We aspired to create chemical recognition units, which bind oligohistidine tags with high affinity and stability, as tools for selectively attaching spectroscopic probes and other functional elements to recombinant proteins. Several supramolecular entities containing 2-4 nitrilotriacetic acid (NTA) moieties were synthesized, which additionally contained an amino group, to which fluorescein was coupled as a sensitive reporter probe. These multivalent chelator heads (MCH) (termed bis-, tris-, and tetrakis-NTA) were characterized with respect to their interaction with hexahistidine (H6)- and decahistidine (H10)-tagged targets. Substantially increased binding stability with increasing number of NTA moieties was observed by analytical size exclusion chromatography. The binding enthalpies as determined by isothermal titration calorimetry increased nearly additively with the number of possible coordinative bonds between chelator heads and tags. Yet, a substantial excess of histidines in the oligohistidine tag was required for obtaining fully additive binding enthalpies. Dissociation kinetics of MCH/oligohistidine complexes measured by fluorescence dequenching showed an increase in stability by 4 orders of magnitude compared to that of mono-NTA, and subnanomolar affinity was reached for tris-NTA. The gain in free energy with increasing multivalency was accompanied by an increasing loss of entropy, which was ascribed to the high flexibility of the binding partners. Numerous applications of these MCHs for noncovalent, high affinity, yet reversible tethering of spectroscopic probes and other functional elements to the recombinant proteins can be envisioned.
Photosensitive silanes containing nitroveratryl (Nvoc)-caged amine groups and protein repellent tetraethylene glycol units were synthesized and used for modification of silica surfaces. Functional surface layers containing different densities of caged amine groups were prepared and activated by UV-irradiation of the surface. The performance of these layers for functional and site-selective immobilization of proteins was tested. For this purpose, biotin and tris-nitrilotriacetic acid (tris-NTA) were fist coupled to the activated surface, and the interaction of streptavidin and His-tagged proteins with the functionalized surfaces was monitored by real-time label-free detection. After optimizing the coupling protocols, highly selective functionalization of the deprotected amine groups was possible. Furthermore, the degree of functionalization (and therefore the amount of immobilized protein) was controlled by diluting the surface concentration of the amine-functionalized silane with a nonreactive (OMe-terminated) tetraethylene glycol silane. Immobilized proteins were highly functional on these surfaces, as demonstrated by protein-protein interaction assays with the type I interferon receptor. Protein micropatterns were successfully generated after masked irradiation and functionalization of the caged surface following the optimized coupling protocols.
Site-specific conjugation of proteins to surfaces, spectroscopic probes, or other functional units is a key task for implementing biochemical assays. The streptavidin-biotin interaction has proven a highly versatile tool for detection, quantification, and functional analysis of proteins. We have developed an approach for site-specific reversible biotinylation of recombinant proteins through their histidine tag using biotin conjugated to the multivalent chelator trisnitrilotriacetic acid (BTtris-NTA). Stable binding of BTtris-NTA to His-tagged proteins was demonstrated, which is readily reversed by addition of imidazole, enabling versatile conjugation schemes in solution as well as at interfaces. Gel filtration experiments revealed that His-tagged proteins bind to streptavidin doped with BTtris-NTA in a 2:1 stoichiometry. Furthermore, an increased binding affinity toward His-tagged proteins was observed for BTtris-NTA linked to streptavidin compared to tris-NTA in solution and on surfaces. These results indicate an efficient cooperative interaction of two adjacent tris-NTA moieties with a single His-tag, yielding an extremely tight complex with a lifetime of several days. We demonstrate several applications of BTtris-NTA including multiplexed capturing of proteins to biosensor surfaces, cell surface labeling, and Western blot detection. The remarkable selectivity of the His-tag-specific biotinylation, as well as the highly stable, yet reversible complex provides the basis for numerous further applications for functional protein analysis.
We have undertaken a structural and functional study of self-assembled monolayers (SAMs) formed on gold from a series of alkylthiol compounds containing terminal multivalent chelators (MCHs) composed of mono-, bis-, and tris-nitrilotriacetic acid (NTA) moieties. SAMs were formed from single-component solutions of the mono-, bis-, and tris-NTA compounds, as well as from mixtures with a tri(ethylene glycol)-terminated alkylthiol (EG(3)). Contact angle goniometry, null ellipsometry, and infrared spectroscopy were used to explore the structural characteristics of the MCH SAMs. Ellipsometric measurements show that the amount of the MCH groups on surfaces increases with increasing mol % of the MCH thiols in the loading solution up to about 80 mol %. We also conclude that mixed SAMs, prepared in the solution composition regime 0-30 mol % of the MCH thiols, consist of a densely packed alkyl layer, an amorphous ethylene glycol layer, and an outermost layer of MCH groups exposed toward the ambient. Above 30 mol %, a significant degree of disorder is observed in the SAMs. Finally, functional evaluation of the three MCH SAMs prepared at 0-30 mol% reveals a consistent increase in binding strength with increasing multivalency. The tris-NTA SAM, in particular, is enabled for stable and functional immobilization of a His6-tagged extracellular receptor subunit, even at low chelator surface concentrations, which makes it suitable for applications when a low surface density of capturing sites is desirable, e.g., in kinetic analyses.
