Saccharomyces cerevisiae oxidosqualene‐lanosterol cyclase: A chemistry–biology interdisciplinary study of the protein's structure–function–reaction mechanism relationships

The oxidosqualene cyclases (EC 5.4.99‐) constitute a family of enzymes that catalyze diverse cyclization/rearrangement reactions of (3S)‐2,3‐oxidosqualene into a distinct array of sterols and triterpenes. The relationship between the cyclization mechanism and the enzymatic structure is extremely complex and compelling. This review covers the historical achievements of biomimetic studies and current progress in structural biology, molecular genetics, and bioinformatics studies to elucidate the mechanistic and structure–function relationships of the Saccharomyces cerevisiae oxidosqualene‐lanosterol cyclase‐catalyzed cyclization/rearrangement reaction. © 2008 The Japan Chemical Journal Forum and Wiley Periodicals, Inc. Chem Rec 8: 302–325; 2008: Published online in Wiley InterScience ( www.interscience.wiley.com ) DOI 10.1002/tcr.20157     

Diffusion of glycosylphosphatidylinositol (GPI)-anchored bovine prion protein (PrP) in supported lipid membranes studied by single-molecule and complementary ensemble methods

In this work cellular bovine prion protein (PrP^c) was incorporated in supported lipid membranes and its lateral diffusion was studied by single-dye tracking (SDT) and a complementary ensemble method, fluorescence recovery after photobleaching (FRAP). PrP^c was purified from calf brain with its native glycosylphosphatidylinositol (GPI) anchor and reconstituted into DMPC lipid vesicles. Homogeneous spreading on solid supports over macroscopic areas was confirmed with fluorescence microscopy. FRAP results demonstrated very high mobile fractions of up to 94%, confirming that most of the GPI-anchored PrP^c are freely diffusive in the fluid supported membrane matrix. Moreover, the lateral diffusivity of PrP^c significantly depends on the pH of the buffer, suggesting that the conformation of PrP^c and thus the frictional drag exerted to the protein molecule (and thus the effective hydrodynamic radius) is influenced by the effective net charge. To complement the ensemble results obtained by FRAP, the statistical variation of lateral diffusion coefficients of individual PrP^c molecules in the supported membranes were measured with SDT. Simulation-based statistical analysis indicated that in addition to the expected statistical scatter there is a significant spread of diffusion coefficients, while the average of the diffusion coefficients of individual proteins obtained by SDT is in excellent agreement with those measured by ensemble FRAP. In further experiments, PrP^c was laterally concentrated in the membrane by the application of tangential electric fields (membrane electrophoresis). However, the equilibrium concentration profile reached after 20 min was different from an exponential gradient. This finding suggests that PrP^c purified from bovine brain possesses non-uniform net charges. As the lateral diffusion coefficient of proteins in two-dimensional lipid membranes sensitively depends upon the frictional drag, the combination of SDT, ensemble FRAP, and membrane electrophoresis can be used as a powerful tool to gain insights into protein–protein binding and oligomer formation that would play a crucial role in infectious protein transmitted diseases such as BSE.     

Crystallization and Preliminary Crystallographic Studies of Cucurmosin 2,a Ribosome-inactivating Protein from the Sarcocarp of Cucurbita moschata

Cucurmosin 2, a type 1 ribosome-inactivating protein （RIP） isolated from sarcocarp of Cucurbita moschata, has been crystallized by the vapor-diffusion method using PEG6000 as the precipitant. The crystals belong to the orthorhombic space group P212121, with unit cell parameters a = 55.853, b = 65.507, c = 91.754 А, and have one molecule per asymmetric unit. X-ray data have been collected to 1.8А, using a synchrotron source.     

Microseparation techniques for the study of the enantioselectivity of drug–plasma protein binding

Stereoselectivity in protein binding can have a significant effect on the pharmacokinetic and pharmacodynamic properties of chiral drugs. The investigation of enantioselectivity of drugs in their binding with human plasma proteins and the identification of the molecular mechanisms involved in the stereodiscrimination by the proteins represent a great challenge for clinical pharmacology. In this review, the separation techniques used for enantioselective protein binding experiments are described and compared. An overview of studies on enantiomer–protein interactions, enantiomer–enantiomer interactions as well as chiral drug–drug interactions, including allosteric effects, is presented. The contribution of individual plasma proteins to the overall enantioselective binding and the animal species variability in drug–plasma protein binding stereoselectivity are reviewed. Copyright © 2008 John Wiley & Sons, Ltd.     

Towards the total chemical synthesis of integral membrane proteins: a general method for the synthesis of hydrophobic peptide-thioester building blocks

Modification of a peptide-^αthioester with a sequence of six arginines on the thioester leaving group can render soluble all peptides derived from a polytopic integral membrane protein. This strategy greatly simplifies the synthesis of peptide-^αthioester building blocks for the total chemical synthesis of integral membrane proteins by native chemical ligation.     

