Protein Side Chains Research Articles

Reaction of cysteine residues with oxidized tyrosine residues mediates cross-linking of photo-oxidized casein proteins

Photo-oxidation of casein proteins is commonplace during milk processing and storage. A major consequence of such light exposure is protein cross-linking and aggregation. Although caseins are key milk components, the nature of the cross-links and the mechanisms involved are poorly characterized, with most previous work having been focused on detecting and quantifying di-tyrosine formed on dimerization of two tyrosine-derived phenoxyl radicals. However, this is only one of a large number of possible cross-links that might be formed. In this study, we have investigated the potential involvement of secondary reactions between oxidized protein side-chains and the thiol group of cysteine (Cys) residues in casein cross-linking. Casein proteins were subjected to photo-oxidation using visible light in the presence of a sensitizer (riboflavin or rose Bengal) and O2, then incubated with a Cys-containing peptide (glutathione, GSH) or protein (κ-casein), and subsequently analyzed by SDS-PAGE, immunoblotting and LC-MS. Our data indicate that that photo-oxidized (but not parent) caseins react efficiently with the Cys-containing species, likely via Michael addition to quinones formed from tyrosine residues to give glutathionylated species or protein adducts. Thus, oxidized α-casein reacts with native κ-casein to give high molecular mass aggregates. This adduct formation was prevented by alkylation of the Cys thiol group. The cross-link site and the residues involved have been confirmed by liquid chromatography-mass spectrometry (LC-MS) proteomic analysis. Together, these data extend our knowledge of the mechanisms involved in casein oxidation and aggregation.

Conserved hydrogen-bond motifs of membrane transporters and receptors

Membrane transporters and receptors often rely on conserved hydrogen bonds to assemble transient paths for ion transfer or long-distance conformational couplings. For transporters and receptors that use proton binding and proton transfer for function, inter-helical hydrogen bonds of titratable protein sidechains that could change protonation are of central interest to formulate hypotheses about reaction mechanisms. Knowledge of hydrogen bonds common at sites of potential interest for proton binding could thus inform and guide studies on functional mechanisms of protonation-coupled membrane proteins. Here we apply graph-theory approaches to identify hydrogen-bond motifs of carboxylate and histidine sidechains in a large data set of static membrane protein structures. We find that carboxylate-hydroxyl hydrogen bonds are present in numerous structures of the dataset, and can be part of more extended H-bond clusters that could be relevant to conformational coupling. Carboxylate-carboxyamide and imidazole-imidazole hydrogen bonds are represented in comparably fewer protein structures of the dataset. Atomistic simulations on two membrane transporters in lipid membranes suggest that many of the hydrogen bond motifs present in static protein structures tend to be robust, and can be part of larger hydrogen-bond clusters that recruit additional hydrogen bonds.

Mechanism of Cooperative Degradation of Gum Arabic Arabinogalactan Protein by Bifidobacterium longum Surface Enzymes.

