Alternatively spliced exon regulates context-dependent MEF2D higher-order assembly during myogenesis.

During muscle cell differentiation, the alternatively spliced, acidic ß-domain potentiates transcription of Myocyte-specific Enhancer Factor 2 (Mef2D). Sequence analysis by the FuzDrop method indicates that the ß-domain can serve as an interaction element for Mef2D higher-order assembly. In accord, we observed Mef2D mobile nuclear condensates in C2C12 cells, similar to those formed through liquid-liquid phase separation. In addition, we found Mef2D solid-like aggregates in the cytosol, the presence of which correlated with higher transcriptional activity. In parallel, we observed a progress in the early phase of myotube development, and higher MyoD and desmin expression. In accord with our predictions, the formation of aggregates was promoted by rigid ß-domain variants, as well as by a disordered ß-domain variant, capable of switching between liquid-like and solid-like higher-order states. Along these lines, NMR and molecular dynamics simulations corroborated that the ß-domain can sample both ordered and disordered interactions leading to compact and extended conformations. These results suggest that ß-domain fine-tunes Mef2D higher-order assembly to the cellular context, which provides a platform for myogenic regulatory factors and the transcriptional apparatus during the developmental process.

Preparation of 3-O-, 5-O- and N-palmitoyl derivatives of fumonisin B1 toxin and their characterisation with NMR and LC-HRMS methods.

We have previously published six esterified O-acyl (EFB1) and three N-acyl fumonisin B1 derivatives extracted from rice cultures inoculated with Fusarium verticillioides, amongst these the identification of N-palmitoyl-FB1 has been clearly established in a spiking experiment. At that time, it was assumed that as in the case of O-acyl-FB1 derivatives, linoleic-, oleic- or palmitic acid esterify through the OH group on the 3C or 5C atom of the carbon chain of the fumonisins. In our most recent experiments, we have synthetically acylated the FB1 toxin and subsequently purified 3-O-palmitoyl- and 5-O-palmitoyl-FB1 toxins in addition to the N-palmitoyl-FB1 toxin. They were identified and characterised using 1H and 13C NMR as well as LC-HRMS. Our aim was the identification of the previously detected O-acyl-FB1 derivatives over the course of a spiking experiment, which were obtained through the solid-phase fermentation of Fusarium verticillioides. By spiking the three synthesized and identified components one-by-one into the fungal culture extract and analysing these cultures using LC-MS, it was clearly demonstrated that the F. verticillioides strain produced both the 5-O-palmitoyl-FB1 and N-palmitoyl-FB1 toxins, but did not produce 3-O-palmitoyl-FB1. Thus, it is highly probable that the components thought to be 3-O-acyl-(linoleoyl-, oleoyl-, palmitoyl-) FB1 derivatives in our previous communication are presumably 10-O-acyl-FB1 derivatives. Since these acylated FB1 derivatives can occur naturally in e.g. maize, the use of these synthesized components as reference materials is of great importance in order to obtain accurate qualitative and quantitative data on the occurrence of acylated fumonisins in different matrices including maize based feed samples. The production of these substances has also made it possible to test their toxicity in cell culture and small animal experiments.

The First Dimeric Derivatives of the Glycopeptide Antibiotic Teicoplanin.

Various dimeric derivatives of the glycopeptide antibiotic teicoplanin were prepared with the aim of increasing the activity of the parent compound against glycopeptide-resistant bacteria, primarily vancomycin-resistant enterococci. Starting from teicoplanin, four covalent dimers were prepared in two orientations, using an α,ω-bis-isothiocyanate linker. Formation of a dimeric cobalt coordination complex of an N-terminal L-histidyl derivative of teicoplanin pseudoaglycone has been detected and its antibacterial activity evaluated. The Co(III)-induced dimerization of the histidyl derivative was demonstrated by DOSY experiments. Both the covalent and the complex dimeric derivatives showed high activity against VanA teicoplanin-resistant enterococci, but their activity against other tested bacterial strains did not exceed that of the monomeric compounds.

Synergy and sensitivity-balance in concatenating experiments in NO relaxation delay NMR (NORD).

