title: Maleimide photolithography for single-molecule protein-protein interaction analysis in micropatterns.
abstract: 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 maleimides, 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.
authors.affiliation: Division of Biophysics, University of Osnabrück, Barbarastrasse 11, 49076 Osnabrück, Germany.
authors.forename: Sharon
authors.initials: S
authors.lastname: Waichman
authors.forename: Changjiang
authors.initials: C
authors.lastname: You
authors.forename: Oliver
authors.initials: O
authors.lastname: Beutel
authors.forename: Maniraj
authors.initials: M
authors.lastname: Bhagawati
authors.forename: Jacob
authors.initials: J
authors.lastname: Piehler
mesh_terms: D003065:Coenzyme A / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055767:Immobilized Proteins / Q000737:chemistry; D016898:Interferon-alpha / Q000737:chemistry; D008301:Maleimides / Q000737:chemistry*; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011506:Proteins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013499:Surface Properties
chemical_list: C471213:His-His-His-His-His-His; D055767:Immobilized Proteins; D016898:Interferon-alpha; D008301:Maleimides; D009842:Oligopeptides; D011506:Proteins; D013438:Sulfhydryl Compounds; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C043592:maleimide; D006639:Histidine; D003065:Coenzyme A