The companion of cellulose synthase 1 confers salt tolerance through a Tau-like mechanism in plants.

Microtubules are filamentous structures necessary for cell division, motility and morphology, with dynamics critically regulated by microtubule-associated proteins (MAPs). Here we outline the molecular mechanism by which the MAP, COMPANION OF CELLULOSE SYNTHASE1 (CC1), controls microtubule bundling and dynamics to sustain plant growth under salt stress. CC1 contains an intrinsically disordered N-terminus that links microtubules at evenly distributed points through four conserved hydrophobic regions.

Structural changes of TasA in biofilm formation of .

Microorganisms form surface-attached communities, termed biofilms, which can serve as protection against host immune reactions or antibiotics. biofilms contain TasA as major proteinaceous component in addition to exopolysaccharides. In stark contrast to the initially unfolded biofilm proteins of other bacteria, TasA is a soluble, stably folded monomer, whose structure we have determined by X-ray crystallography.

Structure of outer membrane protein G in lipid bilayers.

β-barrel proteins mediate nutrient uptake in bacteria and serve vital functions in cell signaling and adhesion. For the 14-strand outer membrane protein G of Escherichia coli, opening and closing is pH-dependent. Different roles of the extracellular loops in this process were proposed, and X-ray and solution NMR studies were divergent.

Direct assessment of substrate binding to the Neurotransmitter:Sodium Symporter LeuT by solid state NMR.

The Neurotransmitter:Sodium Symporters (NSSs) represent an important class of proteins mediating sodium-dependent uptake of neurotransmitters from the extracellular space. The substrate binding stoichiometry of the bacterial NSS protein, LeuT, and thus the principal transport mechanism, has been heavily debated. Here we used solid state NMR to specifically characterize the bound leucine ligand and probe the number of binding sites in LeuT.

Dynamic Nuclear Polarization Provides New Insights into Chromophore Structure in Phytochrome Photoreceptors.

Phytochromes are red/far-red photochromic photoreceptors acting as master regulators of development in higher plants, thereby controlling transcription of about 20 % of their genes. Light-induced isomerization of the bilin chromophore leads to large rearrangements in protein structure, whereby the role of protonation dynamics and charge distribution is of particular interest. To help unravel the inherent mechanisms, we present two-dimensional dynamic nuclear polarization (DNP) enhanced solid-state magic-angle spinning (MAS) NMR spectra of the functional sensory module of the cyanobacterial phytochrome Cph1.

Temperature dependence of cross-effect dynamic nuclear polarization in rotating solids: advantages of elevated temperatures.

Dynamic nuclear polarization exploits electron spin polarization to boost signal-to-noise in magic-angle-spinning (MAS) NMR, creating new opportunities in materials science, structural biology, and metabolomics studies. Since protein NMR spectra recorded under DNP conditions can show improved spectral resolution at 180-200 K compared to 110 K, we investigate the effects of AMUPol and various deuterated TOTAPOL isotopologues on sensitivity and spectral resolution at these temperatures, using proline and reproducibly prepared SH3 domain samples. The TOTAPOL deuteration pattern is optimized for protein DNP MAS NMR, and signal-to-noise per unit time measurements demonstrate the high value of TOTAPOL isotopologues for Protein DNP MAS NMR at 180-200 K.

Structural analysis of a signal peptide inside the ribosome tunnel by DNP MAS NMR.

Proteins are synthesized in cells by ribosomes and, in parallel, prepared for folding or targeting. While ribosomal protein synthesis is progressing, the nascent chain exposes amino-terminal signal sequences or transmembrane domains that mediate interactions with specific interaction partners, such as the signal recognition particle (SRP), the SecA-adenosine triphosphatase, or the trigger factor. These binding events can set the course for folding in the cytoplasm and translocation across or insertion into membranes.

Surface Binding of TOTAPOL Assists Structural Investigations of Amyloid Fibrils by Dynamic Nuclear Polarization NMR Spectroscopy.

Dynamic nuclear polarization (DNP) NMR can enhance sensitivity but often comes at the price of a substantial loss of resolution. Two major factors affect spectral quality: low-temperature heterogeneous line broadening and paramagnetic relaxation enhancement (PRE) effects. Investigations by NMR spectroscopy, isothermal titration calorimetry (ITC), and EPR revealed a new substantial affinity of TOTAPOL to amyloid surfaces, very similar to that shown by the fluorescent dye thioflavin-T (ThT).

