A de novo protein design strategy provides a powerful tool to elucidate how heavy metals interact with proteins. Cysteine derivatives of the TRI peptide family (Ac-G(LKALEEK) 4 G-NH 2 ) have been shown to bind heavy metals in an unusual trigonal geometry. Our present objective was to design binding sites in α-helical scaffolds that are able to form higher coordination number complexes with Cd(II) and Hg(II). Herein, we evaluate the binding of Cd(II) and Hg(II) to double cysteine substituted TRI peptides lacking intervening leucines between sulfurs in the heptads. We compare a -Cys d -X-X-X-Cys a -binding motif found in TRIL12CL16C to the more common -Cys a -X-X-Cys d -sequence of native proteins found in TRIL9CL12C. Compared to TRI, these substitutions destabilize the helical aggregates, leading to mixtures of two and three stranded bundles. The three stranded coiled coils are stabilized by the addition of metals. TRIL9CL12C forms distorted tetrahedral complexes with both Cd(II) and Hg(II), as supported by UV-vis, CD, 113 Cd NMR, 199 Hg NMR and 111m Cd PAC spectroscopy. Additionally, these signatures are very similar to those found for heavy metal substituted rubredoxin. These results suggest that in terms of Hg(II) binding, TRIL9CL12C can be considered as a good mimic of the metallochaperone HAH1, that has previously been shown to form protein dimers. TRIL12CL16C has limited ability to generate homoleptic tetrahedral complexes (Cd (SR) 4 2− ). These type of complexes were identified only for Hg(II). However, the spectroscopic signatures suggest a different geometry around the metal ion, demonstrating that effective metal sequestration into the hydrophobic interior of the bundle requires more than simply adding two sulfur residues in adjacent layers of the peptide core. Thus, proper design of metal binding sites must also consider the orientation of cysteine sidechains in a vs d positions of the heptads.

About 50% of the 1.4 GeV CERN (European Organization for Nuclear Research, www.cern.ch) protons are sent onto targets to produce radioactive beams by online mass separation at the Isotope Separator Online Device (ISOLDE) facility, for a wide range of studies in fundamental and applied physics. CERN-MEDICIS is a spin-off dedicated to R&D in life sciences and medical applications. It is located in an extension of the Class A building presently under construction. It will comprise laboratories to receive the irradiated targets from a new station located at the dump position behind the ISOLDE production targets. An increasing range of innovative isotopes will thus progressively become accessible from the start-up of the facility in 2015 onward; for fundamental studies in cancer research, for new imaging and therapy protocols in cell and animal models and for pre-clinical trials, possibly extended to specific early phase clinical studies up to Phase I trials. Five hundred megabecquerel isotope batches purified by electromagnetic mass separation combined with chemical methods will be collected on a weekly basis. A possible future upgrade with gigabecquerel pharmaceutical-grade i.e., current good manufacturing practices (cGMP) batch production capabilities is finally presented. OPEN ACCESSAppl. Sci. 2014, 4 266

Time differential perturbed angular correlation (TDPAC) of γ-rays spectroscopy has been applied in chemistry and biochemistry for decades. Herein we aim to present a comprehensive review of chemical and biochemical applications of TDPAC spectroscopy conducted at ISOLDE over the past 15 years, including elucidation of metal site structure and dynamics in proteins and model systems. β-NMR spectroscopy is well established in nuclear physics, solid state physics, and materials science, but only a limited number of applications in chemistry have appeared. Current endeavors at ISOLDE advancing applications of β-NMR towards chemistry and biochemistry are presented, including the first experiment on 31Mg2+ in an ionic liquid solution. Both techniques require the production of radioisotopes combined with advanced spectroscopic instrumentation present at ISOLDE.