1. Pb(II) and Hg(II) binding to de novo designed proteins studied by 204m Pb- and 199m Hg-perturbed angular correlation of γ- rays (PAC) spectroscopy: Clues to heavy metal toxicity L. Hemmingsen, Th. Agne, S.K. Das, S.-B. Ruy, V. L. Pecoraro, J.G. Correia Outline Introduction: de novo designed proteins and PAC spectroscopy What do we hope to achieve? Status of research & proposed experiments

2. De novo designed proteins: Heptad repeats 1 10 20 30 g a b c d e f g a b c d e f g a b c d e f g a b c d e f g a LLLL G-LKALEEK-LKALEEK-LKALEEK-LKALEEK-G heptad #1 heptad #2 heptad #3 heptad #4 Inspired by naturally occurring coiled coils Hydrophobic core of leucines

3. De novo designed proteins: Structure determined by X-ray diffraction Tripple-stranded α-helical bundle Hydrophobic core of leucine side chains – as expected Initially used for studies of protein folding Lovejoy et al. Science 1993, 259:1288

4. De novo designed metalloproteins: The L16C TRI-peptide series LCLLCL L16C G-LKALEEK-LKALEEK-CKALEEK-LKALEEK-G Matzapetakis et al. J. Am. Chem. Soc., 2002, 124:8024 UV absorption spectroscopy of Cd(II) binding

5. De novo designed proteins offer a structural framework for... Creation of natural metal ion binding sites, to test essential features necessary for: Electron transfer Metal ion regulated processes Metalloprotein folding, structure, dynamics, and catalysis Creation of metal ion binding sites with properties unprecedented in nature The aim of this work is to create and characterize heavy metal ion binding sites mimicking those found in nature – hoping to capture the molecular basis of heavy metal toxicity

6. Perturbed angular correlation of γ-rays: The influence of extra-nuclear fields 111 Cd Hamilton Phys. Rev., 1940, 58:122, Brady and Deutsch Phys. Rev. 1947, 72:870, Goertzel Phys. Rev., 1946, 70:897, Aeppli et al. Phys. Rev., 1951, 82, 550, Leipert et al. Nature, 1968, 220:907 The nuclear quadrupole interaction is proportional to: Thus, ω 1, ω 2, and ω 3 represent a fingerprint of the local charge- distribution eQ

7. PAC in a nutshell Calculate spectroscopic data based on structure Compare W180 W90 Hemmingsen et al. Chem. Rev., 2004, 104:4027

8. Combination of PAC, NMR, and QM calculations: An exchanging water molecule 113 Cd NMR L16C Matzapetakis et al. J. Am. Chem. Soc., 2002, 124:8024

9. Combination of PAC, NMR, and QM calculations: An exchanging water molecule 113 Cd NMR L16C PAC spectroscopy provides unique information on structure and dynamics

10. What do we hope to achieve? 1.Fundamental knowledge of Pb(II) and Hg(II) interaction with proteins mimicking naturally occuring binding sites: Structure and dynamics at de novo designed metal ion binding sites Selectivity and affinity for these heavy metals 2.Clues to the molecular basis of toxicity of these heavy metals 3.Biotechnological long term goal: Design of high affinity high selectivity heavy metal chelators

11. De novo designed proteins: Pb(II) binding to a and d sites UV-Vis-, CD- and EXAFS spectroscopic studies indicate that Pb(II) binds preferentially to d sites contrary to Cd(II) and Hg(II) Unfortunately, assignment of the spectroscopic signals to a and d sites has not been possible

12. De novo designed proteins: Pb(II) binding to a and d sites 1. Using L16C (a site) and L12C (d site) we will assign 204m Pb- PAC signals to the two sites, and characterize the two sites in terms of structure 2. In proteins with two binding sites (separated by one leucine layer) we will study selectivity for Pb(II) versus Cd(II) and Hg(II)

13. De novo designed proteins: Hg(II) binding to CSL9C UV absorption (and 199 Hg NMR) spectra display a distinct change from addition of 0-2 eq. CSL9C to addition of 2-3 eq. CSL9C to a Hg(II) solution – does this reflect formation of a two-coordinated Hg(CS-L9C) 2 complex and a three-coordinated Hg(CSL9C) 3 -, respectively? 3 – 4.5 eq CSL9C 0 – 2 eq CSL9C

14. De novo designed proteins: Hg(II) binding to CSL9C PAC spectroscopy should allow for discrimination of two- and three- coordinated structures, with considerably higher frequencies for the linear complex  sub-stoichiometric studies is a particular advantage of PAC- spectroscopy 3 – 4.5 eq CSL9C 0 – 2 eq CSL9C

15. De novo designed proteins: Hg(II) binding to CSL9C Is the (potential) three-coordinated structure trigonal planar or T- shaped? PAC spectroscopy is ideally suited for discrimination of these two structures, with an asymmetry parameter of 0 in the prior and 1 in the latter case 3 – 4.5 eq CSL9C 0 – 2 eq CSL9C

16. De novo designed proteins: Hg(II) binding For all the peptides the following equilibrium appears to exist: Hg(peptide) 3 (= HgS 2 SH) ↔ Hg(peptide) 3 - (= HgS 3 ) + H + Does the protonated cysteine coordinate to Hg(II)? PAC spectroscopy is expected to solve this problem as the electric field gradient will depend strongly on the charge state of the SH group

17. De novo designed proteins: Hg(II) binding Proteins have been designed with two metal ion binding sites next to each other (6 cysteines in total) presenting variety of possible coordination geometries PAC spectroscopy should be well suited for discrimination of 2-, 3-, and 4-coordinated Hg(II) ions with thiolate ligands

18. Summary De novo design of proteins is a powerful tool in the study of fundamental heavy metal – protein interactions PAC spectroscopy provides unique information on structure and dynamics of metal ion binding sites We hope to uncover fundamental chemistry of Pb(II) and Hg(II) interaction with proteins: Structure and dynamics at de novo designed metal ion binding sites Selectivity and affinity for these heavy metals Clues to the molecular basis of toxicity of these heavy metals Biotechnology: Design of heavy metal chelators