Chemical biology aims for a perfect control of protein complexes in time and space by their site-specific labeling, manipulation, and structured organization. Here we developed a self-inactivated, lock-and-key recognition element whose binding to His-tagged proteins can be triggered by light from zero to nanomolar affinity. Activation is achieved by photocleavage of a tethered intramolecular ligand arming a multivalent chelator head for high-affinity protein interaction. We demonstrate site-specific, stable, and reversible binding in solution as well as at interfaces controlled by light with high temporal and spatial resolution. Multiplexed organization of protein complexes is realized by an iterative in situ writing and binding process via laser scanning microscopy. This light-triggered molecular recognition should allow for a spatiotemporal control of protein-protein interactions and cellular processes by light-triggered protein clustering.
Functional immobilization and lateral organization of proteins into micro- and nanopatterns is an important prerequisite for miniaturizing bioanalytical and biotechnological devices. Here, we report an approach for efficient site-specific protein immobilization based on enzymatic phosphopantetheinyl transfer (PPT) from coenzyme A (CoA)-functionalized glass-type surfaces to specific peptide tags. We devised a bottom-up surface modification approach for coupling CoA densely to a molecular poly(ethylene glycol) polymer brush. Site-specific enzymatic immobilization of proteins fused to different target peptides for the PPTase Sfp was confirmed by real-time label-free detection. Quantitative protein-protein interaction experiments confirmed that significantly more than 50% of the immobilized protein was fully active. The method was successfully applied with different proteins. However, different immobilization efficiencies of PPT-based immobilization were observed for different peptide tags being fused to the N- and C-termini of proteins. On the basis of this immobilization method, we established photolithographic patterning of proteins into functional binary microstructures.
We describe herein a platform to study protein-protein interactions and to form functional protein complexes in nanoscopic surface domains. For this purpose, we employed multivalent chelator (MCh) templates, which were fabricated in a stepwise procedure combining dip-pen nanolithography (DPN) and molecular recognition-directed assembly. First, we demonstrated that an atomic force microscope (AFM) tip inked with an oligo(ethylene glycol) (OEG) disulfide compound bearing terminal biotin groups can be used to generate biotin patterns on gold achieving line widths below 100 nm, a generic platform for fabrication of functional nanostructures via the highly specific biotin-streptavidin recognition. Subsequently, we converted such biotin/streptavidin patterns into functional MCh patterns for reversible assembly of histidine-tagged (His-tagged) proteins via the attachment of a tris-nitriloacetic acid (trisNTA) biotin derivative. Fluorescence microscopy confirmed reversible immobilization of the receptor subunit ifnar2-His10 and its interaction with interferon-alpha2 labeled with fluorescent quantum dots in a 7 x 7 dot array consisting of trisNTA spots with a diameter of approximately 230 nm. Moreover, we carried out characterization of the specificity, stability, and reversibility as well as quantitative real-time analysis of protein-protein interactions at the fabricated nanopatterns by imaging surface plasmon resonance. Our work offers a route for construction and analysis of functional protein-based nanoarchitectures.
The conformational dynamics of proteins plays a key role in their complex physiological functions. Fluorescence resonance energy transfer (FRET) is a particular powerful tool for studying protein conformational dynamics, but requires efficient site-specific labeling with fluorescent reporter probes. We have employed different tris-NTA/fluorophore conjugates, which bind histidine-tagged proteins with high affinity, for site-specific incorporation of FRET acceptors into proteins, which were covalently labeled with a donor fluorophore. We demonstrate versatile application of this approach for exploring the conformation of the type I interferon receptor ectodomains ifnar1-EC and ifnar2-EC. Substantial ligand-induced conformational changes of ifnar1-EC, but not ifnar2-EC, were observed by monitoring the fluorescence intensity and the fluorescence lifetime of the FRET donor. Time-resolved fluorescence correlation spectroscopy revealed a substantial conformational flexibility of ifnar1-EC and a ligand-induced tightening. Our results demonstrate that protein labeling with tris-NTA/fluorophores enables for efficient quantitative intramolecular FRET analysis.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.