1. Elucidation and Control of Actinide Trafficking Pathways in Mammalian Cells Chuan He University of Chicago Department of Chemistry Mark P. Jensen Argonne National Lab Chemical Sciences and Engineering Division Sixth Argonne – UChicago – Fermilab Collaboration Meeting, October 12, 2009

2. 2 The Nuclear Conundrum  Nuclear Energy is a carbon-free source for 20% of U.S. & world electricity production.  Why not 80%? –Socio-economic reasons Cost and economic risk Proliferation of nuclear technology Public worries about safety –Technical reason How do we handle the waste?

3. 3 Actinides? Really? Recognizing metal ions with high selectivity and sensitivity is essential for life. Nature has evolved various metalloproteins to exploit and control metals.

4. 4 Objectives Discover how cells handle toxic human-made metals, the transuranium actinides 1.Visualize actinide trafficking in cells with X-ray microbeams 2.Identify individual proteins that bind actinides in mammalian cells 3.Biochemically and structurally characterize actinide binding by various proteins Create new ways to efficiently, economically, and safely handle actinides when they are no longer useful

5. 5 Where do Actinides Accumulate in Cells?

6. 6 First Structures of Pu-Containing Proteins Small Angle X-Ray Scattering of Transferrins One form of transferrin containing both plutonium and iron has the same shape as the native protein, which contains only iron.

7. 7 Blocking Pu Uptake by Cells Appears Possible No Plutonium “Bad” Plutonium Pu C Fe N Tf “Bad” Plutonium plus 50 uM chloroquine Images of rat adrenal gland cells Axes - coordinates in mm

8. 8 Could metal-binding proteins be redesigned to bind actinides? NikR is a transcriptional repressor for expression of the nikABCDE operon in the presence of excessive concentrations of intracellular Ni +2 Ref: C. L. Drennan et al, Nat. Struc. Biol., 2003, 10, 794-799 C. L. Drennan et al, PNAS, 2006, 103, 13676-13681 NikR

9. 9 MQRVTITLDD DLLETLDSLS QRRGYNNRSE AIRDILRSAL AQEATQQHGT QGFAVLSYVY EHEKRDLASR IVSTQHHHHD LSVATLHVHI NHDDCLEIAV LKGDMGDVQH FADDVIAQRG VRHGHLQCLP KED Mutations: V72 → SV72SC95→ D, H, SV72S C95D H76→ D,E V72S C95S H76→ D,E Design of an Actinyl Binding Site

10. 10 Design of an Actinyl Binding Site The K d of uranyl binding to NikR’ was determined to be 53 nM

11. 11 Uranyl Binding and Function

12. 12 S. V. Wegner, H. Boyaci, H. Chen, M. P. Jensen, C. He, Angew. Chem. Int. Ed. (2009), 48, 2339. Joint Publication

13. 13 Engineer bacteria to uptake, transport and store actinides

14. 14 Design Peptides that are Selective for Specific Actinides LBT: DTNNDGWYEGDELLA 1 53912