Ligand binding and protein dynamics: a fluorescence depolarization study of aspartate transcarbamylase from Escherichia coli.
The polarization of the fluorescence and the real-time fluorescence intensity decay of the two tryptophan residues of aspartate transcarbamylase from Escherichia coli were studied as a function of temperature. The protein was dissolved in an 80% glycerol/buffer mixture, and temperatures were varied between -40 and 20 degrees C in order to limit the depolarization to local rotations of the tryptophans. Two fluorescent species contribute to over 95% of the emission. They differ in their fluorescence lifetimes by approximately 4 ns depending upon the temperature observed and their fractional contributions to the total intensity. The Y-plot analysis of the polarization and lifetime data allows for the distinction of two rotational species by their critical amplitude of rotation, the first being component 1 and the second being component 2. We suggest that these two species correspond to the two tryptophan residues of the protein. The polarization and lifetime experiments were carried out for ATCase in presence of the bisubstrate analogue N-(phosphonoacetyl)-L-aspartate (PALA) and in presence of the nucleotide effector molecules ATP and CTP. The binding of PALA results in an increase in the thermal coefficient of frictional resistance to rotation of tryptophan 1 and a decrease in that of tryptophan 2. ATP binding does not affect the degree to which the protein hinders tryptophan rotation but does result in a change in the critical amplitude of rotation of tryptophan 2. The results obtained in the presence of CTP are similar to those obtained with PALA.
Royer
C A
CA
Laboratoire d'Enzymologie du CNRS, Gif-sur-Yvette, France.
Tauc
P
P
Hervé
G
G
Brochon
J C
JC
D001221:Aspartate Carbamoyltransferase / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008024:Ligands; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D008024:Ligands; D001221:Aspartate Carbamoyltransferase