Nanosecond dynamics of horse heart apocytochrome c in aqueous solution as studied by time-resolved fluorescence of the single tryptophan residue (Trp-59).
The time-resolved fluorescence emission characteristics of the single tryptophan residue (Trp-59) of horse heart apocytochrome c--the precursor of the intramitochondrial cytochrome c--were studied in aqueous solution. The total fluorescence intensity decay measured over the whole emission spectrum was analyzed as a sum of three or four exponentials by the nonlinear least-squares method, the last model always providing a slight but significant decrease in the chi 2 values. Maximum entropy analysis, recently developed for time-resolved fluorometry (Livesey et al., 1987; Livesey & Brochon, 1987), strongly suggests the existence of a distribution including at least four separate classes of lifetimes. The center values were around 0.1-0.2, 1, 3, and 5 ns, in agreement with the lifetime values obtained by nonlinear least-squares regression analysis. As a function of the emission wavelength, these values remained constant within the experimental error, whereas a redistribution of the fractional amplitudes was observed: the contributions of the short components increased in the blue edge region of the emission spectrum. Temperature increase led essentially to a redistribution of the fractional amplitudes, affecting mostly that of the 5-ns component, which almost totally disappeared at high temperature (35-40 degrees C). The lifetime values were not significantly affected except for the 3-ns component, which decreased by about 15% in the temperature range studied. Such observations strongly suggest that the protein exists under different conformational substates in thermal equilibrium. Time-resolved fluorescence anisotropy measurements evidenced the existence of fast internal rotation of the Trp residue. An average maximum restricted angle of rotation of around 55 degrees was calculated. A second internal motion, slower by 1 order of magnitude, corresponding likely to a local motion of the peptide chain involving the Trp-59 residue, was detected on the anisotropy decay curve. Finally, the longest correlation time (5 ns) should correspond to the average rotation of the overall protein. Its value doubled as a function of the protein concentration, revealing an association process leading most likely to a dimer in the concentration range studied (2-139 microM). The flexibility of the peptide chain was more restrained in the associated than in the monomeric form, but the fast internal rotation of the Trp residue was not.
Vincent
M
M
Laboratoire pour l'Utilisation du Rayonnement Electromagnétique, Centre National de la Recherche Scientifique, Université Paris Sud, Orsay, France.
Brochon
J C
JC
Merola
F
F
Jordi
W
W
Gallay
J
J
D000818:Animals; D001059:Apoproteins / Q000378:metabolism*; D003574:Cytochrome c Group / Q000378:metabolism*; D045304:Cytochromes c; D005454:Fluorescence Polarization; D006736:Horses; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D009206:Myocardium / Q000378:metabolism; D011487:Protein Conformation; D012996:Solutions; D013997:Time Factors; D014364:Tryptophan
D001059:Apoproteins; D003574:Cytochrome c Group; D012996:Solutions; D014364:Tryptophan; D045304:Cytochromes c