Tuning a Protein‐Labeling Reaction to Achieve Highly Site Selective Lysine Conjugation

Activated esters are widely used to label proteins at lysine side chains and N termini. These reagents are useful for labeling virtually any protein, but robust reactivity toward primary amines generally precludes site‐selective modification. In a unique case, fluorophenyl esters are shown to preferentially label human kappa antibodies at a single lysine (Lys188) within the light‐chain constant domain. Neighboring residues His189 and Asp151 contribute to the accelerated rate of labeling at Lys188 relative to the ≈40 other lysine sites. Enriched Lys188 labeling can be enhanced from 50–70 % to >95 % by any of these approaches: lowering reaction temperature, applying flow chemistry, or mutagenesis of specific residues in the surrounding protein environment. Our results demonstrated that activated esters with fluoro‐substituted aromatic leaving groups, including a fluoronaphthyl ester, can be generally useful reagents for site‐selective lysine labeling of antibodies and other immunoglobulin‐type proteins.     

Photoactivatable Myristic Acid Probes for UNC119-Cargo Interactions

Protein myristoylation plays key roles in biological processes, for instance, in membrane attachment and activation of proteins and in mediating protein–protein and protein–lipid interactions. Furthermore, myristoylated proteins are involved in disorders, including cancer and viral infections. Therefore, new tools to study protein myristoylation are in high demand. Herein, we report the development of photoactivatable probes, based on a diazirine-substituted analogue of myristic acid. The probes bind to and, upon irradiation, covalently label the lipid-binding chaperone protein uncoordinated 119 (UNC119). UNC119 increases overall solubility and regulates specifically the transport of myristoylated proteins between intercellular membranes. The binding mode of the probes is similar to that of the myristate moiety, and the residues inside the hydrophobic pocket of UNC119 proteins that are critical for covalent binding have been identified. The interaction with UNC119 was also demonstrated in cell lysate by means of affinity enrichment. Moreover, it is shown that the myristate analogue can be incorporated into peptide substrates by N-myristoyl transferases of Leishmania and Trypanosoma protozoan parasites.     

Sequence and structure determinants of the nonenzymatic deamidation of asparagine and glutamine residues in proteins

The rates of deamidation of Asn and GIn residues in peptidesand proteins depend upon both the identity of other nearby aminoacid residues, some of which can catalyze the deamidation reactionof the Asn and Gln side chains, and upon polypeptide conformation.Proximal amino acids can be contiguous in sequence or broughtclose to Asn or Gln side chains by higher order structure ofthe protein. Local polypeptide conformation can stabilize theoxyanion transition state of the deamidation reaction and alsoenable deamidation through the ß-aspartyl shift mechanism.In this paper, the environments of Asn and Gln residues in knownprotein structures are examined to determine the configurationand identity of groups which participate in deamidation reactions.Sequence information is also analyzed and shown to support evolutionaryselection against the occurrence of certain potentially catalyticamino acids adjacent to Asn and GIn in proteins. This negativeselection supports a functional role for deamidation in thosenon-mutant proteins in which it occurs.     

Current Approaches for RNA Labeling in Vitro and in Cells Based on Click Reactions

Over recent years, click reactions have become recognized as valuable and flexible tools to label biomacromolecules such as proteins, nucleic acids, and glycans. Some of the developed strategies can be performed not only in aqueous solution but also in the presence of cellular components, as well as on (or even in) living cells. These labeling strategies require the initial, specific modification of the target molecule with a small, reactive moiety. In the second step, a click reaction is used to covalently couple a reporter molecule to the biomolecule. Depending on the type of reporter, labeling by the click reaction can be used in many different applications, ranging from isolation to functional studies of biomacromolecules. In this minireview, we focus on labeling strategies for RNA that rely on the click reaction. We first highlight click reactions that have been used successfully to label modified RNA, and then describe different strategies to introduce the required reactive groups into target RNA. The benefits and potential limitations of the strategies are critically discussed with regard to possible future developments.     

A General Method for Artificial Metalloenzyme Formation through Strain‐Promoted Azide–Alkyne Cycloaddition

Strain‐promoted azide–alkyne cycloaddition (SPAAC) can be used to generate artificial metalloenzymes (ArMs) from scaffold proteins containing a p‐azido‐L ‐phenylalanine (Az) residue and catalytically active bicyclononyne‐substituted metal complexes. The high efficiency of this reaction allows rapid ArM formation when using Az residues within the scaffold protein in the presence of cysteine residues or various reactive components of cellular lysate. In general, cofactor‐based ArM formation allows the use of any desired metal complex to build unique inorganic protein materials. SPAAC covalent linkage further decouples the native function of the scaffold from the installation process because it is not affected by native amino acid residues; as long as an Az residue can be incorporated, an ArM can be generated. We have demonstrated the scope of this method with respect to both the scaffold and cofactor components and established that the dirhodium ArMs generated can catalyze the decomposition of diazo compounds and both Si?H and olefin insertion reactions involving these carbene precursors.     

