Traceless Cleavage of Protein–Biotin Conjugates under Biologically Compatible Conditions

Biotinylation of amines is widely used to conjugate biomolecules, but either the resulting label is non‐removable or its removal leaves a tag on the molecule of interest, thus affecting downstream processes. We present here a set of reagents (RevAmines) that allow traceless, reversible biotinylation under biologically compatible, mild conditions. Release following avidin‐based capture is achieved through the cleavage of a (2‐(alkylsulfonyl)ethyl) carbamate linker under mild conditions (200 mm ammonium bicarbonate, pH 8, 16–24 h, room temperature) that regenerates the unmodified amine. The capture and release of biotinylated proteins and peptides from neutravidin, fluorescent labelling through reversible biotinylation at the cell surface and the selective enrichment of proteins from bacterial periplasm are demonstrated. The tags are easily prepared, stable and offer the potential for future application in proteomics, activity‐based protein profiling, affinity chromatography and bio‐molecule tagging and purification.     

A "tag-and-modify" approach to site-selective protein modification

Covalent modification can expand a protein's functional capacity. Fluorescent or radioactive labeling, for instance, allows imaging of a protein in real time. Labeling with an affinity probe enables isolation of target proteins and other interacting molecules. At the other end of this functional spectrum, protein structures can be naturally altered by enzymatic action. Protein-protein interactions, genetic regulation, and a range of cellular processes are under the purview of these post-translational modifications. The ability of protein chemists to install these covalent additions selectively has been critical for elucidating their roles in biology. Frequently the transformations must be applied in a site-specific manner, which demands the most selective chemistry. In this Account, we discuss the development and application of such chemistry in our laboratory. A centerpiece of our strategy is a "tag-and-modify" approach, which entails sequential installation of a uniquely reactive chemical group into the protein (the "tag") and the selective or specific modification of this group. The chemical tag can be a natural or unnatural amino acid residue. Of the natural residues, cysteine is the most widely used as a tag. Early work in our program focused on selective disulfide formation in the synthesis of glycoproteins. For certain applications, the susceptibility of disulfides to reduction was a limitation and prompted the development of several methods for the synthesis of more stable thioether modifications. The desulfurization of disulfides and conjugate addition to dehydroalanine are two routes to these modifications. The dehydroalanine tag has since proven useful as a general precursor to many modifications after conjugate addition of various nucleophiles; phosphorylated, glycosylated, peptidylated, prenylated, and even mimics of methylated and acetylated lysine-containing proteins are all accessible from dehydroalanine. While cysteine is a useful tag for selective modification, unnatural residues present the opportunity for bio-orthogonal chemistry. Azide-, arylhalide-, alkyne-, and alkene-containing amino acids can be incorporated into proteins genetically and can be specifically modified through various transformations. These transformations often rely on metal catalysis. The Cu-catalyzed azide-alkyne addition, Ru-catalyzed olefin metathesis, and Pd-catalyzed cross-coupling are examples of such transformations. In the course of adapting these reactions to protein modification, we learned much about the behavior of these reactions in water, and in some cases entirely new catalysts were developed. Through a combination of these bio-orthogonal transformations from the panel of tag-and-modify reactions, multiple and distinct modifications can be installed on protein surfaces. Multiple modifications are common in natural systems, and synthetic access to these proteins has enabled study of their biological role. Throughout these investigations, much has been learned in chemistry and biology. The demands of selective protein modification have revealed many aspects of reaction mechanisms, which in turn have guided the design of reagents and catalysts that allow their successful deployment in water and in biological milieu. With this ability to modify proteins, it is now possible to interrogate biological systems with precision that was not previously possible.     

Bimodal Detection of Proteins by 129Xe NMR and Fluorescence Spectroscopy

A full understanding of biological phenomena involves sensitive and noninvasive detection. Herein, we report the optimization of a probe for intracellular proteins that combines the advantages of fluorescence and hyperpolarized ¹²⁹Xe NMR spectroscopy detection. The fluorescence detection part is composed of six residues containing a tetracysteine tag (−CCXXCC−) genetically incorporated into the protein of interest and of a small organic molecule, CrAsH. CrAsH becomes fluorescent if it binds to the tetracysteine tag. The part of the biosensor that enables detection by means of ¹²⁹Xe NMR spectroscopy, which is linked to the CrAsH moiety by a spacer, is based on a cryptophane core that is fully suited to reversibly host xenon. Three different peptides, containing the tetracysteine tag and four organic biosensors of different stereochemistry, are benchmarked to propose the best couple that is fully suited for the in vitro detection of proteins.     