Effect of ultrasonic on the structure and quality characteristics of quinoa protein oxidation aggregates

Protein oxidation leads to covalent modification of structure and deterioration of functional properties of quinoa protein. The objective of this study was to investigate the effects of ultrasonic treatment on the functional and physicochemical properties of quinoa protein oxidation aggregates. In this concern, 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH) was selected as oxidative modification of quinoa protein. The microstructure of quinoa protein displayed by scanning electron microscope (SEM) indicated that oxidation induced extensive aggregation, leading to carbonylation and degradation of sulfhydryl groups. Aggregation induced by oxidation had a negative effect on the solubility, turbidity, emulsifying stability. However, according to the analysis of physicochemical properties, ultrasonic significantly improved the water solubility of quinoa protein. The quinoa protein treated by ultrasonic for 30 min exhibited the best dispersion stability in water, which corresponded to the highest ζ-potential, smallest particle size and most uniform distribution. Based on the FT-IR, SDS-PAGE and surface hydrophobicity analysis, the increase of α-helix, β-turn and surface hydrophobicity caused by cavitation effect appeared to be the main mechanism of quinoa protein solubilization. In addition, the hydrophobic region of the protein was re-buried by excessive ultrasonic treatment, and the protein molecules were reaggregated by disulfide bonds. Microstructural observations further confirmed that ultrasonic treatment effectively inhibited protein aggregation and improved the functional properties of quinoa protein.     

Ultrasonic pre-treatment modifies the pH-dependent molecular interactions between β-lactoglobulin and dietary phenolics: Conformational structures and interfacial properties

There is a need to understand the ultrasound-induced changes in the interactions between proteins and phenolic compounds at different pH. This study systematically explored the role of high-intensity ultrasound pre-treatment on the binding mechanisms of β-lactoglobulin (β-LG) to two common phenolic compounds, i.e., (−)-epigallocatechin-3-gallate (EGCG) and chlorogenic acid (CA) at neutral and acidic pH (pH 7.2 and 2.4). Tryptophan fluorescence revealed that compared to proteins sonicated at 20% and 50% amplitudes, 35%-amplitude ultrasound pre-treatment (ULG-35) strengthened the binding affinities of EGCG/CA to β-LG without altering the main interaction force. After phenolic addition, ULG-35 displayed a similar but a greater extent of protein secondary and tertiary structural changes than the native protein, ascribed to the ultrasound-driven hydrophobic stacking among interacted molecules. The dominant form of β-LG (dimer/monomer) played a crucial role in the conformational and interfacial properties of complexes, which can be explained by the distinct binding sites at different pH as unveiled by molecular docking. Combining pre-ultrasound with EGCG interaction notably increased the foaming and emulsifying properties of β-LG, providing a feasible way for the modification of bovine whey proteins. These results shed light on the understanding of protein–phenolic non-covalent binding under ultrasound and help to develop complex systems with desired functionality and delivery.     

Thermosonicated whey protein concentrate blends on quality attributes of reduced fat Panela cheese

Aiming at producing a reduced fat cheese (RFC) as an alternative to full-fat Panela cheese, a highly consumed fresh Mexican dairy product, thermosonication (TS) processes (24 kHz, 400 W nominal power, 2, 4 and 6 min; 50, 55 and 60 °C) were evaluated to treat WPC (80% protein) blended with reduced-fat milk (1 and 2% fat), which were later LTLT pasteurized. TS blends were compared in terms of their technological properties (water holding capacity-WPC, gel firmness- GF, color, pH and titratable acidity) with those of a regular full fat (3%) LTLT pasteurized milk used as a control. Afterwards, a regression analysis was carried out with the obtained data in order to select the most appropriate conditions for cheesemaking purposes (similar GF, higher WHC with respect to the control), minimize both fat content and TS treatment duration to minimize energy expenses. According to these restrictions, the selected conditions were 1.5% fat milk-WPC blend, TS treated at 60 °C for 120 s; 1% fat milk-WPC blend, TS treated at 50 °C for 120 s and 1% fat milk-WPC blend, 50 °C for 144 s, which allowed preparing low fat cheeses (LFCs). These TS treatments were applied in a larger scale to elaborate Panela-type LFCs comparing different technological properties (cheese yield, syneresis, water content, texture profile analysis, color and titratable acidity) with those of a full fat variety, at day 1 and during 14 days of refrigerated storage. Results showed similar texture profiles of LFC cheeses and full fat milk cheeses throughout their storage period with significant changes in composition parameters (higher moisture, protein and salt contents, with low fat percentages), syneresis, selected color parameters (hue, b*), with no observed changes in cheese yield, TA and pH during cheese storage. These promising results are encouraging to develop LFCs with no physicochemical or technological defects using novel processing techniques that may help reducing calorie consumption without compromising sensory acceptability.