The NMR experiment design strategy of NO Relaxation Delay (NORD), introduced mostly as an idealized theoretical approach, is extended and put to practical use by considering synergy and sensitivity-balance in concatenation of experiments. It is illustrated by a novel experiment, NORD {HMBC}-{HSQC}-{TOCSY}, where magnetization of non-13C attached protons effectively is channeled from the TOCSY spectrum toward primarily the least sensitive spectrum of HMBC. The experiment is expected to find its place as a full-package NMR method for metabolomics, carbohydrates, peptides and small-molecules in general.

NORD: NO Relaxation Delay NMR Spectroscopy.

The novel concept of NORD (NO relaxation delay) NMR spectroscopy is introduced. The idea is to design concatenated experiments in a way that the magnetization used in the first relaxes toward equilibrium during the second and vice versa, thus saving instrument time. Applications include complete well-resolved 1 H-1 H and 1 H-13 C one-bond and long-range correlation maps of an 80 mM solution of a trisaccharide recorded in less than two minutes and hydrocortisone with extensive spectral overlap.

Double and adiabatic BANGO for concatenating two NMR experiments relying on the same pool of magnetization.

It is shown how the same pool of magnetization can be tapped twice in two different concatenations of three experiments into a single pulse sequence with only one relaxation delay. This is accomplished by using the BANGO pulse sequence element twice for independent rotations of 1H magnetization attached or not attached to 13C and it includes a refinement of BANGO with an adiabatic 13C inversion pulse resulting in improved tolerance to a spread in 1JCH coupling constants that translates directly into improved sensitivity of the modular experiment relying on 1H magnetization attached to 13C. The two new pulse sequences are SEA XLOC-HMBC-H2OBC/2BOB and SEA XLOC(ZQ)-SEA XLOC(2Q)-H2OBC/2BOB which both represent a rapid route to complete heteronuclear one-bond and long-range JCH correlation maps for small molecules, as is demonstrated on ibuprofen and prednisolone.

BANGO SEA XLOC/HMBC-H2OBC: complete heteronuclear correlation within minutes from one NMR pulse sequence.

Novel NMR experiments, BANGO SEA XLOC-H2OBC or BANGO HMBC-H2OBC, can deliver complete heteronuclear correlation maps on a time scale of minutes for small molecules. By way of example, it is demonstrated that all intra- and inter-residue 1H and 13C correlations and assignments of a trisaccharide are obtained in 20 or 5 minutes of instrument time without or with 25% NUS, respectively.

Photochemical and Structural Studies on Cyclic Peptide Models.

Ultra-violet (UV) irradiation has a significant impact on the structure and function of proteins that is supposed to be in relationship with the tryptophan-mediated photolysis of disulfide bonds. To investigate the correlation between the photoexcitation of Trp residues in polypeptides and the associated reduction of disulfide bridges, a series of small, cyclic oligopeptide models were analyzed in this work. Average distances between the aromatic side chains and the disulfide bridge were determined following molecular mechanics (MM) geometry optimizations. In this way, the possibility of cation⁻π interactions was also investigated. Molecular mechanics calculations revealed that the shortest distance between the side chain of the Trp residues and the disulfide bridge is approximately 5 Å in the cyclic pentapeptide models. Based on this, three tryptophan-containing cyclopeptide models were synthesized and analyzed by nuclear magnetic resonance (NMR) spectroscopy. Experimental data and detailed molecular dynamics (MD) simulations were in good agreement with MM geometry calculations. Selected model peptides were subjected to photolytic degradation to study the correlation of structural features and the photolytic cleavage of disulfide bonds in solution. Formation of free sulfhydryl groups upon illumination with near UV light was monitored by fluorescence spectroscopy after chemical derivatization with 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM) and mass spectrometry. Liquid cromatography-mass spectrometry (LC-MS) measurements indicated the presence of multiple photooxidation products (e.g., dimers, multimers and other oxidated products), suggesting that besides the photolysis of disulfide bonds secondary photolytic processes take place.

Distinguishing between two- and three-bond correlations for all 13C multiplicities in heteronuclear NMR spectroscopy.

A novel two-dimensional method, SEA XLOC, for distinguishing between two- and three-bond correlations in heteronuclear NMR spectroscopy is introduced and demonstrated on ibuprofen and by a complete set of correlations with a simple and most complex quaternary 13C multiplet in strychnine.