Bioorthogonal Peptide Enrichment from Complex Samples Using a Rink-Amide-Based Catch-and-Release Strategy

Uptake and processing of antigens by antigen presenting cells (APCs) is a key step in the initiation of the adaptive immune response. Studying these processes is complex as the identification of low abundant exogenous antigens from complex cell extracts is difficult. Mass-spectrometry based proteomics – the ideal analysis tool in this case – requires methods to retrieve such molecules with high efficiency and low background. Here, we present a method for the selective and sensitive enrichment of antigenic peptides from APCs using click-antigens; antigenic proteins expressed with azidohomoalanine (Aha) in place of methionine residues. We here describe the capture of such antigens using a new covalent method namely, alkynyl functionalized PEG-based Rink amide resin, that enables capture of click-antigens via copper-catalyzed azide-alkyne [2 + 3] cycloaddition (CuAAC). The covalent nature of the thus formed linkage allows stringent washing to remove a-specific background material, prior to retrieval peptides by acid-mediated release. We successfully identified peptides from a tryptic digest of the full APC proteome containing femtomole amounts of Aha-labelled antigen, making this a promising approach for clean and selective enrichment of rare bioorthogonally modified peptides from complex mixtures.     

Properties of a single-chain antibody containing different linker peptides

Single-chain antibodies were constructed using six differentlinker peptides to join the V_H and V_L domains of an anti-2-phenyloxazolone(Ox) antibody. Four of the linker peptides originated from theinterdomain linker region of the fungal cellulase CBHI and consistedof 28, 11, six and two amino acid residues. The two other linkerpeptides used were the (GGGGS)₃ linker with 15 amino acid residuesand a modified IgG2b hinge peptide with 22 residues. Proteolyticstability and Ox binding properties of the six different scFvderivatives produced in Escherichia coli were investigated andcompared with those of the corresponding Fv fragment containingno joining peptide between the V domains. The hapten bindingproperties of different antibody fragments were studied by ELISAand BIAcore^TM. The interdomain linker peptide improved the haptenbinding properties of the antibody fragment when compared withFv fragment, but slightly increased its susceptibility to proteases.Single-chain antibodies with short CBHI linkers of 11, six andtwo residues had a tendency to form multimers which led to ahigher apparent affinity. The fragments with linkers longerthan 11 residues remained monomeric.     

Site-specific chemical modification of proteins with a prelabelled cysteine tag using the artificially split Mxe GyrA intein

The selective modification of proteins with a synthetic probe is of central interest for many aspects of protein chemistry. We have recently reported a new approach in which a short cysteine-containing tag (CysTag) fused to one part of a split intein is first modified with a sulfhydryl-reactive probe. In a second step, protein trans-splicing is used to link the labelled CysTag to a target protein that has been expressed in fusion with the complementary split intein fragment. Here, we present the generation and biochemical characterisation of the artificially split Mycobacterium xenopi GyrA intein. We show that this split intein is active without a renaturation step and that it provides a significant improvement for the CysTag protein-labelling approach in terms of product yields and target protein tolerance. Two proteins with multiple cysteine residues, human growth hormone and a multidomain nonribosomal peptide synthetase, were site-specifically modified with high yields. Our approach combines the benefits of the plethora of commercially available cysteine-reactive probes with a straightforward route for their site-specific incorporation even into complex and cysteine-rich proteins.     

