Methods For Modification Of Proteins Research Articles

Effect of Steam Flash Explosion on Physicochemical and Emulsifying Properties of High-Temperature Rice Bran Meal Protein

High-temperature rice bran meal (HTRBM) is a valuable plant protein resource derived from heat-stabilized rice bran after oil extraction. Steam flash explosion (SFE) is a promising method for protein modification and has been shown to be effective in improving the properties of a variety of proteins. However, the effects of SFE on the Maillard reaction and emulsification of HTRBM remain poorly understood. This research evaluated how different SFE conditions affect the browning degree, physicochemical properties, solubility, emulsifying properties, thermal stability, zeta potential, and particle size of rice bran protein. Samples were labeled as follows: Sample 0 (SFE-untreated HTRBM), Sample 1 (0.8 MPa/140 s), Sample 2 (0.8 MPa/180 s), Sample 3 (1.25 MPa/90 s), Sample 4 (1.25 MPa/180 s), Sample 5 (1.7 MPa/90 s), and Sample 6 (1.7 MPa/180 s). Fourier transform infrared (FTIR) spectroscopy, fluorescence spectroscopy, differential scanning calorimetry (DSC), and other methods were employed to measure the aforementioned properties. The results revealed a substantial elevation in the browning degree of rice bran protein after SFE treatment, with A294 increasing by 82.3% and A420 increasing by 46.3% in Sample 4. The solubility and emulsifying properties exhibited notable enhancements, with an increase of 122.48% in solubility (Sample 5), and 26.51% and 26.58% increment in emulsifying activity index (EAI) and emulsifying stability index (ESI), respectively (Sample 3). FTIR and fluorescence spectra revealed the introduction of sugar groups into rice bran protein molecules, resulting in the Maillard reaction. The observed reduction in particle size and rise in zeta potential of rice bran protein emulsions, along with the reduction in denaturation temperature after SFE treatments, further confirmed the significant enhancements in physicochemical and emulsifying properties of HTRBM, thereby enhancing the utilization value of rice bran protein. This study provides a theoretical basis for the development and utilization of HTRBM and its protein.

Improvement of Surimi Gel Quality Using Protein Cross-Linker, Hydrocolloids and Protease Inhibitor

Surimi and its products have been consumed widely, especially in Japan and Southeast Asia. Market value and consumer acceptability are mainly determined by the textural properties of surimi gel. Textural and gelling properties are governed by the type of fish, lipid content, processing conditions, and presence of endogenous protease and transglutaminase. In addition, several additives such as protein cross-linkers, hydrocolloids, protease inhibitors, etc. have been used in surimi to improve their gelling properties. Cross-linking is the promising method for protein modification to obtain desirable surimi-based gel products possessing unique textural properties. Endogenous transglutaminase (TGase) plays a profound role in protein cross-linking during setting in surimi paste. For surimi from fish with low setting phenomenon, microbial TGase (MTGase) has been widely used. Plant polyphenols have also been employed for protein crosslinking to strengthen the surimi gel. Polysaccharides mainly the hydrocolloids also strengthen the gel network and increase water holding capacity (WHC) of gel. During heating, endogenous heatactivated proteases that are firmly attached to muscle proteins are active in hydrolyzing myofibrillar proteins. This results in a weaker gel with low WHC. Protease inhibitors form plants as well animals could impede the protease activity, thus maintaining the myofibrillar proteins responsible for the gelation. In addition to the additives, processing methods such as highpressure technology or ohmic heating in combination with those additives have a positive impact on the gel properties. For this review, the role of various kinds of additives namely protein crosslinkers, hydrocolloids and protease inhibitors in surimi gel improvement was revisited.

A single backbone amide modification method to achieve single site-specific chemical protein conjugation.

Efficient and precise chemical protein modification methods are highly sought after in biotechnology. However, chemically distinguishing a single site within a large protein is challenging. This study introduces a Copper Assisted Sequence-specific Conjugation Tag (CAST) method, enabling rapid (second order rate 8.1M-1s-1) and site-specific chemical modification of the protein backbone with pinpoint accuracy. The versatility of this method is demonstrated through the preparation of antibody-drug conjugates, showcasing high plasma stability and potent efficacy in both in vitro and in vivo settings. Thus, CAST emerges as an efficient and quantitative approach for attaching payloads to large, native proteins.

