(A) SDS gel (17% total acrylamide; 0.4% cross-linked acrylamide) of the recombinant b30-82 protein of E. coli F₁F_o ATP synthase. (B) Far-UV CD spectrum of the same protein.

Ribbon diagrams of the NMR solution structure of b30-82. (A) Best-fit superimposition of the 10 lowest-energy NMR structures. (B and D) Side and top views, respectively, of the average structure of b30-82, illustrating the relative positions of alanine residues 61, 68, 70, and 72 (blue) inside the α-helix. (C) Different orientations of b30-82, showing the molecular surface with the electrostatic potential of the peptide, drawn in PyMol (11), which uses the Poisson-Boltzmann equation. Positive potentials are shown in blue and negative potentials in red.

(A) SDS-polyacrylamide gel (17% total acrylamide; 0.4% cross-linked acrylamide) of the b22-156 A61C, A68C, A70C, and A72C mutants in the presence of 1 mM DTT. As a marker, the 71-amino-acid truncated N-terminal segment of subunit H of the A-ATP synthase (H_1-71) (3) is shown in the rightmost lane. (B) Far-UV CD spectra of the b22-156 A61C (solid line) and A68C (dashed line) mutants. (C) Cross-linking of the b22-156 A61C (lane 1), A68C (lane 2), A70C (lane 3), and A72C (lane 4) mutants in the presence of 10 μM CuCl₂. The mutant proteins were incubated with 10 μM CuCl₂ for 30 min at 4°C. The reaction was stopped by the addition of 1 mM EDTA. The samples were applied to a gel containing 17% total acrylamide and 0.4% cross-linked acrylamide.

2D ¹H-¹⁵N HSQC spectrum of b30-82 in 25 mM sodium phosphate buffer (pH 6.8) at 288 K. Signals from side chain NH₂ groups are connected by horizontal lines. (B) Secondary chemical shifts (Δ¹³Cα) of b30-82 in 25 mM phosphate buffer (pH 6.8) at 15°C.

Change in diffusion coefficient versus protein concentration for b30-82.

Model structure of a monomeric subunit b of E. coli F₁F_o ATP synthase. (A) The model was generated by fitting the crystallographic structure of b62-122 (orange) (Protein Data Bank entry [pdb] 1L2P) (12) and the NMR solution structures of b30-82 (green) (pdb 2KHK) and b140-156 (yellow) (26) into the envelope of the soluble domain, b22-156, derived from SAXS data (26). These structures have an overall RMSD of 0.498 Å for backbone atoms. The NMR structure of the membrane-embedded domain of subunit b, b1-34, is shown in blue. (Inset) Overlay of structures of residues Ala₆₂ to Ala₇₂, determined by crystallography (orange) and NMR (green). (B) Molecular surface with the electrostatic potential of the structures shown in panel A, which have been rotated by 180°. Positive potentials are shown in blue and negative potentials in red. Hydrophobic residues appear white. Numbers indicate the positions of residues Ala₃₂, Ala₄₅, Ala₅₀, Ala₅₇, Ala₆₁, Ala₆₈, and Ala₇₂. (C) The structures of b62-122 (orange) (pdb 1L2P) (12), b30-82 (green) (pdb 2KHK), and b140-156 (yellow) (26) were superimposed on the low-resolution structure of the dimeric b22-156, derived from solution X-ray scattering data (26). (D) Structures of b30-82 (green) and the N-terminal segment of b62-122 (orange) (pdb 1L2P) (12), revealing the relative positions of residues Ala₆₁, Thr₆₂, and Gln₆₄.