A low-E magic angle spinning probe for biological solid state NMR at 750 MHz

Crossed-coil NMR probes are a useful tool for reducing sample heating for biological solid state NMR. In a crossed-coil probe, the higher frequency ¹H field, which is the primary source of sample heating in conventional probes, is produced by a separate low-inductance resonator. Because a smaller driving voltage is required, the electric field across the sample and the resultant heating is reduced. In this work we describe the development of a magic angle spinning (MAS) solid state NMR probe utilizing a dual resonator. This dual resonator approach, referred to as “low-E,” was originally developed to reduce heating in samples of mechanically aligned membranes. The study of inherently dilute systems, such as proteins in lipid bilayers, via MAS techniques requires large sample volumes at high field to obtain spectra with adequate signal-to-noise ratio under physiologically relevant conditions. With the low-E approach, we are able to obtain homogeneous and sufficiently strong radiofrequency fields for both ¹H and ¹³C frequencies in a 4 mm probe with a ¹H frequency of 750 MHz. The performance of the probe using windowless dipolar recoupling sequences is demonstrated on model compounds as well as membrane-embedded peptides.     

A strip-shield improves the efficiency of a solenoid coil in probes for high-field solid-state NMR of lossy biological samples

A strip-shield inserted between a high inductance double-tuned solenoid coil and the glass tube containing the sample improves the efficiency of probes used for high-field solid-state NMR experiments on lossy aqueous samples of proteins and other biopolymers. A strip-shield is a coil liner consisting of thin copper strips layered on a PTFE (polytetrafluoroethylene) insulator. With lossy samples, the shift in tuning frequency is smaller, the reduction in Q, and RF-induced heating are all significantly reduced when the strip-shield is present. The performance of 800 MHz ¹H/¹⁵N and ¹H/¹³C double-resonance probes is demonstrated on aqueous samples of membrane proteins in phospholipid bilayers.     

A general assignment method for oriented sample (OS) solid-state NMR of proteins based on the correlation of resonances through heteronuclear dipolar couplings in samples aligned parallel and perpendicular to the magnetic field

The acquisition and different appearances observed for wide bandwidth solid-state MAS NMR spectra of low-γ nuclei, using (14)N as an illustrative nucleus and employing two different commercial spectrometers (Varian, 14.1T and Bruker, 19.6T), have been compared/evaluated and optimized from an experimental NMR and an electronic engineering point of view, to account for the huge differences in these spectra. The large differences in their spectral appearances, employing the recommended/standard experimental set-up for the two different spectrometers, are shown to be associated with quite large differences in the electronic design of the two types of preamplifiers, which are connected to their respective probes through a 50Ω cable, and are here completely accounted for. This has led to different opportunities for optimum performances in the acquisition of nearly ideal wide bandwidth spectra for low-γ nuclei on the two spectrometers by careful evaluation of the length for the 50Ω probe-to-preamp cable for the Varian system and appropriate changes to the bandwidth (Q) of the NMR probe used on the Bruker spectrometer. Earlier, we reported quite distorted spectra obtained with Varian Unity INOVA spectrometers (at 11.4 and 14.1T) in several exploratory wide bandwidth (14)N MAS NMR studies of inorganic nitrates and amino acids. These spectra have now been compared/evaluated with fully analyzed (14)N MAS spectra correspondingly acquired at 19.6T on a Bruker spectrometer. It is shown that our upgraded version of the STARS simulation/iterative-fitting software is capable of providing identical sets for the molecular spectral parameters and corresponding fits to the experimental spectra, which fully agree with the electronic measurements, despite the highly different appearances for the MAS NMR spectra acquired on the Varian and Bruker spectrometers.     

Low-E probe for (19)F-(1)H NMR of dilute biological solids

Sample heating induced by radio frequency (RF) irradiation presents a significant challenge to solid state NMR experiments in proteins and other biological systems, causing the sample to dehydrate which may result in distorted spectra and a damaged sample. In this work we describe a large volume, low-E (19)F-(1)H solid state NMR probe, which we developed for the 2D (19)F CPMG studies of dilute membrane proteins in a static and electrically lossy environment at 600MHz field. In (19)FCPMG and related multi-pulse (19)F-(1)H experiments the sample is heated by the conservative electric fields E produced in the sample coil at both (19)F and (1)H frequencies. Instead of using a traditional sample solenoid, our low-E (19)F-(1)H probe utilizes two orthogonal loop-gap resonators in order to minimize the conservative electric fields responsible for sample heating. Absence of the wavelength effects in loop-gap resonators results in homogeneous RF fields and enables the study of large sample volumes, an important feature for the dilute protein preparations. The orthogonal resonators also provide intrinsic isolation between the (19)F and (1)H channels, which is another major challenge for the (19)F-(1)H circuits where Larmor frequencies are only 6% apart. We detail steps to reduce (19)F background signals from the probe, which included careful choice of capacitor lubricants and manufacture of custom non-fluorinated coaxial cables. Application of the probe for two-dimensional (19)F CPMG spectroscopy in oriented lipid membranes is demonstrated with Flufenamic acid (FFA), a non-steroidal anti-inflammatory drug.     

15N and 31P solid-state NMR study of transmembrane domain alignment of M2 protein of influenza A virus in hydrated cylindrical lipid bilayers confined to anodic aluminum oxide nanopores

This communication reports the first example of a high resolution solid-state 15N 2D PISEMA NMR spectrum of a transmembrane peptide aligned using hydrated cylindrical lipid bilayers formed inside nanoporous anodic aluminum oxide (AAO) substrates. The transmembrane domain SSDPLVVA(A-15N)SIIGILHLILWILDRL of M2 protein from influenza A virus was reconstituted in hydrated 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine bilayers that were macroscopically aligned by a conventional micro slide glass support or by the AAO nanoporous substrate. 15N and 31P NMR spectra demonstrate that both the phospholipids and the protein transmembrane domain are uniformly aligned in the nanopores. Importantly, nanoporous AAO substrates may offer several advantages for membrane protein alignment in solid-state NMR studies compared to conventional methods. Specifically, higher thermal conductivity of aluminum oxide is expected to suppress thermal gradients associated with inhomogeneous radio frequency heating. Another important advantage of the nanoporous AAO substrate is its excellent accessibility to the bilayer surface for exposure to solute molecules. Such high accessibility achieved through the substrate nanochannel network could facilitate a wide range of structure-function studies of membrane proteins by solid-state NMR.