NMR SOLUTION STRUCTURE OF THE 205-316 C-TERMINAL FRAGMENT OF THERMOLYSIN - EXAMPLE OF DIMERIZATION COUPLED TO PARTIAL UNFOLDING

The solution structure of the C-terminal fragment 205-316 of thermolys in has been determined by H-1-NMR methods. The fragment forms a dimer in which each subunit has two different regions: the largely disordere d N-terminal segment 205-260 and the structurally well-defined segment 261-316. The structured part of each subunit is composed of three hel ices and is largely coincident with the corresponding region in the so lution structure of the dimer formed by the shorter fragment 255-316, which in turn coincides with the crystallographic structure of intact thermolysin. As with the fragment 255-316, the subunit interface is hi ghly hydrophobic and coincides topologically with the one between the segment 255-316 and the rest of the protein in the intact enzyme. A fo urth helix (residues 235-246), present in the segment 205-316 of nativ e thermolysin, is. mostly disordered in the dimer formed by the fragme nt 205-316. The location of the fourth helix in the native structure o f intact thermolysin does not allow the formation of the dimer interfa ce observed in the solution structure of the fragment 255-316. Under t he NMR conditions, dimer formation is energetically more favorable tha n the dissociated monomers. The latter, based on calorimetric data: wa s proposed to have partial structure in the region 205-254 as in nativ e thermolysin. Thus, it appears that the assembly of the dimer would r equire an initial unfolding in the region 205-254 of the monomer.