ABSTRACTGum arabic is an arabinogalactan protein (AGP) that is effective as a prebiotic for the growth of bifidobacteria in the human intestine. We recently identified a key enzyme in the glycoside hydrolase (GH) family 39, 3-O-α-d-galactosyl-α-l-arabinofuranosidase (GAfase), for the assimilation of gum arabic AGP in Bifidobacterium longum subsp. longum. The enzyme released α-d-Galp-(1→3)-l-Ara and β-l-Arap-(1→3)-l-Ara from gum arabic AGP and facilitated the action of other enzymes for degrading the AGP backbone and modified sugar. In this study, we identified an α-l-arabinofuranosidase (BlArafE; encoded by BLLJ_1850), a multidomain enzyme with both GH43_22 and GH43_34 catalytic domains, as a critical enzyme for the degradation of modified α-l-arabinofuranosides in gum arabic AGP. Site-directed mutagenesis approaches revealed that the α1,3/α1,4-Araf double-substituted gum arabic AGP side chain was initially degraded by the GH43_22 domain and subsequently cleaved by the GH43_34 domain to release α1,3-Araf and α1,4-Araf residues, respectively. Furthermore, we revealed that a tetrasaccharide, α-l-Rhap-(1→4)-β-d-GlcpA-(1→6)-β-d-Galp-(1→6)-d-Gal, was a limited degradative oligosaccharide in the gum arabic AGP fermentation of B. longum subsp. longum JCM7052. The oligosaccharide was produced from gum arabic AGP by the cooperative action of the three cell surface-anchoring enzymes, GAfase, exo-β1,3-galactanase (Bl1,3Gal), and BlArafE, on B. longum subsp. longum JCM7052. Furthermore, the tetrasaccharide was utilized by the commensal bacteria.IMPORTANCE Terminal galactose residues of the side chain of gum arabic arabinogalactan protein (AGP) are mainly substituted by α1,3/α1,4-linked Araf and β1,6-linked α-l-Rhap-(1→4)-β-d-GlcpA residues. This study found a multidomain BlArafE with GH43_22 and GH43_34 catalytic domains showing cooperative action for degrading α1,3/α1,4-linked Araf of the side chain of gum arabic AGP. In particular, the GH43_34 domain of BlArafE was a novel α-l-arabinofuranosidase for cleaving the α1,4-Araf linkage of terminal galactose. α-l-Rhap-(1→4)-β-d-GlcpA-(1→6)-β-d-Galp-(1→6)-d-Gal tetrasaccharide was released from gum arabic AGP by the cooperative action of GAfase, GH43_24 exo-β-1,3-galactanase (Bl1,3Gal), and BlArafE and remained after B. longum subsp. longum JCM7052 culture. Furthermore, in vitro assimilation test of the remaining oligosaccharide using Bacteroides species revealed that cross-feeding may occur from bifidobacteria to other taxonomic groups in the gut.

Pegylation of the Galectin-3 carbohydrate recognition domain creates a kinetic trap protecting the protein from thermal unfolding

Proteins are widely used as therapeutics but often can be unstable, limiting their applications in the clinic. To improve the stability of protein drugs, synthetic polymers such as polyethylene glycol (PEG) can be covalently attached to specific protein side chains, creating protein-polymer conjugates. Though nearly thirty PEGylated proteins have been FDA-approved, our understanding is very limited of how PEG-protein interactions alter the fundamental chemical and physical properties of the conjugate.

Critical Mutations of the SARS-CoV-2 Virus

SARS-CoV-2 presents an opportunity to understand better the role of viral mutations. The Alpha and Delta variants of SARS-CoV-2 provide particular insight. We argue that looking at the mutations through a physical chemistry lens provides a deeper understanding of viral evolutionary trends. We advocate here the use of quantitative (mathematical) methods, based on physical chemistry foundations, to analyze viruses. The behavior of viral proteins depends both on structural properties (how the protein sidechains are configured in three dimensional space) and epistructural properties (how the protein interacts with the enveloping solvent, e.g., water). In both cases, physical chemistry (and ultimately quantum mechanics) plays a dominant role. There are many barriers to entry for quantitative scientists (e.g., mathematicians) to study viruses. At the simplest level, there are multiple ways to describe a virus, by its genomic sequence (RNA or DNA) or its protein sequence. Many papers assume that the context is clear when the word ‘sequence’ is used, but the novice would be forgiven for confusion. But there are much more complicated issues of terminology and interpretation that can make it very hard to understand what is going on. The book [1] was written in part to clarify this. We use the SARS-CoV-2 virus mutations here as the basis for a primer on the tools in [1] and to exhibit the kinds of observations they can yield. We attempt here both to lower the barrier of entry to the subject and to raise the level of rigor in the discussion by showing how a much more quantitative view can be beneficial. We do this by explaining concepts in simple, quantitative terms. In many cases, this involves measuring distances between atoms in PDB files. Thus we quantify what it means to be a hydrogen bond, a salt bridge, to be underwrapped, all of which have rigorous definitions [1]. Our goal here is to look at virus mutation from a mathematical perspective, with a particular focus on the SARS-CoV-2 virus [2]. This is for two reasons. First of all, it has become one of the greatest threats to humanity of all time. But the second is more positive: SARS-CoV-2 is very widely studied, and this allows new opportunities for understanding viruses in general. Any advances could have a very wide impact. We can compare and contrast two closely related viruses by considering mutations of a single virus. This allows us to focus on particular features and their impact on disease. Certain mutations of the SARS-CoV-2 virus have been the focus of attention, especially those in the spike protein [3]. We will limit our attention here to this protein for simplicity. While pure genomic sequence analysis [4] is extremely valuable, and certainly mathematically rigorous, we will stress here a different approach. Instead we focus on the amino acid sequence with a physical chemistry perspective. This brings in new mathematical tools that have yet to be fully utilized [1]. Typical sequence analysis sees all sidechains as the same, whereas physical chemistry allows us to differentiate them, to study their interactions, and to quantify epistructural behavior as well. Using standard sequence analysis helps us understanding what mutations are important, but adding a physical chemistry perspective informs us why they are important. Thus we can see that all mutations are not created equal, but some can be viewed as a “smoking gun” in certain contexts. We examine three mutations in detail, explaining why they play a significant role in enhancing the effectiveness of SARS-CoV-2 mutants. Each one has a different physical chemistry signature, and taken together they provide a blueprint for analysing viruses, and proteins, in general.

