MS Analysis and Molecular Characterization of Botrytis cinerea Protease Prot-2. Use in Bioactive Peptides Production

Prot-2 protease previously purified to homogeneity from Botrytis cinerea showed potentiality to be used in detergency and for production of bioactive peptides. To extend the characterization of Prot-2 protease, antifungal and antibacterial assays were performed in vitro using protein hydrolysates prepared from muscle of mackerel (Scomber scomborus) treated with this enzyme. The most active hydrolysate (degree of hydrolysis of 8 %) exhibited inhibition effect towards bacteria and phytopathogenic fungi, demonstrating that Prot-2 proteolysis generated bioactive peptides. Biochemical and molecular characterization of the purified Prot-2, by SDS-PAGE/Tryptic in gel-digestion and LC-MS/MS analysis, was investigated. The peptide amino acid sequence alignment search in database revealed a moderate homology between the determined amino acid sequence of Prot-2 protease and the known fungal trypsin/chymotrypsin in particular from Glomerella, Metarhizium and Streptomyces. From peptide sequence data obtained by mass spectrometry and sequences homologies, primers were defined and a cDNA fragment of 786 bp was amplified by RT-PCR. The cDNA nucleotide sequence analysis revealed an open reading frame coding for 262 amino acid residues. The deduced amino acid sequence of Prot-2 showed moderate identity with trypsin of Glomerella graminicola (74 %) and with chymotrypsin from Metarhizium anisopliae (71 %). Prot-2 exhibited a Ser protease homology and showed in addition the specific His motif of trypsin/chymotrypsin family.     

Selective Introduction of Sulfhydryl Groups into Recombinant Proteins for Study of Protein–Protein Interactions

In the present work, we proposed to create special sorbents for the study of protein–protein interactions, based on the fixation of cysteine-inserted beta-casein mutants with thiol-Sepharose resin. As a model system, we used bovine beta-casein, which belongs to the family of intrinsically unstructured proteins. Insertion of distal cysteines into the unfolded protein was not found to significantly change beta-casein properties. An amphiphilic beta-casein molecule has one hydrophilic domain and one hydrophobic domain placed on the N- and C-terminus, thus enabling one to exploit its capacity to engage in different types of intermolecular interactions. Two different casein-Sepharose sorbents incorporating either C-4 or C-208 beta-casein mutants bound to thiol-Sepharose were produced, exposing the hydrophobic domain in the case of the C-4 and the hydrophilic domain in the case of the C-208 mutant, respectively. The results obtained using the proposed sorbents with native beta-casein, another partially unfolded protein prion, and an oligomeric globular glyceraldehyde-3-phosphate dehydrogenase were found to be consistent with the data obtained by ELISA on free protein–protein complexes. Thus, Sepharose modified with various proteins is suitable for isolation of proteins interacting with the chromatographic phase bound partners from multicomponent systems such as milk. The obtained results allow the proposing of a fast and convenient method to be used for isolation of proteins, determination of protein-interacting partners, and the study of multi-protein complexes.     

Chemometric study of the aggregation of alcohol dehydrogenase and its suppression by beta-caseins: a mechanistic perspective

Molecular chaperones interact preferentially with certain aggregation-prone intermediates of target protein molecules. An estimation of the chaperone activity based on suppression of aggregation is required to be mechanistically understood. In this study, the multivariate curve resolution chemometric technique was applied on horse alcohol dehydrogenase (ADH) UV-spectra under thermal stress, to obtain the required information about the number and change in concentrations of the species involved. Chemometric analysis of UV-absorption spectra of horse ADH under thermal stress, led to the existence of three different molecular species including native (N), aggregation-prone intermediate (I) and final aggregate (A) species. Appearance and buildup of two molecular species I and A were connected to the disappearance of N-species. In the presence of β-caseins (BCN), however, a new complex between I and BCN (I-BCN) was formed. Meanwhile, by accretion of concentration of I-BCN complex, the light scattering intensity diminished. The data presented in this study clearly demonstrate that the interaction of BCN as a chaperone molecule with I-species takes place in a temperature-dependent manner and leads to a reversible I-BCN complex. In the absence of chaperones, I-state is subsequently converted to the final aggregate species. In the presence of BCN, this molecular species could be converted to the final aggregate state and/or form the I-BCN complex.     

Spectroscopic and Calorimetric Study of 2,2′-Dibipyridin Cu(II) Chloride Binding to Bovine β-Lactoglobulin

The interaction between β-lactoglobulin (BLG) and a newly synthesized Cu(II) complex (2,2′-dibipyridin Cu(II) chloride) was investigated by fluorescence spectroscopy, circular dichroism (CD) and isothermal titration calorimetry (ITC) at temperatures of 27 and 37 °C. The measured heat values of the BLG–Cu(II) complex interaction are reported and analyzed in terms of our previous extended solvation theory for calculating the binding and thermodynamic parameters for the interaction. The Cu(II) complex has a strong ability to quench the intrinsic fluorescence of BLG, to change the microenvironment of tryptophan residues, and to alter the tertiary structure of the protein. Far UV–CD results showed that the complex does not induce any significant changes in the secondary structure of BLG. However, binding of the Cu(II) complex to BLG leads to a significant change in the tertiary structure of BLG, increasing its hydrophobicity and inducing a partial unfolding. This agrees well with ITC data suggesting destabilization of the protein. This finding opens up a way to predict protein destabilization caused by ligand binding, using the extended solvation theory previously proposed.     

Aggregation of sodium dodecyl sulfate in micellar solution of β-casein analyzed by 1H NMR self-diffusion, relaxation and Fourier transform IR spectroscopy

Micellization of anionic sodium dodecyl sulfate (SDS) in the presence of -casein (-CN) micelles in aqueous media was studied by ¹H NMR and Fourier transform IR spectroscopy. At low concentrations SDS molecules incorporate into -CN micelles and modify the protein secondary structure, increasing the portion of helical domains. It was shown that SDS micelles do not appear until binding sites located in the hydrophobic core of -CN micelles are saturated.     

Study of stability of variants of ovine prions with different susceptibilities to scrapie

Well-defined set of sheep PrP polymorphisms at positions 136, 154 and 171 define susceptibility to scrapie, ranging from very high susceptibility observed for V136-R154-Q171 (VRQ) variant to resistance for A136-R154-R171 (ARR).To gain insight into the mechanisms of scrapie susceptibility/resistance, the unfolding pathways of the sheep prion protein variants were analysed by differential scanning calorimetry over a wide range of pH. Thermal unfolding occurs, in the 5.0 to 6.0 pH range, through a reversible one-step process while at pH<4.5 and >6.0 unfolding intermediates are formed, which are stable in the 65–80°C range. The observed differences correlate with ovine susceptibility to scrapie.This revised version was published online in November 2005 with corrections to the Cover Date.