Determining the Optimal Degree of Macromolecular Crowding in Solution Using Fluorescence Recovery after Photobleaching (FRAP)

Abstract

Macromolecular crowding (MMC) is a feature of cellular interiors and, in multicellular organisms, also of extracellular environments. Recreating this crowdedness via employment of macromolecules is a valuable tool in studying protein folding behaviour, supramolecular assembly, acceleration of biochemical reactions and extracellular matrix deposition. Optimal MMC in solution has been mostly determined empirically by quantitating biochemical endpoints (PCR product, amount of matrix deposited). Here, we sought to develop a technique allowing the direct observation of MMC in a purely physical manner in order to make predictions of optimal crowder concentrations. We have shown earlier by dynamic light scattering that above certain concentrations the hydrodynamic radii of Ficolls 70 and 400 shrink (Harve et al 2007). We described this event as self-crowding.Here, we studied this phenomenon with FRAP in order to monitor changes in the molecular diffusion velocity of FITC-tagged Ficoll 70 and Ficoll 400. Both crowders are sucrose polymers, stable at neutral pH, and globular in shape.FRAP allowed us to establish a clear link between molecular speed variation and degree of crowdedness. We verified that beyond a threshold concentration self-crowding occurred as evidenced by a speed increase for the molecules undergoing shrinking. Maximum compaction of Ficoll molecules was reached with increasing concentrations. Further concentration increases led to a rapid drop of diffusion velocity.By purely physical means we can now define the optimal crowding concentration as the greatest possible concentration allowing for the greatest possible diffusion velocity. Interestingly enough, the determined values match those obtained from biological read-outs published earlier (Chen et al 2011) very well. This technique now opens up the possibility to determine for each potential macromolecule the crowding points rapidly, and to predict suitable concentrations for application in biological systems.