High‐Resolution NMR Determination of the Dynamic Structure of Membrane Proteins

¹⁵N spin‐relaxation rates are demonstrated to provide critical information about the long‐range structure and internal motions of membrane proteins. Combined with an improved calculation method, the relaxation‐rate‐derived structure of the 283‐residue human voltage‐dependent anion channel revealed an anisotropically shaped barrel with a rigidly attached N‐terminal helix. Our study thus establishes an NMR spectroscopic approach to determine the structure and dynamics of mammalian membrane proteins at high accuracy and resolution.     

A High‐Pressure NMR Probe for Aqueous Geochemistry

A non‐magnetic piston‐cylinder pressure cell is presented for solution‐state NMR spectroscopy at geochemical pressures. The probe has been calibrated up to 20 kbar using in situ ruby fluorescence and allows for the measurement of pressure dependencies of a wide variety of NMR‐active nuclei with as little as 10 μL of sample in a microcoil. Initial ¹¹B NMR spectroscopy of the H₃BO₃–catechol equilibria reveals a large pressure‐driven exchange rate and a negative pressure‐dependent activation volume, reflecting increased solvation and electrostriction upon boron‐catecholate formation. The inexpensive probe design doubles the current pressure range available for solution NMR spectroscopy and is particularly important to advance the field of aqueous geochemistry.     

The Energetics of a Three‐State Protein Folding System Probed by High‐Pressure Relaxation Dispersion NMR Spectroscopy

The energetic and volumetric properties of a three‐state protein folding system, comprising a metastable triple mutant of the Fyn SH3 domain, have been investigated using pressure‐dependent ¹⁵N‐relaxation dispersion NMR from 1 to 2500 bar. Changes in partial molar volumes (ΔV) and isothermal compressibilities (Δκ_T) between all the states along the folding pathway have been determined to reasonable accuracy. The partial volume and isothermal compressibility of the folded state are 100 mL mol^?1 and 40 μL mol^?1 bar^?1, respectively, higher than those of the unfolded ensemble. Of particular interest are the findings related to the energetic and volumetric properties of the on‐pathway folding intermediate. While the latter is energetically close to the unfolded state, its volumetric properties are similar to those of the folded protein. The compressibility of the intermediate is larger than that of the folded state reflecting the less rigid nature of the former relative to the latter.     

Next‐Generation Heteronuclear Decoupling for High‐Field Biomolecular NMR Spectroscopy

Ultra‐high‐field NMR spectroscopy requires an increased bandwidth for heteronuclear decoupling, especially in biomolecular NMR applications. Composite pulse decoupling cannot provide sufficient bandwidth at practical power levels, and adiabatic pulse decoupling with sufficient bandwidth is compromised by sideband artifacts. A novel low‐power, broadband heteronuclear decoupling pulse is presented that generates minimal, ultra‐low sidebands. The pulse was derived using optimal control theory and represents a new generation of decoupling pulses free from the constraints of periodic and cyclic sequences. In comparison to currently available state‐of‐the‐art methods this novel pulse provides greatly improved decoupling performance that satisfies the demands of high‐field biomolecular NMR spectroscopy.     

Understanding J‐Modulation during Spatial Encoding for Sensitivity‐Optimized Ultrafast NMR Spectroscopy

Ultrafast (UF) NMR spectroscopy is an approach that yields 2D spectra in a single scan. This methodology has become a powerful analytical tool that is used in a large array of applications. However, UF NMR spectroscopy still suffers from an intrinsic low sensitivity, and from the need to compromise between sensitivity, spectral width, and resolution. In particular, the modulation of signal intensities by the spin–spin J‐coupling interaction (J‐modulation) impacts significantly on the intensities of the spectral peaks. This effect can lead to large sensitivity losses and even to missing spectral peaks, depending on the nature of the spin system. Herein, a general simulation package (Spinach) is used to describe J‐modulation effects in UF experiments. The results from simulations match with experimental data and the results of product operator calculations. Several methods are proposed to optimize the sensitivity in UF COSY spectra. The potential and drawbacks of the different strategies are also discussed. These approaches provide a way to adjust the sensitivity of UF experiments for a large range of applications.     

1H‐Detected Solid‐State NMR Studies of Water‐Inaccessible Proteins In Vitro and In Situ

¹H detection can significantly improve solid‐state NMR spectral sensitivity and thereby allows studying more complex proteins. However, the common prerequisite for ¹H detection is the introduction of exchangeable protons in otherwise deuterated proteins, which has thus far significantly hampered studies of partly water‐inaccessible proteins, such as membrane proteins. Herein, we present an approach that enables high‐resolution ¹H‐detected solid‐state NMR (ssNMR) studies of water‐inaccessible proteins, and that even works in highly complex environments such as cellular surfaces. In particular, the method was applied to study the K⁺ channel KcsA in liposomes and in situ in native bacterial cell membranes. We used our data for a dynamic analysis, and we show that the selectivity filter, which is responsible for ion conduction and highly conserved in K⁺ channels, undergoes pronounced molecular motion. We expect this approach to open new avenues for biomolecular ssNMR.     

