STARSS: measuring rotational diffusivity with reversible photo-switching
KTH, SciLifeLab, Stockholm, Sweden
The formation of macromolecular complexes can be measured by detecting changes in rotational mobility using time-resolved fluorescence anisotropy. But 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 more than three orders of magnitude. STARSS is based on long-lived reversible molecular transitions of switchable fluorescent proteins to resolve relatively slow rotational diffusivity of large complexes. Rotational diffusion provides direct information on the size and local environment of molecular complexes in solution and cells. Since STARSS is compatible with live cell imaging and protein tagging it can be applied to study changes in mass, local viscosity or degree of rotational freedom of most proteins in their native environment. We used STARSS to probe the rotational mobility of several molecular complexes in cells, including chromatin, the retroviral Gag lattice and Arc oligomers. Since STARSS can probe arbitrarily large structures it is generally applicable to the entire human proteome.