Extending fluorescence anisotropy to large complexes using reversibly switchable proteins

Abstract

The formation of macromolecular complexes can be measured by detection of changes in rotational mobility using time-resolved fluorescence anisotropy. However, this method is limited to relatively small molecules (~0.1–30 kDa), excluding the majority of the human proteome and its complexes. We describe selective time-resolved anisotropy with reversibly switchable states (STARSS), which overcomes this limitation and extends the observable mass range by more than three orders of magnitude. STARSS is based on long-lived reversible molecular transitions of switchable fluorescent proteins to resolve the relatively slow rotational diffusivity of large complexes. We used STARSS to probe the rotational mobility of several molecular complexes in cells, including chromatin, the retroviral Gag lattice and activity-regulated cytoskeleton-associated protein oligomers. Because STARSS can probe arbitrarily large structures, it is generally applicable to the entire human proteome.

Structural diversity of Arc oligomers within the excitatory synapse

“…Hence, the presence of distinct Arc n -meric states may be necessary to support functions, such as receptors endocytosis, cytoskeletal binding or viral-like interneuronal transfer. In different heterologous systems, we 23 and Hedde et al, 24 have demonstrated that Arc forms, together with monomeric and low-order oligomers, rigid nanometric particles. In primary cortical neurons and in rat dentate gyrus 25 , Arc was reported to form low-abundance dimers and low-order oligomers (trimers, tetramers), which increase upon plasticity induction.…”

Correlation of solute diffusion with dynamic viscosity in lithium salt-added (choline chloride + glycerol) deep eutectic solvents

“…Interestingly, the slopes decrease monotonically with increasing LiCl, suggesting the hydrodynamic volume, V , of perylene to decrease with increasing LiCl concentration in glyceline. 40,41 LiCl appears to detach the H-bond networked DES around the probe perylene, which makes the hydrodynamic volume smaller. This would result in a change in the cybotactic region viscosity ( i.e.…”

Three-dimensional spatio-angular fluorescence microscopy with a polarized dual-view inverted selective-plane illumination microscope (pol-diSPIM)

“…Here we made steady-state measurements, considering only quasi-static fluorescent reporters. Time-resolved orientation measurements provide a large set of possibilities for this field [56, 57], and combining time-resolved measurements with reversibly switchable proteins allows measurement of a wide range of reorientation timescales [58], giving access to reorientation timescales of large protein complexes. We are excited for future developments that probe long-timescale reorientation of fluorophores trapped in 3D angular potentials.…”