Chemical Modification Of Proteins Research Articles

From Protein Structures to Functional Biomimetics

AbstractThe development of complex molecular scaffolds with defined folding properties represents a central challenge in chemical research. Proteins are natural scaffolds defined by a hierarchy of structural complexity and have evolved to manifest unique functional characteristics; for example, molecular recognition capabilities that facilitate the binding of target molecules with high affinity and selectivity. Utilizing these features, proteins have been used as a starting point for the design of synthetic foldamers and enhanced biocatalysts, as well as bioactive reagents in drug discovery. In this account, we describe the strategies used in our group to stabilize protein folds, ranging from the constraint of bioactive peptide conformations to chemical protein engineering. We discuss the evolution of peptides into peptidomimetics to inhibit protein–protein and protein–nucleic acid interactions, and the selective chemical modification of proteins to enhance their properties for biotechnological applications. The reported peptide- and proteomimetic structures cover a broad range of molecular sizes and they highlight the importance of structure stabilization for the design of functional biomimetics.1 Introduction2 Constraining the Conformation of Peptides3 Peptide-Based Covalent Protein Modifiers4 Chemical Protein Engineering5 Conclusions

Study of Monoclonal Antibody Aggregation at the Air-Liquid Interface under Flow by ATR-FTIR Spectroscopic Imaging.

Throughout bioprocessing, transportation, and storage, therapeutic monoclonal antibodies (mAbs) experience stress conditions that may cause protein unfolding and/or chemical modifications. Such structural changes may lead to the formation of aggregates, which reduce mAb potency and may cause harmful immunogenic responses in patients. Therefore, aggregates need to be detected and removed or ideally prevented from forming. Air-liquid interfaces, which arise during various stages of bioprocessing, are one of the stress factors causing mAb aggregation. In this study, the behavior of an immunoglobulin G (IgG) at the air-liquid interface was investigated under flow using macro attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopic imaging. This chemically specific imaging technique allows observation of adsorption of IgG to the air-liquid interface and detection of associated secondary structural changes. Chemical images revealed that IgG rapidly accumulated around an injected air bubble under flow at 45 °C; however, no such increase was observed at 25 °C. Analysis of the second derivative spectra of IgG at the air-liquid interface revealed changes in the protein secondary structure associated with increased intermolecular β-sheet content, indicative of aggregated IgG. The addition of 0.01% w/v polysorbate 80 (PS80) reduced the amount of IgG at the air-liquid interface in a static setup at 30 °C; however, this protective effect was lost at 45 °C. These results suggest that the presence of air-liquid interfaces under flow may be detrimental to mAb stability at elevated temperatures and demonstrate the power of ATR-FTIR spectroscopic imaging for studying the structural integrity of mAbs under bioprocessing conditions.

Probing effects of site-specific aspartic acid isomerization on structure and stability of GB1 through chemical protein synthesis.

Chemical modifications of long-lived proteins, such as isomerization and epimerization, have been evoked as prime triggers for protein-damage related diseases. Deamidation of Asn residues, which results in formation of a mixture of l- and d-Asp and isoAsp via an intermediate aspartyl succinimide, can result in the disruption of cellular proteostasis and toxic protein depositions. In contrast to extensive data on the biological prevalence and functional implications of aspartyl succinimide formation, much less is known about the impact of the resulting altered backbone composition on properties of individual proteins at a molecular level. Here, we report the total chemical synthesis, biophysical characterization, and NMR structural analysis of a series of variants of the B1 domain of protein G from Streptococcal bacteria (GB1) in which all possible Asp isomers as well as an aspartyl succinimide were individually incorporated at a defined position in a solvent-exposed loop. Subtle local structural effects were observed; however, these were accompanied by notable differences in thermodynamic folded stability. Surprisingly, the noncanonical backbone connectivity of d-isoAsp led to a variant that exhibited enhanced stability relative to the natural protein.

