Fig. 1: Condensates based on different LCDs provide different microenvironments for NOX enzymatic activity.
From: Local environment in biomolecular condensates modulates enzymatic activity across length scales
A NOX catalyzes the oxidation of NADH to NAD+ in the presence of oxygen, using FAD as a cofactor. AlphaFold55 structure of Dbp1-NOX, showing the unstructured Dbp1 LCD (blue color) and the globular NOX enzyme (black color). B LCDs derived from the DEAD-box proteins Dbp1, Laf1, and Ddx4 are fused to the N-terminus of the NOX enzyme, creating chimeric proteins with different net charges, which are represented by the bar. The amino acid sequence of each LCD is shown on the right. Basic residues (Arg and Lys) are highlighted in blue, and acidic residues (Asp and Glu) in red. C Representative phase-contrast microscopy images of solutions of 5 µM Ddx4-NOX (top), Dbp1-NOX (bottom, left), and Laf1-NOX (bottom, right) in 25 mM Tris, 20 mM NaCl, pH 7.5 showing the presence of micron-sized condensates when the enzyme is fused to LCDs. The scale bar represents 5 µm. The protein concentration of 5 µM was selected to facilitate visualization of the condensates under the confocal microscope. Each experiment was repeated three times independently with similar results. D Phase separation is abolished at high salt concentration. Green circles and red cross indicate the presence and absence of phase separation, respectively. E Representative fluorescence confocal microscopy images showing the recruitment of NADH (top, blue fluorescence) and FAD (bottom, green fluorescence) in condensates of Ddx4-NOX (left), Laf1-NOX (middle) and Dbp1-NOX (right). NADH and FAD were added individually in two distinct experiments to avoid interference with the rapid reaction. The scale bar represents 5 µm. Each experiment was repeated three times independently with similar results. F Partitioning (Kp) of substrate (top) and cofactor (bottom) as a function of the net charge of the different fusion proteins. The dot lines represent exponential correlations and are a guide to the eyes only. n = 3 independent experiments. Data were presented as mean values ± SEM.
