The binding of metal ions at the interface of protein complexes presents a unique and poorly understood mechanism of molecular assembly. A remarkable example is the Rad50 zinc hook domain, which is highly conserved and facilitates the Zn2+-mediated homodimerization of Rad50 proteins. Here, we present a detailed analysis of the structural and thermodynamic effects governing the formation and stability (logK12 = 20.74) of this evolutionarily conserved protein assembly. We have dissected the determinants of the stability contributed by the small β-hairpin of the domain surrounding the zinc binding motif and the coiled-coiled regions using peptides of various lengths from 4 to 45 amino acid residues, alanine substitutions and peptide bond-to-ester perturbations. In the studied series of peptides, an >650 000-fold increase of the formation constant of the dimeric complex arises from favorable enthalpy because of the increased acidity of the cysteine thiols in metal-free form and the structural properties of the dimer. The dependence of the enthalpy on the domain fragment length is partially compensated by the entropic penalty of domain folding, indicating enthalpy-entropy compensation. This study facilitates understanding of the metal-mediated protein-protein interactions in which the metal ion is critical for the tight association of protein subunits.

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Application of Fourier Transform for processing 3D NMR spectra with random sampling of evolution time space is presented. The 2D FT is calculated for pairs of frequencies, instead of conventional sequence of one-dimensional transforms. Signal to noise ratios and linewidths for different random distributions were investigated by simulations and experiments. The experimental examples include 3D HNCA, HNCACB and (15)N-edited NOESY-HSQC spectra of (13)C (15)N labeled ubiquitin sample. Obtained results revealed general applicability of proposed method and the significant improvement of resolution in comparison with conventional spectra recorded in the same time.

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