Live-cell protein labelling with nanometre precision by cell squeezing

Live-cell labelling techniques to visualize proteins with minimal disturbance are important; however, the currently available methods are limited in their labelling efficiency, specificity and cell permeability. We describe high-throughput protein labelling facilitated by minimalistic probes delivered to mammalian cells by microfluidic cell squeezing. High-affinity and target-specific tracing of proteins in various subcellular compartments is demonstrated, culminating in photoinduced labelling within live cells. Both the fine-tuned delivery of subnanomolar concentrations and the minimal size of the probe allow for live-cell super-resolution imaging with very low background and nanometre precision. This method is fast in probe delivery (~1,000,000 cells per second), versatile across cell types and can be readily transferred to a multitude of proteins. Moreover, the technique succeeds in combination with well-established methods to gain multiplexed labelling and has demonstrated potential to precisely trace target proteins, in live mammalian cells, by super-resolution microscopy

Synthetic protein-conductive membrane nanopores built with DNA

Nanopores are key in portable sequencing and research given their ability to transport elongated DNA or small bioactive molecules through narrow transmembrane channels. Transport of folded proteins could lead to similar scientific and technological benefits. Yet this has not been realised due to the shortage of wide and structurally defined natural pores. Here we report that a synthetic nanopore designed via DNA nanotechnology can accommodate folded proteins. Transport of fluorescent proteins through single pores is kinetically analysed using massively parallel optical readout with transparent silicon-on-insulator cavity chips vs. electrical recordings to reveal an at least 20-fold higher speed for the electrically driven movement. Pores nevertheless allow a high diffusive flux of more than 66 molecules per second that can also be directed beyond equillibria. The pores may be exploited to sense diagnostically relevant proteins with portable analysis technology, to create molecular gates for drug delivery, or to build synthetic cells

Lysosomal targeting of the ABC transporter TAPL is determined by membrane-localized charged residues

The human lysosomal polypeptide ABC transporter TAPL (ABC subfamily B member 9, ABCB9) transports 6-59-amino-acid-long polypeptides from the cytosol into lysosomes. The subcellular localization of TAPL depends solely on its N-terminal transmembrane domain, TMD0, which lacks conventional targeting sequences. However, the intracellular route and the molecular mechanisms that control TAPL localization remain unclear. Here, we delineated the route of TAPL to lysosomes and investigated the determinants of single trafficking steps. By synchronizing trafficking events by a retention using selective hooks (RUSH) assay and visualizing individual intermediate steps through immunostaining and confocal microscopy, we demonstrate that TAPL takes the direct route to lysosomes. We further identified conserved charged residues within TMD0 transmembrane helices that are essential for individual steps of lysosomal targeting. Substitutions of these residues retained TAPL in the endoplasmic reticulum (ER) or Golgi. We also observed that for release from the ER, a salt bridge between Asp-17 and Arg-57 is essential. An interactome analysis revealed that Yip1-interacting factor homolog B membrane-trafficking protein (YIF1B) interacts with TAPL. We also found that YIF1B is involved in ER-to-Golgi trafficking and interacts with TMD0 of TAPL via its transmembrane domain and that this interaction strongly depends on the newly identified salt bridge within TMD0. These results expand our knowledge about lysosomal trafficking of TAPL and the general function of extra transmembrane domains of ABC transporters

A systematic re-examination of processing of MHCI-bound antigenic peptide precursors by ER aminopeptidase 1

