Molecular modeling and functional analysis of the AtoS-AtoC two-component signal transduction system of Escherichia coli.

The AtoS-AtoC two-component signal transduction system positively regulates the expression of the atoDAEB operon in Escherichia coli. Upon acetoacetate induction, AtoS sensor kinase autophosphorylates and subsequently phosphorylates, thereby activating, the response regulator AtoC. In a previous work we have shown that AtoC is phosphorylated at both aspartate 55 and histidine73. In this study, based on known three-dimensional structures of other two component regulatory systems, we modeled the 3D-structure of the receiver domain of AtoC in complex with the putative dimerization/autophosphorylation domain of the AtoS sensor kinase. The produced structural model indicated that aspartate 55, but not histidine 73, of AtoC is in close proximity to the conserved, putative phosphate-donor, histidine (H398) of AtoS suggesting that aspartate 55 may be directly involved in the AtoS-AtoC phosphate transfer. Subsequent biochemical studies with purified recombinant proteins showed that AtoC mutants with alterations of aspartate 55, but not histidine 73, were unable to participate in the AtoS-AtoC phosphate transfer in support of the modeling prediction. In addition, these AtoC mutants displayed reduced DNA-dependent ATPase activity, although their ability to bind their target DNA sequences in a sequence-specific manner was found to be unaltered.

A correlation between the loss of hydrophobic core packing interactions and protein stability.

The hydrophobic core packing in four-alpha-helical bundles appears to be crucial for stabilizing the protein structure. To examine the structural basis of hydrophobic stabilization, the crystal structures of the Leu-->Val (L41V) and Leu-->Ala (L41A) substitutions of the core residue Leu41 of the ROP protein have been determined. Both substitutions are destabilizing and lead to formation of cavities. The main responses to mutations are the collapse of the central part of the alpha-helix containing the site of mutation, shifts of internal water molecules, and in L41A, the trapping of a water molecule in a cavity engineered by the mutation. For both mutants, these effects limit the increase in cavity size to less than 10 A3, while an increase of 37 A3 and 100 A3 is expected for L41V and L41A, respectively, in the absence of any cavity size reducing effects. The mobility of internal side-chains is increased and in L41A, it reaches values typical for exposed residues. A parameter (Deltanh) is introduced as a measure of the number of van der Waals contacts lost. For ROP, barnase and T4 lysozyme mutants, there is a good correlation between Deltanh and the free energy of unfolding DeltaDeltaG relative to wild-type protein. The Deltanh value turns out to be more suitable for analysing structural and energetic responses to mutation than other parameter, such as cavity volumes and packing densities. Possible evolutionary implications of the DeltaDeltaG versus Deltanh relationship are discussed.

Dimer-to-tetramer transformation: loop excision dramatically alters structure and stability of the ROP four alpha-helix bundle protein.

The ROP loop excision mutant RM6 shows dramatic changes in structure and stability in comparison to the wild-type protein. Removal of the five amino acids (Asp30, Ala31, Asp32, Glu33, Gln34) from the loop results in a complete reorganization of the protein as evidenced by single crystal X-ray analysis and thermodynamic unfolding studies. The homodimeric four-alpha-helix motif of the wild-type structure is given up. Instead a homotetrameric four-alpha-helix structure with extended, loop-free helical monomers is formed. This intriguing structural change is associated with the acquisition of hyperthermophilic stability. This is evident in the shift in transition temperature from 71 degreesC characteristic of the wild-type protein to 101 degreesC for RM6. Accordingly the Gibbs energy of unfolding is increased from 71.7 kJ (mol of dimer)-1 to 195.1 kJ (mol of tetramer)-1. The tetramer-to-monomer transition proceeds highly cooperatively involving an enthalpy change of DeltaH=1073+/-30 kJ (mol of tetramer)-1 and a heat capacity change at the transition temperature of DeltaDNCp=14.9(+/-)3% kJ (mol of tetramerxK)-1. The two-state nature of the unfolding reaction is reflected in coinciding calorimetric and van't Hoff enthalpy values.

Identification of residues in the TPR domain of Ssn6 responsible for interaction with the Tup1 protein.

Ssn6, a yeast protein that comprises 10 tandem tetratricopeptide repeat (TPR) motifs, associates with Tup1 repressor protein and acts as a transcriptional corepressor. In this report we identify point mutations in the TPR1 of Ssn6 that disrupt Tup1 interaction. Furthermore, we construct a 3D model of the TPR domain of Ssn6, which is responsible for Tup1 binding, based on the known structure of protein phosphatase 5. According to this model all selected mutations reduce the ability of Ssn6 to interact with Tup1 by affecting the structural integrity of TPR1 and/or the correct spatial arrangement of TPR1 relative to TPR2 and TPR3.

Stability and safety of traditional Greek salami -- a microbiological ecology study.

