B2LiVe, a label-free 1D-NMR method to quantify the binding of amphitropic peptides or proteins to membrane vesicles.

Amphitropic proteins and peptides reversibly partition from solution to membrane, a key process that regulates their functions. Experimental approaches classically used to measure protein partitioning into lipid bilayers, such as fluorescence and circular dichroism, are hardly usable when the peptides or proteins do not exhibit significant polarity and/or conformational changes upon membrane binding. Here, we describe binding to lipid vesicles (B2LiVe), a simple, robust, and widely applicable nuclear magnetic resonance (NMR) method to determine the solution-to-membrane partitioning of unlabeled proteins or peptides. B2LiVe relies on previously described proton 1D-NMR fast-pulsing techniques. Membrane partitioning induces a large line broadening, leading to a loss of protein signals; therefore, the decrease of the NMR signal directly measures the fraction of membrane-bound protein. The method uses low polypeptide concentrations and has been validated on several membrane-interacting polypeptides, ranging from 3 to 54 kDa, with membrane vesicles of different sizes and various lipid compositions.

Identification of Chemical Probes Targeting MBD2.

Epigenetics has received much attention in the past decade. Many insights on epigenetic (dys)regulation in diseases have been obtained, and clinical therapies targeting them are in place. However, the readers of the epigenetic marks are lacking enlightenment behind this revolution, and it is poorly understood how DNA methylation is being read and translated to chromatin function and cellular responses. Chemical probes targeting the methyl-CpG readers, such as the methyl-CpG binding domain proteins (MBDs), could be used to study this mechanism. We have designed analogues of 5-methylcytosine to probe the MBD domain of human MBD2. By setting up a protein thermal shift assay and an AlphaScreen-based test, we were able to identify three fragments that bind MBD2 alone and disrupt the MBD2-methylated DNA interactions. Two-dimensional NMR experiments and virtual docking gave valuable insights into the interaction of the ligands with the protein showing that the compounds interact with residues that are important for DNA recognition. These constitute the starting point for the design of potent chemical probes for MBD proteins.

1H, 15N and 13C resonance assignments and secondary structure of PulG, the major pseudopilin from Klebsiella oxytoca type 2 secretion system.

Bacteria use complex transporters to secrete functionally relevant proteins to the extracellular medium. The type 2 secretion system (T2SS) translocates folded proteins involved in bacterial nutrient acquisition, virulence and adaptation. The T2SS pseudopilus is a periplasmic filament, assembled by the polymerization of PulG subunits, the major pseudopilin. Pseudopilin proteins have a conserved N-terminal hydrophobic segment followed by a more variable C-terminal periplasmic and globular domain. To better understand the mechanism of assembly and function of the T2SS, we have been studying the structure and dynamics of PulG by NMR, as well as its interaction with other components of the secretion machinery. As a first step on this study, here we reported the chemical shift assignments of PulG C-terminal domain and its secondary structure prediction based on NMR data.

Spectral aliasing: a super zoom for 2D-NMR spectra. Principles and applications.

The NMR methodology based on spectral aliasing developed at the University of Geneva is reviewed. Different approaches aimed at increasing the resolution in the indirect carbon dimension of 2D heteronuclear experiments are presented with their respective advantages. Applications to HSQC, HMBC and other 2D heteronuclear experiments to the study of natural products and synthesis intermediates are shown. HSQC-based experiments for diffusion measurements, kinetics studies and titrations experiments all take advantage of spectral aliasing to reduce the experimental time from unrealistically long acquisition times to overnight experiments. The roles of computational methods such as DFT/GIAO and Logic for Structure Determination (LSD) in structure determination are discussed.

Speeding up nuclear magnetic resonance spectroscopy by the use of SMAll Recovery Times - SMART NMR.

A drastic reduction of the time required for two-dimensional NMR experiments can be achieved by reducing or skipping the recovery delay between successive experiments. Novel SMAll Recovery Times (SMART) methods use orthogonal pulsed field gradients in three spatial directions to select the desired pathways and suppress interference effects. Two-dimensional spectra of dilute amino acids with concentrations as low as 2 mM can be recorded in about 0.1 s per increment in the indirect domain.

High-precision heteronuclear 2D NMR experiments using 10-ppm spectral window to resolve carbon overlap.

The acquisition of a complementary heteronuclear 2D NMR experiment with 10-ppm carbon window allows chemists to improve by a factor 20-25 the spectral resolution and determine carbon chemical shifts with five figures from 2D spectra.

Real-time monitoring of a dynamic molecular system using 1H-13C HSQC NMR spectroscopy with an optimized 13C window.

The kinetic and thermodynamic parameters of an equilibrating network involving 8 molecules can be determined from a series of quick and highly resolved (1)H-(13)C HSQC NMR experiments obtained using a reduced carbon spectral window.

NMR diffusion measurements in complex mixtures using constant-time-HSQC-IDOSY and computer-optimized spectral aliasing for high resolution in the carbon dimension.

A new 3D pulse sequence for NMR diffusion measurements in complex mixtures is presented. It is based on the constant-time (CT) HSQC experiment and combines diffusion delay with the carbon evolution time. This combination has great potential to obtain high resolution in the carbon dimension. When using classical sampling of the carbon dimension, maximal resolution would require a large number of time increments, leading to unrealistically long acquisition times. The application of computer-optimized spectral aliasing allows one to reduce the number of time increments and the total acquisition time by 1-2 orders of magnitude by taking advantage of the information content of 1D carbon spectra, HSQC experiments, or both. With the new CT-HSQC-IDOSY experiment, the diffusion rates of the six anomers present in a 0.1 M D2O solution of glucose, maltose, and maltotriose could be obtained at natural abundance in 8 h with standard deviations below 5%.