1H, 13C and 15N resonance assignments of TFIIS LW domain from Saccharomyces cerevisiae.

TFIIS is one of the best-characterized transcription elongation factors, with a domain I (named also as LW domain) in the N-terminus. It can relieve the arrest of RNA polymerase II (RNAP II) when the elongation of RNAP II is impaired. Here we report the resonance assignments of the protein backbone and side chains of the LW domain of TFIIS from S. cerevisiae, the secondary structure prediction indicates the ScTFIIS LW domain contains six α-helices with no β-sheet, which will lay the foundation for the protein structure determination and function elucidation.

Specificity Analysis of Protein Methyltransferases and Discovery of Novel Substrates Using SPOT Peptide Arrays.

Posttranslational methylation of amino acid side chains in proteins mainly occurs on lysine, arginine, glutamine, and histidine residues. It is introduced by different protein methyltransferases (PMTs) and regulates many aspects of protein function including stability, activity, localization, and protein/protein interactions. Although the biological effects of PMTs are mediated by their methylation substrates, the full substrate spectrum of most PMTs is not known. For many PMTs, their activity on a particular potential substrate depends, among other factors, on the peptide sequence containing the target residue for methylation. In this protocol, we describe the application of SPOT peptide arrays to investigate the substrate specificity of PMTs and identify novel substrates. Methylation of SPOT peptide arrays makes it possible to study the methylation of many different peptides in one experiment at reasonable costs and thereby provides detailed information about the specificity of the PMT under investigation. In these experiments, a known substrate sequence is used as template to design a SPOT peptide array containing peptides with single amino acid exchanges at all positions of the sequence. Methylation of the array with the PMT provides detailed preferences for each amino acid at each position in the substratesequence, yielding a substrate sequencespecificity profile. This information can then be used to identify novel potential PMT substrates by in silico data basesearches. Methylation of novel substrate candidates can be validated in SPOT arrays at peptide level, followed by validation at protein level in vitro and in cells.

