Roberto Arreguín-Espinosa

Energetics of protein homodimerization: Effects of water sequestering on the formation of β-lactoglobulin dimer

Transient protein–protein interactions are functionally relevant as a control mechanism in a variety of biological processes. Analysis of the 3D structure of protein–protein complexes indicates that water molecules trapped at the interface are very common; however, their role in the stability and specificity of protein homodimer interactions has been not addressed yet. To provide new insights into the energetic bases that govern the formation of highly hydrated interfaces, the dissociation process of bovine βlg variant A at a neutral pH was characterized here thermodynamically by conducting dilution experiments with an isothermal titration calorimeter. Association was enthalpically driven throughout the temperature range spanned. ΔH and ΔCp were significantly more negative than estimates based on surface area changes, suggesting the occurrence of effects additional to the dehydration of the contact surfaces between subunits. Near‐UV CD spectra proved to be independent of protein concentration, indicating a rigid body‐like association. Furthermore, the process proved not to be coupled to significant changes in the protonation state of ionizable groups or counterion exchange. In contrast, both osmotic stress experiments and a computational analysis of the dimers 3D structure indicated that a large number of water molecules are incorporated into the interface upon association. Numerical estimates considering the contributions of interface area desolvation and water immobilization accounted satisfactorily for the experimental ΔCp. Thus, our study highlights the importance of explicitly considering the effects of water sequestering to perform a proper quantitative analysis of the formation of homodimers with highly hydrated interfaces. Proteins 2008.

Role of vitamin B12 on methylmalonyl-CoA mutase activity

Vitamin B12 is an organometallic compound with important metabolic derivatives that act as cofactors of certain enzymes, which have been grouped into three subfamilies depending on their cofactors. Among them, methylmalonyl-CoA mutase (MCM) has been extensively studied. This enzyme catalyzes the reversible isomerization of L-methylmalonyl-CoA to succinyl-CoA using adenosylcobalamin (AdoCbl) as a cofactor participating in the generation of radicals that allow isomerization of the substrate. The crystal structure of MCM determined in Propionibacterium freudenreichii var. shermanii has helped to elucidate the role of this cofactor AdoCbl in the reaction to specify the mechanism by which radicals are generated from the coenzyme and to clarify the interactions between the enzyme, coenzyme, and substrate. The existence of human methylmalonic acidemia (MMA) due to the presence of mutations in MCM shows the importance of its role in metabolism. The recent crystallization of the human MCM has shown that despite being similar to the bacterial protein, there are significant differences in the structural organization of the two proteins. Recent studies have identified the involvement of an accessory protein called MMAA, which interacts with MCM to prevent MCM’s inactivation or acts as a chaperone to promote regeneration of inactivated enzyme. The interdisciplinary studies using this protein as a model in different organisms have helped to elucidate the mechanism of action of this isomerase, the impact of mutations at a functional level and their repercussion in the development and progression of MMA in humans. It is still necessary to study the mechanisms involved in more detail using new methods.

Purification and properties of a lipase from Cephaloleia presignis (Coleoptera, Chrysomelidae)

A novel lipase from the insect Cephaloleia presignis was purified by a procedure involving ammonium sulphate precipitation, and Phenyl Toyopearl 650M, DEAE‐5PW and hydrophobic‐interaction column chromatographies. The purified lipase was homogeneous with a molecular mass of 31000 Da by SDS/PAGE and of 29000 Da by gel filtration on a Superose 12 column. The enzyme was indentified as a glycoprotein with a pI of 6.9. The enzyme unspecifically liberated short‐chain to long‐chain fatty acids from p‐nitrophenyl esters, methyl esters and triglycerides. The N‐terminal 28 amino acid residues were determined as AGTLGYATRHVLPIFTLDDYTGSNEMWG, which showed no similarity with known proteins, suggesting that the purified lipase may belong to a novel class of hydrolases.

