Metal-coupled folding as the driving force for the extreme stability of Rad50 zinc hook dimer assembly

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

Mitochondrial mutagenesis in<i>Saccharomyces cerevisiae</i>: the origin of<i>mit</i>⁻mutants

SUMMARYYeast cells contain many copies of mitochondrial (mit) genomes. The question we tried to answer was howmit−mutations occurring in one genome as a result of mutagenic treatment might yield homoplasmic mutant cells. Three processes were considered. First, that these cells originate by segregation of mutant and standard alleles during cell division. Secondly, that they originate through intracellular selection, for which cell division is not required. Thirdly, that recombination involving the mutant and standard alleles is non-reciprocal and unidirectionalmit+→mit−so that the mutant allele is spread into the entire population of mitochondrial genomes within a cell, thus making it homoplasmicmit−. The results indicate that the first process, although efficiently producing homoplasmic cells from heteroplasmic zygotes (for review see Birky, 1978), seems to play only a minor, if any, role in producing homoplasmic mutant progenies from mutagenized cells. The most important is the second process, that is, intracellular selection occurring in cells which have one or a few genomes carryingmit−mutations, while the remaining genomes are irreversibly damaged. The third process, unidirectionalmit+→mit−conversion, does not seem to play any part.</jats:p

The TFE-induced transient native-like structure of the intrinsically disordered [Formula: see text] domain of Escherichia coli RNA polymerase.

The transient folding of domain 4 of an E. coli RNA polymerase [Formula: see text] subunit ([Formula: see text]) induced by an increasing concentration of 2,2,2-trifluoroethanol (TFE) in an aqueous solution was monitored by means of CD and heteronuclear NMR spectroscopy. NMR data, collected at a 30 % TFE, allowed the estimation of the population of a locally folded [Formula: see text] structure (CSI descriptors) and of local backbone dynamics ((15)N relaxation). The spontaneous organization of the helical regions of the initially unfolded protein into a TFE-induced 3D structure was revealed from structural constraints deduced from (15)N- to (13)C-edited NOESY spectra. In accordance with all the applied criteria, three highly populated α-helical regions, separated by much more flexible fragments, form a transient HLHTH motif resembling those found in PDB structures resolved for homologous proteins. All the data taken together demonstrate that TFE induces a transient native-like structure in the intrinsically disordered protein

Manganese mutagenesis in yeast: II. Conditions of induction and characteristics of mitochondrial respiratory deficient<i>Saccharomyces cerevisiae</i>mutants induced with manganese and cobalt

SUMMARYManganese and cobalt are capable of inducing ρ−mutations* in non-growing cells ofSaccharomyces cerevisiae, but their mutagenic action is much stronger in growing cells. At a given concentration cobalt and manganese can be either strongly mutagenic or non-mutagenic, depending on the cell density.Most of the ρ−mutants induced with manganese and a considerable proportion of those induced with cobalt are suppressive and/or transmit drug resistance markers, so they must still carry mitochondrial DNA. Cobalt can decrease suppressiveness with low efficiency and eliminate drug resistance markers from established ρ−clones.</jats:p