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The development of near‐infrared (NIR) fluorophores that have both excellent chemical stability and photostability, as well as efficient cell permeability, is highly demanded. In this study, we present phospha‐rhodamine (POR) dyes which display significantly improved performance for protein labeling. This is achieved by incorporating a 2‐carboxy‐3‐benzothiophenyl group at the 9‐position of the xanthene scaffold. The resulting cis and trans isomers were successfully isolated and structurally characterized using X‐ray diffraction. The HaloTag ligand conjugates of the two isomers exhibited different staining abilities in live cells. While the cis isomer showed non‐specific accumulation on the organelle membranes, the trans isomer selectively labeled the HaloTag‐fused proteins, enabling the long‐term imaging of cell division and the 5‐color imaging of cell organelles. Molecular dynamics simulations of the HaloTag ligand conjugates within the lipid membrane suggested that the cis isomer is more prone to forming oligomers in the membrane. In contrast, the oligomerization of the trans isomer is effectively suppressed by its interaction with the lipid molecules. By taking advantage of the superior labeling performance of the trans isomer and its NIR‐emissive properties, multi‐color time‐lapse super‐resolution 3D imaging, namely super‐resolution 5D‐imaging, of the interconnected network between the endoplasmic reticulum and microtubules was achieved in living cells. [ABSTRACT FROM AUTHOR]

Abstract YidC/Alb3/Oxa1 family proteins are involved in the insertion and assembly of membrane proteins. The core five transmembrane regions of YidC, which are conserved in the protein family, form a positively charged cavity open to the cytoplasmic side. The cavity plays an important role in membrane protein insertion. In all reported structural studies of YidC, the second cytoplasmic loop (C2 loop) was disordered, limiting the understanding of its role. Here, we determined the crystal structure of YidC including the C2 loop at 2.8 Å resolution with R / R free = 21.8/27.5. This structure and subsequent molecular dynamics simulation indicated that the intrinsic flexible C2 loop covered the positively charged cavity. This crystal structure provides the coordinates of the complete core region including the C2 loop, which is valuable for further analyses of YidC. [ABSTRACT FROM AUTHOR]

The bacterial peptidoglycan enclosing the cytoplasmic membrane is a fundamental cellular architecture. The integral membrane protein MurJ plays an essential role in flipping the cell wall building block Lipid II across the cytoplasmic membrane for peptidoglycan biosynthesis. Previously reported crystal structures of MurJ have elucidated its V-shaped inward- or outward-facing forms with an internal cavity for substrate binding. MurJ transports Lipid II using its cavity through conformational transitions between these two forms. Here, we report two crystal structures of inward-facing forms from Arsenophonus endosymbiont MurJ and an unprecedented crystal structure of Escherichia coli MurJ in a "squeezed" form, which lacks a cavity to accommodate the substrate, mainly because of the increased proximity of transmembrane helices 2 and 8. Subsequent molecular dynamics simulations supported the hypothesis that the squeezed form is an intermediate conformation. This study fills a gap in our understanding of the Lipid II flipping mechanism. [Display omitted] • Crystal structures of bacterial MurJ reveal its squeezed and inward forms • The squeezed form does not contain an internal cavity required for substrate binding • Transmembrane helices 2, 7, and 8 contribute to MurJ structural transitions • The squeezed form occurs during outward-to-inward conformational changes Kohga et al. present an unprecedented "squeezed" structure of MurJ, a Lipid II flippase that is essential for peptidoglycan biogenesis in gram-negative bacteria. The "squeezed" form is proposed to be an intermediate during outward-to-inward conformational changes after substrate release, which is further supported by molecular dynamics simulations. [ABSTRACT FROM AUTHOR]

Summary Membrane proteins play important roles in various cellular functions. To analyze membrane proteins, nanodisc technology using membrane scaffold proteins allows single membrane protein units to be embedded into the lipid bilayer disc without detergents. Recent advancements in high-speed atomic force microscopy (HS-AFM) have enabled us to monitor the real-time dynamics of proteins in solution at the nanometer scale. In this study, we report HS-AFM imaging of membrane proteins reconstituted into nanodiscs using two membrane protein complexes, SecYEG complex and MgtE dimer. The observed images showed single particles of membrane protein-embedded nanodiscs in an end-up orientation whereby the membrane was fixed parallel to the supporting solid surface and in a side-on orientation whereby the membrane plane was vertically fixed to the solid surface, enabling the elucidation of domain fluctuations in membrane proteins. This technique provides a basic method for the high-resolution imaging of single membrane proteins by HS-AFM. Graphical Abstract Highlights • Membrane protein-embedded nanodiscs were used for high-speed (HS)-AFM observation • Nanodiscs in an end-up and side-on orientation were observed by HS-AFM in real time • HS-AFM combined with nanodiscs is a powerful tool for membrane protein dynamics Haruyama et al. established a procedure for the observation of membrane proteins in nanodiscs by high-speed AFM in real time. Using the two-dimensional crystal of streptavidin as well as mica as a solid support surface, dynamic structural changes of membrane proteins in the lipid environment were visualized from two distinct angles. [ABSTRACT FROM AUTHOR]