Exposed Thiol Groups Research Articles

Rigid, Highly Reactive and Stable DOTA-like Tags Containing a Thiol-Specific Phenylsulfonyl Pyridine Moiety for Protein Modification and NMR Analysis*.

Site specific installation of a paramagnetic ion with magnetic anisotropy in a biomolecule generates valuable structural restraints, such as pseudocontact shifts (PCSs) and residual dipolar couplings (RDCs). These paramagnetic effects can be used to characterize the structures, interactions and dynamics of biological macromolecules and their complexes. Two single-armed DOTA-like tags, BrPSPy-DO3M(S)A-Ln and BrPSPy-6M-DO3M(S)A-Ln, each containing a thiol-specific reacting group, that is, a phenylsulfonyl pyridine moiety, are demonstrated as rigid, reactive and stable paramagnetic tags for protein modification by formation of a reducing resistant thioether bond between the protein and the tag. The two tags present high reactivity with the solvent exposed thiol group in aqueous solution at room temperature. The introduction of Br at the meta-position in pyridine enhances the reactivity of 4-phenylsulfonyl pyridine towards the solvent exposed thiol group in a protein, whereas the ortho-methyl group in pyridine increases the rigidity of the tag in the protein conjugates. The high performance of these two tags has been demonstrated in different cysteine mutants of ubiquitin and GB1. The high reactivity and rigidity of these two tags can be added in the toolbox of paramagnetic tags suitable for the high-resolution NMR measurements of biological macromolecules and their complexes.

Covalent Immobilisation of a Nanoporous Platinum Film onto a Gold Screen-Printed Electrode for Highly Stable and Selective Non-Enzymatic Glucose Sensing

Progress in the development of commercially available non-enzymatic glucose sensors continues to be problematic due to issues regarding selectivity, reproducibility and stability. Overcoming these issues is a research challenge of significant importance. This study reports a novel fabrication process using a double-layer self-assembly of (3 mercaptopropyl)trimethoxysilane (MPTS) on a gold substrate and co-deposition of a platinum–copper alloy. The subsequent electrochemical dealloying of the less noble copper resulted in a nanoporous platinum structure on the uppermost exposed thiol groups. Amperometric responses at 0.4 V vs. Ag/AgCl found the modification to be highly selective towards glucose in the presence of known interferants. The sensor propagated a rapid response time <5 s and exhibited a wide linear range from 1 mM to 18 mM. Additionally, extremely robust stability was attributed to enhanced attachment due to the strong chemisorption between the gold substrate and the exposed thiol of MPTS. Incorporation of metallic nanomaterials using the self-assembly approach was demonstrated to provide a more reproducible and controlled molecular architecture for sensor fabrication. The successful application of the sensor in real blood serum samples displayed a strong correlation with clinically obtained glucose levels.

Parthenolide Destabilizes Microtubules by Covalently Modifying Tubulin

Detyrosination of the α-tubulin C-terminal tail is a post-translational modification (PTM) of microtubules that is key for many biological processes.1 Although detyrosination is the oldest known microtubule PTM,2-7 the carboxypeptidase responsible for this modification, VASH1/2-SVBP, was identified only 3 years ago,8,9 precluding genetic approaches to prevent detyrosination. Studies examining the cellular functions of detyrosination have therefore relied on a natural product, parthenolide, which is widely believed to block detyrosination of α-tubulin in cells, presumably by inhibiting the activity of the relevant carboxypeptidase(s).10 Parthenolide is a sesquiterpene lactone that forms covalent linkages predominantly with exposed thiol groups; e.g., on cysteine residues.11-13 Using mass spectrometry, we show that parthenolide forms adducts on both cysteine and histidine residues on tubulin itself, invitro and in cells. Parthenolide causes tubulin protein aggregation and prevents the formation of microtubules. In contrast to epoY, an epoxide inhibitor of VASH1/2-SVBP,9 parthenolide does not block VASH1-SVBP activity invitro. Lastly, we show that epoY is an efficacious inhibitor of microtubule detyrosination in cells, providing an alternative chemical means to block detyrosination. Collectively, our work supports the notion that parthenolide is a promiscuous inhibitor of many cellular processes and suggests that its ability to block detyrosination may be an indirect consequence of reducing the polymerization-competent pool of tubulin in cells.

