The Ebola virus (EBOV) envelope glycoprotein (GP) mediates the fusion of the virion membrane with the membrane of susceptible target cells during infection. While proteolytic cleavage of GP by endosomal cathepsins and binding of the cellular receptor Niemann-Pick C1 protein (NPC1) are essential steps for virus entry, the detailed mechanisms by which these events promote membrane fusion remain unknown. Here, we applied single-molecule Förster resonance energy transfer (smFRET) imaging to investigate the structural dynamics of the EBOV GP trimeric ectodomain, and the functional transmembrane protein on the surface of pseudovirions. We show that in both contexts, pre-fusion GP is dynamic and samples multiple conformations. Removal of the glycan cap and NPC1 binding shift the conformational equilibrium, suggesting stabilization of conformations relevant to viral fusion. Furthermore, several neutralizing antibodies enrich alternative conformational states. This suggests that these antibodies neutralize EBOV by restricting access to GP conformations relevant to fusion. This work demonstrates previously unobserved dynamics of pre-fusion EBOV GP and presents a platform with heightened sensitivity to conformational changes for the study of GP function and antibody-mediated neutralization.

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Deciphering chromatin regulation at the molecular level is of fundamental importance for an understanding of cellular physiological and pathological processes. Chromatin is an extremely complex system due to its molecular organization, heterogeneous structure and multiscale dynamics induced by post translational modifications on chromatin itself and other regulatory effectors including transcription factors (TFs). One key class of chromatin interacting proteins are pioneer transcription factors. The main characteristic that distinguishes pioneer transcription factors from other TFs is their ability to specifically recognize their target DNA sequences in compacted chromatin and consequently to trigger chromatin opening, thus enabling the cellular machinery to locally access the DNA. In the context of cell fate reprogramming, this pioneer action is crucial, but its molecular mechanism is poorly understood. Known to be an essential protein involved in multiple steps of DNA regulation, Saccharomyces cerevisiae repressor-activator protein 1 (Rap1) is the pioneer transcription factor inspiring us to explore its pioneer role in such complex and dynamic system as chromatin. We recently established a single-molecule Förster resonance energy transfer (FRET) method, using micromirror total internal reflection fluorescence (mmTIRF) microscopy, dedicated to investigate structural dynamics of chromatin fibers. Combining single-molecule FRET and chemically defined synthetic chromatin segments, we uncovered the interconversion kinetics of discrete tetranucleosome units and the impact of the post-translational modifications by ubiquitylation on chromatin structure. Here, we demonstrate that Rap1 invades compacted chromatin and exerts chromatin remodeling. These studies yield fundamental insights into the molecular mechanisms of gene regulation by molecular interactions. 200-Plat Single Molecule Measurements Reveal Conformational TransitionsDuring DNA Clamp Loading and Unloading SeungWon Lee, Eunjin Ryu, Sukhyun Kang, Hajin Kim. UNIST, Ulsan, Republic of Korea. Proliferation cell nuclear antigen (PCNA) is a DNA clamp, playing an important role of providing a ''platform'' for various enzymes during DNA replication. The loading of the closed trimeric ring of PCNA into duplex DNA requires the ATP-dependent activity of replication factor C (RFC) complex. The unloading of PCNA from chromatin is crucial for the regulation of replication process and maintaining genomic stability and it was recently found that ATAD5 protein is complexed with RFC-like complex (RLC) to get involved in the unloading of PCNA. However, the molecular mechanisms of PCNA loading and unloading processes have remained poorly understood. Here, we report direct observation of the loading and unloading dynamics of human PCNA driven by RFC and ATAD5-RLC complexes, respectively, by single molecule fluorescence resonance energy transfer measurements. Distinct conformational stages during PCNA loading were clearly detected that represent open and closed conformations of P...