| Abstract
| - Early steps of unfolding of P43M Calbindin D9k have been evaluated by NMR spectroscopyon the native dicalcium and on the paramagnetic monocerium-substituted derivative. Although at 2 MGdmHCl the protein core maintains its overall folding and structure, amide 15N R2 measurements andcross correlation rates between N−H dipole−dipole relaxation and 15N CSA relaxation reveal a closerand stronger packing of the hydrophobic interactions in the protein as a response to the presence ofdenaturing agents in solution. A complete reorientation of the Met43 side chain toward the hydrophobiccore is accomplished by the disappearance of the millisecond dynamics observed on the native form ofCalbindin D9k, while cross correlation rates provide evidence that the two-way hydrogen bond betweenLeu23 and Val61 is broken or substantially weakened. The substitution of the calcium ion in site II withthe 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, tomonitor subtle structural variations occurring in the non-native state of the protein. Although the averagermsd between the structures of native and non-native states is small (0.48 Å), structural rearrangementscould be reliably identified. Our results provide unprecedented information about the behavior of CalbindinD9k during the early steps of unfolding. Furthermore, they constitute strong evidence of the efficiency ofparamagnetism-based constraints in monitoring subtle structural changes that are beyond the sensitivityof an approach based only on NOE.
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