Structural and functional properties of sodium caseinate glycosylated by dextran aldehyde

Glycosylation is an effective method of protein modification which can improve the functional properties of proteins. Traditional protein glycosylation methods are typically limited by slow reaction rates. A method for improving the efficiency of protein glycosylation from different degrees of dextran oxidation with periodate, to obtain dextran aldehydes, is presented. Increased molar ratio between periodate and glucose unit in dextran led to dextran aldehyde with higher aldehyde group content and reduced molecular weight. This is due to an increased oxidation, which increases the degree of glycosylation by 43.22%. Compared with dextran glycosylated products, the emulsifying activity, stability indices and foaming capacity of dextran aldehyde glycosylated products increased by 30.8, 301 and 50%, respectively, with poor foam stability. Dextran aldehyde-modified sodium caseinate presented lower digestibility and could maintain better stability against salt ions and heat treatment. The polysaccharide aldehyde showed great potential for expanding protein modification methods to promote its industrialization.

Formation of soybean protein isolate-hawthorn flavonoids non-covalent complexes: Linking the physicochemical properties and emulsifying properties

In recent years, more and more attention had been paid to the combination of proteins and flavonoids, and several flavonoids had been reported to improve the physicochemical and emulsifying properties of proteins. This study investigated the effects of ultrasonic treatment (450 W for 10 min, 20 min, and 30 min) on the physicochemical properties, antioxidant activity, and emulsifying properties of soy protein isolate (SPI) -hawthorn flavonoids (HF) non-covalent complexes. The results showed that the addition of HF to SPI and 20 min of ultrasound could reduce α-helix and random coil, increase β-sheet and β-turn, and enhance fluorescence quenching. In addition, it decreased the particle size, zeta potential, surface hydrophobicity, and turbidity to 88.43 or 95.27 nm, −28.80 mV, 1250.42, and 0.23, respectively. The protein solubility, free sulfhydryl group, antioxidant activity, emulsifying activity index, and emulsifying stability index all increased to 73.93%, 15.07 μmol/g, 71.00 or 41.91%, 9.81 m2/g, and 67.71%, respectively. Moreover, high-density small and low-flocculation droplets were formed. Therefore, the combined ultrasound treatment and addition of HF to SPI is a more effective method for protein modification compared to ultrasound treatment alone. It provides a theoretical basis for protein processing and application in the future.

Recent trends in the application of cold plasma for the modification of plant proteins - A review

Plant proteins are gaining attention in recent times. Apart from the greater sustainability and environment-friendliness offered by plant proteins, improved health consciousness among consumers has boosted the market for plant proteins. With the ever-increasing population, the need for alternative sources of protein is urgent. However, plant proteins possess inferior functional properties such as low solubility, emulsification and film-forming properties, which limit their use in food matrices. Cold plasma can be a sustainable and “green” method for modification of proteins, with minimal use of solvents and chemicals. The improvement in mechanical properties and biodegradability of the plant protein-based films by plasma treatment can be a great sustainable option for the industry. Apart from the mechanism of inactivation of enzymes, the immobilization of enzymes and fabrication of plant protein nanoparticles is also possible by cold plasma. Plasma improved the solubility, hydrophilicity, emulsifying and foaming properties of plant proteins. Prolonged exposure to cold plasma treatments had negative impact on the functional properties. However, plasma was successful in inactivating antinutritional factors and allergens in most proteins. Plasma treatments enhanced the adhesiveness of films, improving the composite formation of plant proteins with other films, with improved barrier properties.

Vypal2: A Versatile Peptide Ligase for Precision Tailoring of Proteins.

The last two decades have seen an increasing demand for new protein-modification methods from the biotech industry and biomedical research communities. Owing to their mild aqueous reaction conditions, enzymatic methods based on the use of peptide ligases are particularly desirable. In this regard, the recently discovered peptidyl Asx-specific ligases (PALs) have emerged as powerful biotechnological tools in recent years. However, as a new class of peptide ligases, their scope and application remain underexplored. Herein, we report the use of a new PAL, VyPAL2, for a diverse range of protein modifications. We successfully showed that VyPAL2 was an efficient biocatalyst for protein labelling, inter-protein ligation, and protein cyclization. The labelled or cyclized protein ligands remained functionally active in binding to their target receptors. We also demonstrated on-cell labelling of protein ligands pre-bound to cellular receptors and cell-surface engineering via modifying a covalently anchored peptide substrate pre-installed on cell-surface glycans. Together, these examples firmly establish Asx-specific ligases, such as VyPAL2, as the biocatalysts of the future for site-specific protein modification, with a myriad of applications in basic research and drug discovery.

