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1.
Nat Chem Biol ; 13(7): 697-705, 2017 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-28632705

RESUMEN

The formation of well-defined protein bioconjugates is critical for many studies and technologies in chemical biology. Tried-and-true methods for accomplishing this typically involve the targeting of cysteine residues, but the rapid growth of contemporary bioconjugate applications has required an expanded repertoire of modification techniques. One very powerful set of strategies involves the modification of proteins at their N termini, as these positions are typically solvent exposed and provide chemically distinct sites for many protein targets. Several chemical techniques can be used to modify N-terminal amino acids directly or convert them into unique functional groups for further ligations. A growing number of N-terminus-specific enzymatic ligation strategies have provided additional possibilities. This Perspective provides an overview of N-terminal modification techniques and the chemical rationale governing each. Examples of specific N-terminal protein conjugates are provided, along with their uses in a number of diverse biological applications.


Asunto(s)
Aminoácidos/química , Proteínas/química , Aminoácidos/metabolismo , Humanos , Proteínas/metabolismo
2.
Bioconjug Chem ; 29(9): 3016-3025, 2018 09 19.
Artículo en Inglés | MEDLINE | ID: mdl-30091905

RESUMEN

Protein conjugates of high heterogeneity may contain species with significantly different biological properties, and as a consequence, the focus on methods for production of conjugates of higher quality has increased. Here, we demonstrate an efficient and generic approach for the modification of metal-binding proteins with biocompatible chemical handles without the need for genetic modifications. Affinity-guided small-molecule probes are developed for direct conjugation to off-the-shelf proteins and for installing different chemical handles on the protein surface. While purification of protein conjugates obtained by small molecule conjugation is troublesome, the affinity motifs of the probes presented here allow for purification of the conjugates. The versatility of the probes is demonstrated by conjugation to several His-tagged and natural metal-binding proteins, including the efficient and area-selective conjugation to three therapeutically relevant antibodies.


Asunto(s)
Proteínas Portadoras/química , Metales/química , Sondas Moleculares/química , Proteínas/metabolismo , Anticuerpos/química , Anticuerpos/inmunología , Línea Celular , Línea Celular Tumoral , Humanos , Inmunoglobulina G/inmunología , Dominios Proteicos
3.
Small ; 12(19): 2634-40, 2016 05.
Artículo en Inglés | MEDLINE | ID: mdl-27032044

RESUMEN

DNA origami provides rapid access to easily functionalized, nanometer-sized structures making it an intriguing platform for the development of defined drug delivery and sensor systems. Low cellular uptake of DNA nanostructures is a major obstacle in the development of DNA-based delivery platforms. Herein, significant strong increase in cellular uptake in an established cancer cell line by modifying a planar DNA origami structure with the iron transport protein transferrin (Tf) is demonstrated. A variable number of Tf molecules are coupled to the origami structure using a DNA-directed, site-selective labeling technique to retain ligand functionality. A combination of confocal fluorescence microscopy and quantitative (qPCR) techniques shows up to 22-fold increased cytoplasmic uptake compared to unmodified structures and with an efficiency that correlates to the number of transferrin molecules on the origami surface.


Asunto(s)
ADN/química , ADN/farmacocinética , Nanocápsulas/química , Neoplasias Experimentales/metabolismo , Receptores de Transferrina/metabolismo , Línea Celular Tumoral , Cristalización/métodos , Humanos , Redes y Vías Metabólicas/fisiología , Nanocápsulas/ultraestructura , Neoplasias Experimentales/química , Tamaño de la Partícula , Receptores de Transferrina/química , Fracciones Subcelulares/química , Fracciones Subcelulares/metabolismo
4.
Chembiochem ; 17(14): 1338-42, 2016 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-27168316

RESUMEN

Many medical and biotechnological applications rely on protein labeling, but a key challenge is the production of homogeneous and site-specific conjugates. This can rarely be achieved by simple residue-specific random labeling, but generally requires genetic engineering. Using site-selective DNA-templated reductive amination, we created DNA-protein conjugates with control over labeling stoichiometry and without genetic engineering. A guiding DNA strand with a metal-binding functionality facilitates site-selectivity by directing the coupling of a second reactive DNA strand in the vicinity of a protein metal-binding site. We demonstrate DNA-templated reductive amination for His6 -tagged proteins and metal-binding proteins, including IgG1 antibodies. We also used a cleavable linker between the DNA and the protein to remove the DNA and introduce a single aldehyde on the protein. This functions as a handle for further modifications with desired labels. In addition to directing the aldehyde positioning, the DNA provides a straightforward route for purification between reaction steps.


Asunto(s)
Aldehídos/química , ADN/química , Proteínas/química , Aminación , Sitios de Unión , Reactivos de Enlaces Cruzados , Metales
5.
Angew Chem Int Ed Engl ; 55(30): 8585-9, 2016 07 18.
Artículo en Inglés | MEDLINE | ID: mdl-27239057

RESUMEN

Enzyme-mediated protein modification often requires large amounts of biocatalyst, adding significant costs to the process and limiting industrial applications. Herein, we demonstrate a scalable and straightforward strategy for the efficient capture and recycling of enzymes using a small-molecule affinity tag. A proline variant of an evolved sortase A (SrtA 7M) was N-terminally labeled with lithocholic acid (LA)-an inexpensive bile acid that exhibits strong binding to ß-cyclodextrin (ßCD). Capture and recycling of the LA-Pro-SrtA 7M conjugate was achieved using ßCD-modified sepharose resin. The LA-Pro-SrtA 7M conjugate retained full enzymatic activity, even after multiple rounds of recycling.

