Chemistry Posters Online - Native Beta-C Domain of Lobster : Methods

Determination of the solution three dimensional structure of chemically-synthesized native ¹¹³Cd₃S₉ domain of Lobster Metallothionein by NMR Spectroscopy

Amalia Muñoz^*, F. Holger Forsterling, C. Frank Shaw III⁺ and David H. Petering

METHODS

Peptide synthesis was accomplished using Fmoc-chemistry in an Applied Biosystem (abi) Peptide Synthesizer 430A. S-Acm-cysteine was chosen as the cysteine source to effectively protect its thiol group from possible oxidation process.
Cleavage and Purification of the polypeptides were accomplished by following the standard procedures.
Cd-reconstitution was accomplished by reaction of the apo-b domains with slight excess of Cd⁺² in Tris HCl buffer at pH ~7.5 and in the presence of 2-mercaptoethanol to avoid oxidation of the thiolates formed. Then, it was desalted over a Sephadex G-10 column. Adventitiously bound Cd⁺² was removed by treatment with Chelex-100® resin.
Characterization of the Cd₃beta_C derivatives:
- Amino acid analysis (carried out at the Protein / Nucleic Shared Facility of the Medical College of Wisconsin).
- Cd⁺²/SH content (by AAS and thiol titration with DTNB (5,5'-dithiobis(2-nitrobenzoate) analysis, respectively).
- Molecular weight (gel filtration chromatography, Sephadex G-25).
- ES-MS (samples dissolved in 50% MeOH/H₂O with 1% acetic acid as source of protons were analyzed in a VG AutoSpec, Fison Instruments, equipped with a Harward Syringe Driver).
- UV-spectroscopy (12 µM Cd-beta₃ in 5 mM Tris HCl at pH 7.5 were analyzed in a Perkin-Elmer Lambda 6 UV-vis spectrometer).
- CD-spectropolarimetry (20-30 µM Cd-beta derivatives in 5 mM Tris HCl at pH 7.5 analyzed in a Jasco CD-Spectropolarimeter Model J-715).
¹¹³Cd-NMR (samples reconstituted with ¹¹³Cd⁺² in 5 mM d11-Tris HCl are analyzed in a Brucker DPX300MHz NMR).
2D-NMR: samples reconstituted with ¹¹³Cd⁺² in 5 mM d11-Tris HCl are analyzed in a Brucker DRX500MHz NMR
- In homonuclear experiments quadrature detection in the indirect dimension was obtained using TPPI (COSY, NOESY) or States-TPPI (TOCSY) phase cycling and WATERGATE scheme for water suppression. DQF-COSY used gradients at the magic angle as double quantum filter. TOCSY spectra was acquired using WALTZ17 spin lock. ¹¹³Cd GARP decoupling was used in all experiments except the DQF COSY(used to extract the coupling constants). All data matrices were zero filled to a final size of 2048 x 2048, and multiplied with a 60^o phase shifted squared cousine window function in both dimensions prior to Fourier transform.
- Heteronuclear ¹H{¹¹³Cd} correlation experiments were obtained using pulse field gradients for coherence selection and water suppression. For the ¹H{¹¹³Cd} HSQC-TOCSY a delay of 8 ms for magnetization transfer was used in the INEPT transfer and MLEV17 spin lock of 82 ms for TOCSY transfer, accumulating 256 transients for each of the 64 complex points, t₁. Linear prediction to 128 complex points was applied in f₁, after which zero filling resulted in a total matrix size of 2048 x 1024 points in f₂ and f₁, respectively. A squared cosine window was employed in both dimensions prior to Fourier transformation.
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Amalia Muñoz - April, 2000

Determination of the solution three dimensional structure of chemically-synthesized native 113Cd3S9 domain of Lobster Metallothionein by NMR Spectroscopy

Amalia Muñoz*, F. Holger Forsterling, C. Frank Shaw III+ and David H. Petering

METHODS

Determination of the solution three dimensional structure of chemically-synthesized native ¹¹³Cd₃S₉ domain of Lobster Metallothionein by NMR Spectroscopy

Amalia Muñoz^*, F. Holger Forsterling, C. Frank Shaw III⁺ and David H. Petering