Interaction between polyphenols and amyloids: from the view of prevention of protein misfolding disorders related diseases

Amyloid is the term usually used to describe a particular type of elongated, unbranched protein fibril with cross-<i>β</i>-sheet characteristics, formed through the ordered aggregation of peptides or denatured proteins. Amyloids show high association with many severe neurodegenerative diseases, such as Alzheimer's, Parkinson's, and Huntington's diseases. Unfortunately, there are still no effective medical treatments to cure these neurodegenerative diseases, including the failure of pharmaceutical approaches with amyloids as the targets. Polyphenols, the major phytochemicals in fruits, vegetables, tea and coffee have many beneficial functional properties, such as antioxidant, antimicrobial and anticancer properties. Polyphenols can interact with proteins through non-covalent interactions, including hydrophobic interaction, hydrogen bonding and electrostatic interaction. Polyphenols can also covalently interact with proteins mainly via the interaction between quinones, the oxidized products of polyphenols, and the nucleophilic residues (thiol, amino, guanidine, or imidazole) in protein side chains or peptides. Evidence from previous studies have indicated that polyphenols could reduce amyloid-formation via inhibiting fibril aggregation or steering oligomer formation into unstructured, nontoxic pathways. This effect was realized mainly through: (1) preventing conformational changes of precursor native proteins into amyloidogenic forms, (2) inhibiting the conversion of amyloidogenic precursors to amyloid fibrils, or (3) disrupting preformed fibrils meanwhile destructing the aggregation conditions. These findings imply that polyphenols could play a vital role in inhibiting protein misfolding, which might be useful for the prevention of various neurodegenerative diseases.

Biosynthetic incorporation for visualizing bacterial glycans.

Cell-surface glycans are central to many biological processes, yet methods for their site-selective modification are limited. Strategies for interrogating the structure and function of proteins have been enabled by chemoselective reactions of sidechain functionality for covalent modification, capture, or imaging. However, unlike protein sidechains, glycan building blocks lack distinguishing reactivity. Moreover, glycans are not primary gene products, so encoding glycan variants through genetic manipulation is challenging. Reactive functional groups can be introduced into glycans through metabolic engineering, which involves the generation of modified nucleotide-sugar building blocks. Lipid-linked building blocks, which are also used in glycan biosynthesis, have the advantage that they can be delivered directly to glycosyltransferases to function as surrogate substrates. This process, termed "biosynthetic incorporation," takes advantage of the properties of bacterial glycosyltransferase: they are selective for the products they generate yet promiscuous in their donor preferences. We describe how this strategy can be implemented to label arabinofuranose-containing glycans on the surface of mycobacterial cells. We anticipate that this platform can be expanded to develop chemoselective labeling agents for other important bacterial monosaccharides.

Functional analysis of Ost3p and Ost6p containing yeast oligosaccharyltransferases

The oligosaccharyltransferase (OST) is the central enzyme in the N-glycosylation pathway. It transfers a defined oligosaccharide from a lipid-linker onto the asparagine side chain of proteins. The yeast OST consists of eight subunits and exists in two catalytically distinct isoforms that differ in one subunit, Ost3p or Ost6p. The cryo-electron microscopy structure of the Ost6p containing complex was found to be highly similar to the Ost3p containing OST. OST enzymes with altered Ost3p/Ost6p subunits were generated and functionally analyzed. The three C-terminal transmembrane helices were responsible for the higher turnover-rate of the Ost3p vs. the Ost6p containing enzyme in vitro and the more severe hypoglycosylation in Ost3p lacking strains in vivo. Glycosylation of specific OST target sites required the N-terminal thioredoxin domain of Ost3p or Ost6p. This Ost3p/Ost6p dependence was glycosylation site but not protein specific. We concluded that the Ost3p/Ost6p subunits modulate the catalytic activity of OST and provide additional specificity for OST substrate recognition.

Fungal cellulases: protein engineering and post-translational modifications.

Enzymatic degradation of lignocelluloses into fermentable sugars to produce biofuels and other biomaterials is critical for environmentally sustainable development and energy resource supply. However, there are problems in enzymatic cellulose hydrolysis, such as the complex cellulase composition, low degradation efficiency, high production cost, and post-translational modifications (PTMs), all of which are closely related to specific characteristics of cellulases that remain unclear. These problems hinder the practical application of cellulases. Due to the rapid development of computer technology in recent years, computer-aided protein engineering is being widely used, which also brings new opportunities for the development of cellulases. Especially in recent years, a large number of studies have reported on the application of computer-aided protein engineering in the development of cellulases; however, these articles have not been systematically reviewed. This article focused on the aspect of protein engineering and PTMs of fungal cellulases. In this manuscript, the latest literatures and the distribution of potential sites of cellulases for engineering have been systematically summarized, which provide reference for further improvement of cellulase properties. KEY POINTS: •Rational design based on virtual mutagenesis can improve cellulase properties. •Modifying protein side chains and glycans helps obtain superior cellulases. •N-terminal glutamine-pyroglutamate conversion stabilizes fungal cellulases.