6.
Org Biomol Chem ; 11(45): 7916-22, 2013 Dec 07.
Artículo en Inglés | MEDLINE | ID: mdl-24132123

RESUMEN

Fluoride detection through hydrogen bonding or deprotonation is most commonly achieved using amide, urea or pyrrole derivatives. The sensor molecules are often complex constructs and several synthetic steps are required for their preparation. Here we report the discovery that simple arylaldoximes have remarkable properties as fluoride anion sensors, providing distinct colorimetric or fluorescent readouts, depending on the structure of the arylaldoxime. The oximes showed exceptional selectivity towards fluoride over other typical anions, and low detection limits for fluoride in both DMSO and DMSO-water mixtures were obtained.


Asunto(s)
Dimetilsulfóxido/química , Fluorescencia , Colorantes Fluorescentes/química , Fluoruros/análisis , Oximas/química , Agua/química , Colorimetría , Cristalografía por Rayos X , Modelos Moleculares , Estructura Molecular
7.
Small ; 7(7): 939-49, 2011 Apr 04.
Artículo en Inglés | MEDLINE | ID: mdl-21394906

RESUMEN

Methylation of DNA nucleobases is an important control mechanism in biology applied, for example, in the regulation of gene expression. The effect of methylation on the intermolecular interactions between guanine molecules is studied through an interplay between scanning tunneling microscopy (STM) and density functional theory with empirical dispersion correction (DFT-D). The present STM and DFT-D results show that methylation of guanine can have subtle effects on the hydrogen-bond strength with a strong dependence on the position of methylation. It is demonstrated that the methylation of DNA nucleobases is a precise means to tune intermolecular interactions and consequently enables very specific recognition of DNA methylation by enzymes. This scheme is used to generate four different types of artificial 2D nanostructures from methylated guanine. For instance, a 2D guanine windmill motif that is stabilized by cooperative hydrogen bonding is revealed. It forms by self-assembly on a graphite surface under ambient conditions at the liquid-solid interface when the hydrogen-bonding donor at the N1 site of guanine is blocked by a methyl group.


Asunto(s)
Guanina/química , Nanoestructuras/química , ADN/química , ADN/metabolismo , Grafito/química , Enlace de Hidrógeno , Metilación , Microscopía de Túnel de Rastreo
8.
J Am Chem Soc ; 132(51): 18054-6, 2010 Dec 29.
Artículo en Inglés | MEDLINE | ID: mdl-21133363

RESUMEN

Generation-4 polyamidoamine (PAMAM) dendrimers were surface-functionalized with azides or alkynes and conjugated to one DNA strand. DNA-controlled self-assembly of alternating azide and alkyne dendrimers on a DNA template enabled the coupling of the dendrimers by the azide-alkyne "click" reaction to form covalently coupled dimers, trimers, and tetramers. Polymerization of the DNA-dendrimer conjugates was also demonstrated, as well as assembly in a circular structure on DNA origami and imaging by atomic force microscopy.


Asunto(s)
Alquinos/química , Azidas/química , Química Clic/métodos , ADN/química , Dendrímeros/química , Nanotecnología/métodos , Nylons/química , Biotina/química , Microscopía de Fuerza Atómica , Estreptavidina/química
10.
Nat Biotechnol ; 32(2): 179-81, 2014 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-24441471

RESUMEN

We demonstrate single-molecule, site-specific detection of protein phosphorylation with protein nanopore technology. A model protein, thioredoxin, was phosphorylated at two adjacent sites. Analysis of the ionic current amplitude and noise, as the protein unfolds and moves through an α-hemolysin pore, enables the distinction between unphosphorylated, monophosphorylated and diphosphorylated variants. Our results provide a step toward nanopore proteomics.


Asunto(s)
Biotecnología/métodos , Nanoporos , Fosforilación , Proteínas/análisis , Proteínas/química , Secuencia de Aminoácidos , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas/metabolismo , Proteómica , Alineación de Secuencia
11.
Nat Chem ; 6(9): 804-9, 2014 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-25143216

RESUMEN

DNA-protein conjugates are important in bioanalytical chemistry, molecular diagnostics and bionanotechnology, as the DNA provides a unique handle to identify, functionalize or otherwise manipulate proteins. To maintain protein activity, conjugation of a single DNA handle to a specific location on the protein is often needed. However, preparing such high-quality site-specific conjugates often requires genetically engineered proteins, which is a laborious and technically challenging approach. Here we demonstrate a simpler method to create site-selective DNA-protein conjugates. Using a guiding DNA strand modified with a metal-binding functionality, we directed a second DNA strand to the vicinity of a metal-binding site of His6-tagged or wild-type metal-binding proteins, such as serotransferrin, where it subsequently reacted with lysine residues at that site. This method, DNA-templated protein conjugation, facilitates the production of site-selective protein conjugates, and also conjugation to IgG1 antibodies via a histidine cluster in the constant domain.


Asunto(s)
Anticuerpos/química , Proteínas Portadoras/química , ADN/química , Metales/metabolismo , Transferrina/química , Secuencia de Aminoácidos , Animales , Proteínas Fluorescentes Verdes , Histidina/química , Humanos , Datos de Secuencia Molecular
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