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The interaction between the T4 bacteriophage gp37 adhesin and the bacterial lipopolysaccharide (LPS) is a well-studied system, however, the affinity and strength of the interaction haven’t been analyzed so far. Here, we use atomic force microscopy to determine the strength of the interaction between the adhesin and its receptor, namely LPS taken from a wild strain of E. coli B. As negative controls we used LPSs of E. coli O111:B and Hafnia alvei. To study the interaction an AFM tip modified with the gp37 adhesin was used to scan surfaces of mica covered with one of the three different LPSs. Using the correlation between the surface topography images and the tip-surface interaction we could verify the binding between the specific LPS and the tip in contrast to the very weak interaction between the tip and the non-binding LPSs. Using force spectroscopy we could then measure the binding strength by pulling on the AFM tip until it lifted off from the surface. The force necessary to break the interaction between gp37 and LPS from E. coli B, LPS from E. coli O111:B and LPS from H. alvei were measured to be 70 ± 29 pN, 46 ± 13 pN and 45 ± 14 pN, respectively. The latter values are likely partially due to non-specific interaction between the gp37 and the solid surface, as LPS from E. coli O111:B and LPS from H. alvei have been shown to not bind to gp37, which is confirmed by the low correlation between binding and topography for these samples.
[Nachruf] Hugo Fasold
(2018)
Enhanced labeling density and whole-cell 3D dSTORM imaging by repetitive labeling of target proteins
(2018)
With continuing advances in the resolving power of super-resolution microscopy, the inefficient labeling of proteins with suitable fluorophores becomes a limiting factor. For example, the low labeling density achieved with antibodies or small molecule tags limits attempts to reveal local protein nano-architecture of cellular compartments. On the other hand, high laser intensities cause photobleaching within and nearby an imaged region, thereby further reducing labeling density and impairing multi-plane whole-cell 3D super-resolution imaging. Here, we show that both labeling density and photobleaching can be addressed by repetitive application of trisNTA-fluorophore conjugates reversibly binding to a histidine-tagged protein by a novel approach called single-epitope repetitive imaging (SERI). For single-plane super-resolution microscopy, we demonstrate that, after multiple rounds of labeling and imaging, the signal density is increased. Using the same approach of repetitive imaging, washing and re-labeling, we demonstrate whole-cell 3D super-resolution imaging compensated for photobleaching above or below the imaging plane. This proof-of-principle study demonstrates that repetitive labeling of histidine-tagged proteins provides a versatile solution to break the "labeling barrier" and to bypass photobleaching in multi-plane, whole-cell 3D experiments.
The ATP-binding cassette transporter TAPL translocates polypeptides from the cytosol into the lysosomal lumen. TAPL can be divided into two functional units: coreTAPL, active in ATP-dependent peptide translocation, and the N-terminal membrane spanning domain, TMD0, responsible for cellular localization and interaction with the lysosomal associated membrane proteins LAMP-1 and LAMP-2. Although the structure and function of ABC transporters were intensively analyzed in the past, the knowledge about accessory membrane embedded domains is limited. Therefore, we expressed the TMD0 of TAPL via a cell-free expression system and confirmed its correct folding by NMR and interaction studies. In cell as well as cell-free expressed TMD0 forms oligomers, which were assigned as dimers by PELDOR spectroscopy and static light scattering. By NMR spectroscopy of uniformly and selectively isotope labeled TMD0 we performed a complete backbone and partial side chain assignment. Accordingly, TMD0 has a four transmembrane helix topology with a short helical segment in a lysosomal loop. The topology of TMD0 was confirmed by paramagnetic relaxation enhancement with paramagnetic stearic acid as well as by nuclear Overhauser effects with c6-DHPC and cross-peaks with water.