Identification of novel anti‐Alzheimer’s drugs through inhibition of LRP‐DKK interaction: A computational approach using high throughput virtual screening and molecular dynamics studies

AbstractBackgroundActivation of WNT signalling pathway was involved in a neuroprotective mechanism against amyloid beta toxicity (1). Inhibition of this pathway is associated with GSK‐3 beta activation, tau hyperphosphorylation, neuronal apoptosis (1), (2) and is implicated in Alzheimer’s disease (1). DKK‐1 competes with Wnt for the binding to LRP5/6 receptor (3) and disrupts the Wnt‐induced Fzd‐Wnt‐LRP6 complex (4) and thus antagonizes the neuro‐protective effect of WNT. In this study, we have targeted the DKK‐LRP interaction through virtual screening the first time to develop novel anti‐Alzheimer’s agent.MethodBased upon crystal structure information and protein validation using Ramachandran Plots, a crystal structure (PDB: 3S8V) was selected for further works. In this study, we have targeted the surface of LRP 5/6, which is interacting with DKK and grid was generated on LRP. The virtual screening of Asinex database library (N=19642) was done against this grid. Molecules were selected on the basis of good dock score and binding free energy calculation using MMGBSA in Schrodinger GLIDE (5)(6). The protein ligand complex structure stability were also evaluated by molecular dynamic simulation study (7). ADMET profile of the screened ligands were evaluated using Quick prop module of Schrodinger.ResultAfter running the virtual screening protocol (HTVS: SP: XP), 14 compounds were screened out, which showed significant binding top the DKK1 binding interface of LRP. These compounds were further evaluated on basis of docking score and MMGBSA. On the basis of After then on the basis of ADME profile selected 2 compound id ZINC000100138828 and ZINC000247940500 for further molecular dynamic simulation study which were shown the compound ZINC000100138828 had an avg. RMSD of ∼4.0Å, also the RMSF is 2.4 Å for overall protein side chain residue was reduced with reference to the apo form. It is displays rich interaction profile and strength with core binding residues (A: Pro833, B: Tyr706, B: Glu708, B: Arg751 and B: Ile681).ConclusionZINC000100138828 is a potent binder to the DKK1 binding surface on LRP5/6. It needs further in‐vitro and preclinical validation as a potential anti‐Alzheimer’s agent.

Hindered Rotations of Protein Asparagine/Glutamine Side-Chain NH2 Groups: Impact of Hydrogen Bonding with DNA

Hindered rotation about an sp2 C-N bond is known to occur in arginine (Arg), asparagine (Asn), and glutamine (Gln) side chains of proteins. However, very little is known about the rotational dynamics of Asn and Gln side-chain NH2 groups. Here, using a unique NMR method, we quantitatively characterized the hindered rotations of protein Asn/Gln side-chain NH2 groups. This NMR method yields simple NH2-selective spectra that allow for an accurate determination of the kinetic rate constants for the hindered rotations. Through the NMR measurements at different temperatures, we investigated the energy barriers that restrict the C-N bond rotations of protein side-chain NH2 groups. Through a comparison of the kinetic data for the free and DNA-bound states of the Antp homeodomain, we also examined the impact of hydrogen bonding on the hindered rotations of the side-chain NH2 groups. Our data suggest that the hydrogen bonding increases the energy barriers by 1-6 kJ/mol.

Toward a Computational NMR Procedure for Modeling Dipeptide Side-Chain Conformation.

