Peter Bellstedt

CAPITO—a web server-based analysis and plotting tool for circular dichroism data

MOTIVATIONnCircular dichroism (CD) spectroscopy is one of the most versatile tools to study protein folding and to validate the proper fold of purified proteins. Here, we aim to provide a readily accessible, user-friendly and platform-independent tool capable of analysing multiple CD datasets of virtually any format and returning results as high-quality graphical output to the user.nnnRESULTSnCAPITO (CD Anaylsis and Plotting Tool) is a novel web server-based tool for analysing and plotting CD data. It allows reliable estimation of secondary structure content utilizing different approaches. CAPITO accepts multiple CD datasets and, hence, is well suited for a wide application range such as the analysis of temperature or pH-dependent (un)folding and the comparison of mutants.nnnAVAILABILITYnhttp://capito.nmr.fli-leibniz.de.nnnCONTACTncwiede@fli-leibniz.de or mago@fli-leibniz.dennnSUPPLEMENTARY INFORMATIONnSupplementary data are available at Bioinformatics online.

Solid state NMR of proteins at high MAS frequencies: symmetry-based mixing and simultaneous acquisition of chemical shift correlation spectra

We have carried out chemical shift correlation experiments with symmetry-based mixing sequences at high MAS frequencies and examined different strategies to simultaneously acquire 3D correlation spectra that are commonly required in the structural studies of proteins. The potential of numerically optimised symmetry-based mixing sequences and the simultaneous recording of chemical shift correlation spectra such as: 3D NCAC and 3D NHH with dual receivers, 3D NC′C and 3D C′NCA with sequential 13C acquisitions, 3D NHH and 3D NC′H with sequential 1H acquisitions and 3D CANH and 3D C’NH with broadband 13C–15N mixing are demonstrated using microcrystalline samples of the β1 immunoglobulin binding domain of protein G (GB1) and the chicken α-spectrin SH3 domain.

Preorganization in a Cleft-Type Anion Receptor Featuring Iodo-1,2,3-Triazoles As Halogen Bond Donors

Preorganization via intramolecular hydrogen bonds was applied in a cleft-type receptor by exploiting the excellent halogen bond donor ability as well as hydrogen bond acceptor function of iodo-1,2,3-triazoles. As investigated by isothermal calorimetric titrations, the restriction of conformational freedom causes an enhanced entropic contribution resulting in a strongly increased binding affinity. This efficient way to improve the binding strength of 5-halo-1,2,3-triazoles paves the way for applications of new charge-neutral halogen bond donors in solution.

Core cross-linked nanogels based on the self-assembly of double hydrophilic poly(2-oxazoline) block copolymers

The synthesis of poly(2-oxazoline)-based block copolymers consisting of a cationic and a hydrophilic segment is described. The self-assembly of these macromolecules in organic solvents results in the formation of micelles and vesicles, respectively, depending on the solvent used. To transfer the systems into water, cross-linking using glutaraldehyde was applied, followed by the consumption of excessive aldehyde functions by either diethylamine or 6-aminofluorescein (6AF). The cross-linked assemblies were analyzed regarding their size and shape by electron microscopy and light scattering methods, as well as for their chemical composition by solid state NMR spectroscopy. 6AF associated samples were examined with respect to their absorption and fluorescence behavior in aqueous environment, revealing an alkaline microenvironment within the presented nanostructures. The toxicity of the systems against mouse fibroblast cell line L929 was examined by the XTT assay and was found to be insignificant for concentrations of up to 2.5 mg mL-1. Flow cytometry and fluorescence microscopy analysis revealed an efficient concentration and time dependent cellular uptake of the nanogels.

Sequential protein NMR assignments in the liquid state via sequential data acquisition.

Two different NMR pulse schemes involving sequential (1)H data acquisition are presented for achieving protein backbone sequential resonance assignments: (i) acquisition of 3D {HCCNH and HNCACONH} and (ii) collection of 3D {HNCOCANH and HNCACONH} chemical shift correlation spectra using uniformly (13)C,(15)N labelled proteins. The sequential acquisition of these spectra reduces the overall experimental time by a factor of ≈2 as compared to individual acquisitions. The suitability of this approach is experimentally demonstrated for the C-terminal winged helix (WH) domain of the minichromosome maintenance (MCM) complex of Sulfolobus solfataricus.

