Biology subjects

Roh, S.-H.

Publications and source records attributed to Roh, S.-H..

5 recordsLinked to original sources

bioRxiv2025

CoVET: Cryo-Electron Tomography guided by optical electrophysiology

Neurons exhibit varying electrophysiological properties due to dynamic changes in spatiotemporal molecular networks. In situ cryo-electron tomography (cryo-ET) provides unique advantages for high-resolution visualization of macromolecular complexes within their cellular context. Although correlation with fluorescent labeling allows cryo-ET to target specific cellular regions, it does not adequately reflect the electrophysiological properties of heterogeneous neurons. To bridge high-resolution molecular imaging with electrophysiological properties of individual neurons, we developed a Correlative-Voltage Imaging and cryo-ET (CoVET) technique. The nondestructive nature of voltage imaging is compatible with cryo-ET, enabling a direct correlation between neuronal electrophysiology and molecular structures. Neurons were clustered based on their electrophysiological properties, allowing for single-cell-guided structural analysis using cryo-ET. We analyzed the translational landscapes of individual neurons and found distinct translational profiles and contextual information among ribosomes from different electrophysiological clusters. Our results highlight the importance of the correlation between the electrophysiological properties and molecular structures.

biophysics↗

bioRxiv2025

Monoclonal Nanobodies Alter the Activity and Assembly of the Yeast Vacuolar H+-ATPase

The vacuolar ATPase (V-ATPase; V1Vo) is a multi-subunit rotary nanomotor proton pump that acidifies organelles in virtually all eukaryotic cells, and extracellular spaces in some specialized tissues of higher organisms. Evidence suggests that metastatic breast cancers mislocalize V-ATPase to the plasma membrane to promote cell survival and facilitate metastasis, making the V-ATPase a potential drug target. We have generated a library of camelid single-domain antibodies (Nanobodies; Nbs) against lipid-nanodisc reconstituted yeast V-ATPase Vo proton channel subcomplex. Here, we present an in-depth characterization of three anti-Vo Nbs using biochemical and biophysical in vitro experiments. We find that the Nbs bind Vo with high affinity, with one Nb inhibiting holoenzyme activity and another one preventing enzyme assembly. Using cryoEM, we find that two of the Nbs bind the c subunit ring of the Vo on the lumen side of the complex. Additionally, we show that one of the Nbs raised against yeast Vo can pull down human V-ATPase (HsV1Vo). Our research demonstrates Nb versatility to target and modulate the activity of the V-ATPase, and highlights the potential for future therapeutic Nb development.

biochemistry↗

bioRxiv2023

Unraveling membrane protein localization and stabilization in nanodiscs

Nanodiscs are nanoscale structures consisting of a lipid bilayer surrounded by membrane scaffold proteins (MSPs). They are widely used in the study of membrane proteins (MPs) because they provide a stable lipid environment. However, the precise mechanism governing MP behavior within the nanodisc remains elusive. Here, we examined the cryo-EM structures of various MPs reconstituted in nanodiscs from an electron microscopy database (EMPIAR). By analyzing the heterogeneity and interactions in the nanodiscs, we found that MPs within nanodiscs display a distinct spatial preference toward the edges of the nanodisc shells. Furthermore, we observed that MPs can establish direct, amphipathic interactions with the MSPs, promoting protein stability. These interactions may induce a rearrangement of the MSP-MSP interactions, leading to the formation of MP-MSP interactions Collectively, our study provides structural and biophysical insights into how nanodiscs contribute to MP structural behavior and stability. SIGNIFICANCEBy thoroughly examining multiple deposited datasets of membrane proteins (MPs) reconstituted in nanodiscs, we have gathered compelling evidence that MPs exhibit a clear spatial inclination toward the periphery of the nanodisc shells. Moreover, we have observed that MPs establish direct and amphipathic interactions with membrane scaffold proteins (MSPs). These interactions have the potential to induce a rearrangement of the MSP-MSP interactions, consequently forming MP-MSP interactions. Through quantitative analysis, we have successfully characterized the significant role played by these interactions in ensuring the overall stability of the proteins.

biophysics↗

bioRxiv2023

A structural vista of phosducin-like PhLP2A-chaperonin TRiC cooperation during the ATP-driven folding cycle

Proper cellular proteostasis, essential for viability, requires a network of chaperones and cochaperones. ATP-dependent chaperonin TRiC/CCT partners with cochaperones prefoldin (PFD) and phosducin-like proteins (PhLPs) to facilitate the folding of essential eukaryotic proteins. Using cryoEM and biochemical analyses, we determine the ATP-driven cycle of TRiC-PFD-PhLP2A interaction. In the open TRiC state, PhLP2A binds to the chambers equator while its N-terminal H3-domain binds to the apical domains of CCT3/4, thereby displacing PFD from TRiC. ATP-induced TRiC closure rearranges the contacts of PhLP2A domains within the closed chamber. In the presence of substrate, actin and PhLP2A segregate into opposing chambers, each binding to the positively charged inner surfaces formed by CCT1/3/6/8. Notably, actin induces a conformational change in PhLP2A, causing its N-terminal helices to extend across the inter-ring interface to directly contact a hydrophobic groove in actin. Our findings reveal an ATP-driven PhLP2A structural rearrangement cycle within the TRiC chamber to facilitate folding. Highlights- Structural analysis of TRiC-mediated folding cycle with cochaperones PhLP2A and PFD. - The interactions of PhLP2A and PFD with TRiC are mutually exclusive. - PhLP2A domains interact in a subunit-specific manner with the TRiC chamber. - PhLP2A domains are rearranged in ATP-closed TRiC to contact actin across the ring interface

biophysics↗

bioRxiv2023

Structural insights into ubiquitin chain cleavage by Legionella ovarian tumor deubiquitinases

Although ubiquitin is found only in eukaryotes, several pathogenic bacteria and viruses possess proteins that hinder the host ubiquitin system. Legionella, a gram-negative intracellular bacterium, possesses an ovarian tumor (OTU) family of deubiquitinases (Lot DUBs). Herein, we describe the molecular characteristics of Lot DUBs. We elucidated the structure of LotA OTU1 domain and revealed that entire Lot DUBs possess a characteristic extended helical lobe (EHL) that is not found in other OTU-DUBs. The structural topology of EHL is the same throughout the Lot family, and it provides an S1' ubiquitin-binding site. Moreover, the catalytic triads of Lot DUBs resemble those of the A20-type OTU-DUBs. Furthermore, we revealed a unique mechanism by which LotA OTU domains cooperate together to distinguish the length of the chain and preferentially cleaves longer K48-linked polyubiquitin chains. The LotA OTU1 domain itself cleaves K6-linked ubiquitin chains, while it is also essential for assisting the cleavage of longer K48-linked polyubiquitin chains by the OTU2 domain. Thus, this study provides novel insights into the structure and mechanism of action of Lot DUBs.

biochemistry↗