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This study demonstrates the use of nanoparticles prepared by a gas aggregation source for fabricating structures by combining laser sintering and ablation. At first, the morphology and optical properties of prepared nanoparticle coatings were characterized. Then, the response of coatings to laser irradiation at different powers or exposure times was studied by in situ time-of-flight mass spectrometry, followed by scanning electron microscopy measurements of the resulting structures. By comparing the numbers of detected Ag ions, that were ablated and desorbed, with changes in morphology after irradiation, the optimum conditions for laser sintering and ablation of Ag nanoparticle coatings were found. As a proof of concept we fabricated micromirrors from sintered metal, microwires from both sintered metal and interconnected nanoparticles, and arbitrary metallic bulky or nanoparticle patterns. Vacuum compatibility and the possibility of fabrication of both metallic and nanoparticle structures in one step predetermines applications of developed method in electronics or sensing.

The ATP-independent chaperone SurA protects unfolded outer membrane proteins (OMPs) from aggregation in the periplasm of Gram-negative bacteria, and delivers them to the β-barrel assembly machinery (BAM) for folding into the outer membrane (OM). Precisely how SurA recognises and binds its different OMP clients remains unclear. Escherichia coli SurA comprises three domains: a core and two PPIase domains (P1 and P2). Here, by combining methyl-TROSY NMR, single-molecule Förster resonance energy transfer (smFRET), and bioinformatics analyses we show that SurA client binding is mediated by two binding hotspots in the core and P1 domains. These interactions are driven by aromatic-rich motifs in the client proteins, leading to SurA core/P1 domain rearrangements and expansion of clients from collapsed, non-native states. We demonstrate that the core domain is key to OMP expansion by SurA, and uncover a role for SurA PPIase domains in limiting the extent of expansion. The results reveal insights into SurA-OMP recognition and the mechanism of activation for an ATP-independent chaperone, and suggest a route to targeting the functions of a chaperone key to bacterial virulence and OM integrity., (© 2024. The Author(s).)

The Sec translocon is a highly conserved membrane assembly for polypeptide transport across, or into, lipid bilayers. In bacteria, secretion through the core channel complex-SecYEG in the inner membrane-is powered by the cytosolic ATPase SecA. Here, we use single-molecule fluorescence to interrogate the conformational state of SecYEG throughout the ATP hydrolysis cycle of SecA. We show that the SecYEG channel fluctuations between open and closed states are much faster (~20-fold during translocation) than ATP turnover, and that the nucleotide status of SecA modulates the rates of opening and closure. The SecY variant PrlA4, which exhibits faster transport but unaffected ATPase rates, increases the dwell time in the open state, facilitating pre-protein diffusion through the pore and thereby enhancing translocation efficiency. Thus, rapid SecYEG channel dynamics are allosterically coupled to SecA via modulation of the energy landscape, and play an integral part in protein transport. Loose coupling of ATP-turnover by SecA to the dynamic properties of SecYEG is compatible with a Brownian-rachet mechanism of translocation, rather than strict nucleotide-dependent interconversion between different static states of a power stroke., (© 2023. The Author(s).)