The development of multivalent protein ligands for nanoparticles lags behind that of multidentate polymers and small-molecule ligands largely because of a lack of thorough understanding of the interaction between nanoparticles and multimeric proteins. Guided by protein crystal structures, we have harnessed recombinant technology to develop a collection of mCherry fused multimeric proteins with different spatial distributions of the quantum dot (QD)-binding sequence, hexahistidine tag (histag). All of the proteins can behave as ligands to assemble with ZnS-CdSe QDs through metal-affinity-driven self-assembly. We have observed that protein shape and geometry greatly affect the stoichiometry and stability of their assemblies with QDs. We also demonstrate a peptide-induced structural transition of a nanobelt protein that preorganizes the QD-binding sites and effects a more efficient assembly with QDs. This work reports the first multifaceted investigation on how multivalent proteins, in particular, dimers, tetramers, and linear multidentate proteins, assemble with QDs. It also manifests our capability of harnessing structural and conformational information about proteins to design multivalent protein ligands for QD surface functionalization.

A short, flexible, and unstructured peptide tag that has versatile and facile use in protein labeling applications is highly desirable. Here, we report an 11-residue peptide tag with an internal cysteine (a W-tag, derived from a Comm PY peptide motif that is known to bind with Nedd4 WW3* domain) that can be installed at different regions of the target protein without compromising its covalent reactivity with the reactive label (a 35-residue synthetic Nedd4 WW3* domain derivative). This versatility is explained by the unique structural features of the reaction. NMR analysis reveals that both the W-tag peptide and reactive Nedd4 WW3* protein are unstructured before they encounter each other. The binding interaction of the two induces noticeable structural changes and promotes global folding. Consequently, the reactive cysteine residue at W-tag and the electrophilic chloroacetyl group at Nedd4 WW3* domain are positioned to be in close proximity, inducing an intermolecular covalent cross-linking. The covalent linkage in turn stabilizes the folding of the protein complex. This unique multistep mechanism renders this labeling reaction amenable to different sites of the proteins of interest: installation of the tag at N- and C-termini, in the flexible linker region, in the loop region, and the extracellular terminus of target proteins exhibited comparable reactivity. This work therefore represents the first proximity-induced cysteine reaction based on the unique binding features of WW domains that demonstrates unprecedented versatility.

•Comprehensive review on site-accurate bioconjugation reactions.•Comprehensive review on proximity-induced covalent reactions.To achieve precise control of the signaling events or to achieve unmistakable synthesis of biomolecules, nature has evolved organic reactions involving proteinogenic amino acids with unparalleled site selectivity. For example, dedicated enzymes accurately dictate the site of post-translational modifications in signaling proteins, and ribosomes precisely link the C-terminal carboxylic acid of one unprotected amino acid with the N-terminal amino group of the other amino acid through spatially confined proximity. For many years, chemists have been striving to achieve site selectivity on biomolecules by mimicking nature. Driven by the development of chemoselective protein conjugation reactions, enzymology and protein–protein interactions, the past decade has witnessed a boom in site-selective protein conjugation reactions. (In this review, a site-selective protein conjugation reaction is defined as an organic reaction that targets a single amino acid instead of a kind of amino acids in a protein or a proteome under physiological conditions, for example, a single cysteine residue among all of the cysteines.) In this review, we summarize the recent advancements of bioconjugation reactions that demonstrate this feature of precise site selectivity, focusing on the reactions of the proteinogenic amino acids (excluding those at non-coded or non-proteinogenic amino acids that are introduced to proteins through genetic manipulations).Download high-res image (171KB)Download full-size image

Intracellular reactions on nonenzymatic proteins that activate cellular signals are rarely found. We report one example here that a designed peptide derivative undergoes a nucleophilic reaction specifically with a cytosolic PDZ protein inside cells. This reaction led to the activation of ephrin-B reverse signaling, which subsequently inhibited SDF-1 induced neuronal chemotaxis of human neuroblastoma cells and mouse cerebellar granule neurons. Our work provides direct evidence that PDZ-RGS3 bridges ephrin-B reverse signaling and SDF-1 induced G protein signaling for the first time.