Endoplasmic reticulum aminopeptidase 1 (ERAP1) trims antigenic peptide precursors to generate mature antigenic peptides for presentation by major histocompatibility complex class I (MHCI) molecules and regulates adaptive immune responses. ERAP1 has been proposed to trim peptide precursors both in solution and in pre-formed MHCI-peptide complexes, but which mode is more relevant to its biological function remains controversial. Here, we compared ERAP1-mediated trimming of antigenic peptide precursors in solution or when bound to three MHCI alleles, HLA-B*58, HLA-B*08 and HLA-A*02. For all MHCI-peptide combinations, peptide binding onto MHCI protected against ERAP1-mediated trimming. In only a single MHCI-peptide combination, trimming of an HLA-B*08-bound 12mer progressed at a considerable rate, albeit still slower than in solution. Results from thermodynamic, kinetic and computational analyses suggested that this 12mer is highly labile and that apparent on-MHC trimming rates are always slower than that of MHCI-peptide dissociation. Both ERAP2 and leucine aminopeptidase, an enzyme unrelated to antigen processing, could trim this labile peptide from pre-formed MHCI complexes as efficiently as ERAP1. A pseudopeptide analogue with high affinity for both HLA-B*08 and the ERAP1 active site could not promote the formation of a ternary ERAP1-MHCI-peptide complex. Similarly, no interactions between ERAP1 and purified peptide loading complex (PLC) were detected in the absence or presence of a pseudopeptide trap. We conclude that MHCI binding protects peptides from ERAP1 degradation and that trimming in solution, along with the dynamic nature of peptide binding to MHCI, are sufficient to explain ERAP1 processing of antigenic peptide precursors

Metal-Chelating Amino Acids As Building Blocks For Synthetic Receptors Sensing Metal Ions And Histidine-Tagged Proteins

Protein structure and function rely on a still not fully understood interplay of energetic and entropic constraints defined by the permutation of the twenty genetically encoded amino acids. Many attempts have been undertaken to design peptide ± peptide interaction pairs and synthetic receptors de novo by using this limited number of building blocks. We describe a rational approach to creating a building block based on a tailored metal-chelating amino acid. Ne,Ne-bis(carboxymethyl)-L-lysine can be flexibly introduced into peptides by 9-fluorenylmethoxycarbonyl solidphase chemistry. The corresponding metal-chelating peptides act as metal sensors and synthetic receptors for histidine-tagged proteins. These biochemical tweezers will open new ways to control protein ± protein interactions, to design peptide-based interaction pairs, or to generate switchable protein function

Protein resistant oligo(ethylene glycol) terminated self-assembled monolayers of thiols on gold by vapor deposition in vacuum

Kankate L, Werner U, Turchanin A, Gölzhäuser A, Großmann H, Tampe R. Protein resistant oligo(ethylene glycol) terminated self-assembled monolayers of thiols on gold by vapor deposition in vacuum. Biointerphases. 2010;5(2):30-36.Protein resistant oligo(ethylene glycol) (OEG) terminated self-assembled monolayers (SAMs) of thiols on gold are commonly used for suppression of nonspecific protein adsorption in biology and biotechnology. The standard preparation for these SAMs is the solution method (SM) that involves immersion of the gold surface in an OEG solution. Here the authors present the preparation of 11-(mercaptoundecyl)-triethylene glycol [HS(CH(2))(11)(OCH(2)CH(2))(3)OH] SAMs on gold surface by vapor deposition (VD) in vacuum. They compare the properties of SAMs prepared by VD and SM using x-ray photoelectron spectroscopy (XPS), polarization modulation infrared reflection absorption spectroscopy, and surface plasmon resonance measurements. VD and SM SAMs exhibit similar packing density and show a similar resistance to the nonspecific adsorption of various proteins (bovine serum albumin, trypsin, and myoglobin) under physiological conditions. A very high sensitivity of the OEG SAMs to x-ray radiation is found, which allows tuning their protein resistance. These results show a new path to in situ engineering, analysis, and patterning of protein resistant OEG SAMs by high vacuum and ultrahigh vacuum techniques

Molecular Printboards as a General Platform for Protein Immobilization: A Supramolecular Solution to Nonspecific Adsorption

Be specific: A supramolecular adsorbate consisting of an adamantyl group (red) and an oligo(ethylene glycol) chain has been designed to prevent nonspecific protein adsorption at cyclodextrin molecular printboards. The adamantyl group allows specific and reversible interactions. Specific immobilization of proteins (gray) is possible through multivalent orthogonal linkers by effective replacement of the monovalent adsorbate (Ni2+ ions (green) may be needed; see picture)