Phosphorylation-induced conformational changes in short peptides probed by fluorescence resonance energy transfer in the 10A domain.
Phosphorylation-induced conformational changes in short polypeptides were probed by a fluorescence resonance energy transfer (FRET) method by employing a short-distance FRET pair (R(0) approximately 10 A) based on tryptophan as natural donor and a 2,3-diazabicyclo[2.2.2]oct-2-ene-labeled asparagine (Dbo) as synthetic acceptor. Two substrates for kinases, LeuArgArgTrpSerLeuGly-Dbo (peptide I) and TrpLysArgThrLeuArgArg-Dbo (peptide II), were investigated, with serine and threonine, respectively, as phosphorylation sites. Steady-state and time-resolved fluorescence experiments in H(2)O revealed a decrease in FRET efficiency for peptide I and an increase for peptide II; this suggested that the effective distances between donor and acceptor increased and decreased, respectively. The same trends and similar absolute variations in effective donor-acceptor distances were observed in propylene glycol, a less polar and highly viscous solvent; this suggested that the variations are due to intrinsic structural preferences. Fitting of the time-resolved decay traces according to a distribution function model (Gaussian distribution) provided the mean donor-acceptor distances, which showed an increase upon phosphorylation for peptide I (from 9.7 to 10.5 A) and a decrease for peptide II (from 10.9 to 9.3 A) in H(2)O. The broadness (half-width) of the distributions, which provides a measure of the rigidity of the peptides, remained similar upon phosphorylation of peptide I (3.0 versus 3.1 A), but decreased for peptide II (from 3.1 to 0.73 A in H(2)O); this suggests a more compact, structured conformation upon phosphorylation of the latter peptide. The elongation of the peptide backbone (by ca. 0.7 A) for peptide I is attributed to an increase in steric demand upon phosphorylation, which favors an extended conformation. The contraction (by ca. 1.4 A) and structural rigidification of peptide II is attributed to attractive Coulombic interactions and hydrogen bonding between the phosphate group and the arginine residues.
Sahoo
Harekrushna
H
School of Engineering and Science, Jacobs University Bremen, Campus Ring 1, 28759 Bremen, Germany.
Nau
Werner M
WM
D001120:Arginine / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D008956:Models, Chemical; D015233:Models, Statistical; D016011:Normal Distribution; D010455:Peptides / Q000737:chemistry*; D010710:Phosphates / Q000737:chemistry; D010766:Phosphorylation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012694:Serine / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013237:Stereoisomerism; D013912:Threonine / Q000737:chemistry; D014867:Water / Q000737:chemistry
D010455:Peptides; D010710:Phosphates; D014867:Water; D013912:Threonine; D012694:Serine; D001120:Arginine