Chemical Modification Of Proteins Research Articles

Horseradish-Peroxidase-Catalyzed Tyrosine Click Reaction.

The efficiency of protein chemical modification on tyrosine residues with N-methylluminol derivatives was drastically improved by using horseradish peroxidase (HRP). In the previous method, based on the use of hemin and H2 O2 , oxidative side reactions such as cysteine oxidation were problematic for functionalization of proteins selectively on tyrosine residues. Oxidative activation of N-methylluminol derivatives with a minimum amount of H2 O2 prevented the occurrence of oxidative side reactions under HRP-catalyzed conditions. As probes for HRP-catalyzed protein modification, N-methylluminol derivatives showed much higher efficiency than tyramide without inducing oligomerization of probe molecules. Tyrosine modification also proceeded in the presence of β-nicotinamide adenine dinucleotide (NADH, H2 O2 -free conditions).

Expression and Analysis of Flow-regulated Ion Channels in Xenopus Oocytes.

Mechanically-gated ion channels play key roles in mechanotransduction, a process that translates physical forces into biological signals. Epithelial and endothelial cells are exposed to laminar shear stress (LSS), a tangential force exerted by flowing fluids against the wall of vessels and epithelia. The protocol outlined herein has been used to examine the response of ion channels expressed in Xenopus oocytes to LSS (Hoger et al., 2002; Carattino et al., 2004; Shi et al., 2006). The Xenopus oocyte is a reliable system that allows for the expression and chemical modification of ion channels and regulatory proteins (George et al., 1989; Palmer et al., 1990; Sheng et al., 2001; Carattino et al., 2003). Therefore, this technique is suitable for studying the molecular mechanisms that allow flow-activated channels to respond to LSS.

Cover Picture: Design of S‐Allylcysteine in Situ Production and Incorporation Based on a Novel Pyrrolysyl‐tRNA Synthetase Variant (ChemBioChem 1/2017)

The cover picture shows a journey from garlic to genetic code engineering: In this issue, Budisa and co-workers report in vivo-specific protein olefination with S-allylcysteine (Sac), which is naturally abundant in garlic. Alternatively, it can also be biosynthesized in situ by supplementing the amino acid metabolism of E. coli with allylmercaptan. Sac is recognized and activated by the novel and unique pyrrolysyl-tRNA synthetase-based enzyme S-allylcysteinyl-tRNA synthetase (SacRS), which is capable of specifically accepting a small, polar amino acid instead of the long and bulky aliphatic natural substrate. In this way, a biochemical pathway for the production of proteins containing S-allylcysteine as a minimal tag or label for chemical protein modification has been established. More information can be found in the communication by S. Panke, N. Budisa et al. on page 85 in Issue 1, 2017 (DOI: 10.1002/cbic.201600537).

Identification of Protein Thiazolidination as a Novel Molecular Signature for Oxidative Stress and Formaldehyde Exposure.

Chemical modifications of proteins have been well-documented to play important roles in normal cell physiology such as cell signaling and protein functions. They have also been demonstrated to be one of the milestones in the pathophysiology of many human diseases such as cancer, age-related pathology, and neurodegenerative disorders. Here, we report the initial identification of a novel protein modification, cysteine thiazolidination, through reaction with endogenous and exogenous formaldehyde with cysteine residues in proteins. Using an isotope-dilution liquid chromatography-tandem mass spectrometric (LC-MS3) method, we initiated the study by quantitating thioproline in formaldehyde-treated Escherichia coli (E. coli) protein. The study was then extended to quantitate thioproline in protein obtained from formaldehyde- and oxidant-exposed E. coli. Furthermore, N6-formyllysine, a well-defined formylation product between formaldehyde and lysine, was exploited in a comparative study to evaluate the relative reactivity and amount of cysteine thiazolidination in the reaction of formaldehyde with proteins. It is anticipated that cysteine thiazolidination may serve as a novel biomarker for oxidative stress and formaldehyde exposure.

Transient Induced Molecular Electronic Spectroscopy (TIMES) for study of protein-ligand interactions.