A strong thrombin-inhibitory prourokinase derivative with sequence elements from hirudin and the human thrombin receptor

In order to design plasminogen activators with improved thrombolytic properties, bifunctional proteins with both plasminogen-activating and anticoagulative activity were constructed by fusing a thrombin- inhibitory moiety itself comprises four elements: linker 1, a motif directed to thrombin's active site, linker 2 and a fragment of hirudin which binds to the fibrinogen-recognition site of thrombin. In order to improve further the anticoagulative activity, the thrombin-inhibitory domain was modified by substituting linker 2. Introduction of a linker (FLLRNP) from the human thrombin receptor conferred about a 10-fold increase in anticoagulative activity in protein M37 compared with the parent molecule M23 carrying an aliphatic linker. The increase in anticoagulative activity was also reflected in the shift of the Ki value from 159 +/- 20 nM for M23 to 2.0 +/- 0.5 nM for M37. The increased thrombin-inhibitory activity of M37 may be due to the presence of an arginine in the linker from the thrombin receptor which may interact with one of two glutamic acid residues located at the exit of the thrombin substrate binding pocket. This explanation is supported by the observation that another chimera (M35) carrying a linker sequence with two acidic residues has relatively weak thrombin- inhibitory activity. The thrombin-inhibitory activity of M37 may be strong enough to substitute anticoagulative co-medication during fibrinolytic treatment.      

Solid-Phase Synthesis and Application of a Clickable Version of Epoxomicin for Proteasome Activity Analysis

Degradation of proteins by the proteasome is an essential cellular process and one that many wish to study in a variety of disease types. There are commercially available probes that can monitor proteasome activity in cells, but they typically contain common fluorophores that limit their simultaneous use with other activity-based probes. In order to exchange the fluorophore or incorporate an enrichment tag, the proteasome probe likely has to be synthesized which can be cumbersome. Here, we describe a simple synthetic procedure that only requires one purification step to generate epoxomicin, a selective proteasome inhibitor, with a terminal alkyne. Through a copper-catalyzed cycloaddition, any moiety containing an azide can be incorporated into the probe. Many fluorophores are commercially available that contain an azide that can be “clicked”, allowing this proteasome activity probe to be included into already established assays to monitor both proteasome activity and other cellular activities of interest.     

Deep Sequencing of a Systematic Peptide Library Reveals Conformationally-Constrained Protein Interface Peptides that Disrupt a Protein-Protein Interaction

Disrupting protein-protein interactions is difficult due to the large and flat interaction surfaces of the binding partners. The BLIP and BLIP-II proteins are unrelated in sequence and structure and yet each potently inhibit β-lactamases. High-throughput oligonucleotide synthesis was used to construct a 12,470-member library containing overlapping linear and cyclic peptides ranging in size from 6 to 21 amino acids that scan through the sequences of BLIP and BLIP-II. Phage display affinity selections and deep sequencing revealed that, despite the differences in interaction surfaces with β-lactamases, rapid enrichment of consensus peptide regions originating from both BLIP and BLIP-II contact residues in the binding interface occurred. BLIP and BLIP-II peptides that were enriched by affinity selection were shown to bind β-lactamases and disrupt the BLIP/β-lactamase interaction. The results suggest that peptides that bind at and disrupt PPI interfaces can be identified through systematic peptide library construction, affinity selection, and deep sequencing.     

A Minimalist Substrate for Enzymatic Peptide and Protein Conjugation

Recently a number of nonnatural prenyl groups containing alkynes and azides have been developed as handles to perform click chemistry on proteins and peptides ending in the sequence “CAAX”, where C is a cysteine that becomes alkylated, A is an aliphatic amino acid and X is any amino acid. When such molecules are modified, a tag containing a prenyl analogue and the “CAAX box” sequence remains. Here we report the synthesis of an alkyne‐containing substrate comprised of only nine nonhydrogen atoms. This substrate was synthesized in six steps from 3‐methylbut‐2‐en‐1‐ol and has been enzymatically incorporated into both proteins and peptides by using protein farnesyltransferase. After prenylation the final three amino acids required for enzymatic recognition can be removed by using carboxypeptidase Y, leaving a single residue (the cysteine from the “CAAX box”) and the prenyl analogue as the only modifications. We also demonstrate that this small tag minimizes the impact of the modification on the solubility of the targeted protein. Hence, this new approach should be useful for applications in which the presence of a large tag hinders the modified protein’s solubility, reactivity, or utility.     

