Jose C. Martinez

The folding transition state between SH3 domains is conformationally restricted and evolutionarily conserved.

The protein engineering analysis of the α-spectrin SH3 domain at three different stability conditions (pH 7.0, 3.5 and 2.5) reveals a folding transition state structured around the distal loop β-hairpin and the 310-helix. This region is impervious to overall changes in protein stability, suggesting a transition state ensemble with little conformational variability. Comparison with the Src SH3 domain (36% sequence homology) indicates that the transition state in this protein family may be conserved. Discrepancies at some positions can be rationalized in terms of the different interactions made by the different side chains in both domains. Brønsted plot analysis confirms the φ‡-U results and shows two folding subdomains for this small protein. These results, together with previous data on circular permutants of the α-spectrin SH3 domain, indicate that polypeptide topology and chain connectivity play a major role in the folding reaction of this protein family.

Characteristics of soil after pollution with waste waters from olive oil extraction plants

Abstract Higher levels of soil salinity due to potassium and sodium replacement of soil cations were detected in an alkaline soil after pollution with waste waters from olive oil extraction plants. The pH was practically unchanged and soil C/N ratio was increased. A reversible decrease in the count of sporulated bacteria was noted.

Effect of waste waters from olive oil extraction plants on the bacterial population of soil

Abstract After soil pollution with waste waters from olive oil extraction plants, an increase in total microbial counts was observed. The increase in coryneform bacteria and decrease in Bacillus was correlated with the susceptibility to the antimicrobial effect of these wastes.

Detoxification of semisolid olive-mill wastes and pine-chip mixtures using Phanerochaete flavido-alba

Semisolid olive-mill residues, pine chips, and mixtures of both residues contain phytotoxic components capable of inhibiting germination and vegetative growth in plants. Solid-state cultures of Phanerochate flavido-alba on pine chips or mixtures of both residues reduce these phytotoxic effects in fermented substrates. The phenol and lipid contents in cultures detoxified by this fungus also decreases.

Low molecular weight phenolics attenuation during simulated treatment of wastewaters from olive oil mills in evaporation ponds

Abstract Most Mediterranean countries dispose of olive oil mill wastewater in artificial evaporation ponds. In this study the qualitative and quantitative evaluation of the phenolic content and the antibacterial properties of these residues is performed during evaporation in simulated evaporation ponds. No antibacterial effect was detectable in subsequent evaporation for 90 days. Correlations between antibacterial effect and phenolic content and biodegradation are discussed.

A Thermodynamic and Kinetic Analysis of the Folding Pathway of an SH3 Domain Entropically Stabilised by a Redesigned Hydrophobic Core

The folding thermodynamics and kinetics of the alpha-spectrin SH3 domain with a redesigned hydrophobic core have been studied. The introduction of five replacements, A11V, V23L, M25V, V44I and V58L, resulted in an increase of 16% in the overall volume of the side-chains forming the hydrophobic core but caused no remarkable changes to the positions of the backbone atoms. Judging by the scanning calorimetry data, the increased stability of the folded structure of the new SH3-variant is caused by entropic factors, since the changes in heat capacity and enthalpy upon the unfolding of the wild-type and mutant proteins were identical at 298 K. It appears that the design process resulted in an increase in burying both the hydrophobic and hydrophilic surfaces, which resulted in a compensatory effect upon the changes in heat capacity and enthalpy. Kinetic analysis shows that both the folding and unfolding rate constants are higher for the new variant, suggesting that its transition state becomes more stable compared to the folded and unfolded states. The phi(double dagger-U) values found for a number of side-chains are slightly lower than those of the wild-type protein, indicating that although the transition state ensemble (TSE) did not change overall, it has moved towards a more denatured conformation, in accordance with Hammonds postulate. Thus, the acceleration of the folding-unfolding reactions is caused mainly by an improvement in the specific and/or non-specific hydrophobic interactions within the TSE rather than by changes in the contact order. Experimental evidence showing that the TSE changes globally according to its hydrophobic content suggests that hydrophobicity may modulate the kinetic behaviour and also the folding pathway of a protein.