Theoretical relationships between the vicinal spin–spin coupling constants (SSCCs) and the χ1 torsion angles have been studied to predict the conformations of protein side chains. An efficient computational procedure is developed to obtain the conformation of dipeptides through theoretical and experimental SSCCs, Karplus equations, and quantum chemistry methods, and it is applied to three aliphatic hydrophobic residues (Val, Leu, and Ile). Three models are proposed: unimodal-static, trimodal-static-stepped, and trimodal-static-trigonal, where the most important factors are incorporated (coupled nuclei, nature and orientation of the substituents, and local geometric properties). Our results are validated by comparison with NMR and X-ray empirical data described in the literature, obtaining successful results on the 29 residues considered. Using out trimodal residue treatment, it is possible to detect and resolve residues with a simple conformation and those with two or three staggered conformers. In four residues, a deeper analysis explains that they do not have a unique conformation and that the population of each conformation plays an important role.

Reaction of dirhodium and diruthenium paddlewheel tetraacetate complexes with nucleophilic protein sites: A computational study

The biomolecular affinity of three paddlewheel dinuclear complexes Rh2(µ-O2CCH3)4(H2O)2, Ru2(µ-O2CCH3)4(H2O)Cl, [Ru2(µ-O2CCH3)4(H2O)2]+ for the binding at cancer-specific protein targets was determined by means of DFT approaches. Thermodynamics of axial ligand substitution reaction by models of suitable protein sites was investigated in order to assess the binding selectivity displayed by the paddlewheel complexes. Both Rh2(µ-O2CCH3)4(H2O)2 and Ru2(µ-O2CCH3)4(H2O)Cl resulted to react favorably with most of the examined models of the protein side chains, with higher exergodicity demonstrated in the reaction of Arg, Cys, His, Lys, Sec, thus showing a limited selectivity. On the other hand, we identified Sec and His as the most suitable targets of the examined paddlewheel complexes, while Arg and Lys are expected to be also targetable at higher pH. Therefore, our calculations showed how both dirhodium and, majorly, diruthenium examined complexes undergo to a certain degree of destabilization upon the binding at nucleophilic protein sites. The presented computational results allowed us to shed light onto the protein sites’ affinity and on the therapeutic potential of both Rh2(µ-O2CCH3)4(H2O)2 and Ru2(µ-O2CCH3)4(H2O)Cl, and provided for a structural basis for the development of newly designed paddlewheel complexes.

Structure, Activity, and Function of PRMT1.

PRMT1, the major protein arginine methyltransferase in mammals, catalyzes monomethylation and asymmetric dimethylation of arginine side chains in proteins. Initially described as a regulator of chromatin dynamics through the methylation of histone H4 at arginine 3 (H4R3), numerous non-histone substrates have since been identified. The variety of these substrates underlines the essential role played by PRMT1 in a large number of biological processes such as transcriptional regulation, signal transduction or DNA repair. This review will provide an overview of the structural, biochemical and cellular features of PRMT1. After a description of the genomic organization and protein structure of PRMT1, special consideration was given to the regulation of PRMT1 enzymatic activity. Finally, we discuss the involvement of PRMT1 in embryonic development, DNA damage repair, as well as its participation in the initiation and progression of several types of cancers.

Virtual Homonuclear Decoupling in Direct Detection Nuclear Magnetic Resonance Experiments Using Deep Neural Networks.

Nuclear magnetic resonance (NMR) experiments are frequently complicated by the presence of homonuclear scalar couplings. For the growing body of biomolecular 13C-detected NMR methods, one-bond 13C-13C couplings significantly reduce sensitivity and resolution. The solution to this problem has typically been to perform virtual decoupling by recording multiple spectra and taking linear combinations. Here, we propose an alternative method of virtual decoupling using deep neural networks, which only requires a single spectrum and gives a significant boost in resolution while reducing the minimum effective phase cycles of the experiments by at least a factor of 2. We successfully apply this methodology to virtually decouple in-phase CON (13CO-15N) protein NMR spectra, 13C-13C correlation spectra of protein side chains, and 13Cα-detected protein 13Cα-13CO spectra where two large homonuclear couplings are present. The deep neural network approach effectively decouples spectra with a high degree of flexibility, including in cases where existing methods fail, and facilitates the use of simpler pulse sequences.

