Monochromatic multicomponent fluorescence sedimentation velocity for the study of high-affinity protein interactions

Many proteins in cells combine to form molecular machines or complexes that carry out specific processes inside cells. Analytical ultracentrifugation is a technique commonly used to explore the physical properties of proteins and their complexes and in this way to gain insights into the biological roles of these molecules. The technique involves spinning a sample containing the molecules to generate a strong centrifugal force, while monitoring the movement of the molecules. Under these conditions, molecules with different sizes and masses sink – or “sediment” – at different rates, so individual proteins and their complexes can be clearly distinguished. Analytical ultracentrifugation was recently extended to make it possible to detect fluorescent tags added on to proteins. This advance allowed researchers to study more dilute samples or complexes that are held together especially tightly. However, only tags of a single color can be detected because of physical constraints of the fluorescent detection system. This meant that only one kind of fluorescent signal could be tracked at any one time. However, a group of fluorescent tags called photoswitchable fluorescent proteins (psFPs) offer new opportunities for detecting multiple signals. This is because these psFPs switch between fluorescent and non-fluorescent states while being detected in the ultracentrifuge. Zhao et al. have now exploited this unique photoswitching property by accurately measuring how fast a number of psFPs switched between fluorescent and non-fluorescent states while they were sedimenting. Each different psFPs switched in a distinct way, even for psFPs of the same color, meaning that each psFP could be identified from its switching rate, similar to identifying a person from their fingerprints. This discovery allowed Zhao et al. to distinguish different psFPs in a mixed sample as if they had different colors. Further experiments went on to demonstrate that this approach could identify the binding proteins in a protein mixture made of three components, and be used to study a biologically important protein complex that can itself exist in two distinct forms. The approach will therefore provide a valuable tool to observe different components in a complex individually and will provide researchers the opportunity to study how mixed protein complexes form at very low concentrations. Future developments of the approach may make it possible to study other properties of protein complexes such as their overall shape and their behavior under conditions that mimic those inside the cell.