AS-48: a circular protein with an extremely stable globular structure

The unfolding thermodynamics of the circular enterocin protein AS‐48, produced by Enterococcus faecalis, has been characterized by differential scanning calorimetry. The native structure of the 70‐residue protein is extremely thermally stable. Thus, at pH 2.5 and low ionic strength thermal denaturation occurs under equilibrium at 102°C, while the unfolded state irreversibly aggregates at neutral and alkaline pH. Calorimetric data analysis shows that the specific enthalpy change upon unfolding is unusually small and the heat capacity change is quite normal for a protein of this size, whereas the Gibbs energy change at 25°C is relatively high. At least part of this high stability might be put down to entropic constraints induced by the circular organization of the polypeptide chain.

Characterization of manganese-dependent peroxidase isoenzymes from the ligninolytic fungus Phanerochaete flavido-alba.

Phanerochaete flavido-alba is able to decolorize and detoxify olive oil wastewater (OMW) in a process in which simple and polymeric phenols are removed. An unusual acidic MnP is accumulated during the degradation course. This microorganism produces two families of MnPs. MnP1 has an apparent molecular weight of 45 kDa and is secreted as a mixture of isoenzymes with pI ranging from 5.6 to 4.75. MnP2, which is produced as an unique isoenzyme, has an apparent molecular weight of 55.6 Mr and an unusual acidic pI lower than 2.8. The higher specific peroxidase activity for purified MnP2 was for Mn2+ oxidation. Hydroquinone and methylhydroquinone oxidation by MnP2 was Mn2+ dependent, in reaction mixtures without exogenous H2O2. Conversely, ABTS oxidation was Mn2+ independent. Two different DNA fragments (mnpA and mnpB), amplified by PCR, using MnP2 N-terminal sequence and oligonucleotides deduced from two conserved sequences of other MnPs, code for MnPs that belong to the P. chrysosporium mnp2 subfamily on the basis of intron position. The structure of mnpA and mnpB seems to be related to known manganese peroxidase genes, but mnpA encodes an Alanine instead of a Serine (Ser168) regarded as invariant within typical MnPs.

Effect of carbon and nitrogen limitation on lignin peroxidase and manganese peroxidaseproduction by Phanerochaete flavido‐alba

The ligninolytic enzymes lignin peroxidase (LiP) and manganese dependent peroxidase(MnP), were detected in extracellular fluids of Phanerochaete flavido‐alba FPL 106507cultures under carbon or nitrogen limitation. MnP activities were found to be higher than LiPactivities under all growth conditions tested. Higher titres of both peroxidases were obtainedunder carbon limitation in excess nitrogen. Isoelectric points (pIs) observed after FPLC and IEFof concentrated extracellular fluids revealed more acidic pIs for LiP enzymes obtained innitrogen‐limited cultures than those in carbon‐limited cultures. However, the change in thelimiting growth factor does not significantly affect MnP pIs.

Role of Interfacial Water Molecules in Proline-rich Ligand Recognition by the Src Homology 3 Domain of Abl

The interaction of Abl-Src homology 3 domain (SH3) with the high affinity peptide p41 is the most notable example of the inconsistency existing between the currently accepted description of SH3 complexes and their binding thermodynamic signature. We had previously hypothesized that the presence of interfacial water molecules is partially responsible for this thermodynamic behavior. We present here a thermodynamic, structural, and molecular dynamics simulation study of the interaction of p41 with Abl-SH3 and a set of mutants designed to alter the water-mediated interaction network. Our results provide a detailed description of the dynamic properties of the interfacial water molecules and a molecular interpretation of the thermodynamic effects elicited by the mutations in terms of the modulation of the water-mediated hydrogen bond network. In the light of these results, a new dual binding mechanism is proposed that provides a better description of proline-rich ligand recognition by Abl-SH3 and that has important implications for rational design.