Thermostable Nucleoid Protein Cren7 Slides Along DNA and Rapidly Dissociates From DNA While Not Inhibiting the Sliding of Other DNA-binding Protein.

A nucleoid protein Cren7 compacts DNA, contributing to the living of Crenarchaeum in high temperature environment. In this study, we investigated the dynamic behavior of Cren7 on DNA and its functional relation using single-molecule fluorescence microscopy. We found two mobility modes of Cren7, sliding along DNA and pausing on it, and the rapid dissociation kinetics from DNA.

Single-molecule characterization of target search dynamics of DNA-binding proteins in DNA-condensed droplets.

Target search models of DNA-binding proteins in cells typically consider search mechanisms that include 3D diffusion and 1D sliding, which can be characterized by single-molecule tracking on DNA. However, the finding of liquid droplets of DNA and nuclear components in cells cast doubt on extrapolation from the behavior in ideal non-condensed DNA conditions to those in cells. In this study, we investigate the target search behavior of DNA-binding proteins in reconstituted DNA-condensed droplets using single-molecule fluorescence microscopy.

Suppression of TDP-43 aggregation by artificial peptide binder targeting to its low complexity domain.

TAR DNA-binding protein 43 (TDP-43), aggregation prone protein, is a potential target of drug discovery for amyotrophic lateral sclerosis. The molecular binders, targeting the disordered low complexity domain (LCD) relevant to the aggregation, may suppress the aggregation. Recently, Kamagata et al.

Rational design of phase separating peptides based on phase separating protein sequence of p53.

Artificial phase-separating (PS) peptides can be used in various applications such as microreactors and drug delivery; however, the design of artificial PS peptides remains a challenge. This can be attributed to the limitation of PS-relevant residues that drive phase separation by interactions of their pairs in short peptides and the difficulty in the design involving interaction with target PS proteins. In this study, we propose a rational method to design artificial PS peptides that satisfy the requirements of liquid droplet formation and co-phase separation with target PS proteins based on the target PS protein sequence.

Structure-dependent recruitment and diffusion of guest proteins in liquid droplets of FUS.

Liquid droplets of a host protein, formed by liquid-liquid phase separation, recruit guest proteins and provide functional fields. Recruitment into p53 droplets is similar between disordered and folded guest proteins, whereas the diffusion of guest proteins inside droplets depends on their structural types. In this study, to elucidate how the recruitment and diffusion properties of guest proteins are affected by a host protein, we characterized the properties of guest proteins in fused in sarcoma (FUS) droplets using single-molecule fluorescence microscopy in comparison with p53 droplets.

Molecular principles of recruitment and dynamics of guest proteins in liquid droplets.

Despite the continuous discovery of host and guest proteins in membraneless organelles, complex host-guest interactions hinder the understanding of the molecular grammar governing liquid-liquid phase separation. In this study, we characterized the localization and dynamic properties of guest proteins in liquid droplets using single-molecule fluorescence microscopy. Eighteen guest proteins of different sizes, structures, and oligomeric states were examined in host p53 liquid droplets.

Characterization of design grammar of peptides for regulating liquid droplets and aggregates of FUS.

Liquid droplets of aggregation-prone proteins, which become hydrogels or form amyloid fibrils, are a potential target for drug discovery. In this study, we proposed an experiment-guided protocol for characterizing the design grammar of peptides that can regulate droplet formation and aggregation. The protocol essentially involves investigation of 19 amino acid additives and polymerization of the identified amino acids.

Liquid-like droplet formation by tumor suppressor p53 induced by multivalent electrostatic interactions between two disordered domains.

Early in vivo studies demonstrated the involvement of a tumor-suppressing transcription factor, p53, into cellular droplets such as Cajal and promyelocytic leukemia protein bodies, suggesting that the liquid-liquid phase separation (LLPS) might be involved in the cellular functions of p53. To examine this possibility, we conducted extensive investigations on the droplet formation of p53 in vitro. First, p53 itself was found to form liquid-like droplets at neutral and slightly acidic pH and at low salt concentrations.

Rational design using sequence information only produces a peptide that binds to the intrinsically disordered region of p53.

Intrinsically disordered regions (IDRs) of proteins are involved in many diseases. The rational drug design against disease-mediating proteins is often based on the 3D structure; however, the flexible structure of IDRs hinders the use of such structure-based design methods. Here, we developed a rational design method to obtain a peptide that can bind an IDR using only sequence information based on the statistical contact energy of amino acid pairs.

High Free-Energy Barrier of 1D Diffusion Along DNA by Architectural DNA-Binding Proteins.

Architectural DNA-binding proteins function to regulate diverse DNA reactions and have the defining property of significantly changing DNA conformation. Although the 1D movement along DNA by other types of DNA-binding proteins has been visualized, the mobility of architectural DNA-binding proteins on DNA remains unknown. Here, we applied single-molecule fluorescence imaging on arrays of extended DNA molecules to probe the binding dynamics of three structurally distinct architectural DNA-binding proteins: Nhp6A, HU, and Fis.

The Disordered Linker in p53 Participates in Nonspecific Binding to and One-Dimensional Sliding along DNA Revealed by Single-Molecule Fluorescence Measurements.

The tumor suppressor p53 is a multidomain transcription factor that can quickly bind to its target DNA by sliding along the DNA strand. We hypothesized that the intrinsically disordered and positively charged linker of p53 regulates its search dynamics first by directly interacting with DNA and second by modulating hopping of the core domain. To test the two hypotheses, we prepared five variants of p53 in which the length and charge of the linker were modulated.

One-Dimensional Search Dynamics of Tumor Suppressor p53 Regulated by a Disordered C-Terminal Domain.

Tumor suppressor p53 slides along DNA and finds its target sequence in drastically different and changing cellular conditions. To elucidate how p53 maintains efficient target search at different concentrations of divalent cations such as Ca and Mg, we prepared two mutants of p53, each possessing one of its two DNA-binding domains, the CoreTet mutant having the structured core domain plus the tetramerization (Tet) domain, and the TetCT mutant having Tet plus the disordered C-terminal domain. We investigated their equilibrium and kinetic dissociation from DNA and search dynamics along DNA at various [Mg].

One-Dimensional Sliding of p53 Along DNA Is Accelerated in the Presence of Ca(2+) or Mg(2+) at Millimolar Concentrations.

One-dimensional (1D) sliding of the tumor suppressor p53 along DNA is an essential dynamics required for its efficient search for the binding sites in the genome. To address how the search process of p53 is affected by the changes in the concentration of Mg(2+) and Ca(2+) after the cell damages, we investigated its sliding dynamics at different concentrations of the divalent cations. The 1D sliding trajectories of p53 along the stretched DNA were measured by using single-molecule fluorescence microscopy.