Solution-state NMR investigation of DNA binding interactions in Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg): a dynamic description of the DNA/protein interface

Solution Structure of the Conserved Hypothetical Protein Rv2302 from Mycobacterium tuberculosis

“…This DNA was then transfected into the host Escherichia coli bacterial strain BL21PRO (Clontech, Palo Alto, CA). Uniformly 15 N-and 13 C- 15 N-labeled Rv2302 was obtained by growing the transformed cells (37°C) in minimal medium (Miller) containing 15 NH 4 Cl and D-[ 13 C 6 ]glucose supplemented with thiamine (1 g/ml), Fe 2 Cl 3 (10 M), kanamycin (34 g/ml), and spectinomycin (100 g/ml). At an A 600 reading of ϳ0.8, protein expression was induced by making the broth concentration 1.0 M in isopropyl-␤-D-1-thiogalactopyranoside.…”

“…4B. The core of the structure is a five-strand antiparallel ␤-sheet with a conspicuous ␤-sheet twist (5) qualitatively implies that these residues are undergoing conformational exchange on an intermediate timescale (millisecond to microsecond) (4). The regions between the other ␤-strands in the sheet are more ordered relative to L1, as suggested by the lower pairwise RMSDs of these loop residues in the ensemble structures to the mean structure (Fig.…”

“…4C. The electrostatic nature of the protein's surface often plays a role in biochemical functions (4,10). The electrostatic potentials at the solvent-accessible surface was calculated for Rv2302 using PyMOL (DeLano Scientific, San Carlos, CA) to determine if Rv2302 has any clustering of charges on its solvent-accessible surface that may provide a continuous interface for electrostatic associations.…”

1H, 13C, and 15N resonance assignments of murine amelogenin, an enamel biomineralization protein

“…To assist the assignments, nine residue-specific, 15 N-labeled amino acid (L, V, H, R, K, M, F, Y, and E/Q) samples were prepared by growing the cells in "Redfield-medium" (Griffey et al 1985;Buchko et al 2005), a medium where 19 out of the 20 common amino acids are included. To prevent 15 N-isotopic scrambling of the target, residue-specific, 15 N-labeled amino acid due to endogenous amino acid biosynthesis (Waugh 1996), only ~50% of the unlabeled amino acid of interest was initially added to the medium.…”

“…To prevent 15 N-isotopic scrambling of the target, residue-specific, 15 N-labeled amino acid due to endogenous amino acid biosynthesis (Waugh 1996), only ~50% of the unlabeled amino acid of interest was initially added to the medium. At an OD 600 of ~0.8 the remaining 25% of the targeted amino acid containing 15 N at the amide position (CIL, Andover, MA) was added and protein expression induced at 298 K (Buchko et al 2005(Buchko et al , 2007. The logic in keeping the target amino acid at 75% of the level called for in the "Redfield-medium" recipe (Griffey et al 1985) is that it minimizes the likelihood that the cells will use the 15 N-labeled amino acid as a precursor for other amino acid residues in endogenous amino acid biosynthetic pathways (Waugh 1996).…”