Protein resonance assignment by BSH‐CP‐based 3D solid‐state NMR experiments: A practical guide

Solid-state NMR (ssNMR) spectroscopy has evolved into a powerful method to obtain structural information and to study the dynamics of proteins at atomic resolution and under physiological conditions. The method is especially well suited to investigate insoluble and noncrystalline proteins that cannot be investigated easily by X-ray crystallography or solution NMR. To allow for detailed analysis of ssNMR data, the assignment of resonances to the protein atoms is essential. For this purpose, a set of three-dimensional (3D) spectra needs to be acquired. Band-selective homo-nuclear cross-polarization (BSH-CP) is an effective method for magnetization transfer between carbonyl carbon (CO) and alpha carbon (CA) atoms, which is an important transfer step in multidimensional ssNMR experiments. This tutorial describes the detailed procedure for the chemical shift assignment of the backbone atoms of 13C–15N-labeled proteins by BSH-CP-based 13C-detected ssNMR experiments. A set of six 3D experiments is used for unambiguous assignment of the protein backbone as well as certain side-chain resonances. The tutorial especially addresses scientists with little experience in the field of ssNMR and provides all the necessary information for protein assignment in an efficient, time-saving approach.European Research Council http://dx.doi.org/10.13039/501100000781Max Planck Society http://dx.doi.org/10.13039/501100004189Leibniz‐Forschungsinstitut für Molekulare PharmakologiePeer Reviewe

Measurement of backbone hydrogen-deuterium exchange in the type III secretion system needle protein PrgI by solid-state NMR

In this report we present site-specific measurements of amide hydrogen-deuterium exchange rates in a protein in the solid state phase by MAS NMR. Employing perdeuteration, proton detection and a high external magnetic field we could adopt the highly efficient Relax-EXSY protocol previously developed for liquid state NMR. According to this method, we measured the contribution of hydrogen exchange on apparent 15N longitudinal relaxation rates in samples with differing D2O buffer content. Differences in the apparent T1 times allowed us to derive exchange rates for multiple residues in the type III secretion system needle protein

Accurate Determination of Motional Amplitudes in Biomolecules by Solid-State NMR

Protein dynamics are an intrinsically important factor when considering a protein’s biological function. Understanding these motions is often limited through the use of static structure determination methods, namely, X-ray crystallography and cryo-EM. Molecular simulations have allowed for the prediction of global and local motions of proteins from these static structures. Nevertheless, determining local dynamics at residue-specific resolution through direct measurement remains crucial. Solid-state nuclear magnetic resonance (NMR) is a powerful tool for studying dynamics in rigid or membrane-bound biomolecules without prior structural knowledge with the help of relaxation parameters such as T1 and T1ρ. However, these provide only a combined result of amplitude and correlation times in the nanosecond–millisecond frequency range. Thus, direct and independent determination of the amplitude of motions might considerably improve the accuracy of dynamics studies. In an ideal situation, the use of cross-polarization would be the optimal method for measuring the dipolar couplings between chemically bound heterologous nuclei. This would unambiguously provide the amplitude of motion per residue. In practice, however, the inhomogeneity of the applied radio-frequency fields across the sample leads to significant errors. Here, we present a novel method to eliminate this issue through including the radio-frequency distribution map in the analysis. This allows for direct and accurate measurement of residue-specific amplitudes of motion. Our approach has been applied to the cytoskeletal protein BacA in filamentous form, as well as to the intramembrane protease GlpG in lipid bilayers

High resolution observed in 800 MHz DNP spectra of extremely rigid type III secretion needles

The cryogenic temperatures at which dynamic nuclear polarization (DNP) solid-state NMR experiments need to be carried out cause line-broadening, an effect that is especially detrimental for crowded protein spectra. By increasing the magnetic field strength from 600 to 800 MHz, the resolution of DNP spectra of type III secretion needles (T3SS) could be improved by 22 %, indicating that inhomogeneous broadening is not the dominant effect that limits the resolution of T3SS needles under DNP conditions. The outstanding spectral resolution of this system under DNP conditions can be attributed to its low overall flexibility

