Physicochemical characterization of the heat-stable microtubule-associated protein MAP2

Abstract

MAP2 purified without heat treatment was compared to MAP2 prepared with the usual boiling step. No significant differences were found by isoelectric focusing, peptide mapping, fluorescence spectroscopy and circular dichroism. Hydrodynamically, MAP2 is a monomer with s°_20,w, = 3.5 ± 0.1 S and M_r= 220 000. The molecular mass was measured by sedimentation equilibrium and verified by gel chromatography in denaturing solvent and dodecyl sulfate gel electrophoresis at different acrylamide concentrations. The high frictional ratio, f/f_min= 3.7, indicated that MAP2 was clearly not globular but had either a very elongated shape or an unordered expanded structure. Circular dichroic results were consistent with a predominantly unordered structure independently of the preparation procedure. This supports the notion that MAP2, in solution, is a very flexible and non-compact protein. Examination of the circular dichroism of MAP2 as a function of temperature indicated that the thermal stability of this protein was probably due to its mostly unordered structure and the fact that the little ordered structure present was resistant to thermal denaturation.