Specific and dynamic biological interactions pave the blueprint of signal networks in cell. For example, a great variety of specific protein-ligand interactions define how intracellular signals flow. Taking advantage of the specificity of these interactions, we postulate an “affinity-guided covalent conjugation” strategy to lock binding ligands through covalent reactions between the ligand and the receptor protein. The presence of a nucleophile close to the ligand binding site of a protein is sine qua none of this reaction. Specific noncovalent interaction of a ligand derivative (which contains an electrophile at a designed position) to the ligand binding site of the protein brings the electrophile to the close proximity of the nucleophile. Subsequently, a conjugation reaction spontaneously takes place between the nucleophile and the electrophile, and leads to an intermolecular covalent linkage. This strategy was first showcased in coiled coil peptides which include a cysteine mutation at a selected position. The short peptide sequence was used for covalent labeling of cell surface receptors. The same strategy was then used to guide the design of a set of protein Lego bricks for covalent assembly of protein complexes of unnatural geometry. We finally made “reactive peptides” for natural adaptor proteins that play significant roles in signal transduction. The peptides were designed to react with a single domain of the multidomain adaptor protein, delivered into the cytosol of neurons, and re-directed the intracellular signal of neuronal migration. The trilogy of protein labeling, assembly, and inhibition of intracellular signals, all through a specific covalent bond, fully demonstrated the generality and versatility of “affinity-guided covalent conjugation” in various applications.

A key challenge in bioconjugation is to control the site selectivity of the reaction. Chemical reagents often react with proteineous chemical groups without showing preference to their location or microenvironment in the protein; to confine the reaction to an amino acid at a specific site, one needs to distinguish this residue from others despite their identical chemical properties. Here, we report a strategy that utilizes proximity-driven reactivity to achieve site selective azo coupling between tyrosine and aryldiazonium. A phenylalanine analogue with an aryldiazonium moiety at its side chain was incorporated into a synthetic peptide and was found to react only with tyrosine in its vicinity but also to remain inert to others that are not immediately adjacent, a property attained by fine regulation of the electronic effect of the substituent on the aryl ring. Proximity-driven intramolecular azo coupling was showcased in cyclization of a β-hairpin peptide, structural features of the azo linked cyclic peptide was elucidated by NMR, and intermolecular azo coupling was achieved between an SH3 protein Abl-SH3 and its polyproline peptide ligands at specific tyrosine residues. This approach is generally applicable to develop covalent affinity labels for SH3 proteins because of the high occurrence rate of tyrosine at the peptide-binding site of SH3 proteins.

To label proteins covalently, one faces a trade-off between labeling a protein specifically and using a small tag. Often one must compromise one parameter for the other or use additional components, such as an enzyme, to satisfy both requirements. Here, we report a new reaction that covalently labels proteins by using engineered coiled-coil peptides. Harnessing the concept of “proximity-induced reactivity”, the 21-amino-acid three-heptad peptides CCE/CCK were modified with a nucleophilic cysteine and an α-chloroacetyl group at selected positions. When pairs of coiled coils associated, an irreversible covalent bond spontaneously formed between the peptides. The specificity of the cross-linking reaction was characterized, the probes were improved by making them bivalent, and the system was used to label a protein in vitro and receptors on the surface of mammalian cells.

Specific protein–peptide interactions are prevalent in the living cells and form a tightly regulated signaling network. These interactions, many of which have structural information revealed, provide ideal templates for affinity-guided covalent bioconjugation. Here we report the development of a set of four new reactions that covalently and site-specifically link nonenzymatic scaffolding domains (two PDZ and two SH3 domains) and their ligands through thiol-chloroacetyl SN2 reaction. Guided by the three-dimensional structure of the wild type complex, a selected position of the protein was mutated to cysteine, and at the same time, an α-chloroacetyl group was installed at a corresponding position of the peptide. Specific binding interaction between the two brings the reactive groups into close proximity, converts the nonreactive cysteine residue into a content-dependent reactive site, and induces the nucleophilic reaction that is inert in the absence of the binding event. The specificity, orthogonality, and modularity of the four reactions were characterized, the reaction was applied to label proteins in vitro and receptor on the surface of mammalian cells, and the system was utilized to assemble covalent protein complexes with unnatural geometries.

Multienzyme complexes are of paramount importance in biosynthesis in cells. Yet, how sequential enzymes of cascade catalytic reactions synergize their activities through spatial organization remains elusive. Recent development of site-specific protein–nanoparticle conjugation techniques enables us to construct multienzyme assemblies using nanoparticles as the template. Sequential enzymes in menaquinone biosynthetic pathway were conjugated to CdSe-ZnS quantum dots (QDs, a nanosized particulate material) through metal-affinity driven self-assembly. The assemblies were characterized by electrophoretic methods, the catalytic activities were monitored by reverse-phase chromatography, and the composition of the multienzyme–QD assemblies was optimized through a progressive approach to achieve highly efficient catalytic conversion. Shorter enzyme–enzyme distance was discovered to facilitate intermediate transfer, and a fine control on the stoichiometric ratio of the assembly was found to be critical for the maximal synergy between the enzymes. Multienzyme–QD assemblies thereby provide an effective model to scrutinize the synergy of cascade enzymes in multienzyme complexes.

