Site-Directed Cross-Linking Between Bacterial Flagellar Motor Proteins In Vivo.

The bacterial flagellum employs a rotary motor embedded on the cell surface. The motor consists of the stator and rotor elements and is driven by ion influx (typically H+ or Na+) through an ion channel of the stator. Ion influx induces conformational changes in the stator, followed by changes in the interactions between the stator and rotor. The driving force to rotate the flagellum is thought to be generated by changing the stator-rotor interactions. In this chapter, we describe two methods for investigating the interactions between the stator and rotor: site-directed in vivo photo-crosslinking and site-directed in vivo cysteine disulfide crosslinking.

Oxidation of Whey Proteins during Thermal Treatment Characterized by a Site-Specific LC-MS/MS-Based Proteomic Approach.

Thermal treatment is often employed in food processing to tailor product properties by manipulating the ingredient functionality, but these elevated temperatures may accelerate oxidation and nutrient loss. Here, oxidation of different whey protein systems [α-lactalbumin (α-LA), ß-lactoglobulin (ß-LG), a mix of α-LA and ß-LG (whey model), and a commercial whey protein isolate (WPI)] was investigated during heat treatment at 60-90 °C and a UHT-like treatment by LC-MS-based proteomic analysis. The relative modification levels of each oxidation site were calculated and compared among different heat treatments and sample systems. Oxidation increased significantly in protein systems after heating at ≥90 °C but decreased in systems with higher complexity [pure protein (α-LA > ß-LG) > whey model > WPI]. In α-LA, Cys, Met, and Trp residues were found to be most prone to oxidation. In ß-LG-containing protein systems, Cys residues were suggested to scavenge most of the reactive oxidants and undergo an oxidation-mediated disulfide rearrangement. The rearranged disulfide bonds contributed to protein aggregation, which was suggested to provide physical protection against oxidation. Overall, limited loss of amino acid residues was detected after acidic hydrolysis followed by UHPLC analysis, which showed only a minor effect of heat treatment on protein oxidation in these protein systems.

Preparation and characterization of redox-sensitive glutenin nanoparticles.

In recent thirty years, protein-based nanoparticles have attracted considerable attention, and they are being widely used in the food, pharmaceutical, and biomedical fields. Wheat glutenin, an important natural vegetable protein, has been demonstrated to be nutritive and biocompatible. This study aimed to develop a new type of redox-sensitive protein nanoparticles. The glutenin nanoparticles (GNPs) were synthesized with glutenin concentrations (0.082%, 0.5%, and 0.83%) through the adoption of an antisolvent titration technique and the use of hydrogen peroxide (H2O2) oxidative cross-linking for different periods. At a glutenin concentration of 0.83% and oxidation time of 20 h, the obtained GNPs were spherical in shape and approximately 100-300 nm in size, as measured by transmission electron microscopy and dynamic light scattering. The formation of disulfide was confirmed by Raman spectroscopy. The turbidity values of the GNP suspensions were decreased by half after the addition of ß-mercaptoethanol. Nile blue A, a model hydrophilic substance, was entrapped in the GNPs with 77.67% loading efficiency. The newly developed GNPs can be used as redox-responsive carriers for delivering hydrophilic active substances.

Specific disulfide cross-linking to constrict the mobile carrier domain of nonribosomal peptide synthetases.

Nonribosomal peptide synthetases are large, multi-domain enzymes that produce peptide molecules with important biological activity such as antibiotic, antiviral, anti-tumor, siderophore and immunosuppressant action. The adenylation (A) domain catalyzes two reactions in the biosynthetic pathway. In the first reaction, it activates the substrate amino acid by adenylation and in the second reaction it transfers the amino acid onto the phosphopantetheine arm of the adjacent peptide carrier protein (PCP) domain. The conformation of the A domain differs significantly depending on which of these two reactions it is catalyzing. Recently, several structures of A-PCP di-domains have been solved using mechanism-based inhibitors to trap the PCP domain in the A domain active site. Here, we present an alternative strategy to stall the A-PCP di-domain, by engineering a disulfide bond between the native amino acid substrate and the A domain. Size exclusion studies showed a significant shift in apparent size when the mutant A-PCP was provided with cross-linking reagents, and this shift was reversible in the presence of high concentrations of reducing agent. The cross-linked protein crystallized readily in several of the conditions screened and the best crystals diffracted to ≈8 Å.