Atomic-resolution structure of cytoskeletal bactofilin by solid-state NMR

Bactofilins are a recently discovered class of cytoskeletal proteins of which no atomic-resolution structure has been reported thus far. The bacterial cytoskeleton plays an essential role in a wide range of processes, including morphogenesis, cell division, and motility. Among the cytoskeletal proteins, the bactofilins are bacteria-specific and do not have a eukaryotic counterpart. The bactofilin BacA of the species Caulobacter crescentus is not amenable to study by x-ray crystallography or solution nuclear magnetic resonance (NMR) because of its inherent noncrystallinity and insolubility. We present the atomic structure of BacA calculated from solid-state NMR–derived distance restraints. We show that the core domain of BacA forms a right-handed β helix with six windings and a triangular hydrophobic core. The BacA structure was determined to 1.0 Å precision (heavy-atom root mean square deviation) on the basis of unambiguous restraints derived from four-dimensional (4D) HN-HN and 2D C-C NMR spectra

Protein resonance assignment by BSH‐CP‐based 3D solid‐state NMR experiments: A practical guide

Solid-state NMR (ssNMR) spectroscopy has evolved into a powerful method to obtain structural information and to study the dynamics of proteins at atomic resolution and under physiological conditions. The method is especially well suited to investigate insoluble and noncrystalline proteins that cannot be investigated easily by X-ray crystallography or solution NMR. To allow for detailed analysis of ssNMR data, the assignment of resonances to the protein atoms is essential. For this purpose, a set of three-dimensional (3D) spectra needs to be acquired. Band-selective homo-nuclear cross-polarization (BSH-CP) is an effective method for magnetization transfer between carbonyl carbon (CO) and alpha carbon (CA) atoms, which is an important transfer step in multidimensional ssNMR experiments. This tutorial describes the detailed procedure for the chemical shift assignment of the backbone atoms of 13C–15N-labeled proteins by BSH-CP-based 13C-detected ssNMR experiments. A set of six 3D experiments is used for unambiguous assignment of the protein backbone as well as certain side-chain resonances. The tutorial especially addresses scientists with little experience in the field of ssNMR and provides all the necessary information for protein assignment in an efficient, time-saving approach.European Research Council http://dx.doi.org/10.13039/501100000781Max Planck Society http://dx.doi.org/10.13039/501100004189Leibniz‐Forschungsinstitut für Molekulare PharmakologiePeer Reviewe

Synthetic Access to a Hydrocarbon-Soluble Trifluorinated Ge(II) Compound and its Sn(II) Congener

Trifluorinated germanium anions attracted attention of theoretical chemists already in the late 1990s to predict their physical and chemical properties. However these species were not synthesized in the laboratory, although substantial evidence for their existence was obtained from the mass spectrometry of GeF₄. The present study shows that controlled fluorination of LMNMe₂ (L = PhC­(N^<i>t</i>Bu)₂, M = Ge, Sn) using HF·pyridine in toluene leads to the formation of [LH₂]⁺[MF₃]⁻ under elimination of HNMe₂. The products contain the trifluorinated Ge­(II) and Sn­(II) anionic species which are stabilized by interionic H···F bonds. The new compounds were characterized by single crystal X-ray structural analysis, NMR spectroscopy, and elemental analysis

Atomic Structure and Handedness of the Building Block of a Biological Assembly

Noncovalent supramolecular assemblies possess in general several unique subunit–subunit interfaces.The basic building block of such an assembly consists of several subunits and contains all unique interfaces. Atomic-resolution structures of monomeric subunits are typically accessed by crystallography or solution NMR and fitted into electron microscopy density maps. However, the structure of the intact building block in the assembled state remains unknown with this hybrid approach. Here, we present the solid-state NMR atomic structure of the building block of the type III secretion system needle. The building block structure consists of a homotetrameric subunit complex with three unique supramolecular interfaces. Side-chain positions at the interfaces were solved at atomic detail. The high-resolution structure reveals unambiguously the helical handedness of the assembly, determined to be right-handed for the type III secretion system needle.Additionally, the axial rise per subunit could be extracted from the tetramer structure and independently validated by mass-per-length measurements