BMRB Entry 19491

Assembly:

Entities:

Entity 1, Galectin-3, trans form, 1 (major form) 250 residues - 26150 Da.

Backbone and side chain resonance assignments for apo full-length human Galectin-3 (2-250) with natural occurring P64H & T98P point mutations. The original N-terminal Met1 amino acid is truncated from the amino acid sequence during protein expression (>70% processed), and substituted by an acetylated-Ala2 modification similarly as found in native Gal-3. The DNA sequence of the mutant Gal-3 is derived from human patients having cancer and cloned into a E.Coli plasmid vector for study of the protein. Multiple conformational states of the protein are present in solution caused by slow cis-trans isomerization of multiple prolines in the primary amino acid sequence. Set 1 correspond to the main set of peaks contributed to the major ensemble containing mostly trans-prolines. Exceptions on this rule are: P117 where no trans-proline state can be observed next to the major cis form, and P46/G47 where multiple cis-Pro peaks are observed in the spectra, with no obvious major trans-Pro peaks. Assignments of minor cis proline induced peaks in the NMR spectra are listed in conformer sets 2, 3, 4, and 5 (or resp. b, c,d and e in related figure plots) following set 1. Isomer set 2 represent resonance peaks of associated with the most abundant cis-Pro isomer (5-40%) affecting one particular local region, whereas sets c, d, and e represent minor forms induced by two (nearby) cis-prolines. Note that more than four discrete peaks per resonance signal can exist when multiple prolines are closely located within each other, e.g. at PxP or PxxxP junctions. Galectin-3 contains an N-acetyl alanine (Ala2) at position 2 (according to the original sequence numbering). HAC and CAC atoms of Ac-Ala2 in the BMRB entry represent the CH3 methyl proton and carbon atom of the acetyl group. Chemical shifts are: ----------------------------------------------------- 2 A CAC 13C 24.524 0.062 2 2 A HAC 1H 2.012 0.000 1 -----------------------------------------------------

Samples:

sample_1: Galectin-3, [U-98% 15N], 0.4 mM; DSS 2 uM; potassium phosphate 20 mM; DTT 8 mM; EDTA 0.1 mM

sample_2: Galectin-3, [U-98% 15N], 40 uM; DSS 1 uM; potassium phosphate 20 mM; DTT 8 mM; EDTA 0.1 mM

sample_3: Galectin-3, [U-98% 15N], 20 uM; DSS 1 uM; potassium phosphate 20 mM; DTT 8 mM; EDTA 0.1 mM

sample_4: Galectin-3, [U-98% 13C; U-98% 15N], 0.4 mM; DSS 2 uM; potassium phosphate 20 mM; DTT 8 mM; EDTA 0.1 mM

sample_5: Galectin-3, [U-98% 13C; U-98% 15N], 40 uM; DSS 1 uM; potassium phosphate 20 mM; DTT 8 mM; EDTA 0.1 mM

sample_6: Galectin-3, [U-98% 13C; U-98% 15N], 0.27 mM; DSS 2 uM; potassium phosphate 20 mM; DTT 11 mM; EDTA 0.1 mM; sodium azide 1 mM

sample_conditions_1: ionic strength: 25 mM; pH: 6.8; pressure: 1 atm; temperature: 303 K

Experiments:

Software:

SPARKY v3.114, Goddard - chemical shift assignment, data analysis, peak picking

TOPSPIN v3.2, Bruker Biospin - collection, processing

PACES, Coggins and Zhou - chemical shift assignment

NMR spectrometers:

• Bruker Avance 900 MHz
• Bruker Avance 700 MHz
• Bruker Avance 700 MHz