Efficient Production of Recombinant Brazzein, a Small, Heat-Stable, Sweet-Tasting Protein of Plant Origin

Correlation of the Sweetness of Variants of the Protein Brazzein with Patterns of Hydrogen Bonds Detected by NMR Spectroscopy

“…Sample Preparation-Recombinant brazzein and its variants were prepared as described previously (13 C]glucose (Isotec). Cells were induced for 3 h by the addition of isopropyl-1-thio-␤-D-galactopyranoside to a final concentration of 0.1 mM.…”

“…Brazzein is small, stable, and easily investigated in solution by NMR spectroscopy. We earlier designed a synthetic gene coding for brazzein and developed an efficient bacterial production system (a fusion protein expressed in Escherichia coli) (13) and have used this approach to discover mutants with altered sweetness (14). This system also has allowed us to produce rapidly stable isotope-labeled brazzein variants for structural evaluation by NMR spectroscopy.…”

“…Although trans-hydrogen bond 15 N- 13 C NMR scalar coupling constants are very small (͉ 3h J NCЈ ͉ Ͻ 1 Hz), methods have been developed recently (15)(16)(17)(18) which permit their detection in small proteins (Ͻ30 kDa). These methods employ constant time, long range, two-or three-dimensional NMR correlation experiment that identifies connectivities among backbone 1 H N , 15 N, and 13 CЈ atoms (HNCO) 1 pulse sequences, either without or with the application of the transverse optimized spectroscopy (TROSY) technique (19) for increased sensitivity.…”

“…These methods employ constant time, long range, two-or three-dimensional NMR correlation experiment that identifies connectivities among backbone 1 H N , 15 N, and 13 CЈ atoms (HNCO) 1 pulse sequences, either without or with the application of the transverse optimized spectroscopy (TROSY) technique (19) for increased sensitivity. Identification of hydrogen bonds by detection of trans-hydrogen bond couplings has been used to investigate protein secondary structure (20), protein stability (21), and ligand binding (22).…”

“…1 shows the 1 The abbreviations used are: HNCO, NMR correlation experiment that identifies connectivities among backbone 1 H N , 15 N, and 13 CЈ atoms; COSY, correlated spectroscopy; CS, cysteine-stabilized; DSS, 2,2-dimethyl-2-silapentane-5-sulfonic acid; HSQC, heteronuclear single quantum coherence; NOE, nuclear Overhauser effect; NOE, { 1 H}-15 N steady-state heteronuclear NOE; NOESY, nuclear Overhauser effect spectroscopy; TOCSY, total correlation spectroscopy; SE, sensitivity enhanced; HNCA, NMR correlation experiment that identifies connectivities among backbone 1 H N , 15 N, and 13 C ␣ atoms; HCCH, NMR correlation experiment that identifies connectivities among protons attached to 13 C through 13 C- 13 C correlations; HN(CO)CA, NMR correlation experiment that identifies connectivities among backbone 1 H ␣ , 13 C ␣ , and 13 CЈ atoms of the same residue.…”