Solution structure and function in trifluoroethanol of PP-50, an ATP-binding peptide from F1ATPase

Woei-Jer Chuang, Chitrananda Abeygunawardana, Apostolos G. Gittis, Peter L. Pedersen, Albert S. Mildvan

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12 Citations (Scopus)

Abstract

PP-50, a synthetic peptide, based on residues 141-190 of the β-subunit of mitochondrial F1ATPase, containing the GX4GKT consensus sequence for nucleoside triphosphate binding, binds ATP tightly (Kd = 17.5 μM) as found by fluorescence titration at pH 4.0. CD and 2D proton NMR studies at pH 4.0 revealed two β-turns, regions of extended secondary structure, transient tertiary structure, and flexibility in the GX4GKT 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 that 25% TFE increases the helix content from 5.8 to 28.6%, decreases the β-structure from 30.2 to 20.2% and decreases the coil content from 64 to 51.2%. Fluorescence titrations of H2ATP2- with PP-50 in 25% TFE yields a Kd of 7.3 μM, 2.4-fold tighter than in H2O, probably due to TFE increasing the activity of H2ATP2-. PP-50 completely quenches the fluorescence of H2ATP2- in 25% TFE, while in H2O the fluorescence quenching is only 62%. In H2O the binding of H2ATP2- increases the structure of PP-50 as detected by CD, but in 25% TFE no significant change in CD is found on binding either H2ATP2- or Mg2+HATP (Kd = 14 μM). The complete proton NMR spectrum of PP-50 in 25% TFE has been assigned. The solution structure, determined by distance geometry, molecular dynamics with simulated annealing, and energy minimization, consists of a coil (residues 1-8), a strand (residues 9-12), a loop (residues 13-22) containing the GX4GKT consensus sequence (residues 16-23), an α-helix (residues 23-36), a turn (residues 38-41), and a coil (residues 42-50), similar to that of the corresponding region of the X-ray structure of F1ATPase (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). β,γ-Bidentate Cr3+ ATP binds to PP-50 with the Cr3+ pyrophosphate moiety approaching the ε(lunate)-methylene group of K22 in the GX4GKT consensus sequence, in agreement with the X-ray structure of the Mg2+AMPPNP complex of F1ATPase.

Original languageEnglish
Pages (from-to)110-122
Number of pages13
JournalArchives of Biochemistry and Biophysics
Volume319
Issue number1
DOIs
Publication statusPublished - 1995 May 1

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology

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