Monitoring the early steps of unfolding of dicalcium and mono-Ce3+-substituted forms of P43M calbindin D9k

Early steps of unfolding of P43M Calbindin D-9k have been evaluated by NMR spectroscopy on the native dicalcium and on the paramagnetic monocerium-substituted derivative. Although at 2 M GdmHCl the protein core maintains its overall folding and structure, amide N-15 R-2 measurements and cross correlation rates between N-H dipole-dipole relaxation and N-15 CSA relaxation reveal a closer and stronger packing of the hydrophobic interactions in the protein as a response to the presence of denaturing agents in solution. A complete reorientation of the Met43 side chain toward the hydrophobic core is accomplished by the disappearance of the millisecond dynamics observed on the native form of Calbindin D-9k, while cross correlation rates provide evidence that the two-way hydrogen bond between Leu23 and Va161 is broken or substantially weakened. The substitution of the calcium ion in site 11 with the paramagnetic Ce3+ ion allowed us to obtain a number of long-range nonconventional constraints, namely, pseudocontact shifts, which were used, together with the NOEs collected on the native state, to monitor subtle structural variations occurring in the non-native state of the protein. Although the average rmsd between the structures of native and non-native states is small (0.48 Angstrom), structural rearrangements could be reliably identified. Our results provide unprecedented information about the behavior of Calbindin D-9k during the early steps of unfolding. Furthermore, they constitute strong evidence of the efficiency of paramagnetism-based constraints in monitoring subtle structural changes that are beyond the sensitivity of an approach based only on NOE.