Parthenolide Destabilizes Microtubules by Covalently Modifying Tubulin

Detyrosination of the a-tubulin C-terminal tail is a post-translational modification (PTM) of microtubules that is key for many biological processes. Although detyrosination is the oldest known microtubule PTM, the carboxypeptidase responsible for this modification, VASH1/2-SVBP, was identified only three years ago, precluding genetic approaches to prevent detyrosination. Studies examining the cellular functions of detyrosination have therefore relied on a natural product, parthenolide, that is widely believed to block detyrosination of α-tubulin in cells, presumably by inhibiting the activity of the relevant carboxypeptidase(s). Parthenolide is a sesquiterpene lactone that forms covalent linkages predominantly with exposed thiol groups, e.g., on cysteine residues. Using mass spectrometry, we show that parthenolide forms adducts on both cysteine and histidine residues on tubulin itself, in vitro and in cells. Parthenolide causes tubulin protein aggregation, and prevents the formation of microtubules. In contrast to epoY, an epoxide inhibitor of VASH1/2-SVBP, parthenolide does not block VASH1-SVBP activity in vitro. Lastly, we show that epoY is an efficacious inhibitor of microtubule detyrosination in cells, providing an alternative chemical means to block detyrosination. Collectively, our work supports the notion that parthenolide is a promiscuous inhibitor of many cellular processes, and suggests that its ability to block detyrosination may be an indirect consequence of reducing the polymerization competent pool of tubulin in cells.

The Robust Self-Assembling Tubular Nanostructures Formed by gp053 from Phage vB_EcoM_FV3.

The recombinant phage tail sheath protein, gp053, from Escherichia coli infecting myovirus vB_EcoM_FV3 (FV3) was able to self-assemble into long, ordered and extremely stable tubular structures (polysheaths) in the absence of other viral proteins. TEM observations revealed that those protein nanotubes varied in length (~10–1000 nm). Meanwhile, the width of the polysheaths (~28 nm) corresponded to the width of the contracted tail sheath of phage FV3. The formed protein nanotubes could withstand various extreme treatments including heating up to 100 °C and high concentrations of urea. To determine the shortest variant of gp053 capable of forming protein nanotubes, a set of N- or/and C-truncated as well as poly-His-tagged variants of gp053 were constructed. The TEM analysis of these mutants showed that up to 25 and 100 amino acid residues could be removed from the N and C termini, respectively, without disturbing the process of self-assembly. In addition, two to six copies of the gp053 encoding gene were fused into one open reading frame. All the constructed oligomers of gp053 self-assembled in vitro forming structures of different regularity. By using the modification of cysteines with biotin, the polysheaths were tested for exposed thiol groups. Polysheaths formed by the wild-type gp053 or its mutants possess physicochemical properties, which are very attractive for the construction of self-assembling nanostructures with potential applications in different fields of nanosciences.

Activity of Thioallyl Compounds From Garlic Against Giardia duodenalis Trophozoites and in Experimental Giardiasis.