Effects of FlAsH/Tetracysteine (TC) Tag on PrP Proteolysis and PrPres Formation by TC‐Scanning

Protein–protein interactions associated with proteolytic processing and aggregation are integral to normal and pathological aspects of prion protein (PrP) biology. Characterization of these interactions requires the identification of amino acid residues involved. The FlAsH/tetracysteine (FlAsH/TC) tag is a small fluorescent tag amenable to insertion at internal sites in proteins. In this study, we used serial FlAsH/TC insertions (TC‐scanning) as a probe to characterize sites of protein–protein interaction between PrP and other molecules. To explore this application in the context of substrate–protease interactions, we analyzed the effect of FlAsH/TC insertions on proteolysis of cellular prion protein (PrPsen) in in vitro reactions and generation of the C1 metabolic fragment of PrPsen in live neuroblastoma cells. The influence of FlAsH/TC insertion was evaluated by TC‐scanning across the cleavage sites of each protease. The results showed that FlAsH/TC inhibited protease cleavage only within limited ranges of the cleavage sites, which varied from about one to six residues in width, depending on the protease, providing an estimate of the PrP residues interacting with each protease. TC‐scanning was also used to probe a different type of protein–protein interaction: the conformational conversion of FlAsH‐PrPsen to the prion disease‐associated isoform, PrPres. PrP constructs with FlAsH/TC insertions at residues 90–96 but not 97–101 were converted to FlAsH‐PrPres, identifying a boundary separating loosely versus compactly folded regions of PrPres. Our observations demonstrate that TC‐scanning with the FlAsH/TC tag can be a versatile method for probing protein–protein interactions and folding processes.     

Heterodimer Formation of the Homodimeric ABC Transporter OpuA

Many proteins have a multimeric structure and are composed of two or more identical subunits. While this can be advantageous for the host organism, it can be a challenge when targeting specific residues in biochemical analyses. In vitro splitting and re-dimerization to circumvent this problem is a tedious process that requires stable proteins. We present an in vivo approach to transform homodimeric proteins into apparent heterodimers, which then can be purified using two-step affinity-tag purification. This opens the door to both practical applications such as smFRET to probe the conformational dynamics of homooligomeric proteins and fundamental research into the mechanism of protein multimerization, which is largely unexplored for membrane proteins. We show that expression conditions are key for the formation of heterodimers and that the order of the differential purification and reconstitution of the protein into nanodiscs is important for a functional ABC-transporter complex.     

Fatty Acyl Sulfonyl Fluoride as an Activity-Based Probe for Profiling Fatty Acid-Associated Proteins in Living Cells

Fatty acids play fundamental structural, metabolic, functional, and signaling roles in all biological systems. Altered fatty acid levels and metabolism have been associated with many pathological conditions. Chemical probes have greatly facilitated biological studies on fatty acids. Herein, we report the development and characterization of an alkynyl-functionalized long-chain fatty acid-based sulfonyl fluoride probe for covalent labelling, enrichment, and identification of fatty acid-associated proteins in living cells. Our quantitative chemical proteomics show that this sulfonyl fluoride probe targets diverse classes of fatty acid-associated proteins including many metabolic serine hydrolases that are known to be involved in fatty acid metabolism and modification. We further validate that the probe covalently modifies the catalytically or functionally essential serine or tyrosine residues of its target proteins and enables evaluation of their inhibitors. The sulfonyl fluoride-based chemical probe thus represents a new tool for profiling the expression and activity of fatty acid-associated proteins in living cells.     

Micelle‐Enhanced Bioorthogonal Labeling of Genetically Encoded Azido Groups on the Lipid‐Embedded Surface of a GPCR

Genetically encoded p‐azido‐phenylalanine (azF) residues in G protein‐coupled receptors (GPCRs) can be targeted with dibenzocyclooctyne‐modified (DIBO‐modified) fluorescent probes by means of strain‐promoted [3+2] azide–alkyne cycloaddition (SpAAC). Here we show that azF residues situated on the transmembrane surfaces of detergent‐solubilized receptors exhibit up to 1000‐fold rate enhancement relative to azF residues on water‐exposed surfaces. We show that the amphipathic moment of the labeling reagent, consisting of hydrophobic DIBO coupled to hydrophilic Alexa dye, results in strong partitioning of the DIBO group into the hydrocarbon core of the detergent micelle and consequently high local reactant concentrations. The observed rate constant for the micelleenhanced SpAAC is comparable with those of the fastest bioorthogonal labeling reactions known. Targeting hydrophobic regions of membrane proteins by use of micelle‐enhanced SpAAC should expand the utility of bioorthogonal labeling strategies.     

