Steve Pressé

PNAS December 22, 2020 117 (51) 32189-32191

We are well into the 21st century and even the most fundamental aspects of protein biophysics continue to perplex us. For example, counter to any expectation, proteins are excellent conductors (1); globular proteins, the once hallowed paradigm of structural biology, are now believed to sample their unfolded ensemble multiple times over their lifetime due to their unexpectedly small folding equilibrium constants (2); and proteins, often naïvely depicted as solitary biological actors capable of forming static multimeric assemblies, are now thought to form large transient phase-separated condensates (3). Equally surprising is the observation, counter to any macroscopic hydrodynamic expectation, that catalytic proteins (enzymes) exhibit enhanced diffusion upon catalysis (4). The latter is the focus of “Master curve of boosted diffusion for 10 catalytic enzymes” by Jee et al. (4), where the effect of diffusion enhancement of 10 enzymes is probed by fluorescence correlation spectroscopy (FCS) (Fig. 1).

See https://www.pnas.org/content/117/51/32189

Figure 1: Diffusion coefficients are deduced by correlating photon arrivals. Fluorescently labeled proteins (shown as red dots, Left) are allowed to freely diffuse across an inhomogeneously illuminated confocal volume. Shown in blue (Left) is the overlap between the illuminated (excitation) region and the detected volume. Photon arrivals, {t1,t2,⋯}{t1,t2,⋯}, encode the number of molecules emitting photons, their distance from the center of the spot, their diffusion coefficients, photophysical artifacts, and other parameters (Middle). In principle, this information can be decoded directly from the photon arrivals (8, 9), though traditionally, in a process reminiscent of the Hanbury–Twiss experiments for deducing star sizes, large datasets are collected and photon arrivals are instead correlated in time, τ (Right). The correlation function, G(τ)G(τ), itself is then fitted with an analytical function (red line, Right). Faster correlation function decays coincide with faster diffusion coefficients. It was this setup that has allowed Jee et al. (4) to deduce the diffusion coefficient of 10 different enzymes. Figure adapted from refs. 8 and 9.