We present a method, Transient Induced Molecular Electronic Spectroscopy (TIMES), to detect protein-ligand interactions without any protein engineering or chemical modification. We developed a physics model for the TIMES signal and mathematically formulated the problem to attain physical insight of protein-ligand interactions without any disturbances by molecular probes, fluorescent labels, or immobilization of molecules. To demonstrate the functionality of this method, we have used the TIMES signals to find the dissociation constants for the affinity of reactions, the shear-stress dependent adsorption time of molecules on surface, and other interesting features of protein-ligand interaction in native conditions. As a unique tool, TIMES offers a simple and effective method to investigate fundamental protein chemistry and drug discoveries.

Dairy products and the Maillard reaction: A promising future for extensive food characterization by integrated proteomics studies.

Heating of milk and dairy products is done using various technological processes with the aim of preserving microbiological safety and extending shelf-life. These treatments result in chemical modifications in milk proteins, mainly generated as a result of the Maillard reaction. Recently, different bottom-up proteomic methods have been applied to characterize the nature of these structural changes and the modified amino acids in model protein systems and/or isolated components from thermally-treated milk samples. On the other hand, different gel-based and shotgun proteomic methods have been utilized to assign glycation, oxidation and glycoxidation protein targets in diverse heated milks. These data are essential to rationalize eventual, different nutritional, antimicrobial, cell stimulative and antigenic properties of milk products, because humans ingest large quantities of corresponding thermally modified proteins on a daily basis and these molecules also occur in pharmaceuticals and cosmetics. This review provides an updated picture of the procedures developed for the proteomic characterization of variably-heated milk products, highlighting their limits as result of concomitant factors, such as the multiplicity and the different concentration of the compounds to be detected.

DNA-Based Nanopore Sensing.

Nanopore sensing is an attractive, label-free approach that can measure single molecules. Although initially proposed for rapid and low-cost DNA sequencing, nanopore sensors have been successfully employed in the detection of a wide variety of substrates. Early successes were mostly achieved based on two main strategies by 1) creating sensing elements inside the nanopore through protein mutation and chemical modification or 2) using molecular adapters to enhance analyte recognition. Over the past five years, DNA molecules started to be used as probes for sensing rather than substrates for sequencing. In this Minireview, we highlight the recent research efforts of nanopore sensing based on DNA-mediated characteristic current events. As nanopore sensing is becoming increasingly important in biochemical and biophysical studies, DNA-based sensing may find wider applications in investigating DNA-involving biological processes.

Transition Metal-Free Tryptophan-Selective Bioconjugation of Proteins.

Chemical modifications of native proteins can facilitate production of supernatural protein functions that are not easily accessible by complementary methods relying on genetic manipulations. However, accomplishing precise control over selectivity while maintaining structural integrity and homogeneity still represents a formidable challenge. Herein, we report a transition metal-free method for tryptophan-selective bioconjugation of proteins that is based on an organoradical and operates under ambient conditions. This method exhibits low levels of cross-reactivity and leaves higher-order structures of the protein and various functional groups therein unaffected. The strategy to target less abundant amino acids contributes to the formation of structurally homogeneous conjugates, which may even be suitable for protein crystallography. The absence of toxic metals and biochemically incompatible conditions allows a rapid functional modulation of native proteins such as antibodies and pathogenic aggregative proteins, and this method may thus easily find therapeutic applications.

Epigenetic alterations induced by environmental stress associated with metabolic and neurodevelopmental disorders.

Epigenetics is a gene regulation mechanism that does not depend on genomic DNA sequences but depends on chemical modification of genomic DNA and histone proteins around which DNA is wrapped. The failure of epigenetic mechanisms is known to cause various congenital disorders. It is also known that the failures of epigenetic mechanisms causes various acquired disorders since epigenetic modifications of the genome (i.e., “epigenome”) are more vulnerable to environmental stress, such as malnutrition, environmental chemicals, and mental stress, than the “genome,” especially during the early period of life. However, the epigenome has a reversible property since it is based on removable residues on genomic DNA. Thus, environmentally induced epigenomic alterations can be potentially restored. In fact, some medicines, especially for psychiatric diseases, are known to restore an altered epigenome, resulting in the correction of gene expression. Several lines of evidence suggest that environmentally induced epigenomic alterations are not erased completely during gametogenesis, but are transmitted to subsequent generations with disease phenotypes. In accordance with these understandings, I would like to propose the development of epigenomic-based preemptive medicine that consists of the early detection of the developmental origins of diseases using epigenomic signatures and the early intervention that take advantages of the use of epigenomic reversibility.

