Neutron Resonance Scattering Shows Specific Binding of Plutonium to the Calcium-Binding Sites of the Protein Calmodulin and Yields Precise Distance Information

Abstract

We have successfully substituted 240 Pu 3+ for Ca 2+ in the calcium-binding protein calmodulin and used neutron resonance scattering from the bound 240 Pu to demonstrate that the Pu binds specifically to the Ca 2+ sites and also to measure the distance between the ion binding sites within individual domains of the protein. 240 Pu has a strong nuclear resonance at 0.278 Å, and at this wavelength the coherent scattering from 240 Pu is >1000 times that of any other nucleus present in a protein. The ionic radius of Pu 3+ is very similar to that of Ca 2+ , and hence we chose this species to substitute for Ca 2+ in the protein. We identified solution conditions that stablize Pu 3+ in solution at near neutral pH for 6-7 h in order to form the Pu/calmodulin complex under conditions favorable for both complex formation and maintaining the structural integrity of the protein. We collected small-angle neutron scattering data from solutions of 4( 240 Pu 3+ )‚calmodulin, which contain periodic terms that are directly related to the distances between the Ca 2+ -binding sites. The shorter Pu-Pu distance, i.e., the average distance between the two sites within each globular domain of calmodulin, is found to be 11.8 ( 0.4 Å, in excellent agreement with the value of 11.7 Å from crystallographic determinations. This is the first use of neutron resonance scattering as a structural probe in a protein.

Protein-Based Platform for Purification, Chelation, and Study of Medical Radiometals: Yttrium and Actinium

“…The use of biomacromolecules, such as proteins, has largely been absent from purification strategies targeting radiometals like yttrium and actinium, as most known metal-binding proteins are not selective for f-elements. [19][20][21] Even previous studies investigating interactions of proteins with uranium or heavy actinides (plutonium, americium, curium…) [22][23][24] have largely focused on bacterial and human iron or calcium transport machineries, which not only form relatively weak complexes with trivalent f-elements but also only operate under a very narrow set of conditions (typically pH >6 and requiring a synergistic ligand like a carbonate or a siderophore molecule). By contrast, LanM displays unprecedented selectivity for trivalent lanthanides against most other cations.…”

Uniqueness of models from small-angle scattering data: the impact of a hydration shell and complementary NMR restraints

“…These include pairwise distance restraints between specific residues provided by NMR PREs (Hennig & Sattler, 2014;Carlomagno, 2014;Madl et al, 2011) and also recent developments such as heavy-atom labelling in SAS (Grishaev et al, 2012) or multi-wavelength anomalous X-ray scattering of specific chemical groups (Makowski et al, 2012). The feasibility of the latter approach has also been demonstrated for neutrons using single atoms as labels (Seeger et al, 1997) but is more limited by signal to noise and available labels. The development of new deuterium-labelling schemes of specific chemical groups or parts of proteins, in particular segmental labelling (Hennig & Sattler, 2014), is another promising approach for SANS in the near future.…”

A proteomic approach to identification of plutonium-binding proteins in mammalian cells

“…Similar to calcium, lanthanides and actinides also prefer hard donor ligands and coordination environments with numerous donor atoms (coordination numbers of 8 or 9 are typical). Many calcium-binding proteins are known to bind lanthanides effectively, 65 and the calcium-binding protein, calmodulin, is also reported to bind Pu 3+ in the calcium binding sites 66 . Therefore, the proteins identified in this study are reasonable candidates for plutonium binding in vivo .…”