Marine organisms including bacteria, fungi, algae, sponges, echinoderms, mollusks, and cephalochordates produce a variety of products with antifungal activity including bacterial chitinases, lipopeptides, and lactones; fungal (-)-sclerotiorin and peptaibols, purpurides B and C, berkedrimane B and purpuride; algal gambieric acids A and B, phlorotannins; 3,5-dibromo-2-(3,5-dibromo-2-methoxyphenoxy)phenol, spongistatin 1, eurysterols A and B, nortetillapyrone, bromotyrosine alkaloids, bis-indole alkaloid, ageloxime B and (-)-ageloxime D, haliscosamine, hamigeran G, hippolachnin A from sponges; echinoderm triterpene glycosides and alkene sulfates; molluscan kahalalide F and a 1485-Da peptide with a sequence SRSELIVHQR; and cepalochordate chitotriosidase and a 5026.9-Da antifungal peptide. The antiviral compounds from marine organisms include bacterial polysaccharide and furan-2-yl acetate; fungal macrolide, purpurester A, purpurquinone B, isoindolone derivatives, alterporriol Q, tetrahydroaltersolanol C and asperterrestide A, algal diterpenes, xylogalactofucan, alginic acid, glycolipid sulfoquinovosyldiacylglycerol, sulfated polysaccharide p-KG03, meroditerpenoids, methyl ester derivative of vatomaric acid, lectins, polysaccharides, tannins, cnidarian zoanthoxanthin alkaloids, norditerpenoid and capilloquinol; crustacean antilipopolysaccharide factors, molluscan hemocyanin; echinoderm triterpenoid glycosides; tunicate didemnin B, tamandarins A and B and; tilapia hepcidin 1-5 (TH 1-5), seabream SauMx1, SauMx2, and SauMx3, and orange-spotted grouper β-defensin. Although the mechanisms of antifungal and antiviral activities of only some of the aforementioned compounds have been elucidated, the possibility to use those known to have distinctly different mechanisms, good bioavailability, and minimal toxicity in combination therapy remains to be investigated. It is also worthwhile to test the marine antimicrobials for possible synergism with existing drugs. The prospects of employing them in clinical practice are promising in view of the wealth of these compounds from marine organisms. The compounds may also be used in agriculture and the food industry.

The development of multivalent protein ligands for nanoparticles lags behind that of multidentate polymers and small-molecule ligands largely because of a lack of thorough understanding of the interaction between nanoparticles and multimeric proteins. Guided by protein crystal structures, we have harnessed recombinant technology to develop a collection of mCherry fused multimeric proteins with different spatial distributions of the quantum dot (QD)-binding sequence, hexahistidine tag (histag). All of the proteins can behave as ligands to assemble with ZnS-CdSe QDs through metal-affinity-driven self-assembly. We have observed that protein shape and geometry greatly affect the stoichiometry and stability of their assemblies with QDs. We also demonstrate a peptide-induced structural transition of a nanobelt protein that preorganizes the QD-binding sites and effects a more efficient assembly with QDs. This work reports the first multifaceted investigation on how multivalent proteins, in particular, dimers, tetramers, and linear multidentate proteins, assemble with QDs. It also manifests our capability of harnessing structural and conformational information about proteins to design multivalent protein ligands for QD surface functionalization.

•We prove CE-FL to be a powerful method to resolve M2–FLAG complexes.•We reveal a marked affinity enhancement through multivalency.•We achieve chromatographic separation of antibody–peptide complexes.•We provide a direct observation of 1:1 and 1:2 antibody–peptide complexes.•The second binding constant KD2 is about 103–104 fold higher than KD1.Natural antibodies adopt multivalent constructs to effect superior binding affinities with their antigens. Notwithstanding that the structure of antibodies have been well understood, how antibodies harness multivalency effect to achieve superior binding affinity toward the antigens still remains unclear. Such investigation is often hampered by the difficulty in resolving receptor-ligand complexes with different stoichiometries in the binding solution, especially when the ligand is a small molecule or a short peptide. Here we employed a unique anti-FLAG™ mAb M2 and FLAG™ peptides system, together with fluorescence detection coupled capillary electrophoresis (CE-FL) to reveal how M2 achieves exceptional high affinity with FLAG peptides through multivalency. Complexation of fluorescently labeled FLAG™ peptides with M2 leads to a pronounced mobility shift of the fluorescent peak in CE. Remarkably, CE-FL rendered a base-line separation of 1:1 and 1:2 M2–FLAG™ complexes, allowing the quantification of different M2–FLAG™ species. The quantitative analysis leads to a detailed dissection of the first (functional affinity) and second binding affinities (intrinsic affinity) between M2 and FLAG1 peptide. The marked difference (103–104 fold) between these two affinities indicated that multivalency effect plays a key role for M2 to achieve highly efficient binding to FLAG™ peptides.