Photocatalytic Structures for Protein Modifications

AbstractThe chemical modification of proteins serves as a fundamental tool for understanding biological processes and enables the design of biofunctional materials. Among the available methodologies, photochemical strategies have garnered significant attention because of their remarkable biocompatibility and precise spatiotemporal reaction control. Developing novel reactions tailored to specific applications necessitates a comprehensive understanding of photoreactive properties, including catalyst structures, appropriate modifiers, and reaction conditions. This review discusses chemical modifications of proteins using an array of catalysts, including photoredox catalysts for single‐electron transfer (SET), catalysts for energy transfer, long‐wavelength excitable photocatalysts, genetically encoded photocatalysts, and artificial metalloenzymes. The discussion covers the unique attributes, mechanisms, practical applications, and future prospects of each catalyst‐driven reaction, shedding light on the evolving landscape of protein chemical modifications.

Chemical modification of 5-hydroxytryptophan photoinduced by endogenous sensitizers present in skin

Damage caused to biological targets through sunlight photosensitized reaction is of paramount importance due to their potential effects on human health. In these processes, a chemical alteration of a biomolecule may occur as a result of the initial radiation absorption by another chemical species called photosensitizer. In this respect, pterins are endogenous photosensitizers able of inducing chemical modifications in DNA, proteins and lipids. These molecules are present in many living organisms and play different biological functions. In humans, aromatic pterins (Pt) accumulate in the depigmentation patches on the skin of patients suffering vitiligo. Interestingly, 5-hydroxytryptophan (5-OH-Trp), a common oxidation product of tryptophan acting as a potential endogenous antioxidant, is also present in the skin under oxidative stress conditions, such as those produced by vitiligo. However, the photochemical interaction between Pt and 5-OH-Trp has not been considered yet. With this background, the goal of the present work is to deepen the knowledge of the capability of Pt to photoinduce damage to 5-OH-Trp. By combining different analytical and spectroscopic techniques, we establish that 5-OH-Trp is damaged by Pt through a photosensitized type I process initiated by an electron transfer from the 5-OH-Trp to the Pt triplet excited state that yield the corresponding radical ions. In air-equilibrated aqueous solution four products were identified, two of which correspond to 5-OH-Trp dimers, while the others are a 5-OH-Trp trimer and a dione, in which the 5-OH-Trp has incorporated an oxygen atom. No consumption of Pt was observed in the presence of O2. However, in the absence of O2, further free-radical reactions lead to the reduction of the photosensitizer, and dimers and trimer were the only 5-OH-Trp-derived products detected. The biomedical implications of the generation of this kind of products, in proteins, are discussed.

Mechanochemical responses in β-lactoglobulin-based hydrogels: protein unfolding and chemical modification through compression

Abstract In this study, we investigated mechanical protein unfolding in hydrogels composed of cross-linked β-lactoglobulin. The hydrogels were prepared by the reaction of β-lactoglobulin with polyethylene glycol tethering activated esters at both ends. Compressing the hydrogels enables the modification of β-lactoglobulin in the gel with fluorophores such as a pyridyl disulfide-containing fluorescent protein and a tetraphenylethene derivative. This finding indicates that the unfolding triggered by hydrogel compression induces exposure of a reactive cysteine residue in the protein.

Research progress on chemical modifications of tyrosine residues in peptides and proteins.

The chemical modifications (CMs) of protein is an important technique in chemical biology, protein-based therapy, and material science. In recent years, there has been rapid advances in the development of CMs of peptides and proteins, providing new approaches for peptide and protein functionalization, as well as drug discovery. In this review, we highlight the methods for chemically modifying tyrosine (Tyr) residues in different regions, offering a comprehensive exposition of the research content related to Tyr modification. This review summarizes and provides an outlook on Tyr residue modification, aiming to offer readers assistance in the site-selective modification of macromolecules and to facilitate application research in this field.

Chemical Modification of Silk Proteins via Palladium‐Mediated Suzuki−Miyaura Reactions

AbstractSuzuki−Miyaura cross‐coupling reactions are used to modify the tyrosine residues on Bombyx mori silkworm silk proteins using a water‐soluble palladium catalyst. First, model reactions using tyrosine derivatives are screened to determine optimal reaction conditions. For these reactions, a variety of aryl boronic acids, solvents, buffers, and temperature ranges are explored. Qualitative information on the reaction progress is collected via high‐performance liquid chromatography (HPLC), mass spectrometry (MS), and nuclear magnetic resonance (NMR). Optimized reactions are then applied to silk proteins. It is demonstrated the ability to modify silk fibroin in solution by first iodinating the tyrosine residues on the protein, and then carrying out Suzuki‐Miyaura reactions with a variety of boronic acid derivatives. Modification of silk is confirmed with NMR, ion‐exchange chromatography (IEC), UV‐vis, and infrared spectroscopy (IR).