Purification and characterization of several digestive proteases from the blue abalone, Haliotis fulgens

Abstract Several proteases were found in an aqueous extract from the hepatopancreas of blue abalone. Four, called PH1, PH2, PH3 and PH4, were purified by ammonium sulfate precipitation, low pressure DEAE-Sepharose chromatography and anion exchange HPLC. All these proteases were active on protein substrates (casein and hide powder azure); they showed different activities with synthetic substrates and were inhibited by different compounds. PH1, PH2 and PH3 seemed to be serine-like proteases (PH1, chymotrypsin-like; PH2 and PH3, trypsin-like) while protease PH4 had carboxypeptidase behavior. The purified enzymes showed single bands in SDS-PAGE and IEF minigels showing relative molecular masses of 28,100, 29,500, 32,000 and 30,000, and isoelectric points of 6.3, 3.6, 4.0 and 3.8, respectively. PH1, PH2 and PH3 have common features in their amino acid contents. These enzymes possess different secondary structures as judged from their circular dichroism spectra.

Molecular and Catalytic Properties of the Aldehyde Dehydrogenase of Gluconacetobacter diazotrophicus, a Quinoheme Protein Containing Pyrroloquinoline Quinone, Cytochrome b, and Cytochrome c

Several aldehyde dehydrogenase (ALDH) complexes have been purified from the membranes of acetic acid bacteria. The enzyme structures and the chemical nature of the prosthetic groups associated with these enzymes remain a matter of debate. We report here on the molecular and catalytic properties of the membrane-bound ALDH complex of the diazotrophic bacterium Gluconacetobacter diazotrophicus. The purified ALDH complex is a heterodimer comprising two subunits of 79.7 and 50 kDa, respectively. Reversed-phase high-pressure liquid chromatography (HPLC) and electron paramagnetic resonance spectroscopy led us to demonstrate, for the first time, the unequivocal presence of a pyrroloquinoline quinone prosthetic group associated with an ALDH complex from acetic acid bacteria. In addition, heme b was detected by UV-visible light (UV-Vis) spectroscopy and confirmed by reversed-phase HPLC. The smaller subunit bears three cytochromes c. Aliphatic aldehydes, but not formaldehyde, were suitable substrates. Using ferricyanide as an electron acceptor, the enzyme showed an optimum pH of 3.5 that shifted to pH 7.0 when phenazine methosulfate plus 2,6-dichlorophenolindophenol were the electron acceptors. Acetaldehyde did not reduce measurable levels of the cytochrome b and c centers; however, the dithionite-reduced hemes were conveniently oxidized by ubiquinone-1; this finding suggests that cytochrome b and the cytochromes c constitute an intramolecular redox sequence that delivers electrons to the membrane ubiquinone.

The effect of sulfhydryl groups and disulphide linkage in the thermal aggregation of Z19 α-zein

Zeins are the major storage proteins in corn seeds organized in protein bodies located in the endosperm. They are soluble in alcoholic solution and depict a high tendency to aggregation. The Z19 alpha-zein aggregates obtained by heating show a particular and interesting temperature-dependent behavior. This work was aimed at determining not only the effect of temperature on the aggregation behavior, but also the effect of the sulfhydryl groups and disulphide bonds on the thermal aggregation process under non-aqueous conditions. Z19 alpha-zein was chemically modified to obtain different sulfhydryl groups and disulphide-bonds content. Far-UV CD, ANS emission fluorescence, and dynamic light scattering, as well as differential scanning calorimetry, were performed to characterize this protein. Removal of these disulphide-bonds and alkylation of all the sulfhydryl groups in the protein promoted the lowest T(m) of 57.36 degrees C, eliminated aggregation, enhanced protein flexibility, and diminished thermal stability. These results suggest that the disulphide linkage could be the driving force for the Z19 alpha-zein aggregation.

