Protein complexes are ubiquitous across molecular biology, often found in processes such as transport and translocation of molecules across membranes and signalling, as well as being commonly utilised by bacteria to make toxins. Such processes are vital for the proper functioning of cells and have disastrous consequences when they go wrong, from heart disease to mental illnesses.
The Nanoimager boasts several features that are useful in the study of protein complexes. Single-molecule fluorescence can be used to measure the number of subunits in a complex by intensity based-measurements. An extension of simple intensity measurements involves counting the number of decremental steps as labeling fluorophores in a complex go dark, due to photobleaching.
Moreover, depending on the size of the complex, super-resolution can be used to investigate the structure of the protein complex, as has been rigorously demonstrated in the past with the nuclear pore complex and larger structures such as the cytoskeleton. These methods can also be used to measure the distribution and density of complexes on membranes. In contrast to electron microscopy, single-molecule fluorescence can measure the dynamics of assembling complexes.
The ease of acquiring multiple fields of view with the Nanoimager, as well as the large 50 µm by 80 µm field of view, greatly improves the speed with which protein complexes can be imaged and statistics about their size and properties recorded. In the example below, the Nanoimager was used to study the assembly of the Twin-arginine transport complex. The figure below shows E. coli cells expressing a YFP-labeled component of the Twin-arginine transport (Tat) complex, which transports molecules across the cell membrane.
Each Tat complex contains multiple copies of the YFP-labeled component, so when the complexes assemble they appear as distinct bright spots; each spot in the image contains multiple YFPs. A mutation in the Tat complex prevents it from assembling, therefore in the second phenotype (shown on the right), the YFP are diffusing single molecules which appear as a more homogeneous distribution on the membrane under the same exposure settings. In the 2D projection shown here, this homogeneous distribution gives the effect of a halo around the bacteria. The bacteria were illuminated at 532 nm in HILO mode.
This principle of using the intensity of the spots to determine the composition of the protein complex is broadly applicable. When multiple labelled protein subunits come together, their intensity is summed at the camera and they appear brighter than the monomer. These methods for studying protein complexes have also been applied to other multi-protein phenomena such as the protein aggregates commonly found in neurological diseases like Alzheimers. With the ability to instantaneously present smFRET data, the Nanoimager is well placed to investigate these protein aggregates.