PP-50, a peptide based on residues 141-190 of the π-subunit of mitochondrial FpATPase, contains the GX4GKT consensus region for nucleoside triphosphate binding and has been shown to bind ATP [Garboczi, D.N., Shenbagamurthi, W.K., Hullihen, J., & Pedersen, P.L. (1988) J. Biol. Chem. 263, 812-816]. At pH 4.0, appropriate for NMR studies, PP-50 retains the ability to bind ATP tightly (KD= 17.5 µM) with a 1:1 stoichiometry as shown by titrations measuring the partial quenching of ATP fluorescence by PP-50. CD spectra of PP-50 at pH 4.0 and at low ionic strength show 5.8% helix, 30.2% π-structure, and 64% coil. ATP binding increases the structure of PP-50, changing the CD to 7.5% helix, 44.5% π-structure, and 48% coil. Increasing the ionic strength to 50 mM KC1 also increases the structure, changing the CD to 7.4% helix, 64.4% π-structure, and 28.2% coil. The 600-MHz proton NMR spectrum of PP-50, at pH 4.0 and low ionic strength, has been assigned by 2D methods (TOCSY, DQF-COSY, and NOESY with jump-return water suppression). Based on strong dαN NOEs, JαN values, and NH chemical shifts differing from random coil values, regions of extended structure are detected from residues 1-7 and 43-48. Based on dNN, dNN(i, i+2), and dπN(i, i+2) NOEs and 3JαN values, possible type I‘ and type I turns are found from residues 11-14 and 31-34, respectively. However, the NMR data also show dαN NOEs indicating extended structure throughout the peptide, suggesting that PP-50 in solution may have two principal conformations which interconvert rapidly on the NMR time scale, one involving turns from residues 11-14 and 31-34 and another with an extended conformation. Remote NOEs are found from Val-42 to Gly-13 and to Thr-23, indicating transient tertiary structure. Residues 16-23, consisting of the GX4GKT consensus sequence for nucleoside triphosphate binding show dαN NOEs, unshifted α- and π-proton resonances, and some averaged 3JαN values indicating both extended structure and a random coil, hence flexibility in this region.
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