From cameras to confocal to cytometry: measuring tumbling rates is a general way to reveal protein binding - Raw Data

Molecular interactions are central to understanding any biological system, but they are labor-intensive to measure with existing optical approaches. Tracking "tumbling" (rotational diffusion) is a generalizable strategy for determining particle size, from which the presence and size of binding partners in a complex can be inferred. However, fluorescence-based tumbling measurements have historically been limited to small targets (<30 kDa) due to the short (nanosecond) lifetime of typical fluorophores. This size limit leaves most of the proteome inaccessible, severely limiting the usefulness of the approach. To reveal larger complexes and realize the full potential of optical tumbling measurements, several groups have explored longer-lived states, such as photobleached molecules, reversibly photoswitched proteins, or triggerable triplets. Here, we describe four combinations of photophysics and hardware for measuring tumbling with longer-lived states, gearing each toward a candidate biological use case. We explore these four measurement schemes with simulations and, in one case, experimentally validate the approach. Our work suggests that, with minor reconfiguring, most fluorescence approaches to detect the presence of proteins could also reveal their binding state.
This repository holds the raw data for the main text figures. For the contents of the paper, please see: doi.org/10.5281/zenodo.10028432