Tagged small molecule library approach for facilitated chemical genetics

Chemical genetics is a powerful method which utilizes small molecule regulators to reveal the molecular basis of diverse biological processes. However, the current chemical genetic approach sometimes meets a serious bottleneck during the process of target identification. One faces difficulty in conjugating the active compound to an affinity matrix without losing or reducing its activity that leads to laborious structure-activity relationship (SAR) studies. To facilitate this process, we have developed a tagged triazine library containing a built-in linker that provides a straightforward transition from phenotypic screening to target identification. A strategy for constructing a tagged library and applications with a streamlined target identification and subsequent mechanistic study are discussed in this Account.     

Exploring Non‐obvious Hydrophobic Binding Pockets on Protein Surfaces: Increasing Affinities in Peptide–Protein Interactions

A 42‐residue polypeptide conjugated to a small‐molecule organic ligand capable of targeting the phosphorylated side chain of Ser15 was shown to bind glycogen phosphorylase a (GPa) with a K_D value of 280 nm . The replacement of hydrophobic amino acids by Ala reduced affinities, whereas the incorporation of l ‐2‐aminooctanoic acid (Aoc) increased them. Replacing Nle5, Ile9 and Leu12 by Aoc reduced the K_D value from 280 to 27 nm . “Downsizing” the 42‐mer to an undecamer gave rise to an affinity for GPa an order of magnitude lower, but the undecamer in which Nle5, Ile9 and Leu12 were replaced by Aoc showed a K_D value of 550 nm , comparable with that of the parent 42‐mer. The use of Aoc residues offers a convenient route to increased affinity in protein recognition as well as a strategy for the “downsizing” of peptides essentially without loss of affinity. The results show that hydrophobic binding sites can be found on protein surfaces by comparing the affinities of polypeptide conjugates in which Aoc residues replace Nle, Ile, Leu or Phe with those of their unmodified counterparts. Polypeptide conjugates thus provide valuable opportunities for the optimization of peptides and small organic compounds in biotechnology and biomedicine.     

On-bead chemical synthesis and display of phosphopeptides for affinity pull-down proteomics

We describe a new method for phosphopeptide proteomics based on the solid-phase synthesis of phosphopeptides on beads suitable for affinity pull-down experiments. Peptide sequences containing the Bad Ser112 and Ser136 phosphorylation motifs were used as bait in affinity pull-down experiments to determine their ability to bind 14-3-3 proteins. Support-bound peptides were assembled directly on the solid support (PEGA) by standard solid-phase synthesis through a BAL-type handle. The peptides were varied in length and sequence. This synthetic strategy also allowed introduction of a soft electrophile (aldehyde) at the C terminus for potential activity-based proteomics. The synthetic support-bound Bad phosphopeptides were able to pull down 14-3-3zeta. Furthermore, Bad phosphopeptides bound endogenous 14-3-3 proteins, and all seven members of the 14-3-3 family were identified by mass spectrometry. In control experiments, none of the unphosphorylated Bad peptides bound transfected 14-3-3zeta or endogenous 14-3-3. We conclude that the combined synthesis and display of phosphopeptides on-bead is a fast and efficient method for affinity pull-down proteomics.     

Affinity and Specificity for Binding to Glycosaminoglycans Can Be Tuned by Adapting Peptide Length and Sequence

Although glycosaminoglycan (GAG)–protein interactions are important in many physiological and pathological processes, the structural requirements for binding are poorly defined. Starting with GAG-binding peptide CXCL9(74-103), peptides were designed to elucidate the contribution to the GAG-binding affinity of different: (1) GAG-binding motifs (i.e., BBXB and BBBXXB); (2) amino acids in GAG-binding motifs and linker sequences; and (3) numbers of GAG-binding motifs. The affinity of eight chemically synthesized peptides for various GAGs was determined by isothermal fluorescence titration (IFT). Moreover, the binding of peptides to cellular GAGs on Chinese hamster ovary (CHO) cells was assessed using flow cytometry with and without soluble GAGs. The repetition of GAG-binding motifs in the peptides contributed to a higher affinity for heparan sulfate (HS) in the IFT measurements. Furthermore, the presence of Gln residues in both GAG-binding motifs and linker sequences increased the affinity of trimer peptides for low-molecular-weight heparin (LMWH), partially desulfated (ds)LMWH and HS, but not for hyaluronic acid. In addition, the peptides bound to cellular GAGs with differential affinity, and the addition of soluble HS or heparin reduced the binding of CXCL9(74-103) to cellular GAGs. These results indicate that the affinity and specificity of peptides for GAGs can be tuned by adapting their amino acid sequence and their number of GAG-binding motifs.     