Photosensitized Oxidative Dimerization at Tyrosine by a Water-Soluble 4-Amino-1,8-naphthalimide.

The oxidation of proteins generates reactive amino acid (AA) residue intermediates, leading to protein modification and cross-linking. Aerobic studies with peptides and photosensitizers allow for the controlled generation of reactive oxygen species (ROS) and reactive AA residue intermediates, providing mechanistic insights as to how natural protein modifications form. Such studies have inspired the development of abiotic methods for protein modification and crosslinking, including applications of biomedical importance. Dityrosine linkages derived from oxidation at tyrosine (Tyr) residues represent one of the more well-understood oxidation-induced modifications. Here we demonstrate an aerobic, visible light-dependent oxidation reaction of Tyr-containing substrates promoted by a water-soluble 4-amino-1,8-naphthalimide-based photosensitizer. The developed procedure converts Tyr-containing substrates into o,o'-Tyr-Tyr linked dimers. The regioselectively formed o,o'-Tyr-Tyr linkage is consistent with dimeric standards prepared using a known enzymatic method. A crossover study with two peptides provides a statistical mixture of three distinct o,o'-Tyr-Tyr linked dimers, supporting a mechanism that involves Tyr residue oxidation followed by intermolecular combination.

5-Hydroxy-pyrrolone based building blocks as maleimide alternatives for protein bioconjugation and single-site multi-functionalization.

Recent dramatic expansion in potential uses of protein conjugates has fueled the development of a wide range of protein modification methods; however, the desirable single-site multi-functionalization of proteins has remained a particularly intransigent challenge. Herein, we present the application of 5-hydroxy-1,5-dihydro-2H-pyrrol-2-ones (5HP2Os) as advantageous alternatives to widely used maleimides for the chemo- and site-selective labeling of cysteine residues within proteins. A variety of 5HP2O building blocks have been synthesized using a one-pot photooxidation reaction starting from simple and readily accessible furans and using visible light and oxygen. These novel reagents display excellent cysteine selectivity and also yield thiol conjugates with superior stability. 5HP2O building blocks offer a unique opportunity to introduce multiple new functionalities into a protein at a single site and in a single step, thus, significantly enhancing the resultant conjugate's properties.

Unveiling Druggable Pockets by Site-Specific Protein Modification: Beyond Antibody-Drug Conjugates.

Site-specific modification approaches have been extensively employed in the development of protein-based technologies. In this field, stability and activity integrity are the envisioned features of chemically modified proteins. These methods are especially used in the design of antibody-drug conjugates (ADCs). Nevertheless, a biochemical feature of the target protein in these reactions is often overlooked, residue specificity. Usually, in the course of developing chemical probes to modify a protein of interest (POI), specific amino acids are selected due to their reactivity. It is not critical which residue is modified as long as its modification does not compromise the POI's activity. However, no attention is paid as to why certain residues are preferentially modified over others. Physicochemical and structural constraints are often involved in the reactivity of the residue and account for the preferential modification. We propose that site-specific protein modification approaches can be applied beyond the development of ADCs or protein-drug conjugates, and used as a tool to reveal functionally relevant residues. By preferentially modifying certain side chains in the POI, chemical probes can uncover new binding motifs to investigate. Here we describe methods for protein modification, and how some pitfalls in the field can be turned into tools to reveal and exploit druggable pockets. Thus, allowing the design of innovative inhibitors against disease-relevant POIs. We discuss methodologies for site-specific modification of lysine, tryptophan, cysteine, histidine and tyrosine and comment on instances where the modified residues were used as targets for functionalization or drug design.

Effect of simultaneous treatment combining ultrasonication and pH-shifting on SPI in the formation of nanoparticles and encapsulating resveratrol

Plant protein-based nano-delivery systems have rencently drawn increasing attention, and several physical and chemical methods for improving the functional properties of plant proteins have been reported. In this work, we applied ultrasonication and pH-shifting combined treatment on soy protein isolates (SPI) and investigated the structure changes of SPI and its effects on the formation of SPI- sodium alginate (Alg) electrostatic complex and SPI-resveratrol nanocomplex. It was found that the combined ultrasonication/pH-shifting treatment resulted in a smaller particle size and larger surface hydrophobicity, which were 114.6 ± 1.2 nm and 12741 ± 456, respectively. During the formation of the complex with Alg, compared with pH-shifting or ultrasonication treatment, the combined ultrasonication/pH-shifting treatment expanded stable region, where the particle size was smaller and the binding constant with sodium alginate was higher. Moreover, the encapsulation efficiency of resveratrol in SPI increased to 91.4 ± 4.3% after the combined ultrasonication/pH-shifting treatment. Thus, simultaneous treatment combining ultrasonication and pH-shifting was significantly more effective than ultrasonication alone and was thought as an effective method for protein modification.

