Thermodynamics of the Op18/stathmin-tubulin interaction.
Op18/stathmin (stathmin) is an intrinsically disordered protein involved in the regulation of the microtubule filament system. One function of stathmin is to sequester tubulin dimers into assembly incompetent complexes, and recent studies revealed two tubulin binding sites per stathmin molecule. Using high sensitivity isothermal titration calorimetry, we document that at 10 degrees C and under the conditions of 80 mM PIPES, pH 6.8, 1 mM EGTA, 1 mM MgCl2, 1 mM GTP these two binding sites are of equal affinity with an equilibrium binding constant of K0 = 6.0 x 10(6) m(-1). The obtained large negative molar heat capacity change of deltaCp0 = -860 cal mol(-1) K(-1) (referring to tubulin) for the tubulin-stathmin binding equilibrium suggests that the hydrophobic effect is the major driving force of the binding reaction. Replacing GTP by GDP on beta-tubulin had no significant effect on the thermodynamic parameters of the tubulin-stathmin binding equilibrium. The proposed pH-sensitive dual function of stathmin was further evaluated by circular dichroism spectroscopy and nuclear magnetic resonance. At low temperatures, stathmin was found to be extensively helical but devoid of any stable tertiary structure. However, in complex with two tubulin subunits stathmin adopts a stable conformation. Both the stability and conformation of the individual proteins and complexes were not significantly affected by small changes in pH. A 4-fold decrease in affinity of stathmin for tubulin was revealed at pH 7.5 compared with pH 6.8. This decrease could be attributed to a weaker binding of the C terminus of stathmin. These findings do not support the view that stathmin works as a pH-sensitive protein.
Honnappa
Srinivas
S
Structural Biology, Paul Scherrer Institut, CH-5232 Villigen PSI, Switzerland.
Cutting
Brian
B
Jahnke
Wolfgang
W
Seelig
Joachim
J
Steinmetz
Michel O
MO
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002151:Calorimetry; D002417:Cattle; D002942:Circular Dichroism; D018076:DNA, Complementary / Q000378:metabolism; D019277:Entropy; D005990:Glycerol / Q000737:chemistry; D006153:Guanosine Diphosphate / Q000737:chemistry; D006160:Guanosine Triphosphate / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008854:Microscopy, Electron; D008868:Microtubule Proteins; D008958:Models, Molecular; D015233:Models, Statistical; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D050777:Stathmin; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors; D014404:Tubulin / Q000737:chemistry* / Q000378:metabolism
D018076:DNA, Complementary; D008868:Microtubule Proteins; D010750:Phosphoproteins; C493995:STMN1 protein, human; D050777:Stathmin; D014404:Tubulin; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D005990:Glycerol