Detecting small structural changes in metalloproteins by the use of NMR pseudocontact shifts

The potential use of pseudocontact shifts (PCS) for detecting small structural changes that may occur in a protein in aqueous solution as a function of temperature is demonstrated on lanthanide -substituted calbindin D9k. The protein is a dicalcium protein in which one of the calcium ions can be selectively substituted by lanthanides. The solution structure of the protein had been previously solved at 300 K. New sets of PCS that could readily be obtained at 280 and 310 K from 1H-15N HSQC experiments, together with the other constraints used for the 300 K structure, were used to recalculate the solution structure at the new temperatures, since the new solution structures are consistent with different sets of PCS constraints and a single NOE/dihedral angle set of constraints. Temperature-dependent PCS data were collected and analyzed for as many as eleven different paramagnetic lanthanides, but the relevant structural information could already be obtained by using data from only one [erbium(III)] derivative. It is found that small structural changes occur preferentially in nonhelical regions of the protein, i.e. the N- and C-terminal ends, in the linker region between the two helix-loophelix motifs, as well as at the metal binding loops. This research shows that PCS are most sensitive to small structural rearrangements that may be within the tolerance of the NOE constraints, and can be safely used to monitor changes that would not be detectable by NOE and dihedral angle measurements. From the newly obtained structures, the parameters describing the metal magnetic susceptibility tensors at the different temperatures can be obtained, which represent a further source of information.