- Development of an ON/OFF switchable fluorescent probe targeting His tag fused proteins in living cells
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The fluorescent labeling of target proteins is useful for analyzing their functions and localization in cells, and several fluorescent probes have been developed. However, the fusion of tags such as green fluorescent protein (GFP) to target proteins occas
- Okitsu, Koyo,Misawa, Takashi,Shoda, Takuji,Kurihara, Masaaki,Demizu, Yosuke
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- Poly(N -isopropylacrylamide- co -tris-nitrilotriacetic acid acrylamide) for a combined study of molecular recognition and spatial constraints in protein binding and interactions
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Many biological processes require precise regulation and synergy of proteins, and consequently involve molecular recognition and spatial constraints between biomolecules. Here, a library of poly(N-isopropylacrylamide-co-tris-nitrilotriacetic acid acrylamide) (PNTs) has been synthesized and complexed with Cu2+ in order to serve as models for investigation of the combined effects of molecular recognition and spatial constraints in biomolecular interactions. The average distance between Cu2+-trisNTA binding sites in PNTs polymers was varied from 4.3 to 31.5 nm by adjusting their trisNTA contents. His tag (His6), His-tagged enhanced yellow fluorescent protein (His6-eYFP), and His6-tagged collagenase G (His6-ColG), with sizes ranging from 1 to 11 nm, were used as models to assess whether the binding ability is influenced by a cooperative topology based on molecular recognition interactions with Cu2+-trisNTA binding sites, and spatial constraints created by decreasing average distance between trisNTAs. His-tagged molecules bound to all PNTs polymers due to their molecular recognition interaction involving histidines and Cu2+-trisNTA pockets, but with a binding ability that was highly modulated by the average distance between the trisNTA binding sites. Small molecular mass molecules (His6) exhibit a high binding ability to all PNTs polymers, whereas his-tagged proteins bind to PNTs efficiently only when the average distance between trisNTA binding sites is larger than the protein dimensions.
- Liu, Juan,Spulber, Mariana,Wu, Dalin,Talom, Renee M.,Palivan, Cornelia G.,Meier, Wolfgang
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- Development of a Small Hybrid Molecule That Mediates Degradation of His-Tag Fused Proteins
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In recent years, the induction of target-protein degradation via the ubiquitin-proteasome system (UPS) mediated by small molecules has attracted attention, and this approach has applications in pharmaceutical development. However, this technique requires a ligand for the target protein that can be incorporated into tailor-made molecules, and there are many proteins for which such ligands have not been found. In this study, we developed a protein-knockdown method that recognizes a His-tag fused to a protein of interest. This strategy theoretically allows comprehensive targeting of proteins of interest by a particular molecule recognizing the tag. As expected, our hybrid molecule 10 [SNIPER(CH6)] efficiently degraded His-tagged CRABP-II and Smad2 in cells. This system provides an easy method to determine the susceptibility of proteins of interest to UPS-mediated degradation. Furthermore, we hope that this method will become an efficient tool to analyze the function of the UPS.
- Okitsu, Koyo,Hattori, Takayuki,Misawa, Takashi,Shoda, Takuji,Kurihara, Masaaki,Naito, Mikihiko,Demizu, Yosuke
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- The Scaffold Design of Trivalent Chelator Heads Dictates Affinity and Stability for Labeling His-tagged Proteins in vitro and in Cells
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Small chemical/biological interaction pairs are at the forefront in tracing protein function and interaction at high signal-to-background ratios in cellular pathways. However, the optimal design of scaffold, linker, and chelator head still deserve systematic investigation to achieve the highest affinity and kinetic stability for in vitro and especially cellular applications. We report on a library of N-nitrilotriacetic acid (NTA)-based multivalent chelator heads (MCHs) built on linear, cyclic, and dendritic scaffolds and compare these with regard to their binding affinity and stability for the labeling of cellular His-tagged proteins. Furthermore, we describe a new approach for tracing cellular target proteins at picomolar probe concentrations in cells. Finally, we outline fundamental differences between the MCH scaffolds and define a cyclic trisNTA chelator that displays the highest affinity and kinetic stability of all reported reversible, low-molecular-weight interaction pairs.
- Gatterdam, Karl,Joest, Eike F.,Gatterdam, Volker,Tampé, Robert
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supporting information
p. 12395 - 12399
(2018/09/18)
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- High-Affinity Copolymers Inhibit Digestive Enzymes by Surface Recognition
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This account presents a general method for the construction of polymeric surface binders for digestion enzymes. Two prominent parts, namely, the modification of the copolymer composition and the screening assay for the most powerful inhibitors are both am
- Gilles, Patrick,Wenck, Kirstin,Stratmann, Inga,Kirsch, Michael,Smolin, Daniel A.,Schaller, Torsten,De Groot, Herbert,Kraft, Arno,Schrader, Thomas
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p. 1772 - 1784
(2017/06/19)
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- Hybridization of an Aβ-specific antibody fragment with aminopyrazole-based β-sheet ligands displays striking enhancement of target affinity
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Determining Aβ levels in body fluids remains a powerful tool in the diagnostics of Alzheimer's disease. This report delineates a new supramolecular strategy which increases the affinity of antibodies towards Aβ to make diagnostic procedures more sensitive
- Hellmert, Marco,Müller-Schiffmann, Andreas,Peters, Max Sena,Korth, Carsten,Schrader, Thomas
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p. 2974 - 2979
(2015/04/27)
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- Design of a binuclear Ni(II)-iminodiacetic acid (IDA) complex for selective recognition and covalent labeling of His-tag fused proteins
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Selective protein labeling with a small molecular probe is a versatile method for elucidating protein functions under live-cell conditions. In this Letter, we report the design of the binuclear Ni(II)-iminodiacetic acid (IDA) complex for selective recognition and covalent labeling of His-tag-fused proteins. We found that the Ni(II)-IDA complex 1-2Ni(II) binds to the His6-tag (HHHHHH) with a strong binding affinity (Kd = 24 nM), the value of which is 16-fold higher than the conventional Ni(II)-NTA complex (Kd = 390 nM). The strong binding affinity of the Ni(II)-IDA complex was successfully used in the covalent labeling and fluorescence bioimaging of a His-tag fused GPCR (G-protein coupled receptor) located on the surface of living cells.
