- Specific and stable fluorescence labeling of histidine-tagged proteins for dissecting multi-protein complex formation
-
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
-
-
Read Online
- Multifunctional polymer micelle for jointly conveying chemotherapeutic drugs and gene editing systems as well as preparation method and application thereof (by machine translation)
-
The outer layer of the multifunctional polymer micelle with the three-layer structure, core as a hydrophobic polycaprolactone, inner layer is, and the outer layer of the multifunctional polymer micelle which can be coupled with a gene editing system adopts a layer of three layers and can form a shielding layer NTA, through an intermediate layer click reaction to form a shielding layer. DBCO - PEG20k - DBCO, The, invention discloses a multifunctional polymer micelle which can, be coupled DBCO - PEG with a gene editing system and a preparation method and application of the multifunctional polymer micelle. 20k - DBDBDBCO, the drug and gene editing system, protecting the inner layer load simultaneously have a targeting effect; which can efficiently enter the tumor cell, as the carrier, and enter the lysosome, and then release the chemotherapeutic drug and the gene editing system, to act, together to ensure, no drug resistance of tumor cells is enhanced, at the same time in the case, of chemotherapy drug dosage . (by machine translation)
- -
-
Paragraph 0065; 0067; 0069
(2020/04/29)
-
- Specifically and reversibly immobilizing proteins/enzymes to nitriolotriacetic-acid-modified mesoporous silicas through histidine tags for purification or catalysis
-
Six nitriolotriacetic-acid-modified ordered mesoporous silicas (NTA-OMPSs) with different pore sizes and surface features for specific and reversible protein immobilization were fabricated and characterized. Specific immobilization of a genetically engineered undecaprenyl pyrophosphate synthase (UPPs) from cell lysate and a chemically modified His-tagged horseradish peroxidase (HRP) in these Ni-NTA-OMPSs through histidine coordination to the nickelated NTA was demonstrated and confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Negligible leakage of these enzymes over a wide range of acidic conditions was observed. Moreover, histidine tags with different lengths (His6, His4, His3, and His2) applied to HRP were evaluated to find the minimum length for effective complexation. Enzymatic assessment studies indicated that the pore size of the OMPSs has minimal influence on the enzymatic activity, whereas chemical entities such as unreacted mercapto groups tailored on the interior surfaces of the OMPSs played certain roles in inhibiting the enzymatic activity and stability. On MCF-S-NTA, SBA-S-NTA, and film-S-NTA, which contained unreacted mercaptopropyl groups on the interior surface, immobilized His-tagged HRP showed lower catalytic activity and stability than on MCF-NTA, film-NTA, and SBA-NTA. Selective hydroxylation of optically pure L-tyrosine to (S)-2-amino-3-(3,4-dihydroxyphenyl)propanoic acid (L-DOPA) by the immobilized HRP was also demonstrated. Protein purification and enzyme catalysis: A generic and easily adaptable platform is established for selectively and reversibly immobilizing His-tagged enzymes in ordered mesoporous silicas (OMPSs) through nickelated nitriolotriacetic-acid (NTA)-histidine tag complexation (see figure). The immobilized enzymes exhibit good stability toward heat and pH changes. Negligible leakage of these enzymes over a wide range of acidic conditions was observed. Copyright
- Lin, Yu-Chung,Liang, Ming-Ren,Lin, Yu-Chen,Chen, Chao-Tsen
-
supporting information; experimental part
p. 13059 - 13067
(2012/01/02)
-
- Selective two-step labeling of proteins with an off/on fluorescent probe
-
We present a novel design strategy for off/on fluorescent probes suitable for selective two-step labeling of proteins. To validate this strategy, we designed and synthesized an off/on fluorescent probe, 1-Ni2+, which targets a cysteine-modified hexahistidine (His) tag. The probe consists of dichlorofluorescein conjugated with nitrilotriacetic acid (NTA)-Ni2+ as the His-tag recognition site and a 2,4-dinitrophenyl ether moiety, which quenches the probe's fluorescence by photoinduced electron transfer (PeT) from the excited fluorophore to the 2,4-dinitrophenyl ether (donor-excited PeT; d-PeT) and also has reactivity with cysteine. His-tag recognition by the NTA-Ni2+ moiety is followed by removal of the 2,4-dinitrophenyl ether quencher by proximity-enhanced reaction with the cysteine residue of the modified tag; this results in a marked fluorescence increase. Addition of His-tag peptide bearing a cysteine residue to aqueous probe solution resulted in about 20-fold fluorescence increment within 10 min, which is the largest fluorescence enhancement so far obtained with a visible light-excitable fluorescent probe for a His-based peptide tag. Further, we successfully visualized CysHis6-peptide tethered to microbeads without any washing step. The probe also showed a large fluorescence increment in the presence of His6Cys-tagged enhanced blue fluorescent protein (EBFP), but not His6-tagged EBFP. We consider this system is superior to large fluorescence tags (e.g., green fluorescent protein: 27 kDa), which can perturb protein folding, trafficking and function, and also to existing small tags, which generally show little fluorescence increase upon target recognition and therefore require a washout step. This strategy should also be applicable to other tags.