Chemical Modifications of Peptides and Proteins with Low Concentration Formaldehyde Studied by Mass Spectrometry

Abstract Formaldehyde has been widely employed to immobilize clinical tissue specimens, inactivate toxins and viruses in biomedical fields. Formaldehyde can react with active groups in bio-molecules such as proteins, resulting in protein cross-linking, inactivation and immobilization. By using several standard peptides and tryptic peptides from the matrix protein of influenza virus as experimental models, we studied the chemical modifications of peptides and proteins with formaldehyde by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and nano-electrospray quadruple time-of-flight tandem mass spectrometry. The reaction between formaldehyde and peptides was performed under the conditions consistent with those during inactivation of virus (4 °C, 0.025% Formalin (37% (w/w) formaldehyde solution), and 72 h). The results indicated that under the above conditions, formaldehyde reacted with amino groups of N-termini of standard peptides to generate methylol adducts, which was further condensed into imines to generate +12 Da products. Furthermore, formaldehyde could react with side chain of two amino acids such as arginine and lysine, yielding +12 Da product each. The analysis of the reaction between formaldehyde and tryptic peptides from matrix protein of influenza virus showed that +24 Da products could be detected in most peptides due to the combinational contribution of N-terminus of peptide (+12 Da) and side chain of C-terminal arginine or lysine (+12 Da). Moreover, a +36 Da product was detected for a peptide with an internal miss-cut site. The results indicated that low-concentration formaldehyde primarily reacted with amino groups on N-termini of peptides and proteins, as well as the side chains of arginine and lysine residues. The present study provided an effective mass spectrometry-based method for analyzing the reaction between low-concentration formaldehyde and peptides and proteins, as well as strategies for interpretation for the mass spectra of reaction products.

Polyanions effectively prevent protein conjugation and activity loss during hydrogel cross-linking

In situ encapsulation is a frequently used method to prepare hydrogels loaded with high quantities of therapeutic proteins. However, many cross-linking reactions, such as Michael-type addition or Diels–Alder (DA) reaction are not tolerant toward nucleophiles; therefore, side-reactions with proteins can occur during cross-linking. This may lead to undesired protein conjugation, activity loss and incomplete protein release. In this study, a number of polyanions, namely alginate, dextran sulfate, hyaluronic acid, heparin, and poly(acrylic acid), were screened for their capability to protect proteins during covalent cross-linking. To this end, lysozyme was incubated with furyl- and maleimide-substituted methoxy poly(ethylene glycol); different pH values were tested. The degree of PEGylation and the residual activity of lysozyme were investigated. Without polyanions, 61.1% of the total lysozyme amount was PEGylated at pH7.4; the residual activity was 20.3% of the initial activity. With the most effective polyanion (dextran sulfate), PEGylation could be completely suppressed; the residual activity was 98.4%. The protective effect of polyanions was attributed to electrostatic interactions with proteins; the “shielding” could be reversed by adding high salt concentrations. Furthermore, the protective effect was dependent on the concentration and molecular mass of the polyanion, but almost independent of the protein concentration. As a proof of concept, hydrogels were loaded with lysozyme and bevacizumab during cross-linking via DA reaction. Without polyanions, a large fraction of the protein was covalently bound to the polymer network resulting in degradation-controlled release; the residual activity of lysozyme was 50.0%. With polyanions, the protein molecules were mobile and their release was diffusion-controlled. The residual activity of lysozyme was 88.9%; the released bevacizumab was structurally intact. Polyanions can, therefore, be used as protective additive to prevent chemical protein modification during hydrogel cross-linking.