Chemical methods for modification of proteins.

The library of chemical reactions for C-C and C-heteroatom bond formation is exceptional. The understanding of reactivity and diverse aspects of selectivity facilitates the functional group transformation of high complexity. However, the same is not valid for proteins as an organic substrate. Gratifyingly, we can translate some of the pre-existing reactions for developing methods for the modification of proteins. Also, there is enormous potential to create a new knowledge domain that will be unique to the densely functionalized architecture of proteins. At the outset, we outlined a few concepts that bridge the gap between chemical reactions with small molecules and proteins. Next, we introduced the key attributes and challenges associated with the selectivity that emerges due to the presence of multiple types and copies of functional groups. The examples with nucleophilic amino acids outline the chemoselectivity-associated features. Gradually, the discussion moves toward the concepts that led to the successful realization of site-selectivity and N-terminus residue-specificity. The attributes of organic chemistry that emerge due to the multifunctional organization of the substrate are marked. The last section overviews the analysis of protein bioconjugates by mass spectrometry. Also, the review outlines the unmet needs and opportunities.

Site-Directed Modification of Yeast-Produced Proteins Using Expressed Protein Ligation.

Expressed protein ligation (EPL), using non-self-cleaving inteins, allows for the site-specific addition of customized chemical moieties to the termini of proteins. In this way, protein activity can be preserved while functionalizing the target protein with a wide range of chemical handles. Here, we describe methods for EPL-based modification of proteins produced by yeast, employing an engineered, non-self-cleaving intein known as 202-08. Methods for EPL modification of both yeast surface displayed and secreted proteins with bioorthogonal chemical groups are described. These methods allow for the site-specific modification of intein-fused proteins produced in yeast.

Cysteine-Selective Phosphonamidate Electrophiles for Modular Protein Bioconjugations.

We describe a new technique in protein synthesis that extends the existing repertoire of methods for protein modification: A chemoselective reaction that induces reactivity for a subsequent bioconjugation. An azide-modified building block reacts first with an ethynylphosphonite through a Staudinger-phosphonite reaction (SPhR) to give an ethynylphosphonamidate. The resulting electron-deficient triple bond subsequently undergoes a cysteine-selective reaction with proteins or antibodies. We demonstrate that ethynylphosphonamidates display excellent cysteine-selective reactivity combined with superior stability of the thiol adducts, when compared to classical maleimide linkages. This turns our technique into a versatile and powerful tool for the facile construction of stable functional protein conjugates.

A novel reactive dyeing method for silk fibroin with aromatic primary amine-containing dyes based on the Mannich reaction

Chemical modification of silk fibroin is a useful method for improving its performance. There are many existing methods of protein modification, some of which are limited for application to silk by the disadvantages of severe reaction conditions, poor reaction selectivity and problems with characterization of the product. In this paper, a series of dyes containing aromatic primary amine groups was designed and synthesized for modification of silk fibroin via the Mannich reaction. This three-component Mannich modification showed several advantages, such as mild reaction conditions, good selectivity, easy characterization and high fastness. In particular, the degree of modification could be established visually according to the color yield and the color fixation on silk fibroin. Therefore, the Mannich reaction of silk with dyes containing aromatic primary amine groups may become a novel reactive dyeing method. Studies of structure-performance relationships showed that suitable dyes for dyeing of silk should meet the following criteria: (1) High nucleophilicity of the aromatic primary amine group. (2) No high electron cloud density center around the amino group.

Mechanochemically Induced Controlled Glycation of Lysozyme and Its Effect on Enzymatic Activity and Conformational Changes.

Protein glycation through heating of a mixture of protein and reducing sugars is one of the most commonly used methods of protein modification; however, in most cases, this approach can lead to uncontrolled glycation. The hypothesis that mechanical energy supplied through ball milling can induce glycation of proteins was tested using a well-characterized enzyme lysozyme. The Q-TOF/MS analysis of the milled samples has indicated that the milling of sugar-protein mixtures in stainless steel jars for 30 min and at a frequency of 30 Hz generated mainly monoglycated proteins even with the highly reactive ribose. Increasing the sugar concentration or the milling time did not influence the overall yield or generate more glycoforms. Enzymatic activity measurements, FTIR, and fluorescence spectroscopic studies have indicated that milling of lysozyme alone leads to a significant loss in enzymatic activity and structural integrity in contrast to milling in the presence of sugars.