Living matter is defined by metastability, implying a tightly balanced synthesis and turnover of cellular components. The first step of eukaryotic protein degradation via the ubiquitin-proteasome system (UPS) leads to peptides, which are subsequently degraded to single amino acids by an armada of proteases. A small fraction of peptides, however, escapes further cytosolic destruction and is transported by ATP-binding cassette (ABC) transporters into the endoplasmic reticulum (ER) and lysosomes. The ER-resident heterodimeric transporter associated with antigen processing (TAP) is a crucial component in adaptive immunity for the transport and loading of peptides onto major histocompatibility complex class I (MHC I) molecules. Although the function of the lysosomal resident homodimeric TAPL-like (TAPL) remains, until today, only loosely defined, an involvement in immune defense is anticipated since it is highly expressed in dendritic cells and macrophages. Here, we compare the gene organization and the function of single domains of both peptide transporters. We highlight the structural organization, the modes of substrate binding and translocation as well as physiological functions of both organellar transporters.
The blue light-dependent interaction between the proteins iLID and Nano allows recruiting and patterning proteins on GUV membranes, which thereby capture key features of patterns observed in nature. This photoswitchable protein interaction provides non-invasive, reversible and dynamic control over protein patterns of different sizes with high specificity and spatiotemporal resolution.
Accurate labeling of endogenous proteins for advanced light microscopy in living cells remains challenging. Nanobodies have been widely used for antigen labeling, visualization of subcellular protein localization and interactions. To facilitate an expanded application, we present a scalable and high-throughput strategy to simultaneously target multiple endogenous proteins in living cells with micro- to nanometer resolution. For intracellular protein labeling, we advanced nanobodies by site-specific and stoichiometric attachment of bright organic fluorophores. Their fast and fine-tuned intracellular transfer by microfluidic cell squeezing enabled high-throughput delivery with less than 10% dead cells. This strategy allowed for the dual-color imaging of distinct endogenous cellular structures, and culminated in super-resolution imaging of native protein networks in genetically non-modified living cells. The simultaneous delivery of multiple engineered nanobodies does not only offer exciting prospects for multiplexed imaging of endogenous protein, but also holds potential for visualizing native cellular structures with unprecedented accuracy.
Ribosome recycling orchestrated by ABCE1 is a fundamental process in protein translation and mRNA surveillance, connecting termination with initiation. Beyond the plenitude of well-studied translational GTPases, ABCE1 is the only essential factor energized by ATP, delivering the energy for ribosome splitting via two nucleotide-binding sites by a yet unknown mechanism. Here, we define how allosterically coupled ATP binding and hydrolysis events in ABCE1 empower ribosome recycling. ATP occlusion in the low-turnover control site II promotes formation of the pre-splitting complex and facilitates ATP engagement in the high-turnover site I, which in turn drives the structural reorganization required for ribosome splitting. ATP hydrolysis and ensuing release of ABCE1 from the small subunit terminate the post-splitting complex. Thus, ABCE1 runs through an allosterically coupled cycle of closure and opening at both sites, consistent with a processive clamp model. This study delineates the inner mechanics of ABCE1 and reveals why various ABCE1 mutants lead to defects in cell homeostasis, growth, and differentiation.
The immune system makes use of major histocompatibility complex class I (MHC I) molecules to present peptides to other immune cells, which can evoke an immune response. Within this process of antigen presentation, the MHC I peptide loading complex, consisting of a transporter associated with antigen processing TAP, MHC I, and chaperones, is key to the initiation of immune response by shuttling peptides from the cytosol into the ER lumen. However, it is still enigmatic how the flux of antigens is precisely coordinated in time and space, limiting our understanding of antigen presentation pathways. Here, we report on the development of a synthetic viral TAP inhibitor that can be cleaved by light. This photo-conditional inhibitor shows temporal blockade of TAP-mediated antigen translocation, which is unleashed upon illumination. The recovery of TAP activity was monitored at single-cell resolution both in human immune cell lines and primary cells. The development of a photo-conditional TAP inhibitor thus expands the repertoire of chemical intervention tools for immunological processes.