Refining S-acylation: Structure, regulation, dynamics, and therapeutic implications.

With a limited number of genes, cells achieve remarkable diversity. This is to a large extent achieved by chemical posttranslational modifications of proteins. Amongst these are the lipid modifications that have the unique ability to confer hydrophobicity. The last decade has revealed that lipid modifications of proteins are extremely frequent and affect a great variety of cellular pathways and physiological processes. This is particularly true for S-acylation, the only reversible lipid modification. The enzymes involved in S-acylation and deacylation are only starting to be understood, and the list of proteins that undergo this modification is ever-increasing. We will describe the state of knowledge on the enzymes that regulate S-acylation, from their structure to their regulation, how S-acylation influences target proteins, and finally will offer a perspective on how alterations in the balance between S-acylation and deacylation may contribute to disease.

Evaluation of laboratory and environmental exposure systems for protein modification upon gas pollutants and environmental factors

Chemical modifications of proteins induced by ambient ozone (O3) and nitrogen oxides (NOx) are of public health concerns due to their potential to trigger respiratory diseases. The laboratory and environmental exposure systems have been widely used to investigate their relevant mechanism in the atmosphere. Using bovine serum albumin (BSA) as a model protein, we evaluated the two systems and aimed to reduce the uncertainties of both the reactants and products in the corresponding kinetic study. In the laboratory simulation system, the generated gaseous pollutants showed negligible losses. Ten layers of BSA were coated on the flow tube with protein extraction recovery of 87.4%. For environmental exposure experiment, quartz fiber filter was selected as the upper filter with low gaseous O3 (8.0%) and NO2 (1.7%) losses, and cellulose acetate filter was appropriate for the lower filter with protein extraction efficiency of 95.2%. The protein degradation process was observed without the exposure to atmospheric oxidants and contributed to the loss of protein monomer mass fractions, while environmental factors (e.g., molecular oxygen and ultraviolet) may cause greater protein monomer losses. Based on the evaluation, the study exemplarily applied the two systems to protein modification and both showed that O3 promotes the protein oligomerization and nitration, while increased temperature can accelerate the oligomerization and increased relative humidity can inhibit the nitration in the environmental exposure samples. The developed laboratory and environmental systems are suitable for studying protein modifications formed under different atmospheric conditions. A combination of the two will further reveal the actual mechanism of protein modifications.

Multiphase Kinetic Modeling of Air Pollutant Effects on Protein Modification and Nitrotyrosine Formation in Epithelial Lining Fluid.

Exposure to ambient air pollution is a major risk factor for human health. Inhalation of air pollutants can enhance the formation of reactive species in the epithelial lining fluid (ELF) of the respiratory tract and can lead to oxidative stress and oxidative damage. Here, we investigate the chemical modification of proteins by reactive species from air pollution and endogenous biological sources using an extended version of the multiphase chemical kinetic model KM-SUB-ELF 2.0 with a detailed mechanism of protein modification. Fine particulate matter (PM2.5) and nitrogen dioxide (•NO2) act synergistically and increase the formation of nitrotyrosine (Ntyr), a common biomarker of oxidative stress. Ozone (O3) is found to be a burden on the antioxidant defense system but without substantial influence on the Ntyr concentration. In simulations with low levels of air pollution, the Ntyr concentration in the ELF is consistent with the range of literature values for bronchoalveolar lavage fluid from healthy individuals. With high levels of air pollution, however, we obtain strongly elevated Ntyr concentrations. Our model analysis shows how chemical reactions of air pollutants can modify proteins and thus their functionality in the human body, elucidating a molecular pathway that may explain air pollutant effects on human health.

Simultaneous RNA and DNA Adductomics Using Single Data-Independent Acquisition Mass Spectrometry Analysis.

