SOLUTION STRUCTURE AND FUNCTION IN TRIFLUOROETHANOL OF PP-50, AN ATP-BINDING PEPTIDE FROM F(1)ATPASE

PP-50, a synthetic peptide, based on residues 141-190 of the beta-subu nit of mitochondrial F(1)ATPase, containing the GX(4)GKT consensus seq uence for nucleoside triphosphate binding, binds ATP tightly (K-d = 17 .5 mu M) as found by fluorescence titration at pH 4.0. CD and 2D proto n NMR studies at pH 4.0 revealed two beta-turns, regions of extended s econdary structure, transient tertiary structure, and flexibility in t he GX(4)GKT region (W. J. Chuang, C. Abeygunawardana, P. L. Pedersen, and A. S. Mildvan, 1992, Biochemistry 31, 7915-7921). CD titration of PP-50 with trifluoroethanol (TFE) reveals a decrease in ellipticity at 208 and 222 nm, saturating at 25% TFE. Computer analysis indicates th at 25% TFE increases the helix content from 5.8 to 28.6%, decreases th e beta-structure from 30.2 to 20.2% and decreases the coil content fro m 64 to 51.2%. Fluorescence titrations of H(2)ATP(2-) with PP-50 in 25 % TFE yields a K-d of 7.3 mu M, 2.4-fold tighter than in H2O, probably due to TFE increasing the activity of H(2)ATP(2-). PP-50 completely q uenches the fluorescence of H(2)ATP(2-) in 25% TFE, while in H2O the f luorescence quenching is only 62%. In H2O the binding of H(2)ATP(2-) i ncreases the structure of PP-50 as detected by CD, but in 25% TFE no s ignificant change in CD is found on binding either H(2)ATP(2-) or Mg(2 +)HATP (K-d = 14 mu M). The complete proton NMR spectrum of PP-50 in 2 5% TFE has been assigned. The solution structure, determined by distan ce geometry, molecular dynamics with simulated annealing, and energy m inimization, consists of a coil (residues 1-8), a strand (residues 9-1 2), a loop (residues 13-22) containing the GX(4)GKT consensus sequence (residues 16-23), an alpha-helix (residues 23-36), a turn (residues 3 8-41), and a coil (residues 42-50), similar to that of the correspondi ng region of the X-ray structure of F(1)ATPase (J. P. Abrahams, A. G. W. Leslie, R. Lutter, and J. E. Walker, 1994 Nature 370, 621-628) and to the structure of a homologous peptide from the ATP-binding site of adenylate kinase (D. C. Fry, D. M. Byler, H. Susi, E. M. Brown, S. A. Kuby, and A. S. Mildvan, 1988 Biochemistry 27, 3588-3598). beta,gamma- Bidentate Cr3+ ATP binds to PP-50 with the Cr3+ pyrophosphate moiety a pproaching the E-methylene group of K22 in the GX(4)GKT consensus sequ ence, in agreement with the X-ray structure of the Mg(2+)AMPPNP comple x of F(1)ATPase. (C) 1995 Academic Press, Inc.