Maleimide Photolithography for Single-Molecule Protein-Protein Interaction Analysis in Micropatterns
Autor(en): | Waichman, Sharon You, Changjiang Beutel, Oliver Bhagawati, Maniraj Piehler, Jacob |
Stichwörter: | BINDING; BIOTIN; Chemistry; Chemistry, Analytical; FUNCTIONAL IMMOBILIZATION; HISTIDINE-TAGGED PROTEINS; MOTOR PROTEINS; RECEPTOR INTERACTIONS; REFLECTOMETRIC INTERFERENCE SPECTROSCOPY; SFP PHOSPHOPANTETHEINYL TRANSFERASE; STREPTAVIDIN; SURFACE | Erscheinungsdatum: | 2011 | Herausgeber: | AMER CHEMICAL SOC | Journal: | ANALYTICAL CHEMISTRY | Volumen: | 83 | Ausgabe: | 2 | Startseite: | 501 | Seitenende: | 508 | Zusammenfassung: | Spatial organization of proteins into microscopic structures has important applications in fundamental and applied research. Preserving the function of proteins in such microstructures requires generic methods for site-specific capturing through affinity handles. Here, we present a versatile bottom-up surface micropatterning approach based on surface functionalization with male-imides, which selectively react with organic thiols. Upon UV irradiation through a photomask, the functionality of illuminated maleimide groups was efficiently destroyed. Remaining maleimides in nonilluminated regions were further reacted with different thiol-functionalized groups for site-specific protein immobilization under physiological conditions. Highly selective immobilization of His-tagged proteins into tris(nitrilotriacetic acid) functionalized microstructures with very high contrast was possible even by direct capturing of proteins from crude cell lysates. Moreover, we employed phosphopantetheinyl transfer from surface-immobilized coenzyme A to ybbR-tagged proteins in order to implement site-specific, covalent protein immobilization into microstructures. The functional integrity of the immobilized protein was confirmed by monitoring protein-protein interactions in real time. Moreover, we demonstrate quantitative single-molecule analysis of protein-protein interactions with proteins selectively captured into these high-contrast micropatterns. |
ISSN: | 00032700 | DOI: | 10.1021/ac1021453 |
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geprüft am 02.05.2024