Protein Spin Labeling with a Photocaged Nitroxide Using Diels–Alder Chemistry

EPR spectroscopy of diamagnetic bio-macromolecules is based on site-directed spin labeling (SDSL). Herein, a novel labeling strategy for proteins is presented. A nitroxide-based spin label has been developed and synthesized that can be ligated to proteins by an inverse-electron-demand Diels–Alder (DA_inv) cycloaddition to genetically encoded noncanonical amino acids. The nitroxide moiety is shielded by a photoremovable protecting group with an attached tetra(ethylene glycol) unit to achieve water solubility. SDSL is demonstrated on two model proteins with the photoactivatable nitroxide for DA_inv reaction (PaNDA) label. The strategy features high reaction rates, combined with high selectivity, and the possibility to deprotect the nitroxide in Escherichia coli lysate.     

Specific isotope labeling of colicin E1 and B channel domains for membrane topological analysis by oriented solid-state NMR spectroscopy

An approach is presented to selectively label the methionines of the colicin E1 and B channel domains, each about 200 residues in size, and use them for oriented solid-state NMR investigations. By combining site-directed mutagenesis, bacterial overexpression in a methionine auxotroph E. coli strain and biochemical purification, quantitative amounts of the proteins for NMR structural investigations were obtained. The proteins were selectively labeled with (15)N at only one, or at a few, selected sites. Multidimensional heteronuclear correlation high-resolution NMR spectroscopy and mass spectrometry were used to monitor the quality of isotopic labeling. Thereafter the proteins were reconstituted into oriented phospholipid bilayers and investigated by proton-decoupled (15)N solid-state NMR spectroscopy. The colicin E1 thermolytic fragment that carries a single (15)N methionine within its hydrophobic helix 9 region exhibited (15)N resonances that are characteristic of helices that are oriented predominantly parallel to the membrane surface at low temperature, and a variety of alignments and conformations at room temperature. This suggests that the protein can adopt both umbrella and pen-knife conformations.     

A simple approach for protein structure discrimination based on the network pattern of conserved hydrophobic residues

Evolutionarily conserved hydrophobic residues at the core of protein structures are generally assumed to play a structural role in protein folding and stability. Recent studies have implicated that their importance to protein structures is uneven, with a few of them being crucial and the rest of them being secondary. In this work, we explored the possibility of employing this feature of native structures for discriminating non-native structures from native ones. First, we developed a network tool to quantitatively measure the structural contributions of individual amino acid residues. We systematically applied this method to diverse fold-type sets of native proteins. It was confirmed that this method could grasp the essential structural features of native proteins. Next, we applied it to a number of decoy sets of proteins. The results indicate that such an approach indeed identified non-native structures in most test cases. This finding should be of help for the investigation of the fundamental problem of protein structure prediction.     

Nitric oxide reduction by char and carbon monoxide: Fundamental kinetics of nitric oxide reduction in fluidizedbed combustion of coal

The reduction of nitric oxide during combustion of coal char in a fluidized-bed combustor was examined with respect to two reactions : a char-catalysed reaction; and a char-consuming reaction, which control nitric oxide emissions. The relative importance of the two reactions was investigated by measuring detailed material balances for the reactions. The product distribution was explained in terms of three fundamental parallel reaction paths. Reaction rates were investigated with a fixed-bed flow reactor over the temperature range 883–1194 K, the same as the fluidized bed combustor.     

The Covalent Reactions of Primary Amino Side–chains and Disulphide Bonds in Wool Keratin

The reactions with alkali of the disulphide bonds and the primary amino side–chains in wool have been investigated. Formation of dehydroalamine has been postulated and it has been conclusively identified in an alkali–treated, cystine–containing polypeptide. The mechanism of degradation of cystine in such a peptide has been further investigated and it has been shown that cysteine residues, produced from cystine during degradation, are also capable of degrading further to dehydroalanyl residues. The dehydroalanyl residues so produced are capable of reacting not only with other residues to form LAN and LAL, but also with other species present in the alkaline medium. The influence of amines on the cold setting of wool fibres with thioglycollates and on the dyeability of wool has been studied. Mechanisms are postulated to explain these effects.     

The Role of Short-Chain Conjugated Poly-(R)-3-Hydroxybutyrate (cPHB) in Protein Folding

Poly-(R)-3-hydroxybutyrate (PHB), a linear polymer of R-3-hydroxybutyrate (R-3HB), is a fundamental constituent of biological cells. Certain prokaryotes accumulate PHB of very high molecular weight (10,000 to >1,000,000 residues), which is segregated within granular deposits in the cytoplasm; however, all prokaryotes and all eukaryotes synthesize PHB of medium-chain length (~100–200 residues) which resides within lipid bilayers or lipid vesicles, and PHB of short-chain length (<12 residues) which is conjugated to proteins (cPHB), primarily proteins in membranes and organelles. The physical properties of cPHB indicate it plays important roles in the targeting and folding of cPHB-proteins. Here we review the occurrence, physical properties and molecular characteristics of cPHB, and discuss its influence on the folding and structure of outer membrane protein A (OmpA) of Escherichia coli.     