- Hirabayashi, Kazuhisa,Hanaoka, Kenjiro,Shimonishi, Manabu,Terai, Takuya,Komatsu, Toru,Ueno, Tasuku,Nagano, Tetsuo
-
experimental part
p. 14763 - 14771
(2012/02/06)
-
- Enhanced drug loading in polymerized micellar cargo
-
A new drug carrier system based on self-assembly and polymerization of polydiacetylenic amphiphiles is described. Although classical amphiphiles can help in solubilizing hydrophobic molecules upon self-arrangement into a variety of nanometric structures, a greater effect on drug loading was observed for our polymerized micelles as compared to the non-polymerized analogues. This permitted higher aqueous solubilization of lipophilic drugs with low micelle concentration. 14C labeling of a model drug on one side and of the amphiphile on the other side permitted assessment, after intravenous injection, of biodistribution and excretion profiles of the drug cargo.
- Ogier, Julien,Arnauld, Thomas,Carrot, Geraldine,Lhumeau, Antoine,Delbos, Jean-Marie,Boursier, Claire,Loreau, Olivier,Lefoulon, Francois,Doris, Eric
-
experimental part
p. 3902 - 3907
(2010/09/17)
-
- Supramolecular self-assembly of amphiphiles on carbon nanotubes: A versatile strategy for the construction of CNT/metal nanohybrids, application to electrocatalysis
-
Homogeneous coating of carbon nanotubes with metallic nanoparticles was achieved using supramolecular auto-organization of amphiphilic molecules as template. The resulting Pd nanoparticles/carbon nanotube nanohybrids were then evaluated in electrocatalysis experiments, showing superior activity in ethanol oxidation compared to analogous systems. Copyright
- Mackiewicz, Nicolas,Surendran, Geetarani,Remita, Hynd,Keita, Bineta,Zhang, Guangjin,Nadjo, Louis,Hagege, Agnes,Doris, Eric,Mioskowski, Charles
-
supporting information; body text
p. 8110 - 8111
(2009/02/02)
-
- Gold nanoparticle size controlled by polymeric Au(I) thiolate precursor size
-
We developed a method in preparing size-controllable gold nanoparticles (Au NPs, 2-6 nm) capped with glutathione by varying the pH (between 5.5 and 8.0) of the solution before reduction. This method is based on the formation of polymeric nanoparticle precursors, Au(I)-glutathione polymers, which change size and density depending on the pH. Dynamic light scattering, size exclusion chromatography, and UV-vis spectroscopy results suggest that lower pH values favor larger and denser polymeric precursors and higher pH values favor smaller and less dense precursors. Consequently, the larger precursors led to the formation of larger Au NPs, whereas smaller precursors led to the formation of smaller Au NPs. Using this strategy, Au NPs functionalized with nickel(II) nitriloacetate (Ni-NTA) group were prepared by a mixed-ligand approach. These Ni-NTA functionalized Au NPs exhibited specific binding to 6x-histidine-tagged Adenovirus serotype 12 knob proteins, demonstrating their utility in biomolecular labeling applications.
- Brinas, Raymond P.,Hu, Minghui,Qian, Luping,Lymar, Elena S.,Hainfeld, James F.
-
p. 975 - 982
(2008/09/20)
-
- Assembly of nanoparticle-protein binding complexes: From monomers to ordered arrays
-
(Figure Presented) Tag it: Well-defined monomers, dimers, trimers, three-dimensional spherical shells, and two-dimensional ordered arrays of genetically engineered proteins were constructed by using functionalized gold nanoparticles. Nanoparticle size, fu
- Hu, Minghui,Qian, Luping,Brinas, Raymond P.,Lymar, Elena S.,Hainfeld, James F.