The microbiological and physicochemical changes which occurred during the industrial fermentation and ripening of four batches of Greek dry salami manufactured without starter cultures were followed. Moderated dehydration rates, monitored by slowly decreasing relative humidity from 94 to 90% during fermentation, prevented the production of insufficiently acidified batches by maintaining microbial activity for longer when the natural inoculum was low. The terminal pH values (5.0-5.2) and water contents (27.7-30.3%) of the sausages were narrowly ranged. Fermentation was governed by an active (> 10(8) cfu g(-1)) lactic flora, consisting of 'wild' strains of Lactobacillus sake. Gram-negative bacteria and aerobic sporeformers decreased below 10(2) and l0(3) cfu g(-1), respectively, while yeasts did not significantly increase during ripening and were below 10(5) cfu g(-1) in the ripened product. Sausages were substantially free of sulfite-reducing clostridia and coagulase-positive staphylococci during the whole process. Listeria spp., occurred in the fresh sausage mix, but disappeared from all batches at the latest by the end of fermentation. Enterococci exceeded 10(5) cfu g(-1) during the first days and remained at this level during ripening. Novobiocin-resistant staphylococci matching Staphylococcus saprophyticus (mainly) and S. xylosus dominated Micrococcaceae populations, ranged between 10(5) and 10(6) cfu g(-1). This is the first report of such a large contribution from S. saprophyticus to the production of dry salami of good quality. It is concluded that to keep or improve the traditional 'sensory type' of Greek salamis, suitable strains of L. sake, S. xylosus and possibly nitrate-reducing S. saprophyticus should be selected and validated as starter cultures in experimentally inoculated salamis.

Structural parameters for proteins derived from the atomic resolution (1.09 A) structure of a designed variant of the ColE1 ROP protein.

The crystal structure of a designed variant of the ColE1 repressor of primer (ROP) protein has been refined with SHELXL93 to a resolution of 1.09 A. The final model with 510 non-H protein atoms, 576 H atoms in calculated positions and 114 water molecules converged to a standard R factor of 10% using unrestrained blocked full-matrix refinement. For all non-H atoms six-parameter anisotropic thermal parameters have been refined. The majority of atomic vibrations have a preferred orientation which is approximately perpendicular to the bundle axis; analysis with the TLS method [Schomaker & Trueblood (1968). Acta Cryst. B24, 63-77] showed a relatively good agreement between the individual atomic displacements and a rigid-body motion of the protein. Disordered residues with multiple conformations form clusters on the surface of the protein; six C-terminal residues have been omitted from the refined model due to disorder. Part of the solvent structure forms pentagonal or hexagonal clusters which bridge neighbouring protein molecules. Some water molecules are also conserved in wild-type ROP. The unrestrained blocked full-matrix least-squares refinement yielded reliable estimates of the standard deviations of the refined parameters. Comparison of these parameters with the stereochemical restraints used in various protein refinement programs showed statistically significant differences. These restraints should be adapted to the refinement of macromolecules by taking into account parameters determined from atomic resolution protein structures.

Crystal structure of PvuII endonuclease reveals extensive structural homologies to EcoRV.

The crystal structure of the dimeric PvuII restriction endonuclease (R.PvuII) has been determined at a resolution of 2.4A. The protein has a mixed alpha/beta architecture and consists of two subdomains. Despite a lack of sequence homology, extensive structural similarities exist between one R.PvuII subdomain and the DNA-binding subdomain of EcoRV endonuclease (R.EcoRV); the dimerization subdomains are unrelated. Within the similar domains, flexible segments of R.PvuII are topologically equivalent to the DNA-binding turns of R.EcoRV; potential catalytic residues can be deduced from the structural similarities to R.EcoRV. Conformational flexibility is important for the interaction with DNA. A possible classification of endonuclease structures on the basis of the positions of the scissile phosphates is discussed.

Restored heptad pattern continuity does not alter the folding of a four-alpha-helix bundle.

The sequences of alpha-helical coiled-coils and bundles are characterized by a specific pattern of hydrophobic and hydrophilic residues which is repeated every seven residues. Highly conserved breaks in this pattern frequently occur in segments of otherwise continuous heptad substructures. The hairpin bend of the ROP protein coincides with such a break and provides a model system for the study of the structural effects induced by heptad discontinuities. The structure of a ROP mutant which re-establishes a continuous heptad pattern, shows insignificant changes relative to the wild-type protein, as is also reflected in its conformational stability, spectroscopic properties and unfolding behaviour. Thus, formation of alpha-alpha-hairpin bends may occur both in the presence and absence of heptad breaks.

Correlation between protein stability and crystal properties of designed ROP variants.

Six variants of the ROP protein, designed with the aim to analyze by X-ray crystallography loop formation and core packing interactions in 4-alpha-helical bundles, have been purified and a search of their crystallization conditions has been carried out. Five mutants yield crystals that are suitable for medium to high resolution X-ray diffraction studies. For all mutants crystal size, sensitivity to X-irradiation and diffraction limit are correlated to their stability as determined by differential scanning calorimetry, in a manner which is not yet understood in detail.