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. (C) 2010 Elsevier Inc. All rights reserved

Applications du repliement spectral en RMN à deux dimensions

La résonance magnétique nucléaire est devenue un outil indispensable aux chimistes de synthèse. Basé sur les propriétés nucléaires des atomes, elle apporte des informations cruciales sur la structure, la composition, la dynamique des systèmes qui peuvent aller de la simple molécule organique, à la protéine, en passant par des mélanges complexes hors-équilibres. Ces informations extraites d'expériences de plus en plus complexes nécessitent une résolution suffisante afin de discriminer les signaux correspondants à chacun des atomes. Dans le cas d'expériences multidimensionnelles, l'obtention de la résolution nécessaire augmente considérablement la durée d'expérience

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%

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 1H-13C HSQC NMR experiments obtained using a reduced carbon spectral window

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

International audienceBacteria 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

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

Posted December 16, 2022 on bioRxiv.Amphitropic proteins and peptides reversibly partition from solution to membrane, a key process that regulates their functions. Experimental approaches, such as fluorescence and circular dichroism, are classically used to measure the partitioning of amphitropic peptides and proteins into lipid bilayers, yet hardly usable when the peptides or proteins do not exhibit significant polarity and/or conformational changes upon membrane binding. Here, we describe B2LiVe ( i . e ., Binding to Lipid Vesicles), a simple, robust, and widely applicable NMR method to determine the solution-to-membrane partitioning of unlabeled proteins or peptides. The experimental strategy proposed here relies on previously described proton 1D NMR fast pulsing techniques with selective adiabatic pulses. Membrane partitioning induces a large line broadening leading to a progressive loss of protein signals, and therefore, the decrease of the NMR signal directly measures the fraction of membrane-bound protein. The B2LiVe method uses low polypeptide concentrations and has been validated on several membrane-interacting peptides and proteins, ranging from 3 to 54 kDa, with membrane vesicles of different sizes and various lipid compositions. Motivation Characterization of the interaction of peptides and proteins with lipid membranes is involved in various biological processes and is often challenging for polypeptides which do not possess intrinsic fluorophores, do not exhibit significant structural content changes, as well as for those characterized by low affinities for membranes. To meet these challenges, we have developed a simple and robust label-free NMR-based experimental approach, named B2LiVe, to measure the binding of polypeptides to lipid vesicles. The experimental strategy relies on previously described proton 1D NMR fast pulsing techniques with selective adiabatic pulses to excite the amide resonances. B2LiVe is a label-free method based on the observation of amide hydrogen nuclei which are naturally present in all protein and peptide backbones. Our results validate the B2LiVe method and indicate that it compares well with established technics to quantify polypeptide-membrane interactions. Overall, B2LiVe should efficiently complement the arsenal of label-free biophysical assays available to characterize protein-membrane interactions. In brief We describe a robust label-free NMR-based experimental approach (B2LiVe) to measure interactions between peptides or proteins with membranes. The validity of this approach has been established on several polypeptides and on various membrane vesicles. The B2LiVe method efficiently complements the arsenal of label-free biophysical techniques to characterize protein-membrane interactions. Highlights B2LiVe is a simple and robust NMR-based method to quantify affinity of proteins and peptides for membranes B2LiVe is a label-free approach that relies on proton 1D NMR fast pulsing techniques with selective excitation of amide resonances B2LiVe has been validated on several membrane-interacting peptides and proteins B2LiVe coupled to DOSY can pinpoint the presence within a membrane-bound protein of polypeptide segments remaining in solutio

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

Withanolides from Withania adpressa

From the leaves of Withania adpressa, a plant endemic to Sahara of Morocco and Algeria, the novel steroidal lactone (22R)-14 alpha;,15 alpha;,17ß,20ß-tetrahydroxy-1-oxowitha-2,5,24-trien-26,22-olide (= (15S,17S)-14,15,17,20-tetrahydroxy-22,26-epoxyergosta-2,5,24-triene-1,26-dione; 1), was isolated, along with three known compounds, withanolides F (2), J (3), and oleanolic acid. Their structures were mainly solved by in-depth 1D- and 2D-NMR (including ADEQUATE) experiments, as well as by HR-MS analyses and chemical evidence

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