2-DIMENSIONAL NMR, CIRCULAR-DICHROISM, AND FLUORESCENCE STUDIES OF PP-50, A SYNTHETIC ATP-BINDING PEPTIDE FROM THE BETA-SUBUNIT OF MITOCHONDRIAL ATP SYNTHASE

PP-50, a peptide based on residues 141-190 of the beta-subunit of mitochondrial F1-ATPase, 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, 812-8161. At pH 4.0, appropriate for NMR studies, PP-50 retains the ability to bind ATP tightly (K(D) = 17.5-mu-M) 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% beta-structure, and 64% coil. ATP binding increases the structure of PP-50, changing the CD to 7.5% helix, 44.5% beta-structure, and 48% coil. Increasing the ionic strength to 50 mM KCl also increases the structure, changing the CD to 7.4% helix, 64.4% beta-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 d(alpha-N) NOEs, J(alpha-N) 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 d(NN), d(NN(i,i+2)), and d(alpha-N(i,i+2)) NOEs and 3J(alpha-N) values, possible type I' and type I turns are found from residues 11-14 and 31-34, respectively. However, the NMR data also show d(alpha-N) 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 d(alpha-N) NOEs, unshifted alpha- and beta-proton resonances, and some averaged 3J(alpha-N) values indicating both extended structure and a random coil, hence flexibility in this region.