N-terminal specific fluorescence labeling of proteins through incorporation of fluorescent hydroxy acid and subsequent ester cleavage

We have developed a novel method to attach a fluorescent label at the N terminus of proteins through a four-base codon-mediated incorporation of a fluorescent hydroxy acid and subsequent cleavage of the ester bond in a cell-free translation system. We found that a fluorescent-labeled p-amino-L-phenyllactic acid was successfully incorporated downstream of N-terminal tag peptides in response to a CGGG codon, and the tag peptides could be removed through ester cleavage to leave the fluorescent hydroxy acid at the N terminus of the proteins. Immunoprecipitation analysis revealed that ester cleavage occurred spontaneously during the translation reaction. The efficiency of the ester cleavage and the yield of the labeled proteins were dependent on the peptide tag sequence. We demonstrate that the insertion of an asparagine residue between the N-terminal T7 tag and the fluorescent hydroxy acid achieved both quantitative ester cleavage and efficient expression of the labeled proteins. The present method is a potential tool for N-terminal specific labeling of proteins with various compounds.     

Versatile Interacting Peptide (VIP) Tags for Labeling Proteins with Bright Chemical Reporters

Fluorescence microscopy is an essential tool for the biosciences, enabling the direct observation of proteins in their cellular environment. New methods that facilitate attachment of photostable synthetic fluorophores with genetic specificity are needed to advance the frontiers of biological imaging. Here, we describe a new set of small, selective, genetically encoded tags for proteins based on a heterodimeric coiled‐coil interaction between two peptides: CoilY and CoilZ. Proteins expressed as a fusion to CoilZ were selectively labeled with the complementary CoilY fluorescent probe peptide. Fluorophore‐labeled target proteins were readily detected in cell lysates with high specificity and sensitivity. We found that these versatile interacting peptide (VIP) tags allowed rapid and specific delivery of bright organic dyes or quantum dots to proteins displayed on living cells. Additionally, we validated that either CoilY or CoilZ could serve as the VIP tag, which enabled us to observe two distinct cell‐surface protein targets with this one heterodimeric pair.     

Engineering a de novo-designed coiled-coil heterodimerization domain for the rapid detection, purification and characterization of recombinantly expressed peptides and proteins

Using the techniques of genetic engineering and the principlesof protein de novo design, we have developed a unique affinitymatrix protein tag system as a rapid, convenient and sensitivemethod to detect, purify and characterize newly expressed recombinantpeptides or proteins from cell extracts. The method utilizestwo de novo-designed linear peptide sequences that can selectivelydimerize to form the stable protein motif, the two-stranded-helical coiled-coil. In this method, a recombinant bacterialexpression vector pRLDE has been engineered so that one of thedimerization strands (E-coil) is expressed as a C-terminal fusiontag on newly expressed peptides or proteins, while the other(K-coil) is either biotin-labeled for detection in a Westernblot-type format or immobilized on an insoluble silica supportfor selective dimerization affinity chromatography. Recombinantlyexpressed peptides from Escherichia coli containing the dimerizationtag have been produced, detected and purified using this method.The recombinant peptides were easily and clearly identifiedusing the biotin-labeled coil, while the single-step affinitypurification results indicated the purity of the affinity purifiedexpressed peptides to be >95%, as assessed by reversed-phasechromatography. The stability of the dimerization domain alsoallows for the purified peptide to be left attached to the matrix,thus creating a new peptide-bound column that can be used tostudy peptide–protein or peptide–ligand interactions.Therefore this system offers a new alternative to existing peptideor protein fusion tags and demonstrates the utility of a denovo-designed system.     