NMR Backbone Assignment of Large Proteins by Using 13Cα‐Only Triple‐Resonance Experiments

Nuclear magnetic resonance (NMR) is a powerful tool to interrogate protein structure and dynamics residue by residue. However, the prerequisite chemical‐shift assignment remains a bottleneck for large proteins due to the fast relaxation and the frequency degeneracy of the ¹³C_α nuclei. Herein, we present a covariance NMR strategy to assign the backbone chemical shifts by using only HN(CO)CA and HNCA spectra that has a high sensitivity even for large proteins. By using the peak linear correlation coefficient (LCC), which is a sensitive probe even for tiny chemical‐shift displacements, we correctly identify the fidelity of approximately 92 % cross‐peaks in the covariance spectrum, which is thus a significant improvement on the approach developed by Snyder and Brüschweiler (66 %) and the use of spectral derivatives (50 %). Thus, we calculate the 4D covariance spectrum from HN(CO)CA and HNCA experiments, in which cross‐peaks with LCCs above a universal threshold are considered as true correlations. This 4D covariance spectrum enables the sequential assignment of a 42 kDa maltose binding protein (MBP), in which about 95 % residues are successfully assigned with a high accuracy of 98 %. Our LCC approach, therefore, paves the way for a residue‐by‐residue study of the backbone structure and dynamics of large proteins.     

Disentangling Complex Mixtures of Compounds with Near‐Identical 1H and 13C NMR Spectra using Pure Shift NMR Spectroscopy

The thorough analysis of highly complex NMR spectra using pure shift NMR experiments is described. The enhanced spectral resolution obtained from modern 2D HOBS experiments incorporating spectral aliasing in the ¹³C indirect dimension enables the distinction of similar compounds exhibiting near‐identical ¹H and ¹³C NMR spectra. It is shown that a complete set of extremely small Δδ(¹H) and Δδ(¹³C) values, even below the natural line width (1 and 5 ppb, respectively), can be simultaneously determined and assigned.     

Quadruple‐Resonance Magic‐Angle Spinning NMR Spectroscopy of Deuterated Solid Proteins

¹H‐detected magic‐angle spinning NMR experiments facilitate structural biology of solid proteins, which requires using deuterated proteins. However, often amide protons cannot be back‐exchanged sufficiently, because of a possible lack of solvent exposure. For such systems, using ²H excitation instead of ¹H excitation can be beneficial because of the larger abundance and shorter longitudinal relaxation time, T₁, of deuterium. A new structure determination approach, “quadruple‐resonance NMR spectroscopy”, is presented which relies on an efficient ²H‐excitation and ²H‐¹³C cross‐polarization (CP) step, combined with ¹H detection. We show that by using ²H‐excited experiments better sensitivity is possible on an SH3 sample recrystallized from 30 % H₂O. For a membrane protein, the ABC transporter ArtMP in native lipid bilayers, different sets of signals can be observed from different initial polarization pathways, which can be evaluated further to extract structural properties.     

Accessing Tetravalent Transition‐Metal Nitridophosphates through High‐Pressure Metathesis

Advancing the attainable composition space of a compound class can lead to fascinating materials. The first tetravalent metal nitridophosphate, namely Hf_9?xP₂₄N_52?4xO_4x (x≈1.84), was prepared by high‐pressure metathesis. The Group 4 nitridophosphates are now an accessible class of compounds. The high‐pressure metathesis reaction using a multianvil setup yielded single crystals that were suitable for structure analysis. Magnetic properties of the compound indicate Hf in oxidation state +IV. Optical measurements show a band gap in the UV region. The presented route unlocks the new class of Group 4 nitridophosphates by significantly improving the understanding of this nitride chemistry. Hf_9?xP₂₄N_52?4xO_4x (x≈1.84) is a model system and its preparation is the first step towards a systematic exploration of the transition‐metal nitridophosphates.     

Solid‐State NMR of PEGylated Proteins

PEGylated proteins are widely used in biomedicine but, in spite of their importance, no atomic‐level information is available since they are generally resistant to structural characterization approaches. PEGylated proteins are shown here to yield highly resolved solid‐state NMR spectra, which allows assessment of the structural integrity of proteins when PEGylated for therapeutic or diagnostic use.