NEUTRON RESONANCE SCATTERING SHOWS SPECIFIC BINDING OF PLUTONIUM TO THE CALCIUM-BINDING SITES OF THE PROTEIN CALMODULIN AND YIELDS PRECISE DISTANCE INFORMATION

We have successfully substituted Pu-240(3+) for Ca2+ in the calcium-bi nding protein calmodulin and used neutron resonance scattering from th e bound Pu-240 to demonstrate that the Pu binds specifically to the Ca 2+ sites and also to measure the distance between the ion binding site s within individual domains of the protein. Pu-240 has a strong nuclea r resonance at 0.278 Angstrom, and at this wavelength the coherent sca ttering from Pu-240 is > 1000 times that of any other nucleus present in a protein. The ionic radius of Pu3+ is very similar to that of Ca2, and hence eve chose this species to substitute for Ca2+ in the prote in. We identified solution conditions that stabilize Pu3+ in solution at near neutral pH for 6-7 h in order to form the Pu/calmodulin comple x under conditions favorable for both complex formation and maintainin g the structural integrity of the protein. We collected small-angle ne utron scattering data from solutions of 4(Pu-240(3+)). calmodulin, whi ch contain periodic terms that are directly related to the distances b etween the Ca2+-binding sites. The shorter Pu-Pu distance, i.e., the a verage distance between the two sites within each globular domain of c almodulin, is found to be 11.8 +/- 0.4 Angstrom, in excellent agreemen t with the value of 11.7 Angstrom from crystallographic determinations . This is the first use of neutron resonance scattering as a structura l probe in a protein.