Fresh aqueous extracts (AGEs) and several thioallyl compounds (TACs) from garlic have an important antimicrobial activity that likely involves their interaction with exposed thiol groups at single aminoacids or target proteins. Since these groups are present in Giardia duodenalis trophozoites, in this work we evaluated the anti-giardial activity of AGE and several garlic's TACs. In vitro susceptibility assays showed that AGE affected trophozoite viability initially by a mechanism impairing cell integrity and oxidoreductase activities while diesterase activities were abrogated at higher AGE concentrations. The giardicidal activities of seven TACs were related to the molecular descriptor HOMO (Highest Occupied Molecular Orbital) energy and with their capacity to modify the –SH groups exposed in giardial proteins. Interestingly, the activity of several cysteine proteases in trophozoite lysates was inhibited by representative TACs as well as the cytopathic effect of the virulence factor giardipain-1. Of these, allicin showed the highest anti-giardial activity, the lower HOMO value, the highest thiol-modifying activity and the greatest inhibition of cysteine proteases. Allicin had a cytolytic mechanism in trophozoites with subsequent impairment of diesterase and oxidoreductase activities in a similar way to AGE. In addition, by electron microscopy a marked destruction of plasma membrane and endomembranes was observed in allicin-treated trophozoites while cytoskeletal elements were not affected. In further flow cytometry analyses pro-apoptotic effects of allicin concomitant to partial cell cycle arrest at G2 phase with the absence of oxidative stress were observed. In experimental infections of gerbils, the intragastric administration of AGE or allicin decreased parasite numbers and eliminated trophozoites in experimentally infected animals, respectively. These data suggest a potential use of TACs from garlic against G. duodenalis and in the treatment of giardiasis along with their additional benefits in the host's health.

Effect of pH on the reaction mechanism of thermal denaturation and aggregation of bovine β-lactoglobulin

New insights in the aggregation mechanisms of the whey protein β-lactoglobulin were gained by detailed investigation of the denaturation behavior (80–120 °C) at variable pH values (5.1, 6.8, 8.0). Denaturation followed a 1.5 order kinetic and reaction velocity was depending on pH and temperature. Characterization of aggregates formed at 80 °C was correlated to the denaturation progress and comprised particle size measurement, quantification of free thiol groups and analysis of quaternary structures as well as stabilizing forces by various types of gel electrophoresis. Due to low electrostatic repulsion at pH 5.1, a fast and steady aggregate growth was observed, where intermolecular disulfide bonds gain more importance with increasing degree of denaturation. Contrary to that, denaturation at pH 6.8 and 8.0 yielded lower reaction rate constants and a steady increase of the amount of exposed thiol groups, resulting in non-native oligomers which were mainly non-covalently connected to small aggregates.

Cadmium Causes Misfolding and Aggregation of Cytosolic Proteins in Yeast.

Cadmium is a highly poisonous metal and is classified as a human carcinogen. While its toxicity is undisputed, the underlying in vivo molecular mechanisms are not fully understood. Here, we demonstrate that cadmium induces aggregation of cytosolic proteins in living Saccharomyces cerevisiae cells. Cadmium primarily targets proteins in the process of synthesis or folding, probably by interacting with exposed thiol groups in not-yet-folded proteins. On the basis of in vitro and in vivo data, we show that cadmium-aggregated proteins form seeds that increase the misfolding of other proteins. Cells that cannot efficiently protect the proteome from cadmium-induced aggregation or clear the cytosol of protein aggregates are sensitized to cadmium. Thus, protein aggregation may contribute to cadmium toxicity. This is the first report on how cadmium causes misfolding and aggregation of cytosolic proteins in vivo The proposed mechanism might explain not only the molecular basis of the toxic effects of cadmium but also the suggested role of this poisonous metal in the pathogenesis of certain protein-folding disorders.

Tumor Microenvironment-Triggered Aggregation of Antiphagocytosis 99m Tc-Labeled Fe3 O4 Nanoprobes for Enhanced Tumor Imaging In Vivo.

