Supplementary Material

Purification and Characterization of Rhodobactin: a Mixed Ligand Siderophore from Rhodococcus rhodochrous strain OFS

Suraj Dhungana1#, Ryszard Michalczyk1, Hakim Boukhalfa2, Joseph G. Lack1, Andrew T. Koppisch1, Jason M. Fairlee1, Mitchell T. Johnson2, Christy E. Ruggiero2, Seth G. John2, Matthew M. Cox2, Cindy C. Browder3, Jennifer H. Forsythe1, Laura A. Vanderberg1,2, Mary P. Neu2*, and Larry E. Hersman1*

1Bioscience and 2Chemistry Divisions, Los Alamos National Laboratory, Los Alamos NM 87545

3 Department of Chemistry, 1000 Rim Dr., Fort Lewis College, Durango, CO 81301

# Current Address: National Institute of Environmental Health Sciences (NIEHS), 111 T W Alexander Drive, RTP, NC 27709

1


*Address correspondence to this author:

L.E. Hersman

Mailstop M888

Ph. (505) 667-2779

Fax. (505) 665-3024

M.P. Neu

Mailstop J514

Ph. (505) 667-9313

Fax. (505) 665-4624

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Experimental Details for 1D and 2D NMR Analysis

One-dimensional 1H spectra were obtained using the standard one-pulse sequence. 13C and 15N 1H-decoupled spectra and 1H 15N-decoupled spectra were obtained using the inverse gated pulse program with WALTZ-16 decoupling during acquisition. CH, NH, CH2, and NH2 groups were assigned using DEPT spectra with flip angles of 45 and 90 degrees and WALTZ-16 proton decoupling.

Through-bond 1H correlations were obtained using 2D Double-Quantum-Filtered (DQF) COSY spectra and gradient selected TOCSY (Hurd 1990) spectra collected with 13.6, 27.6 and 55.2 ms mixing time. Spectral width in the experiments was set to 5.3 kHz in both dimensions, and the spectra were collected with 2048 complex points in the direct dimension and 512 complex points in the indirect dimension. Quadrature detection in t1 was achieved using TPPI method. 15N-decouping was achieved with a 180 degree pulse during t1 period and with WALTZ-16 decoupling sequence during acquisition. For TOCSY spectra the DIPSI-2 (Rucker & Shaka 1989) mixing sequence was used with 6.25 kHz radiofrequency field. 2D NOESY spectra were recorded using the pulse sequence with gradient selection and 15N-decoupling (180 degree pulse during t1 period and WALTZ-16 decoupling sequence during acquisition) (Wagner & Berger 1996). Spectra were obtained at 100 ms, 200 ms, 300 ms and 500 ms mixing times, with 5.3 kHz spectral width in both dimensions and 2048x512 complex points. Quadrature detection in t1 was achieved using TPPI method.

Assignment of 13C resonances was obtained from 1H-13C HSQC and long-range (LR) HSQC spectra. Spectra were recorded using modified pulse sequence with purge gradients (Grzesiek & Bax 1993), in which selective water pulses were removed. Spectral width was set to 4 kHz in 1H dimension and 17.6 kHz in the 13C dimension (22.6 kHz for LR-HSQC), with 2048 and 800 (240 for LR-HSQC) complex points in 1H and 13C dimensions, respectively. The INEPT transfer delay, (4J)-1, was set to 1.6 ms for HSQC and 56.6 ms for LR-HSQC experiments and the spectra were collected with 64 and 800 scans per t1 increment, respectively. The one-bond INEPT transfer was not suppressed in the LR-HSQC experiment.

Assignment of 15N resonances was obtained from 1H-15N HSQC and LR-HSQC spectra. Spectra were recorded using the same pulse sequence as 1H-13C HSQC. Spectral width was set to 4.5 kHz in 1H dimension and 2.6 kHz in the 15N dimension, with 2048 and 512 complex points (256 for LR-HSQC), respectively. The INEPT transfer delay was set to 2.5 ms for HSQC and 28 ms for LR-HSQC experiments and the spectra were collected with 16 and 128 scans per t1 increment, respectively. The one-bond INEPT transfer was not suppressed in the LR-HSQC experiment.

Sequential correlations between amide protons and carbonyl carbons of preceding residues were confirmed using a two-dimensional version of a HNCO experiment (Kay et al 1994) with 15N evolution omitted. Spectral width was set to 5 kHz in 1H dimension and 7.5 kHz in the 13C dimension (13C carrier at 150 ppm), with 1024 and 96 complex points, respectively, and 128 scans per t1 increment.

References

Grzesiek S, Bax A. 1993. The Importance of Not Saturating H2O in Protein Nmr - Application to Sensitivity Enhancement and Noe Measurements. Journal of the American Chemical Society 115:12593-4

Hurd RE. 1990. Gradient-Enhanced Spectroscopy. Journal of Magnetic Resonance 87:422-8

Kay LE, Xu GY, Yamazaki T. 1994. Enhanced-Sensitivity Triple-Resonance Spectroscopy with Minimal H2O Saturation. Journal of Magnetic Resonance Series A 109:129-33

Rucker SP, Shaka AJ. 1989. Broad-Band Homonuclear Cross Polarization in 2D Nmr Using Dipsi-2. Molecular Physics 68:509-17

Wagner R, Berger S. 1996. Gradient-selected NOESY - A fourfold reduction of the measurement time for the NOESY experiment. Journal of Magnetic Resonance Series A 123:119-21


Figure S1 15N-decoupled natural abundance 13C spectrum of 15N -labeled rhodobactin in DMSO-d6.

Solvent resonance is marked with #; peaks marked with asterisks (*) originate from impurities in the sample.


Figure S2 15N-decoupled 1H-1H TOCSY spectrum, recorded with 27 ms mixing time, showing through-bond correlations for the rhodobactin.


Figure S3 15N-decoupled 1H-1H NOESY spectrum (300 ms mixing time) of the rhodobactin


Figure S4 1H-15N HSQC spectrum of the rhodobactin showing direct one-bond N-H correlations.


Figure S5 1H-13C HSQC spectrum (15N-decoupled) of the rhodobactin showing direct one-bond C-H correlations. The cross-peak containing regions were expanded for greater detail.


Figure S6 1H NMR spectra of hydrolyzed rhodobactin (bottom, solid line) and 2,3-dihydroxybenzoic acid ( top, dashed line)


Figure S7 ESI-MS spectrum of rhodobactin.


Figure S8 ESI-MS CID spectra for rhodobactin.


Figure S9 ESI-MS spectrum of 15N-labeled rhodobactin.


Figure S10 ESI-MS CID spectra for 15N-labeled rhodobactin.

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