Resonance assignment for a particularly challenging protein based on systematic unlabeling of amino acids to complement incomplete NMR data sets

NMR-based structure determination of a protein requires the assignment of resonances as indispensable first step. Even though heteronuclear through-bond correlation methods are available for that purpose, challenging situations arise in cases where the protein in question only yields samples of limited concentration and/or stability. Here we present a strategy based upon specific individual unlabeling of all 20 standard amino acids to complement standard NMR experiments and to achieve unambiguous backbone assignments for the fast precipitating 23xa0kDa catalytic domain of human aprataxin of which only incomplete standard NMR data sets could be obtained. Together with the validation of this approach utilizing the protein GB1 as a model, a comprehensive insight into metabolic interconversion (scrambling”) of NH and CO groups in a standard Escherichia coli expression host is provided.

Solvent Removal Induces a Reversible β-to-α Switch in Oligomeric Aβ Peptide

Solvation and hydration are key factors for determining the stability and folding of proteins, as well as the formation of amyloid fibrils and related polypeptide aggregates. Using attenuated total reflectance Fourier-transform infrared and solid-state NMR spectroscopy, we find that the Aβ peptide experiences a remarkable conformational switch from β to α secondary structure upon solvent removal by lyophilization of oligomers. This transition is, contrary to Aβ fibrils, independent of concentration of organic co-solvents or co-solutes and is reversible upon re-addition of the solvent. Our data illuminate a previously unnoted secondary structural plasticity of the Aβ peptide in amyloid oligomers that could bear relevance for Aβs interactions with cellular structures of low polarity.

Fluorinated Boronic Acid-Appended Pyridinium Salts and 19F NMR Spectroscopy for Diol Sensing

The identification and discrimination of diols is of fundamental importance in medical diagnostics, such as measuring the contents of glucose in the urine of diabetes patients. Diol sensors are often based on fluorophore-appended boronic acids, but these severely lack discriminatory power and their response is one-dimensional. As an alternative strategy, we present the use of fluorinated boronic acid-appended pyridinium salts in combination with 19F NMR spectroscopy. A pool of 59 (bio)analytes was screened, containing monosaccharides, phosphorylated and N-acetylated sugars, polyols, carboxylic acids, nucleotides, and amines. The majority of analytes could be clearly detected and discriminated. In addition, glucose and fructose could be distinguished up to 1:9 molar ratio in mixtures. Crucially, the receptors feature high sensitivity and selectivity and are water-soluble, and their 19F-NMR analyte fingerprint is pH-robust, thereby making them particularly well-suited for medical application. Finally, to demonstrate this applicability, glucose could be detected in synthetic urine samples down to 1 mM using merely a 188 MHz NMR spectrometer.

Sequential acquisition of multi-dimensional heteronuclear chemical shift correlation spectra with 1H detection

RF pulse schemes for the simultaneous acquisition of heteronuclear multi-dimensional chemical shift correlation spectra, such as {HA(CA)NH & HA(CACO)NH}, {HA(CA)NH & H(N)CAHA} and {H(N)CAHA & H(CC)NH}, that are commonly employed in the study of moderately-sized protein molecules, have been implemented using dual sequential 1H acquisitions in the direct dimension. Such an approach is not only beneficial in terms of the reduction of experimental time as compared to data collection via two separate experiments but also facilitates the unambiguous sequential linking of the backbone amino acid residues. The potential of sequential 1H data acquisition procedure in the study of RNA is also demonstrated here.

MAS solid state NMR of proteins: simultaneous 15N–13CA and 15N–13CO dipolar recoupling via low-power symmetry-based RF pulse schemes

The generation of efficient RNnνs,νk symmetry-based low-power RF pulse schemes for simultaneous 15N–13CA and 15N–13CO dipolar recoupling is demonstrated. The method involves mixing schemes employing phase and amplitude-modulated dual band-selective 180° pulses as basic “R” element and tailoring of the RF field-modulation profile of the 180° pulses so as to obtain efficient magnetisation transfer characteristics over the resonance offset range of the nuclei involved. Mixing schemes leading to simultaneous 15N–13CA and 15N–13CO dipolar recoupling would permit the one-shot acquisition of different chemical shift correlation spectra that are typically utilized for protein backbone resonance assignments and thereby save data acquisition time. At representative MAS frequencies the efficacies of the mixing schemes presented here have been experimentally demonstrated via the simultaneous acquisition of {3D CONH and 3D CANH}, {3D CONH and 3D CO(CA)NH} and {3D CONH, 3D CANH, 3D CO(CA)NH and 3D CA(CO)NH} spectra generated via the magnetisation transfer pathways 1Hxa0→xa013COxa0→xa015Nxa0→xa01H (CONH), 1Hxa0→xa013CAxa0→xa015Nxa0→xa01H (CANH) and 1Hxa0→xa013COxa0→xa013CAxa0→xa015Nxa0→xa01H (CO(CA)NH) and 1Hxa0→xa013CAxa0→xa013COxa0→xa015Nxa0→xa01H (CA(CO)NH).