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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A BSTR ACTA 12-residue peptide AcDKDGDGY-ISAAENH 2 analogous to the third calcium-binding loop of calmodulin strongly coordinates lanthanide ions (K ‫؍‬ 10 5 M ؊1 ). When metal saturated, the peptide adopts a very rigid structure, the same as in the native protein, with three last residues AAE fixed in the ␣-helical conformation. Therefore, the peptide provides an ideal helix nucleation site for peptide segments attached to its C terminus. NMR and CD investigations of peptide AcDKDGDGYISAAEAAAQNH 2 presented in this paper show that residues A13-Q16 form an ␣-helix of very high stability when the La 3؉ ion is bound to the D1-E12 loop. In fact, the lowest estimates of the helix content in this segment give values of at least 80% at 1°C and 70% at 25°C. This finding is not compatible with existing helix-coil transition theories and helix propagation parameters, s, reported in the literature. We conclude, therefore, that the initial steps of helix propagation are characterized by much larger s values, whereas helix nucleation is even more unfavorable than is believed. In light of our findings, thermodynamics of the nascent ␣-helices is discussed. The problem of CD spectra of very short ␣-helices is also addressed.

S100 proteins play a crucial role in multiple important biological processes in vertebrate organisms acting predominantly as calcium signal transmitters. S100A1 is a typical representative of this family of proteins. After four Ca(2+) ions bind, it undergoes a dramatic conformational change, resulting in exposure, in each of its two identical subunits, a large hydrophobic cleft that binds to target proteins. It has been shown that abnormal expression of S100A1 is strongly correlated with a number of severe human diseases: cardiomyopathy and neurodegenerative disorders. A few years ago, we found that thionylation of Cys 85, the unique cysteine in two identical S100A1 subunits, leads to a drastic increase of the affinity of the protein for calcium. We postulated that the protein activated by thionylation becomes a more efficient calcium signal transmitter. Therefore, we decided to undertake, using nuclear magnetic resonance methods, a comparative study of the structure and dynamics of native and thionylated human S100A1 in its apo and holo states. In this paper, we present the results obtained for both forms of this protein in its holo state and compare them with the previously published structure of native apo-S100. The main conclusion that we draw from these results is that the increased calcium binding affinity of S100A1 upon thionylation arises, most probably, from rearrangement of the hydrophobic core in its apo form.

S100A1 belongs to the EF-hand superfamily of calcium binding proteins. It is a representative of the S100 protein family based on amino acid sequence, three-dimensional structure, and biological function as a calcium signal transmitter. It is a homodimer of noncovalently bound subunits. S100A1, like most of other members of the S100 protein family, is a multifunctional, regulatory protein involved in a large variety of biological processes and closely associated with several human diseases. The three-dimensional structure of human apo-(i.e. calcium free)-S100A1 protein was determined by NMR spectroscopy (PDB 2L0P) and its backbone dynamics established by ¹⁵N magnetic relaxation. Comparison of these results with the structure and backbone dynamics previously determined for bovine apo-S100A1 protein modified by disulfide formation with β-mercaptoethanol at Cys 85 revealed that the secondary structure of both these proteins was almost identical, whereas the global structure of the latter was much more mobile than that of human apo-S100 protein. Differences between the structures of human and rat apo-S100A1 are also discussed.

The binding of mono- and oligo-vanadates to sarcoplasmic reticulum was analysed by 51V-n.m.r. spectroscopy. The observations indicate that, in addition to monovanadate, the di-, tetra- and deca-vanadates are also bound to sarcoplasmic-reticulum membranes with high affinity. The binding of the vanadate oligoanions may explain some of the effects of vanadates on the conformation and crystallization of Ca2+-transport ATPase.

Complexation of single-wall carbon nanotubes with 12-membered cyclodextrins enables not only their solubilization in water but also their partial separation with respect to diameters and determination of the number of nanotube types on the basis of NMR spectra.

1H and (13)C NMR spectra of the complexes of camphor enantiomers with alpha-cyclodextrin in D(2)O manifest splittings due to chiral recognition. The complexes were found to be of 1:2 guest-to-host stoichiometry. Free energies of the complex formation obtained from (1)H NMR titration data are equal to -7.95 +/- 0.09 kcal mol(-)(1) for the complex with (1S,4S)- and -7.61 +/- 0.06 kcal mol(-)(1) for that with (1R,4R)-enantiomer. Thus, the free energy difference between the complexes is equal to 0.34 +/- 0.11 kcal mol(-)(1), with the complex involving the (1S,4S)-camphor more stable. A strong positive cooperativity of the guests binding has been found. In agreement with experimental results, molecular dynamics simulations yielded greater stability of the complex with (1S,4S)-camphor. However, they reproduced only qualitatively the experimental trend since the corresponding difference in average energies obtained from molecular dynamic simulations carried out in a water solution is equal to 5 kcal/mol with the CVFF force field.