A proton magnetic resonance and a circular dichroism study of the solvent dependent conformation of the synthetic tubulin fragment Ac tubulin, alpha (430-441) amide and its interaction with substance-P.
Proton magnetic resonance techniques were used to study the conformation of the synthetic tubulin fragment Ac-tubulin (430-441) amide in H2O and 80% CD3OH/20% D2O solutions, using water suppression techniques. Proton assignments are based on two-dimensional COSY experiments combined with one-dimensional spin decoupling. A comparison of the NH proton shifts between the two solvents, namely delta(CD3OH/H2O-H2O) shows a small solvent effect for the Lys1 to Val6 region of the molecule, whereas for Gly7 to Glu12 the solvent effect is much larger. The smaller effects in the region of Lys1 to Val6 may be due to some hydrogen bonding as these protons are shielded from the solvent. These conclusions are in agreement with the circular dichroism results in 80% methanol/20% water where the alpha helix is present to the extent of 30%, whereas the peptide is completely unstructured in water with some aggregation. The temperature dependence of the NH proton shifts was also carried out. In water these shifts are of the order of 7-9 X 10(-3) ppm/K indicating that most of the protons are not involved in hydrogen bonding. In CD3OH/H2O, these values range from about 4-6 X 10(-3) ppm/K, which are compatible with the presence of hydrogen bonds. Finally, binding studies were carried out between the tubulin peptide and the undecapeptide neutrotransmitter substance P. The largest shifts are for the Tyr3 NH proton of the tubulin fragment, whereas for substance P it is for the Lys3, Gln5 and Leu10 NH protons, indicating a change in conformation of both peptides on interaction.
Sugiura
M
M
Department of Chemistry, University of Alberta, Edmonton, Canada.
Maccioni
R B
RB
Cann
J R
JR
York
E J
EJ
Stewart
J M
JM
Kotovych
G
G
D002942:Circular Dichroism; D009682:Magnetic Resonance Spectroscopy; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D012996:Solutions; D012997:Solvents / Q000494:pharmacology; D013373:Substance P / Q000378:metabolism*; D014404:Tubulin / Q000031:analogs & derivatives / Q000378:metabolism / Q000648:ultrastructure*
D010446:Peptide Fragments; D012996:Solutions; D012997:Solvents; D014404:Tubulin; D013373:Substance P