Accurate DOSY measure of out-of-equilibrium systems by permutated DOSY (p-DOSY)

NMR spectroscopy is an excellent tool for monitoring in-situ chemical reactions. In particular, DOSY measurement is well suited to characterize transient species by the determination of their sizes. However, here we bring to light a difficulty in the DOSY experiments performed in out-of-equilibrium systems. On such a system, the evolution of the concentration of species interferes with the measurement process, and creates a bias on the diffusion coefficient determination that may lead to erroneous interpretations. We show that a random permutation of the series of gradient strengths used during the DOSY experiment allows to average out this bias. This approach, that we name p-DOSY does not require changes in the the pulse sequences nor in the processing software, and restores completely the full accuracy of the measure. This technique is demonstrated on the monitoring of the anomerization reaction of \alpha- to \beta-glucose.Comment: Revised version - 15 pages, 8 figures program archived at 10.5281/zenodo.1926

Automatic differential analysis of NMR experiments in complex samples

Liquid state NMR is a powerful tool for the analysis of complex mixtures of unknown molecules. This capacity has been used in many analytical approaches: metabolomics, identification of active compounds in natural extracts, characterization of species, and such studies require the acquisition of many diverse NMR measurements on series of samples. While acquisition can easily be performed automatically, the number of NMR experiments involved in these studies increases very rapidly and this data avalanche requires to resort to automatic processing and analysis. We present here a program that allows the autonomous, unsupervised processing of a large corpus of 1D, 2D and DOSY experiments from a series of samples acquired in different conditions. The program provides all the signal processing steps, as well as peak-picking and bucketing of 1D and 2D spectra, the program and its components are fully available. In an experiment mimicking the search of an active species in natural extract, we use it for the automatic detection of small amounts of artemisin added to a series of plant extracts, and for the generation of the spectral fingerprint of this molecules. This program called Plasmodesma is a novel tool which should be useful to decipher complex mixtures, particularly in the discovery of biologically active natural products from plants extracts, but can also in drug discovery or metabolomics studies.Comment: 35 pages, 36 figures, 26 reference

Characterization and molecular basis of the oligomeric structure of HIV-1 Nef protein

International audienceThe Nef protein of human immunodeficiency virus type I~HIV-1! is an important determinant for the onset of AIDS disease. The self-association properties of HIV-1 Nef are analyzed by chemical cross-linking, dynamic light scattering, equilibrium analytical ultracentrifugation, and NMR spectroscopy. The experimental data show that the HIV-1 Nef core domain forms stable homo-dimers and trimers in solution, but not higher oligomers. These Nef homomers are not covalently linked by disulfide bridges, and the equilibrium between these forms is dependent on the Nef concentration. We further provide the molecular basis for the Nef core dimers and trimers obtained by analysis of crystallographic models. Oligomerization of biological polypeptides is a common tool used to trigger events in cellular signaling and endocytosis, both of which are targeted by Nef. The quaternary structure of Nef may be of physiological importance and may help to connect its cellular targets or to increase affinity of the viral molecule for its ligands. The herein described models for Nef dimers and trimers will allow further mutational studies to elucidate their role in vivo. These results provide novel insight into the structural and functional relationships of this important viral protein. Moreover, the oligomer interface may represent a novel target for the design of antiviral agents

Application of tandem two-dimensional mass spectrometry for top-down deep sequencing of calmodulin

Two-dimensional mass spectrometry (2DMS) involves simultaneous acquisition of the fragmentation patterns of all the analytes in a mixture by correlating their precursor and fragment ions by modulating precursor ions systematically through a fragmentation zone. Tandem two-dimensional mass spectrometry (MS/2DMS) unites the ultra-high accuracy of Fourier transform ion cyclotron resonance (FT-ICR) MS/MS and the simultaneous data-independent fragmentation of 2DMS to achieve extensive inter-residue fragmentation of entire proteins. 2DMS was recently developed for top-down proteomics (TDP), and applied to the analysis of calmodulin (CaM), reporting a cleavage coverage of about ~23% using infrared multiphoton dissociation (IRMPD) as fragmentation technique. The goal of this work is to expand the utility of top-down protein analysis using MS/2DMS in order to extend the cleavage coverage in top-down proteomics further into the interior regions of the protein. In this case, using MS/2DMS, the cleavage coverage of CaM increased from ~23% to ~42%

An Amyloidogenic Sequence at the N-Terminus of the Androgen Receptor Impacts Polyglutamine Aggregation

The human androgen receptor (AR) is a ligand inducible transcription factor that harbors an amino terminal domain (AR-NTD) with a ligand-independent activation function. AR-NTD is intrinsically disordered and displays aggregation properties conferred by the presence of a poly-glutamine (polyQ) sequence. The length of the polyQ sequence as well as its adjacent sequence motifs modulate this aggregation property. AR-NTD also contains a conserved KELCKAVSVSM sequence motif that displays an intrinsic property to form amyloid fibrils under mild oxidative conditions. As peptide sequences with intrinsic oligomerization properties are reported to have an impact on the aggregation of polyQ tracts, we determined the effect of the KELCKAVSVSM on the polyQ stretch in the context of the AR-NTD using atomic force microscopy (AFM). Here, we present evidence for a crosstalk between the amyloidogenic properties of the KELCKAVSVSM motif and the polyQ stretch at the AR-NTD