Could Raman spectroscopy distinguish between P. falciparum and P. vivax Infection?

The erythrocytes obtained from patients diagnosed with Plasmodium falciparum or Plasmodium vivax infection (two groups of five patients each) and treated in the Department of Infectious Diseases, University Hospital in Kraków, were measured using the Raman spectroscopy method (1–2 cells of each patient) and then compared with the results from these patients during their convalescence. Principal Component Analysis (PCA) was used to determine the variance between the Raman spectra. Changes in the heme structure were observed by the v4 oxidation state marker and by other heme and hemozoin marker bands, as well as protein side chains marker bands. The recognized Raman bands allow to differentiate changes taking place in the red blood cells during the development of Plasmodium falciparum from Plasmodium vivax. The 1385 cm−1 Asp band along with the 1587 cm−1 Gln vibrations at the beginning of hospitalization specify the invasion of P. falciparum that occurs with the basigin receptor which requires respective protein glycosylation. The 1361 cm−1 and 1544 cm−1 Trp bands indicate the invasion stage of the P. vivax parasite, and the formation of the ligand-receptor complex. To our knowledge, this is the first Raman spectroscopic observation that we can distinguish Plasmodium species.

Chemical approaches for the preparation of ubiquitinated proteins via natural linkages.

Ubiquitination is an important posttranslation modification (PTM) that regulates a variety of cellular processes, including protein degradation, DNA repair, and viral infections. In this process, the C-terminal carboxyl group of ubiquitin (Ub) or poly-Ub is attached to the ε-amine of lysine (Lys) side chain of an acceptor protein through an isopeptide bond. Studying a molecular mechanism of ubiquitination and deubiquitination is fundamental for unraveling its precise role in health and disease and hence crucial for drug development. Enzymatic approaches for protein ubiquitination possess limited ability to selectivity install Ub or Ub chain on the desired position of an acceptor protein and often lead to heterogeneous mixtures. In the past decades, chemical protein (semi)synthesis has been proved to be an efficient tool to facilitate site-specific protein ubiquitination, which significantly contributes to decode the Ub signal at molecular and structural levels. In this review, we summarize the synthetic strategies developed for protein ubiquitination, and the achievements to generate monoubiquitinated, di-ubiquitinated, and tetraubiquitinated proteins with native isopeptide and ester bonds.

Minimizing Pervasive Artifacts in 4D Covariance Maps for Protein Side Chain NMR Assignments.

Nuclear magnetic resonance (NMR) is a mainstay of biophysical studies that provides atomic level readouts to formulate molecular mechanisms. Side chains are particularly important to derive mechanisms involving proteins as they carry functional groups, but NMR studies of side chains are often limited by challenges in assigning their signals. Here, we designed a novel computational method that combines spectral derivatives and matrix square-rooting to produce reliable 4D covariance maps from routinely acquired 3D spectra and facilitates side chain resonance assignments. Thus, we generate two 4D maps from 3D-HcccoNH and 3D-HCcH-TOCSY spectra that each help overcome signal overlap or sensitivity losses. These 4D maps feature HC-HSQCs of individual side chains that can be paired to assigned backbone amide resonances of individual aliphatic signals, and both are obtained from a single modified covariance calculation. Further, we present 4D maps produced using conventional triple resonance experiments to easily assign asparagine side chain amide resonances. The 4D covariance maps encapsulate the lengthy manual pattern recognition used in traditional assignment methods and distill the information as correlations that can be easily visualized. We showcase the utility of the 4D covariance maps with a 10 kDa peptidyl carrier protein and a 52 kDa cyclization domain from a nonribosomal peptide synthetase.