Recent Progress in Chemical Modification of Proteins.

Chemical modification of proteins is important for creating a myriad of engineered proteins and for elucidating the function and dynamics of proteins in live cells. A wide variety of chemical protein modification methods have been developed and can be categorized into three classes: (i) modification of proteins using the reactivity of naturally occurring amino acids; (ii) modification by bioorthogonal reactions using unnatural amino acids, most of which can be site-selectively incorporated into proteins-of-interest using genetic codon expansion techniques; and (iii) recognition driven chemical modification, which is the only approach that allows modification of endogenous proteins without any genetic manipulation even under heavily crowded and multi-molecular conditions, as in live cells and organisms. All of these approaches have merits and limitations. In this review, we summarize these approaches and discuss their characteristics with respect to specificity, reaction rate and versatility.

Chemo- and Regioselective Lysine Modification on Native Proteins.

Site-selective chemical conjugation of synthetic molecules to proteins expands their functional and therapeutic capacity. Current protein modification methods, based on synthetic and biochemical technologies, can achieve site selectivity, but these techniques often require extensive sequence engineering or are restricted to the N- or C-terminus. Here we show the computer-assisted design of sulfonyl acrylate reagents for the modification of a single lysine residue on native protein sequences. This feature of the designed sulfonyl acrylates, together with the innate and subtle reactivity differences conferred by the unique local microenvironment surrounding each lysine, contribute to the observed regioselectivity of the reaction. Moreover, this site selectivity was predicted computationally, where the lysine with the lowest pKa was the kinetically favored residue at slightly basic pH. Chemoselectivity was also observed as the reagent reacted preferentially at lysine, even in those cases when other nucleophilic residues such as cysteine were present. The reaction is fast and proceeds using a single molar equivalent of the sulfonyl acrylate reagent under biocompatible conditions (37 °C, pH 8.0). This technology was demonstrated by the quantitative and irreversible modification of five different proteins including the clinically used therapeutic antibody Trastuzumab without prior sequence engineering. Importantly, their native secondary structure and functionality is retained after the modification. This regioselective lysine modification method allows for further bioconjugation through aza-Michael addition to the acrylate electrophile that is generated by spontaneous elimination of methanesulfinic acid upon lysine labeling. We showed that a protein–antibody conjugate bearing a site-specifically installed fluorophore at lysine could be used for selective imaging of apoptotic cells and detection of Her2+ cells, respectively. This simple, robust method does not require genetic engineering and may be generally used for accessing diverse, well-defined protein conjugates for basic biology and therapeutic studies.

A Method for SUMO Modification of Proteins in vitro.

The Small Ubiquitin-related Modifier (SUMO) is a protein that is post-translationally added to and reversibly removed from other proteins in eukaryotic cells. SUMO and enzymes of the SUMO pathway are well conserved from yeast to humans and SUMO modification regulates a variety of essential cellular processes including transcription, chromatin remodeling, DNA damage repair, and cell cycle progression. One of the challenges in studying SUMO modification in vivo is the relatively low steady-state level of a SUMO-modified protein due in part to the activity of SUMO deconjugating enzymes known as SUMO Isopeptidases or SENPs. Fortunately, the use of recombinant SUMO enzymes makes it possible to study SUMO modification in vitro. Here, we describe a sensitive method for detecting SUMO modification of target human proteins using an in vitro transcription and translation system derived from rabbit reticulocyte and radiolabeled amino acids.

Genetic Incorporation of a Reactive Isothiocyanate Group into Proteins.

Methods for the site-specific modification of proteins are useful for introducing biological probes into proteins and engineering proteins with novel activities. Herein, we genetically encode a novel noncanonical amino acid (ncAA) that contains an aryl isothiocyanate group which can form stable thiourea crosslinks with amines under mild conditions. We show that this ncAA (pNCSF) allows the selective conjugation of proteins to amine-containing molecular probes through formation of a thiourea bridge. pNCSF was also used to replace a native salt bridge in myoglobin with an intramolecular crosslink to a proximal Lys residue, leading to increased thermal stability. Finally, we show that pNCSF can form stable intermolecular crosslinks between two interacting proteins.