The oligomerization state of bacterial enzyme I (EI) determines EI's allosteric stimulation or competitive inhibition by α-ketoglutarate.

The bacterial phosphotransferase system (PTS) is a signal transduction pathway that couples phosphoryl transfer to active sugar transport across the cell membrane. The PTS is initiated by phosphorylation of enzyme I (EI) by phosphoenolpyruvate (PEP). The EI phosphorylation state determines the phosphorylation states of all other PTS components and is thought to play a central role in the regulation of several metabolic pathways and to control the biology of bacterial cells at multiple levels, for example, affecting virulence and biofilm formation. Given the pivotal role of EI in bacterial metabolism, an improved understanding of the mechanisms controlling its activity could inform future strategies for bioengineering and antimicrobial design. Here, we report an enzymatic assay, based on Selective Optimized Flip Angle Short Transient (SOFAST) NMR experiments, to investigate the effect of the small-molecule metabolite α-ketoglutarate (αKG) on the kinetics of the EI-catalyzed phosphoryl transfer reaction. We show that at experimental conditions favoring the monomeric form of EI, αKG promotes dimerization and acts as an allosteric stimulator of the enzyme. However, when the oligomerization state of EI is shifted toward the dimeric species, αKG functions as a competitive inhibitor of EI. We developed a kinetic model that fully accounted for the experimental data and indicated that bacterial cells might use the observed interplay between allosteric stimulation and competitive inhibition of EI by αKG to respond to physiological fluctuations in the intracellular environment. We expect that the mechanism for regulating EI activity revealed here is common to several other oligomeric enzymes.

NMRlib: user-friendly pulse sequence tools for Bruker NMR spectrometers.

We present NMRlib, a suite of jython-based tools designed for Bruker spectrometers (TopSpin versions 3.2-4.0) that allow easy setup, management, and exchange of NMR experiments. A NMR experiment can be set up and executed in a few clicks by navigating through the NMRlib GUI tree structure, without any further parameter adjustment. NMRlib is magnetic-field independent, and thus particularly helpful for laboratories operating multiple NMR spectrometers. NMRlib is easily personalized by adding, deleting, or reorganizing experiments. Additional tools are provided for data processing, visualization, and analysis. In particular, NMRlib contains all the polarization-enhanced fast-pulsing NMR experiments (SOFAST, BEST, HADAMAC,…) developed in our laboratory over the last decade. We also discuss some specific features that have been implemented to make these experiments most efficient and user friendly.

SOFAST-HMQC-an efficient tool for metabolomics.

Nuclear magnetic resonance (NMR)-based metabolomics relies mostly on 1D NMR; however, the technique is limited by overlap of the signals from the metabolites. In order to circumvent this problem, 2D 1H-13C correlation spectroscopy techniques are often used. However owing to poorer natural abundance and gyromagnetic ratio of 13C, the acquisition time for 2D 1H-13C heteronuclear single quantum coherence spectroscopy (HSQC) is long. This makes it almost impossible to be used in high throughput study. We have reported the application of selective optimized flip angle short transient (SOFAST) technique coupled to heteronuclear multiple quantum correlation (HMQC) along with nonlinear sampling (NUS) in urine and serum samples. This technique takes sevenfold less experimental time than the conventional 1H-13C HSQC experiment with retention of almost all molecular information. Hence, this can be used for high throughput study. Graphical abstract SOFAST-HMQC is a two-dimensional NMR technique that significantly decreases experimental time without loss of information. This technique is applied in complex biofluid samples that are used for high throughput metabolomics studies and shows promise of better information recovery than conventional two-dimensional NMR technique in shorter time.

15N and 13C- SOFAST-HMQC editing enhances 3D-NOESY sensitivity in highly deuterated, selectively [1H,13C]-labeled proteins.

The ongoing NMR method development effort strives for high quality multidimensional data with reduced collection time. Here, we apply 'SOFAST-HMQC' to frequency editing in 3D NOESY experiments and demonstrate the sensitivity benefits using highly deuterated and 15N, methyl labeled samples in H2O. The experiments benefit from a combination of selective T 1 relaxation (or L-optimized effect), from Ernst angle optimization and, in certain types of experiments, from using the mixing time for both NOE buildup and magnetization recovery. This effect enhances sensitivity by up to 2.4× at fast pulsing versus reference HMQC sequences of same overall length and water suppression characteristics. Representative experiments designed to address interesting protein NMR challenges are detailed. Editing capabilities are exploited with heteronuclear 15N,13C-edited, or with diagonal-free 13C aromatic/methyl-resolved 3D-SOFAST-HMQC-NOESY-HMQC. The latter experiment is used here to elucidate the methyl-aromatic NOE network in the hydrophobic core of the 19 kDa FliT-FliJ flagellar protein complex. Incorporation of fast pulsing to reference experiments such as 3D-NOESY-HMQC boosts digital resolution, simplifies the process of NOE assignment and helps to automate protein structure determination.

Aromatic SOFAST-HMBC for proteins at natural 13C abundance.

