Evolution of CPEB4 Dynamics Across its Liquid–Liquid Phase Separation Transition

Abstract

Knowledge about the structural and dynamic properties of proteins that form membrane-less organelles in cells via liquid–liquid phase separation (LLPS) is required for understanding the process at a molecular level. We used spin labeling and electron paramagnetic resonance (EPR) spectroscopy to investigate the dynamic properties (rotational diffusion) of the low complexity N-terminal domain of cytoplasmic polyadenylation element binding-4 protein (CPEB4 NTD ) across its LLPS transition, which takes place with increasing temperature. We report the coexistence of three spin labeled CPEB4 NTD (CPEB4*) populations with distinct dynamic properties representing different conformational spaces, both before and within the LLPS state. Monomeric CPEB4* exhibiting fast motion defines population I and shows low abundance prior to and following LLPS. Populations II and III are part of CPEB4* assemblies where II corresponds to loose conformations with intermediate range motions and population III represents compact conformations with strongly attenuated motions. As the temperature increased the population of component II increased reversibly at the expense of component III , indicating the existence of an III ⇌ II equilibrium. We correlated the macroscopic LLPS properties with the III ⇌ II exchange process upon varying temperature and CPEB4* and salt concentrations. We hypothesized that weak transient intermolecular interactions facilitated by component II lead to LLPS, with the small assemblies integrated within the droplets. The LLPS transition, however, was not associated with a clear discontinuity in the correlation times and populations of the three components. Importantly, CPEB4 NTD exhibits LLPS properties where droplet formation occurs from a preformed microscopic assembly rather than the monomeric protein molecules.

Peptide-RNA Coacervates as a Cradle for the Evolution of Folded Domains

“…Increasing to 2 mg/mL polyU and reducing the PA-12 concentration to 100 μM increased the population of II to 95% (Figures B and S15G). Such restricted motion and dynamic components have also been reported for proteins undergoing phase separation. , Mixing unlabeled scrambled peptide with 1 mg/mL polyU, in contrast, did not form coacervates but rather aggregates, as previously reported …”

“…Such restricted motion and dynamic components have also been reported for proteins undergoing phase separation. 38 , 39 Mixing unlabeled scrambled peptide with 1 mg/mL polyU, in contrast, did not form coacervates but rather aggregates, as previously reported. 17 …”

Peptide-RNA Coacervates as a Cradle for the Evolution of Folded Domains

“…Increasing to 2 mg/mL polyU and reducing the PA-12 concentration to 100 µM increased the population of II to 95% (Figure 3B and S15G). Such restricted motion and dynamic components have also been reported for proteins undergoing phase separation 38,39 . Mixing unlabeled scrambled peptide with 1 mg/mL polyU, in contrast, did not form coacervates but rather aggregates, as previously reported 17 .…”

Allostery‐driven changes in dynamics regulate the activation of bacterial copper transcription factor

“…The spectrum of the M101R1 mutant (Figure 4a) exhibits a two‐component spectrum, characterized by a mobile (marked as “m” in Figure 4A) and an immobile component (marked as “im” in Figure 4A)—such complex lineshape is not unusual for R1 attached to proteins 32–34 . Figures 4B–D show overlays of the experimental data at various Cu(I) concentrations.…”

“…For example, the M101R1 has a The spectrum of the M101R1 mutant (Figure 4a) exhibits a two-component spectrum, characterized by a mobile (marked as "m" in Figure 4A) and an immobile component (marked as "im" in Figure 4A)-such complex lineshape is not unusual for R1 attached to proteins. [32][33][34] Figures 4B-D show overlays of the experimental data at various Cu(I) concentrations. Between 0 to 1 equivalents of Cu(I), we observe systematic changes in the lineshapes (Figure 4B), whereas the lineshape is stable between 1 and 2 Cu(I):CueR (Figure 4C), and in presence of excess Cu(I), at a ratio above 3 Cu(I):CueR (Figure 4D).…”