A tumor microenvironment responsive nanoprobe is developed for enhanced tumor imaging through in situ crosslinking of the Fe3 O4 nanoparticles modified with a responsive peptide sequence in which a tumor-specific Arg-Gly-Asp peptide for tumor targeting and a self-peptide as a "mark of self" are linked through a disulfide bond. Positioning the self-peptide at the outmost layer is aimed at delaying the clearance of the nanoparticles from the bloodstream. After the self-peptide is cleaved by glutathione within tumor microenvironment, the exposed thiol groups react with the remaining maleimide moieties from adjacent particles to crosslink the particles in situ. Both in vitro and in vivo experiments demonstrate that the aggregation substantially improves the magnetic resonance imaging (MRI) contrast enhancement performance of Fe3 O4 particles. By labeling the responsive particle probe with 99m Tc, single-photon emission computed tomography is enabled not only for verifying the enhanced imaging capacity of the crosslinked Fe3 O4 particles, but also for achieving sensitive dual modality imaging of tumors in vivo. The novelty of the current probe lies in the combination of tumor microenvironment-triggered aggregation of Fe3 O4 nanoparticles for boosting the T2 MRI effect, with antiphagocytosis surface coating, active targeting, and dual-modality imaging, which is never reported before.

Regulation of protein-binding activities of molecularly imprinted polymers via post-imprinting modifications to exchange functional groups within the imprinted cavity.

We prepared lysozyme-imprinted polymers bearing modifiable sites within the imprinted cavity to introduce various functional groups via post-imprinting modifications. For this purpose, ({[2-(2-methacrylamido)-ethyldithio]-ethylcarbamoyl}-methoxy)acetic acid (MDTA), which has a carboxy group to interact with the target protein, lysozyme, and a disulfide linkage for post-imprinting modifications, was used as a functional monomer. A lysozyme-imprinted polymer film was prepared by copolymerization of MDTA with a cross-linker, N,N'-methylenebisacrylamide, in the presence of lysozyme. After removing lysozyme, followed by reducing the disulfide linkage, various functional groups, such as carboxy, amino, sulfonate, and oligo-ethylene oxide, were introduced to the exposed thiol groups via a disulfide exchange reaction using the pyridyldisulfide derivatives of these functional groups. Various functional groups could be introduced reversibly via this post-imprinting disulfide exchange reaction after the construction of the lysozyme-imprinted cavities. The modification regulated the protein-binding activity. The proposed post-imprinting modification system, based on a molecular imprinting process, is expected to lead to the development of advanced materials for fine-tuning and/or introducing desired functions.

One-step post-imprint modification achieve dual-function of glycoprotein fluorescent sensor by "Click Chemistry"

A novel molecular imprinting fluorescent sensor synthesized avoiding complicated design and synthesis of a fluorescent functional monomer, and without the extra cost of additional fluorescence materials (quantum dots, carbon dots and fluorescein), only by a click reaction making the common protein imprinting polymers upgraded to a fluorescent sensing material which can be applied to the fast detection of glycoproteins. In this study, a common protein imprinted polymer (protein-IP) which containing a disulfide bond in the imprinted cavities was first synthesized, then the disulfide linkage was cleaved by reduction, the exposed thiol groups were used for the click reaction to introduce 4-vinyl phenylboronic acid to improve the imprinted recognition for glycoproteins and convert recognition behavior into fluorescent signal simultaneously in one-step site-specific post-imprinting modifications (PIMs). Good linear relationship was obtained in the range of 0.1–10mgmL−1 by fluorescence assay, comparison of the adsorption capacity of the MIPs before and after modification proved that the method has no negative impact on the imprinting effect and exhibit good imprinting effect for glycoproteins of different molecular size and weight.

Molecularly imprinted protein recognition cavities bearing exchangeable binding sites for postimprinting site-directed introduction of reporter molecules for readout of binding events.