Glucose-6-Phosphate Dehydrogenase: Update and Analysis of New Mutations around the World

Glucose-6-phosphate dehydrogenase (G6PD) is a key regulatory enzyme in the pentose phosphate pathway which produces nicotinamide adenine dinucleotide phosphate (NADPH) to maintain an adequate reducing environment in the cells and is especially important in red blood cells (RBC). Given its central role in the regulation of redox state, it is understandable that mutations in the gene encoding G6PD can cause deficiency of the protein activity leading to clinical manifestations such as neonatal jaundice and acute hemolytic anemia. Recently, an extensive review has been published about variants in the g6pd gene; recognizing 186 mutations. In this work, we review the state of the art in G6PD deficiency, describing 217 mutations in the g6pd gene; we also compile information about 31 new mutations, 16 that were not recognized and 15 more that have recently been reported. In order to get a better picture of the effects of new described mutations in g6pd gene, we locate the point mutations in the solved three-dimensional structure of the human G6PD protein. We found that class I mutations have the most deleterious effects on the structure and stability of the protein.

Controlling the morphology of silica–carbonate biomorphs using proteins involved in biomineralization

Silica–carbonate biomorphs are inorganic self-organized structures that mimic the morphology of living organisms. In this study, we present the effect that proteins involved in the in vivo biomineralization of silica and calcium carbonate have on the formation of silica–carbonate biomorphs. We tested four different sources of protein: (1) struthiocalcin-1, (2) the catalytic domain of silicatein-α of Tethya aurantia, (3) a protein extract obtained from the spicules of a vitreous sponge (Protosuberitis sp.), and (4) a protein extract obtained from the spines of the sea urchin Echinometra lucunter. In addition to the well-established role that pH plays in biomorph formation, all the proteins tested controlled the morphology of these aggregates. Biomorphs obtained in the presence of the catalytic domain of silicatein-α were similar in shape to those observed in the control though considerably smaller in size. Struthiocalcin-1 affected the availability of carbonate ions and completely inhibited the formation of biomorphs resulting only in worm-like aggregates. Finally, novel biomorphs with shapes such as twisting rods, sunflowers, and mitotic cells were obtained in the presence of protein extracts from the marine sponge spicules and sea urchin spines.

Biological and taxonomic perspective of triterpenoid glycosides of sea cucumbers of the family Holothuriidae (Echinodermata, Holothuroidea).

Since the discovery of saponins in sea cucumbers, more than 150 triterpene glycosides have been described for the class Holothuroidea. The family Holothuriidae has been increasingly studied in search for these compounds. With many species awaiting recognition and formal description this family currently consists of five genera and the systematics at the species-level taxonomy is, however, not yet fully understood. We provide a bibliographic review of the triterpene glycosides that has been reported within the Holothuriidae and analyzed the relationship of certain compounds with the presence of Cuvierian tubules. We found 40 species belonging to four genera and 121 compounds. Holothurin A and B are the most common saponins for Actinopyga, Holothuria, and Pearsonothuria. The genus Bohadschia presents mainly bivittoside C and D. Actinopyga has only sulfated saponins mainly oxidized, Bohadschia non-sulfated ones mainly non-oxidized, Holothuria and Pearsonothuria contain both types of compounds, mainly oxidized. Within the genus Holothuria, the subgenus Panningothuria only has non-sulfated saponins. The presence of sulfated and non-sulfated compounds seemingly relates to the expellability or the absence of Cuvierian tubules and the temporal or permanent concealing habits of the species. Our study concludes that better insights into the systematic distribution of saponins in Holothuriidae will only be possible if the identifications of the investigated species are confirmed by a taxonomist, especially in this group wherein cryptic species and variation between life-history stages are common and yet poorly understood. Understanding of saponin distribution within the Holothuriidae would also benefit from a stabilization of triterpene glycoside nomenclature.

An electrically assisted device for protein crystallization in a vapor‐diffusion setup

A new easy-to-use device has been designed and implemented for electric field-induced protein crystallization in a vapor-diffusion configuration. The device not only controls crystal nucleation by means of the electrical current, but also favors crystal growth owing to its vapor-diffusion setup. Crystallization was conducted in the presence of an internal electric field and direct current. The proteins investigated were lysozyme, as model protein, and 2TEL-lysozyme (a synthetic protein consisting of two tandem alpha helix motifs connected to a lysozyme moiety). Lysozyme crystals that grew attached to the cathode were larger than those grown attached to the anode or in the absence of an electric current. On the other hand, crystals of 2TEL-lysozyme qualitatively showed a better X-ray diffraction pattern when grown in the presence of an electric current.