Protein modification has entered the limelight of chemical and biological sciences, since, by appending small molecules into proteins surfaces, fundamental biological and biophysical processes may be studied and even modulated in a physiological context. Herein we present a new strategy to modify the lysine's ε-amino group and the protein's N-terminal, based on the formation of stable iminoboronates in aqueous media. This functionality enables the stable and complete modification of these amine groups, which can be reversible upon the addition of fructose, dopamine, or glutathione. A detailed DFT study is also presented to rationalize the observed stability toward hydrolysis of the iminoboronate constructs.
Extracts of leaves, fruits and seeds of olive tree cultivars of Trás-os-Montes e Alto Douro (Portugal) were analysed by reverse phase HPLC with diode array detection and mass spectrometry (MS). This methodology allowed the identification of some common phenolic compounds, namely, verbascoside, rutin, luteolin-7-glucoside, oleuropein and hydroxytyrosol. Moreover the hyphenation of HPLC with MS enabled the identification of nüzhenide in olive seeds. An oleuropein glucoside was also detected in olive tree leaves. The total phenolic content was determined with the Folin Denis reagent and the total antioxidant activity with the ABTS method. There is a correlation between total antioxidant activity and total phenolic content with the exception of the seed extracts analysed. The apparent high antioxidant activity of seed extracts may be due to nüzhenide, a secoiridoid that is the major phenolic component of olive seeds. These results suggest a possible application of olive seeds as sources of natural antioxidants.
The genome of Desulfovibrio vulgaris Hildenborough (DvH) encodes for six hydrogenases (Hases), making it an interesting organism to study the role of these proteins in sulphate respiration. In this work we address the role of the [NiFeSe] Hase, found to be the major Hase associated with the cytoplasmic membrane. The purified enzyme displays interesting catalytic properties, such as a very high H(2) production activity, which is dependent on the presence of phospholipids or detergent, and resistance to oxygen inactivation since it is isolated aerobically in a Ni(II) oxidation state. Evidence was obtained that the [NiFeSe] Hase is post-translationally modified to include a hydrophobic group bound to the N-terminal, which is responsible for its membrane association. Cleavage of this group originates a soluble, less active form of the enzyme. Sequence analysis shows that [NiFeSe] Hases from Desulfovibrionacae form a separate family from the [NiFe] enzymes of these organisms, and are more closely related to [NiFe] Hases from more distant bacterial species that have a medial [4Fe4S](2+/1+) cluster, but not a selenocysteine. The interaction of the [NiFeSe] Hase with periplasmic cytochromes was investigated and is similar to the [NiFe](1) Hase, with the Type I cytochrome c (3) as the preferred electron acceptor. A model of the DvH [NiFeSe] Hase was generated based on the structure of the Desulfomicrobium baculatum enzyme. The structures of the two [NiFeSe] Hases are compared with the structures of [NiFe] Hases, to evaluate the consensual structural differences between the two families. Several conserved residues close to the redox centres were identified, which may be relevant to the higher activity displayed by [NiFeSe] Hases.
Sea urchins are common inhabitants of wave-swept shores. To withstand the action of waves, they rely on highly specialized independent adhesive organs, the adoral tube feet. The latter are extremely well-designed for temporary adhesion being composed by two functional subunits: (1) an apical disc that produces an adhesive secretion to fasten the sea urchin to the substratum, as well as a deadhesive secretion to allow the animal to move and (2) a stem that bears the tensions placed on the animal by hydrodynamism. Despite their technological potential for the development of new biomimetic underwater adhesives, very little is known about the biochemical composition of sea urchin adhesives. A characterization of sea urchin adhesives is presented using footprints. The latter contain inorganic residues (45.5%), proteins (6.4%), neutral sugars (1.2%), and lipids (2.5%). Moreover, the amino acid composition of the soluble protein fraction revealed a bias toward six amino acids: glycine, alanine, valine, serine, threonine, and asparagine/aspartic acid, which comprise 56.8% of the total residues. In addition, it also presents higher levels of proline (6.8%) and half-cystine (2.6%) than average eukaryotic proteins. Footprint insolubility was partially overcome using strong denaturing and reducing buffers, enabling the visualization of 13 proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The conjugation of mass spectrometry with homology-database search allowed the identification of six proteins: alpha and beta tubulin, actin, and histones H2B, H3, H2A, and H4, whose location and function in the adhesive are discussed but require further investigation. For the remaining unidentified proteins, five de novo-generated peptide sequences were found that were not present in the available protein databases, suggesting that they might be novel or modified proteins.
Sheep and goats differ in diet selection, which may reflect different abilities to deal with the ingestion of plant secondary metabolites. Although saliva provides a basis for immediate oral information via sensory cues and also a mechanism for detoxification, our understanding of the role of saliva in the pre-gastric control of the intake of herbivores is rudimentary. Salivary proteins have important biological functions, but despite their significance, their expression patterns in sheep and goats have been little studied. Protein separation techniques coupled to mass spectrometry based techniques have been used to obtain an extensive comprehension of human saliva protein composition but far fewer studies have been undertaken on animals' saliva. We used twodimensional electrophoresis gel analysis to compare sheep and goats parotid saliva proteome. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) and liquid chromatography tandem mass spectrometry (LC-MS/MS) were used to identify proteins. From a total of 260 sheep and 205 goat saliva protein spots, 117 and 106 were identified, respectively. A high proportion of serum proteins were found in both salivary protein profiles. Major differences between the two species were detected for proteins within the range of 25-35 kDa. This study presents the parotid saliva proteome of sheep and goat and highlights the potential of proteomics for investigation relating to intake behavior research.
BackgroundInsulin is a hormone that regulates blood glucose homeostasis and is a central protein in a medical condition termed insulin injection amyloidosis. It is intimately associated with glycaemia and is vulnerable to glycation by glucose and other highly reactive carbonyls like methylglyoxal, especially in diabetic conditions. Protein glycation is involved in structure and stability changes that impair protein functionality, and is associated with several human diseases, such as diabetes and neurodegenerative diseases like Alzheimer's disease, Parkinson's disease and Familiar Amyloidotic Polyneuropathy. In the present work, methylglyoxal was investigated for their effects on the structure, stability and fibril formation of insulin.ResultsMethylglyoxal was found to induce the formation of insulin native-like aggregates and reduce protein fibrillation by blocking the formation of the seeding nuclei. Equilibrium-unfolding experiments using chaotropic agents showed that glycated insulin has a small conformational stability and a weaker dependence on denaturant concentration (smaller m-value). Our observations suggest that methylglyoxal modification of insulin leads to a less compact and less stable structure that may be associated to an increased protein dynamics.ConclusionsWe propose that higher dynamics in glycated insulin could prevent the formation of the rigid cross-β core structure found in amyloid fibrils, thereby contributing to the reduction in the ability to form fibrils and to the population of different aggregation pathways like the formation of native-like aggregates.
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