Adductomics studies are used for the detection and characterization of various chemical modifications (adducts) of nucleic acids and proteins. The advancements in liquid chromatography coupled with high-resolution tandem mass spectrometry (HRMS/MS) have resulted in efficient methods for qualitative and quantitative adductomics. We developed an HRMS-based method for the simultaneous analysis of RNA and DNA adducts in a single run and demonstrated its application using Baltic amphipods, useful sentinels of environmental disturbances, as test organisms. The novelty of this method is screening for RNA and DNA adducts by a single injection on an Orbitrap HRMS instrument using full scan and data-independent acquisition. The MS raw files were processed with an open-source program, nLossFinder, to identify and distinguish RNA and DNA adducts based on the characteristic neutral loss of ribonucleosides and 2'-deoxyribonucleosides, respectively. In the amphipods, in addition to the nearly 150 putative DNA adducts characterized earlier, we detected 60 putative RNA adducts. For the structural identification of the detected RNA adducts, the MODOMICS database was used. The identified RNA adducts included simple mono- and dimethylation and other larger functional groups on different ribonucleosides and deaminated product inosine. However, 54 of these RNA adducts are not yet structurally identified, and further work on their characterization may uncover new layers of information related to the transcriptome and help understand their biological significance. Considering the susceptibility of nucleic acids to environmental factors, including pollutants, the developed multi-adductomics methodology with further advancement has the potential to provide biomarkers for diagnostics of pollution effects in biota.

Protein Modification via Nitrile Oxide-Dehydroalanine Cycloaddition: Formation of Isoxazoline Ring on the Protein Backbone.

Here we describe a novel catalyst-free 1,3-dipolar cycloaddition bioconjugation approach for chemical modification of proteins. The dehydroalanine (Dha)-containing protein reacts with nitrile oxides generated in situ through 1,3-dipolar cycloaddition in fully aqueous-buffered systems. This leads to the formation of a new isoxazoline ring at a pre-defined site (Dha) of the protein. Furthermore, the 1-pyrene isoxazoline-installed annexin V acts as a fluorescent probe, which successfully labels the outer cellular membranes of human cholangiocarcinoma (HuCCA-1) cells for detection of apoptosis.

Chemical Modification of Peptides and Proteins Using Spirooxindole Oxirane Derivatives

AbstractMethods for chemical modification of proteins and peptides at certain positions are required for the preparation of antibody‐drug conjugates, protein‐ or peptide‐biotin conjugates, and conjugates used as therapeutics or as tools in biomedical research. We have developed chemical modification methods that use spirooxindole oxirane derivatives for the synthesis of protein‐ and peptide‐conjugates. In most cases, the epoxide group of the spirooxindole oxirane derivatives reacted to form a covalent bond with certain histidine residues and/or the N‐terminal amino group of peptides and proteins. The modification reactions of various proteins resulted in the formation of proteins modified at only one or two positions.

A New Epigenetic Crosstalk: Chemical Modification Information Flow.

Central dogma is the most fundamental hypothesis in the field of molecular biology and explains the genetic information flow from DNA to protein. Beyond residue-by-residue transmission of sequential information, chemical modifications of DNA, RNA, and protein are also relayed in the course of gene expression. Here, this work presents recent evidence supporting bidirectional interplay between chromatin modifications and RNA modifications. Furthermore, several RNA modifications likely affect chemical modifications of proteins. The relay of chemical modifications occurs co-transcriptionally or co-translationally, ensuring crosstalk among chemical modifications at the DNA, RNA, and protein levels. Overall, this work proposes a hypothetical framework that represents transmission of chemical modification information among chromatin, RNA, and proteins.

A Versatile Strategy to Manipulate and Probe Native Carbonic Anhydrases In Cellulo Utilizing Two-Step Ligand-Directed Functionalization.

Elucidating the biological logistics and functional interplay of proteins in their natural context has long been a great challenge in biological research. Chemical modification of proteins allows understanding of their roles and their interactions. Over decades, numerous strategies have been developed to modify target proteins with desired probes in test tubes and even biological systems. Nevertheless, these approaches require the design and synthesis of different probes for different applications, even for the same target protein, which is very time- and labor-consuming. Herein, we developed a general two-step protein functionalization strategy that utilizes ligand-directed chemistry to modify a clickable tag on the intact protein in the first step. Then, the desired functional moiety can be conjugated onto the target protein via a simple bioorthogonal click reaction in the second step, thus achieving probing and activity regulation of the target protein. In this work, carbonic anhydrase (CA) was chosen as our model protein for functionalization. We successfully labeled endogenous CAs with fluorophores to allow cellular imaging. In addition, a photoswitchable ligand was conjugated to CAs such that its activity could be manipulated in a light-responsive manner.