Conversion of Low‐Affinity Peptides to High‐Affinity Peptide Binders by Using a β‐Hairpin Scaffold‐Assisted Approach

Affinity maturation of protein‐targeting peptides is generally accomplished by homo‐ or heterodimerization of known peptides. However, applying a heterodimerization approach is difficult because it is not clear a priori what length or type of linker is required for cooperative binding to a target. Thus, an efficient and simple affinity maturation method for converting low‐affinity peptides into high‐affinity peptides would clearly be advantageous for advancing peptide‐based therapeutics. Here, we describe the development of a novel affinity maturation method based on a robust β‐hairpin scaffold and combinatorial phage‐display technology. With this strategy, we were able to increase the affinity of existing peptides by more than four orders of magnitude. Taken together, our data demonstrate that this scaffold‐assisted approach is highly efficient and effective in generating high‐affinity peptides from their low‐affinity counterparts.     

Chemical tags for labeling proteins inside living cells

To build on the last century's tremendous strides in understanding the workings of individual proteins in the test tube, we now face the challenge of understanding how macromolecular machines, signaling pathways, and other biological networks operate in the complex environment of the living cell. The fluorescent proteins (FPs) revolutionized our ability to study protein function directly in the cell by enabling individual proteins to be selectively labeled through genetic encoding of a fluorescent tag. Although FPs continue to be invaluable tools for cell biology, they show limitations in the face of the increasingly sophisticated dynamic measurements of protein interactions now called for to unravel cellular mechanisms. Therefore, just as chemical methods for selectively labeling proteins in the test tube significantly impacted in vitro biophysics in the last century, chemical tagging technologies are now poised to provide a breakthrough to meet this century's challenge of understanding protein function in the living cell. With chemical tags, the protein of interest is attached to a polypeptide rather than an FP. The polypeptide is subsequently modified with an organic fluorophore or another probe. The FlAsH peptide tag was first reported in 1998. Since then, more refined protein tags, exemplified by the TMP- and SNAP-tag, have improved selectivity and enabled imaging of intracellular proteins with high signal-to-noise ratios. Further improvement is still required to achieve direct incorporation of powerful fluorophores, but enzyme-mediated chemical tags show promise for overcoming the difficulty of selectively labeling a short peptide tag. In this Account, we focus on the development and application of chemical tags for studying protein function within living cells. Thus, in our overview of different chemical tagging strategies and technologies, we emphasize the challenge of rendering the labeling reaction sufficiently selective and the fluorophore probe sufficiently well behaved to image intracellular proteins with high signal-to-noise ratios. We highlight recent applications in which the chemical tags have enabled sophisticated biophysical measurements that would be difficult or even impossible with FPs. Finally, we conclude by looking forward to (i) the development of high-photon-output chemical tags compatible with living cells to enable high-resolution imaging, (ii) the realization of the potential of the chemical tags to significantly reduce tag size, and (iii) the exploitation of the modular chemical tag label to go beyond fluorescent imaging.     

Rapid isolation of high-affinity protein binding peptides using bacterial display

A robust bacterial display methodology was developed that allows the rapid isolation of peptides that bind to arbitrarily selected targets with high affinity. To demonstrate the utility of this approach, a large library (5 x 10(10) clones) was constructed composed of random 15-mer peptide insertions constrained within a flexible, surface exposed loop of the Escherichia coli outer membrane protein A (OmpA). The library was screened for binding to five unrelated proteins, including targets previously used in phage display selections: human serum albumin, anti-T7 epitope mAb, human C-reactive protein, HIV-1 GP120 and streptavidin. Two to four rounds of enrichment (2-4 days) were sufficient to enrich peptide ligands having high affinity for each of the target proteins. Strong amino acid consensus sequences were apparent for each of the targets tested, with up to seven consensus residues. Isolated peptide ligands remained functional when expressed as insertional fusions within a monomeric fluorescent protein. This bacterial display methodology provides an efficient process for identifying peptide affinity reagents and should be useful in a variety of molecular recognition applications.