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A Time-Resolved Diffusion Technique for Detection of the Conformational Changes and Molecular Assembly/Disassembly Processes of Biomolecules.

ABSTRACT: Biological liquid-liquid phase separation (LLPS) is driven by dynamic and multivalent interactions, which involves conformational changes and intermolecular assembly/disassembly processes of various biomolecules. To understand the molecular mechanisms of LLPS, kinetic measurements of the intra- and intermolecular reactions are essential. In this review, a time-resolved diffusion technique which has a potential to detect molecular events associated with LLPS is presented. This technique can detect changes in protein conformation and intermolecular interaction (oligomer formation, protein-DNA interaction, and protein-lipid interaction) in time domain, which are difficult to obtain by other methods. After the principle and methods for signal analyses are described in detail, studies on photoreactive molecules (intermolecular interaction between light sensor proteins and its target DNA) and a non-photoreactive molecule (binding and folding reaction of α-synuclein upon mixing with SDS micelle) are presented as typical examples of applications of this unique technique.

SUBMITTER: Nakasone Y

PROVIDER: S-EPMC8278059 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:A relatively stable and flexible capsid is critical to the viral life cycle. However, the capsid dynamics and cytosol trafficking of porcine circovirus type 2 (PCV2) during its infectious cycle are poorly understood. Here, we report the structural stability and conformation flexibility of PCV2 virions by genome labeling and the use of three monoclonal antibodies (MAbs) against the native capsid of PCV2. Genome labeling showed that the infectivity of the PCV2 virion was not affected by conjugation with deoxy-5-ethynylcytidine (EdC). Heat stability experiments indicated that PCV2 capsids started to disassemble at 65°C, causing binding incompetence for all antibodies, and the viral genome was released without capsid disassembly upon heating at 60°C. Antibody binding experiments with PCV2 showed that residues 186 to 192 were concealed in the early endosomes of epithelial PK-15 and monocytic 3D4/31 cells with or without chloroquine treatment and then exposed in PK-15 cytosol and the 3D4/31 nucleus. Viral propagation and localization experiments showed that PCV2 replication and cytosol trafficking were not significantly affected by microtubule depolymerization in monocytic 3D4/31 cells treated with nocodazole. These findings demonstrated that nuclear targeting of viral capsids involved conformational changes, the PCV2 genome was released from the assembled capsid, and the transit of PCV2 particles was independent of microtubules in 3D4/31 cells.IMPORTANCE Circovirus is the smallest virus known to replicate autonomously. Knowledge of viral genome release may provide understanding of viral replication and a method to artificially inactivate viral particles. Currently, little is known about the release model of porcine circovirus type 2 (PCV2). Here, we report the release of the PCV2 genome from assembled capsid and the intracellular trafficking of infectious PCV2 by alterations in the capsid conformation. Knowledge of PCV2 capsid stability and dynamics is essential to understanding its infectious cycle and lays the foundation for discovering powerful targets for therapeutic and prophylactic intervention.

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A Time-Resolved Diffusion Technique for Detection of the Conformational Changes and Molecular Assembly/Disassembly Processes of Biomolecules.

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