Matthias Hiller

Rapid Proton-Detected NMR Assignment for Proteins with Fast Magic Angle Spinning

Using a set of six 1H-detected triple-resonance NMR experiments, we establish a method for sequence-specific backbone resonance assignment of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectra of 5–30 kDa proteins. The approach relies on perdeuteration, amide 2H/1H exchange, high magnetic fields, and high-spinning frequencies (ωr/2π ≥ 60 kHz) and yields high-quality NMR data, enabling the use of automated analysis. The method is validated with five examples of proteins in different condensed states, including two microcrystalline proteins, a sedimented virus capsid, and two membrane-embedded systems. In comparison to contemporary 13C/15N-based methods, this approach facilitates and accelerates the MAS NMR assignment process, shortening the spectral acquisition times and enabling the use of unsupervised state-of-the-art computational data analysis protocols originally developed for solution NMR.

Assigning large proteins in the solid state: a MAS NMR resonance assignment strategy using selectively and extensively 13C-labelled proteins.

In recent years, solid-state magic-angle spinning nuclear magnetic resonance spectroscopy (MAS NMR) has been growing into an important technique to study the structure of membrane proteins, amyloid fibrils and other protein preparations which do not form crystals or are insoluble. Currently, a key bottleneck is the assignment process due to the absence of the resolving power of proton chemical shifts. Particularly for large proteins (approximately >150 residues) it is difficult to obtain a full set of resonance assignments. In order to address this problem, we present an assignment method based upon samples prepared using [1,3-13C]- and [2-13C]-glycerol as the sole carbon source in the bacterial growth medium (so-called selectively and extensively labelled protein). Such samples give rise to higher quality spectra than uniformly [13C]-labelled protein samples, and have previously been used to obtain long-range restraints for use in structure calculations. Our method exploits the characteristic cross-peak patterns observed for the different amino acid types in 13C-13C correlation and 3D NCACX and NCOCX spectra. An in-depth analysis of the patterns and how they can be used to aid assignment is presented, using spectra of the chicken α-spectrin SH3 domain (62 residues), αB-crystallin (175 residues) and outer membrane protein G (OmpG, 281 residues) as examples. Using this procedure, over 90% of the Cα, Cβ, C′ and N resonances in the core domain of αB-crystallin and around 73% in the flanking domains could be assigned (excluding 24 residues at the extreme termini of the protein).

Solid-State Magic-Angle Spinning NMR of Outer-Membrane Protein G from Escherichia coli

Uniformly 13C‐,15N‐labelled outer‐membrane protein G (OmpG) from Escherichia coli was expressed for structural studies by solid‐state magic‐angle spinning (MAS) NMR. Inclusion bodies of the recombinant, labelled protein were purified under denaturing conditions and refolded in detergent. OmpG was reconstituted into lipid bilayers and several milligrams of two‐dimensional crystals were obtained. Solid‐state MAS NMR spectra showed signals with an apparent line width of 80–120 Hz (including homonuclear scalar couplings). Signal patterns for several amino acids, including threonines, prolines and serines were resolved and identified in 2D proton‐driven spin‐diffusion (PDSD) spectra.

Structural changes of TasA in biofilm formation of Bacillus subtilis

Significance Understanding the formation and structure of protective bacterial biofilms will help to design and identify antimicrobial strategies. Our experiments with the secreted major biofilm protein TasA characterize on a molecular level in vivo the transition of a folded protein into protease-resistant biofilm-stabilizing fibrils. Such conformational changes from a globular state into fibrillar structures are so far not seen for other biofilm-forming proteins. In this context, TasA can serve as a model system to study functional fibril formation from a globular state. Microorganisms form surface-attached communities, termed biofilms, which can serve as protection against host immune reactions or antibiotics. Bacillus subtilis 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. Subsequently, we characterized in vitro different oligomeric forms of TasA by NMR, EM, X-ray diffraction, and analytical ultracentrifugation (AUC) experiments. However, by magic-angle spinning (MAS) NMR on live biofilms, a swift structural change toward only one of these forms, consisting of homogeneous and protease-resistant, β-sheet–rich fibrils, was observed in vivo. Thereby, we characterize a structural change from a globular state to a fibrillar form in a functional prokaryotic system on the molecular level.

Structure of outer membrane protein G in lipid bilayers

Abstractβ-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. Here, we report the structure of outer membrane protein G investigated in bilayers of E. coli lipid extracts by magic-angle-spinning NMR. In total, 1847 inter-residue 1H–1H and 13C–13C distance restraints, 256 torsion angles, but no hydrogen bond restraints are used to calculate the structure. The length of β-strands is found to vary beyond the membrane boundary, with strands 6–8 being the longest and the extracellular loops 3 and 4 well ordered. The site of barrel closure at strands 1 and 14 is more disordered than most remaining strands, with the flexibility decreasing toward loops 3 and 4. Loop 4 presents a well-defined helix.Porins, like OmpG, are embedded in the outer membrane of bacteria and facilitate uptake and secretion of nutrients and ions. Here the authors present a protocol for solid state NMR structure determination of proteins larger than 25 kDa and use it to structurally characterize membrane embedded OmpG.

Multifunctional Benzoxazines Feature Low Polymerization Temperature and Diverse Polymer Structures

3,4-dihydro-3-phenyl-2H-1,3-benzoxazines derived from phenol-, resorcinol-, and phloroglucinol give monomers with one, two, and three oxazine units at a single benzene ring, respectively. Aside from the synthesis and characterization of such multifunctional benzoxazines, reactivity and polymerization behavior is studied in dependence of the oxazine functionality. Monomer reactivities are directly related to the number of oxazine functionalities present at the benzene ring yielding the lowest polymerization temperature for the trifunctional phloroglucinol-based benzoxazine. Comparing the polymerization processes and resulting structures, the trifunctional benzoxazine derivative enter new polymerization pathways, which include methylene linkages bridging aniline units, as well as the formation of carbonyl-derived structures.

Quantitative and Qualitative Analysis of Surface Modified Cellulose Utilizing TGA-MS

With the aim to enhance interfacial adhesion of a hydrophobic polymer matrix and cellulosic fibers and fillers, chemical surface modifications with silane coupling agents are performed. Thermogravimetric analysis (TGA) could be used to determine the degree of surface functionalization. However, similar thermal properties of treated and untreated cellulose hamper a precise determination of silane loading. This contribution deals with quantitative determination of silane loading combining both TGA and elemental analysis. Firstly, silane modified celluloses were studied by FT-IR, Raman, solid state NMR spectroscopy, and polarized light microscopy in order to determine functional groups and to study the impact of chemical treatment on cellulose morphology. Secondly, thermal stability and pyrolysis processes were studied by TG-MS analysis. In order to determine the exact silane loading, the mass percentages of the appropriate elements were quantified by elemental analysis and correlated with the charred residues determined by TGA yielding a linear dependency. With that correlation, it was possible to determine silane loadings for additional samples utilizing simple TGA measurements. The main advantage of that approach is that only one calibration is necessary for routine analyses of further samples and TGA-MS coupling gives additional information on thermal stability and pyrolysis routes, simultaneously.