Zinc(II)cyclen–peptide conjugates interacting with the weak effector binding state of Ras

Zinc(II)cyclen–peptide hybrid compounds and bis-zinc(II)cyclen complexes are prepared as potential binders of the guanine nucleotide binding protein Ras, an important molecular switch in cellular signal transduction. The design of the compounds is based on the previous observation that zinc(II)cyclen complexes could serve as lead compounds for inhibitors of Ras-effector interaction and thus be able to interrupt Ras induced signal transduction. Zinc(II)cyclen selectively stabilizes conformational state 1 of active Ras, a conformational state with drastically decreased affinity to effector proteins like Raf-kinase. To achieve higher binding affinities of such Ras–Raf interaction inhibitors, zinc(II)cyclen conjugates with short peptides, derived from the sequence of the Ras-activator SOS, were prepared by solid phase synthesis protocols. Dinuclear bis-zinc(II)cyclen complexes were obtained from alkyne-azide cycloaddition reactions. NMR investigations of the prepared compounds revealed that the peptide conjugates do not lead to an increase in Ras binding affinity of the metal complex–peptide conjugates. The dinuclear zinc complexes lead to an immediate precipitation of the protein prohibiting spectroscopic investigations of their binding

Cu2+-cyclen as Probe to Identify Conformational States in Guanine Nucleotide Binding Proteins

(31)P NMR spectroscopy is a suitable method for identifying conformational states in the active site of guanine nucleotide binding proteins detecting the nucleotide placed there. Because there is no labeling necessary, this method is gaining increasing interest. By (31)P NMR spectroscopy two major conformational states, namely state 1(T) and state 2(T), can be detected in active Ras protein characterized by different chemical shifts. Depending on the conformational state Ras shows clearly different physiological properties. Meanwhile analogous conformational equilibria could also be shown for other members of the Ras superfamily. It is often difficult to determine the conformational states of the proteins on the basis of chemical shift alone; therefore, direct detection would be a great advantage. With the use of Cu(2+)-cyclen which selectively interacts only with one of the major conformational states (state 1) one has a probe to distinguish between the two states, because only proteins existing in conformational state 1 interact with the Cu(2+)-cyclen at low millimolar concentrations. The suitability was proven using Ras(wt) and Ras mutants, Ras complexed with GTP, GppNHp, or GTPγS, as well as two further members of the Ras superfamily namely Arf1 and Ran