MSFragger-Labile: A Flexible Method to Improve Labile PTM Analysis in Proteomics

Posttranslational modifications of proteins play essential roles in defining and regulating the functions of the proteins they decorate, making identification of these modifications critical to understanding biology and disease. Methods for enriching and analyzing a wide variety of biological and chemical modifications of proteins have been developed using mass spectrometry-based proteomics, largely relying on traditional database search methods to identify the resulting mass spectra of modified peptides. These database search methods treat modifications as static attachments of a mass to particular position in the peptide sequence, but many modifications undergo fragmentation in tandem mass spectrometry experiments alongside, or instead of, the peptide backbone. While this fragmentation can confound traditional search methods, it also offers unique opportunities for improved searches that incorporate modification-specific fragment ions. Here, we present a new labile mode in the MSFragger search engine that provides the flexibility to tailor modification-centric searches to the fragmentation observed. We show that labile mode can dramatically improve spectrum identification rates of phosphopeptides, RNA-crosslinked peptides, and ADP-ribosylated peptides. Each of these modifications presents distinct fragmentation characteristics, showcasing the flexibility of MSFragger labile mode to improve search for a wide variety of biological and chemical modifications.

Benchmarking In Silico Tools for Cysteine pKa Prediction.

Accurate estimation of the pKa's of cysteine residues in proteins could inform targeted approaches in hit discovery. The pKa of a targetable cysteine residue in a disease-related protein is an important physiochemical parameter in covalent drug discovery, as it influences the fraction of nucleophilic thiolate amenable to chemical protein modification. Traditional structure-based in silico tools are limited in their predictive accuracy of cysteine pKa's relative to other titratable residues. Additionally, there are limited comprehensive benchmark assessments for cysteine pKa predictive tools. This raises the need for extensive assessment and evaluation of methods for cysteine pKa prediction. Here, we report the performance of several computational pKa methods, including single-structure and ensemble-based approaches, on a diverse test set of experimental cysteine pKa's retrieved from the PKAD database. The dataset consisted of 16 wildtype and 10 mutant proteins with experimentally measured cysteine pKa values. Our results highlight that these methods are varied in their overall predictive accuracies. Among the test set of wildtype proteins evaluated, the best method (MOE) yielded a mean absolute error of 2.3 pK units, highlighting the need for improvement of existing pKa methods for accurate cysteine pKa estimation. Given the limited accuracy of these methods, further development is needed before these approaches can be routinely employed to drive design decisions in early drug discovery efforts.

Cryo-EM reconstruction of the human 40S ribosomal subunit at 2.15 Å resolution.

The chemical modification of ribosomal RNA and proteins is critical for ribosome assembly, for protein synthesis and may drive ribosome specialisation in development and disease. However, the inability to accurately visualise these modifications has limited mechanistic understanding of the role of these modifications in ribosome function. Here we report the 2.15 Å resolution cryo-EM reconstruction of the human 40S ribosomal subunit. We directly visualise post-transcriptional modifications within the 18S rRNA and four post-translational modifications of ribosomal proteins. Additionally, we interpret the solvation shells in the core regions of the 40S ribosomal subunit and reveal how potassium and magnesium ions establish both universally conserved and eukaryote-specific coordination to promote the stabilisation and folding of key ribosomal elements. This work provides unprecedented structural details for the human 40S ribosomal subunit that will serve as an important reference for unravelling the functional role of ribosomal RNA modifications.

Chemical Modifications of Proteins In Vivo: Selected Examples Important to Cellular Regulation

The synthesis and degradation pathways for individual proteins and functional peptides often involve numerous co- and post-translational modifying steps. This review focuses on the diversity of such reactions. The reactions are organized into functional categories. An overall goal is to highlight post-translational events as often having the same importance to functional protein production as the transcriptional and translational events that initiate their synthesis.