Membrane topologies of the PGLa antimicrobial peptide and a transmembrane anchor sequence by Dynamic Nuclear Polarization/solid-state NMR spectroscopy

Abstract

Dynamic Nuclear Polarization (DNP) has been introduced to overcome the sensitivity limitations of nuclear magnetic resonance (NMR) spectroscopy also of supported lipid bilayers. When investigated by solid-state NMR techniques the approach typically involves doping the samples with biradicals and their investigation at cryo-temperatures. Here we investigated the effects of temperature and membrane hydration on the topology of amphipathic and hydrophobic membrane polypeptides. Although the antimicrobial PGLa peptide in dimyristoyl phospholipids is particularly sensitive to topological alterations, the DNP conditions represent well its membrane alignment also found in bacterial lipids at ambient temperature. With a novel membrane-anchored biradical and purpose-built hardware a 17-fold enhancement in NMR signal intensity is obtained by DNP which is one of the best obtained for a truly static matrix-free system. Furthermore, a membrane anchor sequence encompassing 19 hydrophobic amino acid residues was investigated. Although at cryotemperatures the transmembrane domain adjusts it membrane tilt angle by about 10 degrees, the temperature dependence of two-dimensional separated field spectra show that freezing the motions can have beneficial effects for the structural analysis of this sequence.Solid-state NMR spectroscopy is unique in providing information at atomic resolution of membrane polypeptides in a native bilayer environment. The technique has been used for investigations of their structure, topology, dynamics and heterogeneous nature in phospholipid bilayers [1][2][3][4][5][6][7][8][9] . However, NMR suffers from its inherently low signal intensity and the investigation of membrane-associated polypeptides turns out particularly difficult due to the dilution of peptide in lipid (typically around 1-2 mole%) and the increased line width due to inhomogeneous line broadening. Although these limitations are most pronounced when static samples are investigated the large anisotropy of NMR chemical shift, dipolar and quadrupolar interactions has been successively used to determine the structure and/or topology of a number of peptides in oriented lipid bilayers 1,2,8,9 . These could be refined by additional distance restraints for example from magic angle sample spinning (MAS) solid-state NMR spectra 1,2,8,9 . During the last years considerable progress has been made in dynamic nuclear polarization (DNP) solid-state NMR and the technique has been shown to overcome many of the sensitivity limitations of solid-state NMR spectroscopy [10][11][12][13] . During DNP/solid-state NMR experiments the radicals dispersed in the sample result in a high electron polarization which is transferred to the 1 H bath through microwave irradiation. In a next step cross-polarization to the heteronuclei assures enhancements of the NMR signal typically by about two orders of magnitude 10,14 . Also in the case of membrane samples 4,11,15,16 or of membrane proteins in cellular environments considerable improvements have been obtained 17 .

Dynamic Nuclear Polarization/Solid‐State NMR Spectroscopy of Membrane Polypeptides: Free‐Radical Optimization for Matrix‐Free Lipid Bilayer Samples

“…In conclusion, a new palmitoyl analogue of AMUPOL, namely, PyPol–C16, was introduced for dynamic nuclear polarization (DNP)/solid‐state NMR spectroscopy studies of membrane samples, and it allowed signal enhancements that were increased by factors of 1.2 and 2 relative to those of AMUPOL for POPC and DMPC lipids, respectively (Tables and S1) and by a factor of 7 if compared to the signal enhancements observed for previous polarizing agents designed for membrane studies ,. The combination of biradicals designed for improved g ‐tensor orientation and inter‐radical distance together with homogenous distribution within the bilayer assured by their membrane anchor resulted in large improvements that led to signals that were up to 49‐fold more intense, which is the highest value so far obtained in matrix‐free environments, including stacks of mechanically oriented lipid bilayers . An enhancement of this size led to an approximate >18 000‐fold reduction in the experiment time relative to the time required at room temperature.…”

“…Our principal goal was to develop and investigate biradicals and protocols for optimal DNP enhancement in solvent‐free membrane samples. In particular, we aimed to develop biradicals and strategies for NMR structural investigations on supported lipid bilayers, for which enhancements factors are typically greatly reduced relative to those of glass‐forming solvent mixtures . Although the concept of locating a biradical in the membrane through a fatty acyl chain was previously presented, much higher enhancement factors (≫10) are desirable .…”

“…DMSO, glycerol), so that they could thus also be used for NMR structural investigations on supported lipid bilayers, for which enhancements factors are typically multifold reduced relative to those of glass‐forming solvent mixtures . In this paper, we demonstrate DNP enhancements of nearly 50 for the transmembrane hΦ19W peptide, which thereby enables a new range of experiments that are not feasible without DNP . This improvement in solvent‐free membrane DNP experiments was possible through the detailed study of a large number of interdependent parameters.…”

“…Our principal goal was to develop polarizing agents and protocols for improved DNP enhancement in membranes and for systems free of cryoprotectants (e.g. DMSO, glycerol), so that they could thus also be used for NMR structural investigations on supported lipid bilayers, for which enhancements factors are typically multifold reduced relative to those of glass‐forming solvent mixtures . In this paper, we demonstrate DNP enhancements of nearly 50 for the transmembrane hΦ19W peptide, which thereby enables a new range of experiments that are not feasible without DNP .…”

“…DNP has the potential to overcome sensitivity limitations, which often constitute the dominant bottleneck, and this thereby enables a new range of structural investigations of proteins and peptides embedded in anisotropic bilayer environments. Indeed, biomacromolecules have been studied by using DNP/solid‐state NMR spectroscopy in cells or membranes but with enhancement factors far below those obtained in isotropic environments ,,,…”