Untangling a Repetitive Amyloid Sequence: Correlating Biofilm-Derived and Segmentally Labeled Curli Fimbriae by Solid-State NMR Spectroscopy.

Curli are functional bacterial amyloids produced by an intricate biogenesis machinery. Insights into their folding and regulation can advance our understanding of amyloidogenesis. However, gaining detailed structural information of amyloids, and their tendency for structural polymorphisms, remains challenging.

Assignment and secondary structure of the YadA membrane protein by solid-state MAS NMR.

We report the complete solid-state MAS NMR resonance assignment of a medium-sized, trimeric membrane protein, YadA-M. The protein YadA (Yersinia adhesin A) is an important virulence factor of enteropathogenic Yersinia species (such as Yersinia enterocolitica and Yersinia pseudotuberculosis). YadA is localized on the bacterial cell surface and is involved in adhesion to host cells and tissues.

Membrane-protein structure determination by solid-state NMR spectroscopy of microcrystals.

Membrane proteins are largely underrepresented among available atomic-resolution structures. The use of detergents in protein purification procedures hinders the formation of well-ordered crystals for X-ray crystallography and leads to slower molecular tumbling, impeding the application of solution-state NMR. Solid-state magic-angle spinning NMR spectroscopy is an emerging method for membrane-protein structural biology that can overcome these technical problems.

Efficient modeling of symmetric protein aggregates from NMR data.

An efficient approach to determine the structures of symmetric protein aggregates from liquid and solid-state NMR data is presented. Any symmetry can be used (cyclic or dihedral point symmetries, helical symmetries, crystallographic symmetries). Because the starting point is the random structure of the monomer, the knowledge of the 3D structure of the monomer is not required.

Broadband excitation pulses for high-field solid-state nuclear magnetic resonance spectroscopy.

In nuclear magnetic resonance spectroscopy, experimental limits due to the radiofrequency transmitter and/or coil means that conventional radiofrequency pulses ("hard pulses") are sometimes not sufficiently powerful to excite magnetization uniformly over a desired range of frequencies. Effects due to nonuniform excitation are most frequently encountered at high magnetic fields for nuclei with a large range of chemical shifts. Using optimal control theory, we have designed broadband excitation pulses that are suitable for solid-state samples under magic-angle-spinning conditions.

Practical aspects of high-sensitivity multidimensional ¹³C MAS NMR spectroscopy of perdeuterated proteins.

The double nucleus enhanced recoupling (DONER) experiment employs simultaneous irradiation of protons and deuterons to promote spin diffusion processes in a perdeuterated protein. This results in 4-5 times higher sensitivity in 2D (13)C-(13)C correlation experiments as compared to PDSD [1]. Here, a quantitative comparison of PDSD, (1)H-DARR, (2)H-DARR, and (1)H+(2)H DONER has been performed to analyze the influence of spin diffusion on polarization transfer processes.

The effect of biradical concentration on the performance of DNP-MAS-NMR.

With the technique of dynamic nuclear polarization (DNP) signal intensity in solid-state MAS-NMR experiments can be enhanced by 2-3 orders of magnitude. DNP relies on the transfer of electron spin polarization from unpaired electrons to nuclear spins. For this reason, stable organic biradicals such as TOTAPOL are commonly added to samples used in DNP experiments.

Neurotoxin II bound to acetylcholine receptors in native membranes studied by dynamic nuclear polarization NMR.

Methods enabling structural studies of membrane-integrated receptor systems without the necessity of purification provide an attractive perspective in membrane protein structural and molecular biology. This has become feasible in principle since the advent of dynamic nuclear polarization (DNP) magic-angle-spinning NMR spectroscopy, which delivers the required sensitivity. In this pilot study, we observed well-resolved solid-state NMR spectra of extensively (13)C-labeled neurotoxin II bound to the nicotinic acetylcholine receptor (nAChR) in native membranes.

Solid-state magic-angle spinning NMR of membrane proteins and protein-ligand interactions.