-
p. 5111 - 5114
(2008/09/16)
-
- Hydrogels of water soluble polymers crosslinked by protein domains
-
A stimuli-responsive, hybrid hydrogel wherein the bulk of the polymer is made up of relatively inexpensive water soluble polymer strands crosslinked by protein domains. The responsiveness of the gel is controlled or modulated by the protein component. The physical and biological properties of the hydrogel are determined by specifically designed or engineered protein domains. The crosslinking of the protein domains to the water soluble polymers is by means of non-covalent bonding such as chelation or coordination bonding, biotin-avidin bonding, protein—protein interaction and protein-ligand interaction, or by means of covalent bonding. Methods of making and using the polymer-protein hydrogels are disclosed in this application.
- -
-
Page/Page column 10; 11
(2010/11/25)
-
- Metal affinity capture tandem mass spectrometry for the selective detection of phosphopeptides
-
We report a new method called metal affinity capture that when coupled with tandem mass spectromery (MAC-MSMS) allows for the selective detection and identification of phosphopeptides in complex mixtures. Phosphopeptides are captured as ternary complexes with GaIII or FeIII and Nα,Nα-bis(carboxymethyl)lysme (LysNTA) in solution and electrosprayed as doubly or triply charged positive ions. The gas-phase complexes uniformly dissociate to produce a common (LysNTA + H) + ion that is used as a specific marker in precursor ion scans. The advantages of MAC-MSMS over the current methods of phosphopeptide detection are as follows. (1) MAC-MSMS uses metal complexes that self-assemble in solution at pH 69Ga-71Ga isotope pattern for selective recognition in mixtures. Detection by MAC-MSMS of singly and multiply phosphorylated peptides in tryptic digests is demonstrated at low-nanomolar protein concentrations.
- Blacken, Grady R.,Gelb, Michael H.,Turecek, Frantisek
-
p. 6065 - 6073
(2008/02/10)
-
- High-affinity adaptors for switchable recognition of histidine-tagged proteins
-
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
-
p. 10205 - 10215
(2007/10/03)
-
- Grafting nitrilotriacetic groups onto carboxylic acid-terminated self-assembled monolayers on gold surfaces for immobilization of histidine-tagged proteins
-
In this paper, we report a common intermediate method to present nitrilotriacetic acid (NTA) groups on gold surfaces for immobilizing His-tagged proteins onto the surfaces, and a full characterization of self-assembled monolayers (SAMs) terminating in carboxylic acids [HS(CH2) 15COOH (C15-COOH), HS(CH2)11(OCH 2CH2)3-OCH2COOH (EG 3-COOH), and HS(CH2)11(OCH2CH 2)5OCH2COOH (EG5-COOH)] and coupling reactions of an NTA-containing primary amine [(1S)-N-(5-amino-1-carboxypentyl) iminodiacetic acid; NTA-NH2] with the carboxylic acid on surfaces. The lateral packing densities of the COOH-terminated SAMs were calculated to be 4.32 (for C15-COOH), 3.49 (for EG3-COOH), and 2.65 (for EG5-COOH) molecules/nm2. The packing densities were decreased by incorporating a relatively flexible ethylene glycol (EG) group into the backbone of alkanethiols and increasing the number of the EG groups in the backbone of alkanethiols. The NTA group was then attached by coupling NTA-NH2 with the COOH group on the surfaces, followed by a Ni(II) complexation. The coupling reaction was characterized by FT-IR spectroscopy, ellipsometry, and XPS, and the coupling efficiency ("yield") was estimated by comparing the experimentally determined N Is to S 2p (N/S) ratio of XPS data with the N/S ratio calculated for the functionalization of the SAMs presenting NTA-Ni(II): the coupling yields were 30% (for C15-COOH) and 25% (for EG3-COOH and EG5-COOH). Preliminary experiments on the binding of His-tagged proteins onto the surfaces were also performed.
- Lee, Jungkyu K.,Kim, Yang-Gyun,Chi, Young Shik,Yun, Wan Soo,Choi, Insung S.
-
p. 7665 - 7673
(2007/10/03)
-
- Convergent and sequential synthesis of dendritic, multivalent complexing agents
-
Two series of macrocyclic polyamine derivatives with various length of linkers were synthesized as dendritic ligands containing two, three, four, five or six terminal nitrilotriacetic acid (NTA) groups through convergent and sequential pathways. Tetrafluorophenyl esters were employed as activating reagents in the coupling step of assembling NTA groups to the cyclic polyamine head. A key step of the sequential synthesis is the use of the coupling reagent, 2-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) for the formation of amide bonds between the cyclic polyamine and pendant groups. Finally, two representative compounds were used to demonstrate the formation of stoichiometric protein assemblies.
- Liu, Cuihua,Hudson, Robert H. E.,Petersen, Nils O.
-
p. 1398 - 1406
(2007/10/03)
-
