Live-cell three-dimensional single-molecule tracking reveals modulation of enhancer dynamics by NuRD.

To understand how the nucleosome remodeling and deacetylase (NuRD) complex regulates enhancers and enhancer-promoter interactions, we have developed an approach to segment and extract key biophysical parameters from live-cell three-dimensional single-molecule trajectories. Unexpectedly, this has revealed that NuRD binds to chromatin for minutes, decompacts chromatin structure and increases enhancer dynamics. We also uncovered a rare fast-diffusing state of enhancers and found that NuRD restricts the time spent in this state. Hi-C and Cut&Run experiments revealed that NuRD modulates enhancer-promoter interactions in active chromatin, allowing them to contact each other over longer distances. Furthermore, NuRD leads to a marked redistribution of CTCF and, in particular, cohesin. We propose that NuRD promotes a decondensed chromatin environment, where enhancers and promoters can contact each other over longer distances, and where the resetting of enhancer-promoter interactions brought about by the fast decondensed chromatin motions is reduced, leading to more stable, long-lived enhancer-promoter relationships.

Structure-specific amyloid precipitation in biofluids.

The composition of soluble toxic protein aggregates formed in vivo is currently unknown in neurodegenerative diseases, due to their ultra-low concentration in human biofluids and their high degree of heterogeneity. Here we report a method to capture amyloid-containing aggregates in human biofluids in an unbiased way, a process we name amyloid precipitation. We use a structure-specific chemical dimer, a Y-shaped, bio-inspired small molecule with two capture groups, for amyloid precipitation to increase affinity. Our capture molecule for amyloid precipitation (CAP-1) consists of a derivative of Pittsburgh Compound B (dimer) to target the cross ß-sheets of amyloids and a biotin moiety for surface immobilization. By coupling CAP-1 to magnetic beads, we demonstrate that we can target the amyloid structure of all protein aggregates present in human cerebrospinal fluid, isolate them for analysis and then characterize them using single-molecule fluorescence imaging and mass spectrometry. Amyloid precipitation enables unbiased determination of the molecular composition and structural features of the in vivo aggregates formed in neurodegenerative diseases.

Substrate aggregation and cooperative enzyme kinetics: consideration of enzyme access with large aggregates.

Consideration was given to a system in which an enzyme substrate indefinitely self-associates according to a general model with f sites of aggregation and a single intrinsic binding constant k. Where enzyme attack occurs at random points on the substrate surface, surface regions will be obscured from enzyme access by aggregate formation. The significance of reduced surface access for the infinite array of different size aggregates which co-exist was explored through use of a reacted site probability function, PA: the proportion of total substrate surface which is accessible to the enzyme was estimated as the fraction of total sites for aggregation which are unoccupied. The effective substrate concentration was thereby specified in terms of total substrate concentration (mA) by the simple expression mA = (1 - PA)mA. Plots of simulated v versus mA data were examined for a Michaelis-Menten enzyme of maximal velocity Vm and Michaelis constant Km to reveal deviations from expected enzyme behaviour; corresponding Hofstee (v/mA versus v) plots were found to be convex to the v axis as is characteristic of a negatively cooperative enzyme. As self-association is known to occur widely with large or small molecules in solution, the experimenter should be aware of the potential for these phenomena in kinetic studies to produce pseudo-allosteric effects, or to mask true allosteric behaviour.