Structural biology is developing into a universal tool for visualizing biological processes in space and time at atomic resolution. The field has been built by established methodology like X-ray crystallography, electron microscopy and solution NMR and is now incorporating new techniques, such as small-angle X-ray scattering, electron tomography, magic-angle-spinning solid-state NMR and femtosecond X-ray protein nanocrystallography. These new techniques all seek to investigate non-crystalline, native-like biological material.

A software framework for analysing solid-state MAS NMR data.

Solid-state magic-angle-spinning (MAS) NMR of proteins has undergone many rapid methodological developments in recent years, enabling detailed studies of protein structure, function and dynamics. Software development, however, has not kept pace with these advances and data analysis is mostly performed using tools developed for solution NMR which do not directly address solid-state specific issues. Here we present additions to the CcpNmr Analysis software package which enable easier identification of spinning side bands, straightforward analysis of double quantum spectra, automatic consideration of non-uniform labelling schemes, as well as extension of other existing features to the needs of solid-state MAS data.

Cryogenic temperature effects and resolution upon slow cooling of protein preparations in solid state NMR.

X-ray crystallography using synchrotron radiation and the technique of dynamic nuclear polarization (DNP) in nuclear magnetic resonance (NMR) require samples to be kept at temperatures below 100 K. Protein dynamics are poorly understood below the freezing point of water and down to liquid nitrogen temperatures. Therefore, we investigate the α-spectrin SH3 domain by magic angle spinning (MAS) solid state NMR (ssNMR) at various temperatures while cooling slowly.

Triple resonance cross-polarization for more sensitive 13C MAS NMR spectroscopy of deuterated proteins.

Save the last WALTZ for me: the use of simultaneous proton and deuterium cross-polarization for (13)C CPMAS NMR spectroscopy in highly deuterated proteins is discussed. The aim of the new method introduced herein, triple-resonance cross-polarization, is to increase the sensitivity of the carbon-detected methods in such systems.

Radio frequency assisted homonuclear recoupling - a Floquet description of homonuclear recoupling via surrounding heteronuclei in fully protonated to fully deuterated systems.

We present a Floquet theory approach for the analysis of homonuclear recoupling assisted by radio frequency (RF) irradiation of surrounding heteronuclear spins. This description covers a broad range of systems from fully protonated to deuterated proteins, focusing in detail on recoupling via protons and deuterons separately as well as simultaneously by the double nucleus enhanced recoupling (DONER) scheme. The theoretical description, supported by numerical simulations and compared to experimental results from a partially deuterated model compound, indicates that in perdeuterated systems setting the RF amplitude equal to the magic angle spinning (MAS) frequency is not necessarily optimal for recoupling via (1)H and/or (2)H nuclei and modified recoupling conditions are identified.

Solid-state NMR and SAXS studies provide a structural basis for the activation of alphaB-crystallin oligomers.

The small heat shock protein alphaB-crystallin (alphaB) contributes to cellular protection against stress. For decades, high-resolution structural studies on oligomeric alphaB have been confounded by its polydisperse nature. Here, we present a structural basis of oligomer assembly and activation of the chaperone using solid-state NMR and small-angle X-ray scattering (SAXS).

Intermolecular protein-RNA interactions revealed by 2D 31P-15N magic angle spinning solid-state NMR spectroscopy.

The structural investigation of large RNP complexes by X-ray crystallography can be a difficult task due to the flexibility of the RNA and of the protein-RNA interfaces, which may hinder crystallization. In these cases, NMR spectroscopy is an attractive alternative to crystallography, although the large size of typical RNP complexes may limit the applicability of solution NMR. Solid-state NMR spectroscopy, however, is not subject to any intrinsic limitations with respect to the size of the object under investigation, with restrictions imposed solely by the sensitivity of the instrumentation.

A MAS NMR study of the bacterial ABC transporter ArtMP.

ATP-binding cassette (ABC) transport systems facilitate the translocation of substances, like amino acids, across cell membranes energised by ATP hydrolysis. This work describes first structural studies on the ABC transporter ArtMP from Geobacillus stearothermophilus in native lipid environment by magic-angle spinning NMR spectroscopy. The 2D crystals of ArtMP and 3D crystals of isolated ArtP were prepared in different nucleotide-bound or -unbound states.