In Situ Metabolism of Cinnamyl Alcohol in Reconstructed Human Epidermis: New Insights into the Activation of This Fragrance Skin Sensitizer.

Chemical modification of epidermal proteins by skin sensitizers is the molecular event which initiates the induction of contact allergy. However, not all chemical skin allergens react directly as haptens with epidermal proteins but need either a chemical (prehaptens) or metabolic (prohaptens) activation step to become reactive. Cinnamyl alcohol has been considered a model prohapten, as this skin sensitizer has no intrinsic reactivity. Therefore, the prevailing theory is that cinnamyl alcohol is enzymatically oxidized into the protein-reactive cinnamaldehyde, which is the sensitizing agent. Knowing that reconstructed human epidermis (RHE) models have been demonstrated to be quite similar to the normal human epidermis in terms of metabolic enzymes, use of RHE may be useful to investigate the in situ metabolism/activation of cinnamyl alcohol, particularly when coupled with high-resolution magic angle spinning nuclear magnetic resonance. Incubation of carbon-13 substituted cinnamyl derivatives with RHE did not result in the formation of cinnamaldehyde. The metabolites formed suggest the formation of an epoxy-alcohol and an allylic sulfate as potential electrophiles. These data suggest that cinnamyl alcohol is inducing skin sensitization through a route independent of the one involving cinnamaldehyde and should therefore be considered as a skin sensitizer on its own.

Epigenetics: The origins and evolution of a fashionable topic

The term “epigenetics” was introduced in 1942 by embryologist Conrad Waddington, who, relating it to the 17th century concept of “epigenesis”, defined it as the complex of developmental processes between the genotype and phenotype. While in the years that followed, these processes – in particular gene regulation – were tackled, not in the frame of epigenetics but of genetics, research labelled “epigenetics” rose strongly only in the 21st century. Then it consisted of research on chromatin modifications, i.e. chemical modifications of DNA or histone proteins around DNA that do not change the base sequence. This rise was accompanied by far-reaching claims, such as that epigenetics provides a mechanism for “Lamarckian” inheritance. This article highlights the origin of epigenetics, the major phases of epigenetic research, and the changes in the meaning of the term. It also calls into question some of the far-reaching claims that have accompanied the recent rise of epigenetics.

Histidine-Directed Arylation/Alkenylation of Backbone N-H Bonds Mediated by Copper(II).

Chemical modification of proteins and peptides represents a challenge of reaction design as well as an important biological tool. In contrast to side-chain modification, synthetic methods to alter backbone structure are extremely limited. In this communication, copper-mediated backbone N-alkenylation or N-arylation of peptides and proteins by direct modification of natural sequences is described. Histidine residues direct oxidative coupling of boronic acids at the backbone NH of a neighboring amino acid. The mild reaction conditions in common physiological buffers, at ambient temperature, are compatible with proteins and biological systems. This simple reaction demonstrates the potential for directed reactions in complex systems to allow modification of N-H bonds that directly affect polypeptide structure, stability, and function.

Adding an appropriate amino acid during crosslinking results in more stable crosslinked enzyme aggregates

Carrier free immobilization, especially crosslinked enzyme aggregates (CLEAs), has become an important design for biocatalysis in several areas. Adding amino acids during formation of CLEAs was found to give biocatalysts more stable at 55 °C and in the presence of 60% acetonitrile. The half-lives of CLEAs prepared with and without Arg addition were 21 and 15 h (subtilisin) and 4 and 1.6 h (α-chymotrypsin) at 55 °C, respectively. The corresponding half-lives during acetonitrile presence were 4.1 and 3.0 h (subtilisin) and 39 and 22 min (α-chymotrypsin), respectively. CLEAs made with Arg had higher percentages of alpha helix. CLEAs made by adding Lys, Ala, or Asp also were more stable. In the case of Thermomyces lanuginosus lipase (TLL), CLEA with Ala was even more stable than CLEA with Arg. The addition of a suitable amino acid, thus, enhances CLEA stabilities. The results are discussed in the light of earlier results on chemical modification of proteins and the observation that the Arg/Lys ratio is invariably high in the case of enzymes from thermophiles.

