Nitroxide spin–labeled peptides for DNP‐NMR in‐cell studies

Abstract

Antimicrobial peptides (AMPs) that target lipid membranes show promise as alternatives to conventional antibiotics. However, the molecular mechanisms of membrane perturbation, as most studies are performed in model systems and in‐cell structural studies, have yet to be achieved. Solid‐state NMR spectroscopy is a valuable technique to investigate peptide‐membrane interactions and to determine the structure of peptides, but the short lifespan of bacteria, especially under magic angle spinning conditions, has not permitted in‐cell structural studies. Here, we present the first dynamic nuclear polarization (DNP)‐NMR in‐cell studies of Escherichia coli bacteria incubated with the AMP maculatin 1.1 (Mac1) in combination with novel nitroxide spin‐labeled peptides 2,2,6,6‐tetramethylpi‐peridine‐N‐oxyl‐4‐amino‐4‐carboxylic acid (TOAC)–[F3W]‐Mac1 (MacW) and TOAC‐TOAC‐MacW. The in‐cell 13C and 15N signal NMR enhancements, and 1H spin‐lattice T1 relaxation times showed that TOAC‐MacW and TOAC‐TOAC‐MacW performed better than the more hydrophilic biradical AMUPol used for DNP studies. Furthermore, the pores formed by the AMP increased the signal enhancements and decreased T1 values of specifically 13C‐ and 15N‐labeled Mac1. This approach has a great potential for determining the first in situ structures of AMPs in bacteria.—Sani, M.‐A., Zhu, S., Hofferek, V., Separovic, F. Nitroxide spin–labeled peptides for DNP‐NMR in‐cell studies. FASEB J. 33, 11021–11027 (2019). http://www.fasebj.org

Targeted DNP for biomolecular solid-state NMR

“…1b ) at the N-terminus of a maculatin mutant in order to investigate with MAS-DNP the interaction of this antimicrobial peptide with the bacterial cell membrane of E. coli . 74 Although neither the monoradical TOAC nor the biradical TOAC–TOAC are optimized PAs for MAS-DNP, DNP enhancement factors of 10 and 13, respectively, were obtained on cell samples.…”

“…Making use of spin labels in the context of MAS-DNP was thus a natural next step in the development of the technique, and several groups in the last years have investigated the benefits of tagging their biomolecular system with the PA required for hyperpolarization. 57,58 The motivation and objectives of the different approaches were diverse, going from optimization of the sample preparation for best sensitivity 59,60 and biocompatibility, [61][62][63] investigation of the bleaching/quenching effect of the paramagnetic tag [64][65][66] under DNP conditions for its potential use as a structural probe, [67][68][69] selection of a particular protein in a complex environment, 62,63,[70][71][72][73][74][75] to the detection of protein-protein interactions, 76 and the development of highresolution localized techniques through selective DNP. 77…”

“…59 (b) Spin-label amino acid TOAC compatible with solidphase peptide synthesis. 74 (c) and (d) MTS derivatives of TOTAPOL and AMUPol, respectively, which reacts with accessible cysteine thiol groups of proteins. 64,67 The original radical or biradical moiety is highlighted in red throughout the figure.…”

NMR Chemical Shift and Methylation of 4-Nitroimidazole: Experiment and Theory

“…DNP-enhanced NMR measurements [27] were carried out on a 400 MHz Bruker Avance III spectrometer, equipped with a 263 GHz gyrotron and a cryogenic triple-resonance 3. magic angle spinning (MAS) probe, at a spinning rate of 8 kHz and temperature of 100 K. An equal volume to mass ratio of 10 mM AMUPol (d 6 -glycerol/D 2 O/H 2 O 6 : 3 : 1) [28] was added to 4-NI and packed into a 3.2 mm sapphire rotor. DNP-enhanced 13 C and 15 N cross polarization (CP) MAS NMR measurements were carried out using a 1 H excitation pulse of 102 kHz, followed by a CP contact time of 1.5 ( 13 C) or 3 ms ( 15 N) using a 50 % ramped field on 1 H and a 62 kHz field for 13 15 N were acquired and 50 Hz line broadening applied before Fourier transformation.…”

In Situ Detection of Endogenous HIV Activation by Dynamic Nuclear Polarization NMR and Flow Cytometry

“…This unique reagent additionally provided control over the cellular localization of the DNP radical in addition to the direct assessment of radical localization by microscopy [24]. Other laboratories have also demonstrated the use of radical tagged biomolecules, including the antimicrobial peptides used to observe the membrane-associated resonances in intact bacteria [26]. Petzold and colleagues used DNP-NMR to observe antisense oligonucleotide drugs delivered to HEK 293T cells [27].…”