. A. and Prigge, S. T. (2011), Plasmodium falciparum Apicoplast Transit Peptides are Unstructured in vitro and During Apicoplast Import. Traffic, 12: 1124–1138. doi: 10.1111/j.1600-0854.2011.01232.x
- Issue online: 11 AUG 2011
- Version of Record online: 7 JUL 2011
- Accepted manuscript online: 10 JUN 2011 08:34AM EST
- Received 7 September 2010, revised and accepted for publication 9 June 2011, uncorrected manuscript published online 10 June 2011, published online 7 July 2011
Additional Supporting Information may be found in the online version of this article:
Figure S1: Comparison to the previously published chemical shifts for the ACP domain of P. falciparum ACP. Comparison of ACP domain chemical shifts reported here with previously reported chemical shifts of P. falciparum ACP from BMRB accession number 6516. The overall agreement between chemical shifts is excellent despite some variations in the experimental conditions. Significant deviations at the N-terminus are expected because TP-ACP presented here has 42 additional amino acids at the N-terminus. The N- and C-termini are close in space in the folded structure, perhaps explaining a moderate difference observed in Cβ assignment at the C-terminus, and apparent in several nuclei at positions 59–63. The largest differences are observed in close proximity to the prosthetic group, which is attached at residue 95. The phosphopantetheine prosthetic group may have been more sensitive to the differences in experimental conditions. For each of the regions described, the NMR spectra were re-examined. For the termini, we confirmed that the cross-peaks nearest to the previously reported chemical shifts were inconsistent with our data, and that the cross-peaks reported here were consistent with our data. For the prosthetic group region, no cross-peaks were found in our data near the spectral location previously reported, and again self-consistency of our assignments was confirmed.
Figure S2: Chemical shift differences analyzed by the program TALOS+. Bar height indicates the confidence score assigned by TALOS+ for a particular secondary structure assignment, and reflects the scoring of the individual secondary structure type, combined with the scores of other competing secondary structure types. Secondary structure assignments are indicated by color: helix (red), coil (green), extended (blue). The backbone order parameter S2 is predicted from chemical shifts and plotted as a solid black line. A) TALOS+ analysis of the full-length TP-ACP construct. Helix assignments are in excellent agreement with (top) the crystal structure helices indicated by cylinders. Also assigned are the short 310 helix and single turn of alpha helix between main helices 1 and 2. Predicted S2 values between 0.8 and 0.9 are consistent with a well-folded domain. B) TALOS+ analysis of the transit peptide and IV. A short helix is predicted at positions 28–30, but confidence scores for secondary structure assignment are lower. Predicted S2 values vary between 0.4 and 0.6, which are significantly lower than for the ACP domain.
Figure S3: Predicted HSP70 affinity for TP constructs. HSP70 affinity predicted using the DnaK binding algorithm described by Rüdiger et al. A horizontal line drawn at −5 kJ/mol corresponds to approximately two times thermal energy, 2kT, of binding energy. Although chaperone affinity is partially reduced in some TP constructs, they are still predicted to bind effectively.
Table S1: Oligonucleotides used for this study.
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