Subtle changes in the chemical structure or the composition of surface bound ligands on quantum dots (QDs) remain difficult to detect. Here we describe a facile setup for fluorescence detection coupled capillary electrophoresis (CE-FL) and its application in monitoring ligand displacement on QDs through metal-affinity driven assembly. We also describe the use of CE-FL to monitor amide bond cleavage by a specific protease, based on Förster resonance energy transfer (FRET) between Cy5 and QDs spaced by a hexahistidine peptide (H6–Cy5). CE-FL allowed separation of unbound QDs and ligand bound QDs and also revealed an ordered assembly of H6–Cy5 on QDs. In a ligand displacement experiment, unlabeled hexahistidine peptide gradually displaced surface bound H6–Cy5 until finally reaching equilibrium. The displacement intermediates were clearly separated on CE-FL. Proteolytic cleavage of surface bound H6–Cy5 by thrombin was monitored by CE-FL through mobility shift, peak broadening, and FRET changes. Enzymatic parameters thus obtained were comparable with those measured by fluorescence spectroscopy.Graphical abstractHighlights► Capillary electrophoresis reveals details in QD–oligohistidine peptide binding. ► An ordered assembly of peptides on QDs was revealed. ► Intermediates of QD–peptide binding were found. ► Detailed displacement kinetics was revealed. ► Proteolysis of surface ligands causes mobility shift and peak broadening in CE.

Polyhistidine peptide dendrimer self-assembles on CdSe/ZnS quantum dots (QDs) with very high affinity and stability, a property ascribable to its multivalent geometry. Here we designed a fluorescent protein, GCN-mCherry, that exists as an oligomeric bundled structure in solution as well as on the surface to imitate the structure of a synthetic dendrimer. GCN-mCherry forms a very stable assembly with QDs, which can resist displacement by 500 mM imidazole and the dendrimer peptide, as measured by the Förster resonance energy transfer from QD to mCherry. Our work manifested a prominent stability enhancement of protein–nanoparticle assembly through directional ligand–ligand interaction on the surface.

Engineered peptide ligands with exceptionally high affinity for metal can self-assemble with nanoparticles in biological fluids. A high-affinity dendrimeric peptide ligand for CdSe-ZnS quantum dots (QDs) exhibited very fast association kinetics with QDs and reached equilibrium within 2 s. Here, we have combined a droplet-based microfluidic device with fluorescence detection based on Förster resonance energy transfer (FRET) to provide subsecond resolution in dissecting this fast self-assembly kinetics in solution. This work represents the first application of microfluidic devices to ligand–particle assembly for the measurement of fast assembly kinetics in solution.

Fluorescence detection coupled to capillary electrophoresis (CE-FL) effectively separates molecules in solution and at the same time allows monitoring of the fluorescence spectrum of each individual species. The integration of separation and fluorescence detection results in a powerful method superior to the ensemble in-cuvette fluorescence measurement, in probing the binding interaction between ligands and quantum dots (QDs) in complex solutions. Förster resonance energy transfer (FRET) between fluorescent ligands and QDs could be readily detected by CE-FL, which together with the migration times of the fluorescent peaks provides an indication of the binding interaction between ligands and QDs. In the present study, the binding interaction between a multivalent ligand, polyhistidine peptide denderimer (PHPD), and CdSe-ZnS QDs was probed by CE-FL using the monovalent hexahistidine peptide as a control. Cy5 labeled PHPD assembles on glutathione capped QDs, showing a higher FRET signal than that of the assembly between Cy5 labeled hexahistidine peptide and QDs. Capillary electrophoresis further revealed that PHPD outcompetes other QD binding small molecules, peptides, and proteins in cell lysate. Our study demonstrates the power of CE-FL in analyzing the binding interaction between ligands and QDs in a complex binding solution. It also shows that clustering surface binding motifs yields multivalent ligands that can preferentially assemble with nanoparticles.

Gold nanoparticles functionalized with amphiphilic peptides can encapsulate Bodipy and release it by diffusion. Proteolytic cleavage of the peptide monolayer triggers the rapid release of the encapsulated Bodipy from nanoparticles to mammalian cells, representing a prototypical model of protease-promoted drug delivery using gold nanoparticles.