Solution structure and function in trifluoroethanol of PP-50, an ATP-binding peptide from F1ATPase.
PP-50, a synthetic peptide, based on residues 141-190 of the beta-subunit of mitochondrial F1ATPase, containing the GX4GKT consensus sequence for nucleoside triphosphate binding, binds ATP tightly (Kd = 17.5 microM) as found by fluorescence titration at pH 4.0. CD and 2D proton NMR studies at pH 4.0 revealed two beta-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 beta-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 microM, 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 microM). 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 alpha-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), beta, gamma-Bidentate Cr3+ ATP binds to PP-50 with the Cr3+ pyrophosphate moiety approaching the epsilon-methylene group of K22 in the GX4GKT consensus sequence, in agreement with the X-ray structure of the Mg2+ AMPPNP complex of F1ATPase.
Chuang
W J
WJ
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185, USA.
Abeygunawardana
C
C
Gittis
A G
AG
Pedersen
P L
PL
Mildvan
A S
AS
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D003198:Computer Simulation; D009682:Magnetic Resonance Spectroscopy; D008928:Mitochondria / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012996:Solutions; D013050:Spectrometry, Fluorescence; D014270:Trifluoroethanol
D010446:Peptide Fragments; D012996:Solutions; D014270:Trifluoroethanol; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases