Andreas Hunkeler

Characterization of different water pools in solid-state NMR protein samples

We observed and characterized two distinct signals originating from different pools of water protons in solid-state NMR protein samples, namely from crystal water which exchanges polarization with the protein (on the NMR timescale) and is located in the protein-rich fraction at the periphery of the magic-angle spinning (MAS) sample container, and supernatant water located close to the axis of the sample container. The polarization transfer between the water and the protein can be probed by two-dimensional exchange spectroscopy, and we show that the supernatant water does not interact with protein on the timescale of the experiments. The two water pools have different spectroscopic properties, including resonance frequency, longitudinal, transverse and rotating frame relaxation times. The supernatant water can be removed almost completely physically or can be frozen selectively. Both measures lead to an enhancement of the quality factor of the probe circuit, accompanied by an improvement of the experimental signal/noise, and greatly simplify solvent-suppression by substantially reducing the water signal. We also present a tool, which allows filling solid-state NMR sample containers in a more efficient manner, greatly reducing the amount of supernatant water and maximizing signal/noise.

A Sedimented Sample of a 59 kDa Dodecameric Helicase Yields High‐Resolution Solid‐State NMR Spectra

Crystal clear: Preparing solid-state NMR samples that yield high-resolution spectra displaying high sensitivity is time-consuming and complicated. A sample of the 59 kDa protein DnaB, prepared simply by preparative centrifugation, provides spectra that are as good as the ones from carefully grown microcrystals.

A multi-sample 94 GHz dissolution dynamic-nuclear-polarization system

We describe the design and initial performance results of a multi-sample dissolution dynamic-nuclear-polarization (DNP) polarizer based on a Helium-temperature NMR cryostat for use in a wide-bore NMR magnet with a room-temperature bore. The system is designed to accommodate up to six samples in a revolver-style sample changer that allows changing samples at liquid-Helium temperature and at pressures ranging from ambient pressure down to 1 mbar. The multi-sample setup is motivated by the desire to do repetitive in vivo measurements and to characterize the DNP process by investigating samples of different chemical composition. The system can be loaded with up to six samples simultaneously to reduce sample loading and unloading. Therefore, series of experiments can be carried out faster and more reliably. The DNP probe contains an oversized microwave cavity and includes EPR and NMR capabilities for monitoring the DNP process. In the solid state, DNP enhancements corresponding to ∼45% polarization for [1-(13)C]pyruvic acid with a trityl radical have been measured. In the initial liquid-state acquisition experiments described here, the polarization was found to be ∼13%, corresponding to an enhancement factor exceeding 16,000 relative to thermal polarization at 9.4 T and ambient temperature.

Switched-angle spinning applied to bicelles containing phospholipid-associated peptides

In a model study, the proton NMR spectrum of the opioid pentapeptide leucine-enkephalin associated with bicelles is investigated. The spectral resolution for a static sample is limited due to the large number of anisotropic interactions, in particular strong proton–proton couplings, but resolution is greatly improved by magic-angle sample spinning. Here we present two-dimensional switched-angle spinning NMR experiments, which correlate the high-resolution spectrum of the membrane-bound peptide under magic-angle spinning with its anisotropic spectrum, leading to well-resolved spectra. The two-dimensional spectrum allows the exploitation of the high resolution of the isotropic spectrum, while retaining the structural information imparted by the anisotropic interactions in the static spectrum. Furthermore, switched-angle spinning techniques are demonstrated that allow one to record the proton spectrum of ordered bicellar phases as a function of the angle between the rotor axis and the magnetic field direction, thereby scaling the dipolar interactions by a predefined factor.

One‐ and Two‐Dimensional NMR Spectroscopy with a Magnetic‐Resonance Force Microscope

Magnetic-resonance imaging (MRI) is a non-invasive method to generate three-dimensional images which have a high information content and is used in various fields, ranging from human medicine to material science. In microimaging, the spatial resolution of MRI can approach one micrometer in favorable systems. Magnetic-resonance force microscopy (MRFM) has opened an avenue for extending imaging to the nanometer range. Two-dimensional images mapping the spin density with 90 nm resolution have recently been obtained and single-spin resolution, as reported for electrons, can be envisioned. As with MRI, the MRFM method is not limited to the three spatial dimensions. Spectroscopic dimensions can be added, providing detailed chemical and structural information at the atomic level. Such experiments are routinely performed in clinical MRI and are denoted as MR spectroscopic imaging (MRSI) or chemical-shift imaging (CSI). Spectral information, for example, from dipolar and quadrupolar interactions, has been used in MRFM experiments, in particular for generating new image contrast. The most important interaction—the chemical shift—however, has not been employed in MRFM, because of the difficulty of combining high spatial with high spectral resolution. Mechanical detection of chemical shifts, without spatial resolution, has been demonstrated on millimeter-sized samples with a setup where the field gradient vanishes at the sample position. MRFM provides an image of the object4s spin density by using the spatial variation of the resonance frequency in a magnetic field gradient, in full analogy to MRI. In contrast to MRI, the magnetization is detected mechanically with a micromechanical cantilever that measures the force on the spin magnetic moment in a magnetic field gradient. Spatial resolution and detection sensitivity can be significantly improved over inductively detected MRI, but the permanent presence of a gradient complicates spectroscopy. This problem is particularly true for chemical-shift spectroscopy, because the interaction has the same symmetry properties as the interaction with the magnetic field (gradient). In principle, it is conceivable to extract chemical-shift information in a gradient by recording zero-quantum spectra. Other, related methods have also been discussed; all of them, however, have limitations and the full information content of a regular NMR spectrum is not reproduced. An alternative approach, presented herein, is to temporarily move away the gradient source during the experiment (see Figure 1). The spectroscopic information can then be collected in a nearly homogeneous field. We shall demonstrate below that this method allows for chemical-shift imaging.

Digital feedback controller for force microscope cantilevers

We present a fast, digital signal processor (DSP)-based feedback controller that allows active motion damping of low-k, high-Q cantilevers in magnetic resonance force microscopy. A setup using a piezoelement attached to the cantilever base for actuation and a beam deflection sensor for tip motion detection is employed for controller demonstration. Controller parameters, derived according to stochastic optimal control theory, are formulated in a simple form readily implemented on a DSP, and extensions to other detection and actuation schemes are indicated. The controller is combined with an automated calibration scheme allowing for adaptive parameter adjustment. With the digital device operating at a sampling rate of 625kHz and 16bits of dynamic range, we were able to obtain closed-loop quality factors Qcl 103 at undiminished signal to noise, and reduces response time and vibration amplitu...