- Takahira, Ikuko,Fuchida, Hirokazu,Tabata, Shigekazu,Shindo, Naoya,Uchinomiya, Shohei,Hamachi, Itaru,Ojida, Akio
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supporting information
p. 2855 - 2858
(2014/06/10)
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- Identifying modulators of protein-protein interactions using photonic crystal biosensors
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(Figure Presented) Inhibitors and activators of protein-protein interactions are valuable as biological probes and medicinal agents but are often difficult to identify. Herein we describe a high-throughput assay, based upon photonic crystal (PC) biosensor
- Heeres, James T.,Kim, Seok-Ho,Leslie, Benjamin J.,Lidstone, Erich A.,Cunningham, Brian T.,Hergenrother, Paul J.
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supporting information; experimental part
p. 18202 - 18203
(2010/04/25)
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- Utilizing reversible copper(II) peptide coordination in a sequence-selective luminescent receptor
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Although vast information about the coordination ability of amino acids and peptides to metal ions is available, little use of this has been made in the rational design of selective peptide receptors. We have combined a copper(II) nitrilotriacetato (NTA) complex with an ammonium-ion-sensitive and luminescent benzocrown ether. This compound revealed micromolar affinities and selectivities for glycine- and histidine-containing sequences, which closely resembles those of copper(II) ion peptide binding: the two free coordination sites of the copper(II) NTA complex bind to imidazole and amido nitrogen atoms, replicating the initial coordination steps of non-complexed copper(II) ions. The benzocrown ether recognizes the N-terminal amino moiety intramolecularly, and the significantly increased emission intensity signals the binding event, because only if prior coordination of the peptide has taken place is the intramolecular ammonium ion-benzocrown ether interaction of sufficient strength in water to trigger an emission signal. Intermolecular ammonium ion-benzocrown ether binding is not observed. Isothermal titration calorimetry confirmed the binding constants derived from emission titrations. Thus, as deduced from peptide coordination studies, the combination of a truncated copper(II) coordination sphere and a luminescent benzocrown ether allows for the more rational design of sequence-selective peptide receptors.
- Stadlbauer, Stefan,Riechers, Alexander,Spaeth, Andreas,Koenig, Burkhard
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experimental part
p. 2536 - 2541
(2009/04/11)
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- MULTIVALENT CHELATORS FOR MODIFYING AND ORGANIZING OF TARGET MOLECULES
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New compounds of the general formula: Xm-G-Cln are described as well as methods for their production. These constitute multivalent chelator-compounds with an affinity-tag binding to metal-chelator-complexes which can selectively modify and/or immobilize target molecules by a multitude of probes or functional units.
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Page/Page column 29
(2008/06/13)
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- Specific and stable fluorescence labeling of histidine-tagged proteins for dissecting multi-protein complex formation
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Labeling of proteins with fluorescent dyes offers powerful means for monitoring protein interactions in vitro and in live cells. Only a few techniques for noncovalent fluorescence labeling with well-defined localization of the attached dye are currently available. Here, we present an efficient method for site-specific and stable noncovalent fluorescence labeling of histidine-tagged proteins. Different fluorophores were conjugated to a chemical recognition unit bearing three NTA moieties (tris-NTA). In contrast to the transient binding of conventional mono-NTA, the multivalent interaction of tris-NTA conjugated fluorophores with oligohistidine-tagged proteins resulted in complex lifetimes of more than an hour. The high selectivity of tris-NTA toward cumulated histidines enabled selective labeling of proteins in cell lysates and on the surface of live cells. Fluorescence labeling by tris-NTA conjugates was applied for the analysis of a ternary protein complex in solution and on surfaces. Formation of the complex and its stoichiometry was studied by analytical size exclusion chromatography and fluorescence quenching. The individual interactions were dissected on solid supports by using simultaneous mass-sensitive and multicolor fluorescence detection. Using these techniques, formation of a 1:1:1 stoichiometry by independent interactions of the receptor subunits with the ligand was shown. The incorporation of transition metal ions into the labeled proteins upon labeling with tris-NTA fluorophore conjugates provided an additional sensitive spectroscopic reporter for detecting and monitoring protein-protein interactions in real time. A broad application of these fluorescence conjugates for protein interaction analysis can be envisaged.
- Lata, Suman,Gavutis, Martynas,Tampe, Robert,Piehler, Jacob
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p. 2365 - 2372
(2007/10/03)
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- High-affinity adaptors for switchable recognition of histidine-tagged proteins
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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.
- Lata, Suman,Reichel, Annett,Brock, Roland,Tampe, Robert,Piehler, Jacob
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p. 10205 - 10215
(2007/10/03)
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