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. To mimic nuclear condensates, we reconstituted DNA-condensed droplets using dextran and PEG polymers. In the DNA-condensed droplets, we measured the translational movement of four DNA-binding proteins (p53, Nhp6A, Fis and Cas9) and p53 mutants possessing different structures, sizes, and oligomeric states. Our results demonstrate the presence of fast and slow mobility modes in DNA-condensed droplets for the four DNA-binding proteins. The slow mobility mode capability is correlated strongly to the molecular size and the number of DNA-binding domains on DNA-binding proteins, but only moderately to the affinity to single DNA segments in non-condensed conditions. The slow mobility mode in DNA-condensed droplets is interpreted as a multivalent interaction mode of the DNA-binding protein to multiple DNA segments.

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. The salt dependence analysis suggests a sliding with continuous contact to DNA, rather than hopping/jumping. The mutational analysis demonstrates that Cren7 slides along DNA while Trp (W26) residue interacts with the DNA. Furthermore, Cren7 does not impede the target search by a model transcription factor p53, implying no significant interference to other DNA-binding proteins on DNA. At high concentration of Cren7, the molecules form large clusters on DNA via bridging, which compacts DNA. We discuss how the dynamic behavior of Cren7 on DNA enables DNA-compaction and protein-bypass functions.

Supplier Satisfaction as a Mediating Effect on Delivery and Service: A PLS-SEM Approach in India During COVID-19 Pandemic.

PURPOSE: The role of suppliers is emerging to be of considerable importance given the current state of competition in times of globalization and technological advancements. Past studies have depicted considerable importance in determining supplier satisfaction for generating client satisfaction. The purpose of this article is to investigate the role that Supplier Satisfaction (SS) plays in partnering with manufacturers and traders in connection with the SME in India during the difficult COVID-19 era of business and to propose and demonstrate these collaborations. METHODS: I present a partial least-squares structural equation model. Production units and manufacturers are attracted to the satisfaction of both distributors as well as distributors, leading to B2B loyalty. RESULTS: This paper supports the idea that the benefits derived from the relationship between the four explanatory points of the solution are a significant extension of the first type. Ironically, the response to corporate policies and regulations has declined dramatically. This decision was surprising, because a strong communication strategy had already affected the image of the service provider. CONCLUSION: To be sure, the experiment has a system in place to update the performance of these suppliers by examining the effect of supplier certification on supplier verification and engagement.

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. Unlike p53 droplets, disordered guest proteins were recruited into FUS droplets more efficiently than folded guest proteins, suggesting physical exclusion of the folded proteins from the small voids of the droplet. The recruitment did not appear to depend on the physical parameters (electrostatic or cation-π) of guests, implying that molecular size exclusion limits intermolecular interaction-assisted uptake. The diffusion of disordered guest proteins was comparable to that of the host FUS, whereas that of folded proteins varied widely, similar to the results for host p53. The scaling exponent of diffusion highlights the molecular sieving of large folded proteins in droplets. Finally, we proposed a molecular recruitment and diffusion model for guest proteins in FUS droplets.

Engineering of the genome editing protein Cas9 to slide along DNA.

The genome editing protein Cas9 faces engineering challenges in improving off-target DNA cleavage and low editing efficiency. In this study, we aimed to engineer Cas9 to be able to slide along DNA, which might facilitate genome editing and reduce off-target cleavage. We used two approaches to achieve this: reducing the sliding friction along DNA by removing the interactions of Cas9 residues with DNA and facilitating sliding by introducing the sliding-promoting tail of Nhp6A. Seven engineered mutants of Cas9 were prepared, and their performance was tested using single-molecule fluorescence microscopy. Comparison of the mutations enabled the identification of key residues of Cas9 to enhance the sliding along DNA in the presence and absence of single guide RNA (sgRNA). The attachment of the tail to Cas9 mutants enhanced sliding along DNA, particularly in the presence of sgRNA. Together, using the proposed approaches, the sliding ability of Cas9 was improved up to eightfold in the presence of sgRNA. A sliding model of Cas9 and its engineering action are discussed herein.

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. Recruitment did not significantly depend on the structural properties of the guest proteins, but was moderately correlated with their length, total charge, and number of R and Y residues. In contrast, the diffusion of disordered guest proteins was comparable to that of host p53, whereas that of folded proteins varied widely. Molecular dynamics simulations suggest that folded proteins diffuse within the voids of the liquid droplet while interacting weakly with neighboring host proteins, whereas disordered proteins adapt their structures to form tight interactions with the host proteins. Our study provides insights into the key molecular principles of the localization and dynamics of guest proteins in liquid droplets.