In situ assembly of macromolecular complexes triggered by light

Abstract

Chemical biology aims for a perfect control of protein complexes in time and space by their site-specific labeling, manipulation, and structured organization. Here we developed a self-inactivated, lock-and-key recognition element whose binding to His-tagged proteins can be triggered by light from zero to nanomolar affinity. Activation is achieved by photocleavage of a tethered intramolecular ligand arming a multivalent chelator head for high-affinity protein interaction. We demonstrate site-specific, stable, and reversible binding in solution as well as at interfaces controlled by light with high temporal and spatial resolution. Multiplexed organization of protein complexes is realized by an iterative in situ writing and binding process via laser scanning microscopy. This light-triggered molecular recognition should allow for a spatiotemporal control of protein-protein interactions and cellular processes by light-triggered protein clustering.

Spatiotemporal control of microtubule nucleation and assembly using magnetic nanoparticles

“…Perturbations using small chemicals 4 or lightsensitive molecules 5,6 are now exploited to understand biological functions 7 . For instance, gating activity by light-switchable interactions (allostery, scaffolding) using optogenetic tools has become a promising approach for localizing signalling pathways 8,9 .…”

Cobalt(III) als stabiles und inertes Vermittlerion zwischen Nitrilotriessigsäure und Proteinen mit His6‐Tag

“…Obwohl ursprünglich für die Aufreinigung rekombinanter Proteine entwickelt, [1][2][3][4] kommt diese Methode in zahlreichen anderen Anwendungen zum Einsatz. Diese Technik ermçg-lichte zum Beispiel die spezifische Immobilisierung von Proteinen auf Proteinchips, [5,6] die Inkorporierung aktiver Proteine in Nanomaterialien [7,8] und Oberflächen, [9,10] die Markierung von Proteinen mit Fluorophoren [11][12][13][14] und die spezifische Konjugation von Biomolekülen mit Proteinen. [15][16][17] Im Komplex [18][19][20] sowie die Bildung kovalenter Bindungen durch photochemische [21,22] (oder weniger spezifische) [23,24] [28,29] Außerdem werden Komplexe von bioaktiven Liganden mit substitutionsinerten Metallen als Prodrug eingesetzt, die erst nach dem Eindringen in die Zelle aktiviert werden.…”

His-tags lighten up and lose their inhibitions

“…Protein microarrays have been fabricated using a variety of ligand pairs, including azide-alkyne click chemistry, double-stranded DNA hybridization, and avidin-biotin interactions. An appealing feature of the Grunwald et al (3) study is the use of the widely available Histag system, where an immobilized metal ion recognizes an oligo-His affinity tag introduced into a protein using genetic engineering. The Ni-NTA (nitrilotriacetic acid)·oligo-His complex can be reversed by the addition of imidazole or EDTA.…”

“…Ideally, protein microarrays could be prepared via iterative light-based assembly (9), because light offers the advantage that the domains of individual proteins that can be fabricated are in the size range of 10-50 μm (related to the near-UV light wavelength), much smaller than the physical delivery techniques (spotting, jetting) that can be iterated. Until the methodology of Grunwald et al (3), such a capability had not been demonstrated.…”