Protein-imprinted cavities bearing exchangeable domains to be used for postimprinting fluorophore introduction to transform binding events into fluorescence changes were constructed in molecularly imprinted polymer (MIPs) matrixes prepared on glass substrates. Copolymerization was performed with acrylamide, N,N'-methylenebisaclylamide, and a newly designed functional group-exchangeable monomer, ({[2-(2-methacrylamido)ethyldithio]ethylcarbamoyl}methoxy)acetic acid (MDTA), in the presence of a model basic protein, lysozyme (Lyso); MDTA can interact with Lyso and assemble close to Lyso in the resulting polymer. After removal of Lyso, followed by a disulfide reduction to cleave the (ethylcarbamoylmethoxy)acetic acid moiety from the MDTA residues, the exposed thiol groups within the imprinted cavities were modified by aminoethylpyridyldisulfide to be transformed into aminoethyl groups that function as active sites for amine-reactive fluorophores. Fluorescein isothiocyanate (FITC) was then coupled with the aminoethyl groups, yielding site specifically FITC-modified signaling imprinted cavities for Lyso binding. Because the in-cavity fluorescent labeling was achieved via a disulfide linkage, it was easy to remove, exchange, and/or replace amine-reactive fluorophores. This facilitated the screening of fluorophores to select the highest readout for binding events, replace fluorophores when photobleaching occurred, and introduce other functions. The proposed molecular imprinting process, combined with postimprinting modifications, is expected to provide an affordable route to develop multifunctional MIPs for specific detection of protein binding events.

Abstract 321: Obesity Causes Enhanced Sensitivity Of The Mitochondrial Permeability Transition Pore

Several mechanisms have been implicated in heart failure (HF) development due to obesity, including altered Ca2+ homeostasis and mitochondrial increased reactive oxygen species (ROS). Besides their metabolic role, mitochondria are important cell death regulators, since their disruption induces apoptosis. The mitochondrial permeability transition pore (MPTP) formation is key in this process. Ca2+ and ROS are known inducers of MPTP, and mitochondria are the main ROS generators. However, it has not been demonstrated that MPTP formation is involved in cardiac cell death due to obesity. Therefore, the aim of this work was to determine whether Ca2+ alterations and/or MPTP opening underlie cardiac dysfunction. We used obese Zucker fa/fa rats (32 weeks old), displaying concentric hypertrophy and cardiac dysfunction. We measured: i) Systolic and diastolic Ca2+ signaling in isolated myocytes, in basal conditions and upon β-adrenergic stimulation (β-AS), and ii) in vitro mitochondrial function: respiration, ROS production and MPTP opening. We found that the main alteration in Ca2+ signaling in fa/fa myocytes was a decrease in SERCA Ca2+ removal capacity, since Ca2+ transient amplitude and spark frequency were unchanged. Furthermore, in fa/fa myocytes, β-AS response was preserved. On the other hand, fa/fa mitochondria respiration, in state 3 decreased, but was unchanged in state 4, when glutamate/malate were used as substrate, resulting in an small decrease in respiratory control. In addition, fa/fa mitochondria were more sensitive to MPTP opening, induced by Ca2+ and carboxyatractiloside (CAT). Moreover, fa/fa mitochondria showed increased H2O2 production, and in exposed thiol groups in the adenine nucleotide translocase, a regulatory MPTP component. Since Ca2+ signaling is relatively normal in fa/fa cells, it does not seem to be the main contributor to the cardiac contractile dysfunction. However, given that fa/fa mitochondria showed decrease respiratory performance, were more susceptible to MPTP opening, and showed enhanced H2O2 production. We conclude that fa/fa mitochondria were more vulnerable to enhanced oxidative stress, causing MPTP opening, which could be exacerbated by SERCA slower Ca2+ removal capacity, leading to myocyte apoptosis.