ChemInform Abstract: Chemical Protein Modification Through Cysteine

AbstractReview: 271 refs.

Epigenetic Effect of Environmental Factors on Autism Spectrum Disorders.

Both environmental factors and genetic factors are involved in the pathogenesis of autism spectrum disorders (ASDs). Epigenetics, an essential mechanism for gene regulation based on chemical modifications of DNA and histone proteins, is also involved in congenital ASDs. It was recently demonstrated that environmental factors, such as endocrine disrupting chemicals and mental stress in early life, can change epigenetic status and gene expression, and can cause ASDs. Moreover, environmentally induced epigenetic changes are not erased during gametogenesis and are transmitted to subsequent generations, leading to changes in behavior phenotypes. However, epigenetics has a reversible nature since it is based on the addition or removal of chemical residues, and thus the original epigenetic status may be restored. Indeed, several antidepressants and anticonvulsants used for mental disorders including ASDs restore the epigenetic state and gene expression. Therefore, further epigenetic understanding of ASDs is important for the development of new drugs that take advantages of epigenetic reversibility.

The physics of epigenetics

In higher organisms, all cells share the same genome, but every cell expresses only a limited and specific set of genes that defines the cell type. During cell division, not only the genome, but also the cell type is inherited by the daughter cells. This intriguing phenomenon is achieved by a variety of processes that have been collectively termed epigenetics: the stable and inheritable changes in gene expression patterns. This article reviews the extremely rich and exquisitely multi-scale physical mechanisms that govern the biological processes behind the initiation, spreading and inheritance of epigenetic states. These include not only the changes in the molecular properties associated with the chemical modifications of DNA and histone proteins, such as methylation and acetylation, but also less conventional ones, such as the physics that governs the three-dimensional organization of the genome in cell nuclei. Strikingly, to achieve stability and heritability of epigenetic states, cells take advantage of many different physical principles, such as the universal behavior of polymers and copolymers, the general features of non-equilibrium dynamical systems, and the electrostatic and mechanical properties related to chemical modifications of DNA and histones. By putting the complex biological literature under this new light, the emerging picture is that a limited set of general physical rules play a key role in initiating, shaping and transmitting this crucial "epigenetic landscape". This new perspective not only allows to rationalize the normal cellular functions, but also helps to understand the emergence of pathological states, in which the epigenetic landscape becomes dysfunctional.

Chemical Protein Modification through Cysteine.

The modification of proteins with non-protein entities is important for a wealth of applications, and methods for chemically modifying proteins attract considerable attention. Generally, modification is desired at a single site to maintain homogeneity and to minimise loss of function. Though protein modification can be achieved by targeting some natural amino acid side chains, this often leads to ill-defined and randomly modified proteins. Amongst the natural amino acids, cysteine combines advantageous properties contributing to its suitability for site-selective modification, including a unique nucleophilicity, and a low natural abundance--both allowing chemo- and regioselectivity. Native cysteine residues can be targeted, or Cys can be introduced at a desired site in a protein by means of reliable genetic engineering techniques. This review on chemical protein modification through cysteine should appeal to those interested in modifying proteins for a range of applications.

Bioorthogonal Modification of Proteins Using Genetically Encoded Non-Natural Amino Acids

Chemical modification of proteins with bioorthogonal reaction and site-selectivity has been increasingly recognized for exquisite control over homogeneity and geometry, allowing more complex and various applications of proteins. The development of novel bioorthogonal reactions and their adaptation to protein chemistry in the form of genetically encoded non-natural amino acids are fueling widespread use of the so-called ‘bioorthogonal protein conjugation’. Site-specific incorporation of a non-natural amino acid technique precisely delivers a bioorthogonal group to any defined site in response to an expanded genetic code where a selective bond-forming reaction equips proteins with novel functionalities in a chemically well-defined and homogeneous manner. Keywords: Non-natural amino acid, bioorthogonal reaction, site-specific incorporation, bioconjugation, protein evolution, genetic code.