Monitoring the cellular surface display of recombinant proteins by cysteine labeling and flow cytometry

A general method is described that allows one to follow the surface display of recombinant proteins in Escherichia coli without having to use specific antibodies or enzymatic reactions. The method is based on cysteine-specific labeling through Michael addition to the double bond of maleimide and its derivatives, and takes advantage of the fact that naturally occurring surface proteins in E. coli contain no accessible cysteine residues. The method is easy to perform and could be simply applied to different analytic procedures including Western blot, spectral photometry, and flow cytometry. By using this new labeling method, single cells bearing a distinct protein at the surface could be selected by fluorescence-activated cell sorting. The data were obtained by using autodisplay, an efficient surface display system established for E. coli, but the method presented here represents rather a general solution for analyzing the surface display of recombinant proteins, independent of the cellular system used.     

Effects of Charge and Charge Distribution on the Cellular Uptake of Multivalent Arginine‐Containing Peptide–Polymer Conjugates

Copolymers of N‐(2‐hydroxypropyl)methacrylamide (HPMA) and N‐methacryloyl‐β‐alaninyl‐S‐benzyl thioester were prepared by employing free radical or RAFT conditions and denominated as “NCL polymers”. The copolymer with a polydispersity index of 1.2–1.3 was used for the direct conjugation of unprotected peptides and peptide mixtures bearing differentially loaded side chains by native chemical ligation reactions conducted in aqueous buffer. Uptake into human HeLa cells was correlated with the overall surface charge and the ζ potentials of the peptide–polymer conjugates. Most notable were the differential effects found for various multivalent peptide–polymer conjugates containing arginine residues. Although positive ζ potentials were required for cellular uptake of the peptide–polymer conjugates, this sole charge effect was strongly dominated by the effect exerted by the relative distribution of arginine residues. Polymers conjugated with nona‐arginine peptides were over‐proportionally taken up, relative to their surface charge, compared to polymers with random distribution of single arginine residues. In view of these findings, peptide–polymer compositions suitable for efficient cellular uptake with negligible toxicity at polymer concentrations relevant for intracellular functional studies were determined.     

Single Labeled DNA FIT Probes for Avoiding False‐Positive Signaling in the Detection of DNA/RNA in qPCR or Cell Media

Oligonucleotide hybridization probes that fluoresce upon binding to complementary nucleic acid targets allow the real‐time detection of DNA or RNA in homogeneous solution. The most commonly used probes rely on the distance‐dependent interaction between a fluorophore and another label. Such duallabeled oligonucleotides signal the change of the global conformation that accompanies duplex formation. However, undesired nonspecific binding events and/or probe degradation also lead to changes in the label–label distance and, thus, to ambiguities in fluorescence signaling. Herein, we introduce singly labeled DNA probes, “DNA FIT probes”, that are designed to avoid false‐positive signals. A thiazole orange (TO) intercalator dye serves as an artificial base in the DNA probe. The probes show little background because the attachment mode hinders 1) interactions of the “TO base” in cis with the disordered nucleobases of the single strand, and 2) intercalation of the “TO nucleotide” with double strands in trans. However, formation of the probe–target duplex enforces stacking and increases the fluorescence of the TO base. We explored open‐chain and carbocyclic nucleotides. We show that the incorporation of the TO nucleotides has no effect on the thermal stability of the probe–target complexes. DNA and RNA targets provided up to 12‐fold enhancements of the TO emission upon hybridization of DNA FIT probes. Experiments in cell media demonstrated that false‐positive signaling was prevented when DNA FIT probes were used. Of note, DNA FIT probes tolerate a wide range of hybridization temperature; this enabled their application in quantitative polymerase chain reactions.     

Promiscuous Enzymes for Residue-Specific Peptide and Protein Late-Stage Functionalization

The late-stage functionalization of peptides and proteins holds significant promise for drug discovery and facilitates bioorthogonal chemistry. This selective functionalization leads to innovative advances in in vitro and in vivo biological research. However, it is a challenging endeavor to selectively target a certain amino acid or position in the presence of other residues containing reactive groups. Biocatalysis has emerged as a powerful tool for selective, efficient, and economical modifications of molecules. Enzymes that have the ability to modify multiple complex substrates or selectively install nonnative handles have wide applications. Herein, we highlight enzymes with broad substrate tolerance that have been demonstrated to modify a specific amino acid residue in simple or complex peptides and/or proteins at late-stage. The different substrates accepted by these enzymes are mentioned together with the reported downstream bioorthogonal reactions that have benefited from the enzymatic selective modifications.