The Effect of Multilayer Gold Nanoparticles on the Electrochemical Response of Ammonium Ion Biosensor Based on Alanine Dehydrogenase Enzyme

The use of multilayer of gold nanoparticles (AuNPs) attached on gold electrode surface via thiol chemistry to fabricate an ammonium (NH4+) ion biosensor based on alanine dehydrogenase (AlaDH) was investigated. The approach of the study was based on construction of biosensor by direct deposition of AuNPs and 1,8-octanedithiol (C8-DT) onto the gold electrode surface. For the immobilisation of enzyme, 2-mercaptoethanol (2BME) was first covalently attached to AlaDH via esther bonding and then followed by chemically attached the 2BME-modified AlaDH (2BME-AlaDH) moiety onto the AuNPs electrode via the exposed thiol group of 2BME. The resulting biosensor response was examined by means of amperometry for the quantification ofNH4+ion. In the absence of enzyme attachment, the use of three layers of AuNPs was found to improve the electrochemistry of the gold electrode when compared with no AuNPs was coated. However, when more than three layers of AuNPs were coated, the electrode response deteriorated due to excessive deposition of C8-DT. When AlaDH was incoporated into the AuNPs modified electrode, a linear response toNH4+ion over the concentration range of 0.1–0.5 mM with a detection limit of 0.01 mM was obtained. In the absence of AuNPs, theNH4+ion biosensor did not exhibit any good linear response range although the current response was observed to be higher. This work demonstrated that the incorporation of AuNPs could lead to the detection of higherNH4+ion concentration without the need of dilution for highNH4+ion concentration samples with a rapid response time of <1 min.

Sunlight mediated disruption of peptide-based soft structures decorated with gold nanoparticles

This communication reports morphological studies of a novel C(3)-symmetric thiolated, tren-based ditryptophan conjugate. The exposed thiol groups on the soft structures interacted with gold nanoparticles and mediated the release of encapsulated fluorescent dye, when exposed to natural sunlight.

Complexation of bovine β-lactoglobulin with 11S protein fractions of soybean (Glycine max) and sesame (Sesamum indicum)

β-Lactoglobulin (β-Lg) comprises 50% of the whey component of bovine milk. Protein–protein interactions between bovine β-Lg and 11S protein fractions of soybean and sesame were investigated by turbidity, solubility behaviour and by evaluation of functional properties in the mixed systems. In this work, the aggregation behaviour of soybean and the whey protein (β-Lg) showed the formation of soluble complexes. Turbidity and solubility studies showed that the proteins interacted at temperatures between 60 and 90±5°C. Heating a mixture of β-Lg and 11S proteins of soybean at higher temperatures formed soluble complexes with β-Lg. It also reduced the self aggregation behaviour, especially that of 11S protein fraction of soybean. This reduced the precipitation of soybean proteins at higher temperature. The complex formed was resolved by gel filtration using high-performance liquid chromatography. Upon heating β-Lg at neutral pH, native dimer starts to dissociate into monomers leading to the exposure of previously buried hydrophobic amino acids and the free thiol group. The soluble complex is formed by the exposed thiol groups. But interaction of β-Lg with sesame 11S protein fractions did not form any soluble complexes. The mechanism of interaction indicates that hydrophobic interactions were preferred over disulfide linkages at the high salt concentrations of the buffer used. During thermal treatment the molecules are unfolded, leading to an exposure of the hydrophobic groups that further enhance the protein–protein interactions that are entropically driven hydrophobic interactions.

Alternative Modes of Binding of Recombinant Human Histone Deacetylase 8 to Colloidal Gold Nanoparticles

Histone deacetylases are intimately involved in the transcriptional regulation of genes, and they are high priority drug targets for cancer therapy. Due to prevalence of several sulfhydryl groups on the surface of histone deacetylase 8, we explored the possibility of its binding to colloidal gold nanoparticles by determining its potentials to inhibit the flocculation as well as retaining the enzyme activity. It was observed that although both these processes conformed to the binding affinity of the gold-histone deacetylase 8 conjugate as being equal to 15-20 nM, only 30% of the nanoparticle-bound enzyme exhibited the enzymatic activity. In the light of the structural features of histone deacetylase 8, we propose that the enzyme interacts with the gold nanoparticles via the surface exposed thiol groups, and such interaction occurs in two alternative modes. Whereas the enzyme bound via mode-1 is catalytically inactive (presumably due to the orientation of the enzyme's active site toward the gold nanoparticle surface), and it prevents the flocculation of the nanoparticles, the enzyme bound via mode-2 shows the full catalytic activity (as its active site is believed to be oriented away from the nanoparticle surface). Although the histone deacetylase 8 bound to AuNP via mode-2 exhibits the same inhibitory potency against Trichostatin A as the free enzyme, the former is more susceptible to thermal denaturation. The potential of potent interaction between gold nanoparticles and histone deacetylase 8 via alternative modes may find diagnostic and/or therapeutic applications for different forms of cancers.

