The ability of amphipathic polymers to self-assemble with lipids and form nanodiscs has been a boon for the field of functional reconstitution of membrane proteins. In a field dominated by detergent micelles, a unique feature of polymer nanodiscs is their much-desired ability to align in the presence of an external magnetic field. Magnetic alignment facilitates the application of solidstate NMR spectroscopy and aids in the measurement of residual dipolar couplings (RDCs) via well-established solution NMR spectroscopy. In this study, we comprehensively investigate the magnetic-alignment properties of SMA-QA polymer based nanodiscs by using 31 P and 14 N solidstate NMR experiments under static conditions. The results reported herein demonstrate the spontaneous magnetic-alignment of large-size (≥ 20 nm diameter) SMA-QA nanodiscs (also called as macro-nanodiscs) with the lipid-bilayer-normal perpendicular to the magnetic field direction. Consequently, the orientation of macro-nanodiscs are further shown to flip their alignment axis parallel to the magnetic field direction upon the addition of a paramagnetic lanthanide salt. These results demonstrate the use of SMA-QA polymer nanodiscs for solid-state NMR applications including structural studies on membrane proteins..
Edited by Wolfgang PetiThe recent development of plants that overexpress antimicrobial peptides (AMPs) provides opportunities for controlling plant diseases. Because plants employ a broad-spectrum antimicrobial defense, including those based on AMPs, transgenic modification for AMP overexpression represents a potential way to utilize a defense system already present in plants. Herein, using an array of techniques and approaches, we report on VG16KRKP and KYE28, two antimicrobial peptides, which in combination exhibit synergistic antimicrobial effects against plant pathogens and are resistant against plant proteases. Investigating the structural origin of these synergistic antimicrobial effects with NMR spectroscopy of the complex formed between these two peptides and their mutated analogs, we demonstrate the formation of an unusual peptide complex, characterized by the formation of a bulky hydrophobic hub, stabilized by aromatic zippers. Using three-dimensional structure analyses of the complex in bacterial outer and inner membrane components and when bound to lipopolysaccharide (LPS) or bacterial membrane mimics, we found that this structure is key for elevating antimicrobial potency of the peptide combination. We conclude that the synergistic antimicrobial effects of VG16KRKP and KYE28 arise from the formation of a well-defined amphiphilic dimer in the presence of LPS and also in the cytoplasmic bacterial membrane environment. Together, these findings highlight a new application of solution NMR spectroscopy to solve complex structures to study peptidepeptide interactions, and they underscore the importance of structural insights for elucidating the antimicrobial effects of AMP mixtures.Despite progress in the field of antimicrobial therapeutics (1, 2), plant production remains profoundly affected by microbial disease outbreaks caused by pathogens of Xanthomonas and Pseudomonas species, leading to considerable losses in crop production and challenges for global food security (3). Finding new approaches for the management of microbe-borne diseases in plants remains a challenge, further complicated by the increasing occurrence of multidrug-resistant pathogens ("superbugs") that are immune to available treatments (4, 5). Additionally, commonly used bactericides and fungicides pose considerable environmental constraints (6), necessitating the development of sustainable management techniques that can effectively combat such pathogens.Antimicrobial peptides (AMPs) 3 represent the oldest domain of the evolutionary tree, being structurally and functionally conserved across all forms of life (7-10). While having attracted research interest for several decades, peptide-based antimicrobial therapy has seen accelerated attention during the last few years, motivated by its potential applicability against drug-resistant strains (11)(12)(13)(14)(15). The direct interaction of AMP with bacterial membranes and membrane components provides broad-spectrum antimicrobial effects (16), and for many AMPs also anti-inflammatory and oth...
The ability of amphipathic polymers to self-assemble with lipids and form nanodiscs has been a boon for the field of functional reconstitution of membrane proteins. In a field dominated by detergent micelles, a unique feature of polymer nanodiscs is their much-desired ability to align in the presence of an external magnetic field. Magnetic alignment facilitates the application of solidstate NMR spectroscopy and aids in the measurement of residual dipolar couplings (RDCs) via well-established solution NMR spectroscopy. In this study, we comprehensively investigate the magnetic-alignment properties of SMA-QA polymer based nanodiscs by using 31 P and 14 N solidstate NMR experiments under static conditions. The results reported herein demonstrate the spontaneous magnetic-alignment of large-size (≥ 20 nm diameter) SMA-QA nanodiscs (also called as macro-nanodiscs) with the lipid-bilayer-normal perpendicular to the magnetic field direction. Consequently, the orientation of macro-nanodiscs are further shown to flip their alignment axis parallel to the magnetic field direction upon the addition of a paramagnetic lanthanide salt. These results demonstrate the use of SMA-QA polymer nanodiscs for solid-state NMR applications including structural studies on membrane proteins. 14 N NMR Experiments: Nitrogen-14 NMR spectra were acquired using a Bruker 400 MHz solidstate NMR spectrometer and a 5 mm double-resonance probe operating at the 14 N resonance frequency of 28.910 MHz. 14 N NMR spectra were recorded using the quadrupole-echo pulse sequence 46 with a 90° pulse length of 8 µs and an echo-delay of 80 µs. 14 N magnetization was acquired using 25 ms acquisition time, 20,000 scans and a recycle delay of 0.9 s with no 1 H decoupling.
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