Methanethiolation of the Liberated Cysteine Residues of Human α2-Macroglobulin Treated with Methylamine Generates a Derivative with Similar Functional Characteristics as Native α2-Macroglobulin

The thiol-modifying reagent methyl methanethiosulfonate reacts with the cysteine residues of thiol esters released upon treatment of human α2-macroglobulin with methylamine. This methanethiolation generates a derivative of α2-macroglobulin, with an ‘open trap’ and slow mobility in non-denaturing PAGE, similar to native α2-macroglobulin. This similarity is further substantiated by surface hydrophobicity determinations and by the fact that neither the derivative nor native α2-macroglobulin are cleared from the circulation in mice. Cleavages of bait regions in the derivative and native α2-macroglobulin, however, result in electrophoretically fast forms which are cleared from the circulation in mice. In contrast to native α2-macroglobulin, which can bind 2 mol chymotrypsin/mol, α2-macroglobulin treated with methylamine and methylmethanethiosulfonate binds only 0.8 mol chymotrypsin/mol. Protection of trypsin against inhibition by soybean trypsin inhibitor is significantly better when α2-macroglobulin is modified by methylamine and methylmethanethiosulfonate than when it is modified by dinitrophenyl thiocyanate, which cyanylates the exposed thiol group. The methanethiolated derivative is also more stable than the corresponding cyanylated derivative in that it is transformed to an electrophoretically fast form with a half-life of 9 h as compared to a half-life of 7 h for the latter. The transformation to the fast form is not due to instability of the thiol modification.

Molecular Level Protein Composition of Flour Mill Streams from a Pilot‐Scale Flour Mill and Its Relationship to Product Quality

ABSTRACTFlour mill streams prepared from two Australian and two New Zealand wheat cultivars using a pilot‐scale roller mill were analyzed for rheological and baking quality characteristics and for protein composition using size‐exclusion HPLC. Differences in mill stream protein composition, on an industrially relevant scale, and the relationships between the distribution of proteins (and their degree of thiol exposure) and the technological quality of the flour mill streams were examined. Consistent, significant differences were observed in the physicochemical and processing characteristics of the flour streams. Between mill streams, changes in the quantities of the storage protein groups were more marked than for nonstorage protein groups. Changes in protein composition differed between the break and reduction stream flours. In contrast, the degree of exposure of thiol groups on the various protein groups followed different patterns between mill streams. Numerous significant relationships were observed between dough mixing and product baking tests and the composition and thiol exposure state of the various protein classes. These relationships are discussed in context of manipulating the processing quality of flour‐based products using mill streaming. A possible role for exposed thiol groups on storage proteins in the phenomenon of flour “aging” is suggested.

Fluorescence resonance energy transfer induced by conjugation of metalloproteins to nanoparticles

We show the possibility of realizing a hybrid system composed of a semiconductor nanoparticle (NP) and a metalloprotein, in which the photophysical properties of the two species can be exploited to elicit fluorescence resonance energy transfer mechanisms from the biomolecule to the NP. A specific conjugation process between CdSe/ZnS core/shell water soluble NPs, functionalized with surface exposed thiol groups (–SH), and the apo form of the metalloprotein azurin (Az) has been achieved, resulting in a fixed distance in the donor-acceptor pairs. The increase in the NP fluorescence intensity was found to be dependent on the Az to NP molar ratio.