We propose here SOFAST-HMBC as a new complementary NMR tool for aromatic side chain assignment of protein samples at natural 13C abundance. The characteristic peak patterns detected in SOFAST-HMBC for each aromatic side chain allow straightforward assignment of all protons and carbons (including quaternary ones) of the aromatic ring, and for tyrosine and phenylalanine, connection to the CB of the aliphatic chain. The performance of SOFAST-HMBC is demonstrated for three small proteins (7-14 kDa) at millimolar sample concentration using modern high-field NMR instruments equipped with cryogenically cooled probes. Despite the low amount of NMR-active 13C nuclei in these samples, 1H-13C multiple-bond correlation spectra of good quality were obtained in reasonable experimental times of typically less than 24 h.

Toolkit for NMR Studies of Methyl-Labeled Proteins.

Selective methyl labeling is an extremely powerful approach to study the structure, dynamics, and explore mechanistic insights of large biomolecules by solution NMR. Methyls are relatively insensitive to chemical exchange-induced depolarization and provide superior probes of supramolecular interactions and allostery in such systems. In this chapter, we describe our systematic approach and contributions in the areas of sample preparation, data collection, and data analysis that streamline the application of methyl labeling in solution NMR studies of large proteins. We focus our effort on the initial and often onerous task of methyl resonance assignment and here we detail our approaches to simplify the process. We produce new methyl labeling combinations using Escherichia coli auxotrophs, increase speed, sensitivity, and resolution of NOESY experiments by employing 3D SOFAST-NOESY, and assign methyl resonances from raw data with spectral simulation tools and(or) automatically with minimal expert supervision using the MAGIC algorithm.

Optimized fast mixing device for real-time NMR applications.

We present an improved fast mixing device based on the rapid mixing of two solutions inside the NMR probe, as originally proposed by Hore and coworkers (J. Am. Chem. Soc. 125 (2003) 12484-12492). Such a device is important for off-equilibrium studies of molecular kinetics by multidimensional real-time NMR spectrsocopy. The novelty of this device is that it allows removing the injector from the NMR detection volume after mixing, and thus provides good magnetic field homogeneity independently of the initial sample volume placed in the NMR probe. The apparatus is simple to build, inexpensive, and can be used without any hardware modification on any type of liquid-state NMR spectrometer. We demonstrate the performance of our fast mixing device in terms of improved magnetic field homogeneity, and show an application to the study of protein folding and the structural characterization of transiently populated folding intermediates.

Amiloride as a new RNA-binding scaffold with activity against HIV-1 TAR.

Diversification of RNA-targeted scaffolds offers great promise in the search for selective ligands of therapeutically relevant RNA such as HIV-1 TAR. We herein report the establishment of amiloride as a novel RNA-binding scaffold along with synthetic routes for combinatorial C(5)- and C(6)-diversification. Iterative modifications at the C(5)- and C(6)- positions yielded derivative 24, which demonstrated a 100-fold increase in activity over the parent dimethylamiloride in peptide displacement assays. NMR chemical shift mapping was performed using the 2D SOFAST- [1H-13C] HMQC NMR method, which allowed for facile and rapid evaluation of binding modes for all library members. Cheminformatic analysis revealed distinct differences between selective and non-selective ligands. In this study, we evolved dimethylamiloride from a weak TAR ligand to one of the tightest binding selective TAR ligands reported to date through a novel combination of synthetic methods and analytical techniques. We expect these methods to allow for rapid library expansion and tuning of the amiloride scaffold for a range of RNA targets and for SOFAST NMR to allow unprecedented evaluation of small molecule:RNA interactions.

A new NMR technique to probe protein-ligand interaction.

Non covalent grafting of proteins on affinity phases is a very common approach for isolation, purification and re-concentration of tagged proteins. Many biophysical studies are conducted on these grafted proteins (surface plasmon resonance, quartz crystal microbalance, etc.) showing that the integrity and function of the protein is usually maintained. However, NMR studies of such samples were not undertaken so far, due to the broadening observed on this kind of heterogeneous samples. We present here the use of the HR-MAS technology to obtain 2D NMR spectra of the MAGI-1 PDZ2/6 protein domain, C13-labeled, tagged with a His-tag and grafted on a Nickel affinity resin. We optimized the C13 Methyl SOFAST HMQC experiment allowing important gains in terms of signal-to-noise. The gain comes from the gathering of proton magnetization from the resin material to the protein under study. Several methyl signals from the unstructured C-terminal tail, which is involved in the binding of the PDZ domain to C-terminal peptides of its partners, were observed and measured. The interaction of the bound PDZ domain with cognate peptides was monitored using <500µg of protein sample. A response proportional to the peptide Kd is obtained, indicating that the method can be used to rapidly and efficiently monitor protein-ligand interactions.

Oligomeric states along the folding pathways of ß2-microglobulin: kinetics, thermodynamics, and structure.

The transition of proteins from their soluble functional state to amyloid fibrils and aggregates is associated with the onset of several human diseases. Protein aggregation often requires some structural reshaping and the subsequent formation of intermolecular contacts. Therefore, the study of the conformation of excited protein states and their ability to form oligomers is of primary importance for understanding the molecular basis of amyloid fibril formation. Here, we investigated the oligomerization processes that occur along the folding of the amyloidogenic human protein ß2-microglobulin. The combination of real-time two-dimensional NMR data with real-time small-angle X-ray scattering measurements allowed us to derive thermodynamic and kinetic information on protein oligomerization of different conformational states populated along the folding pathways. In particular, we could demonstrate that a long-lived folding intermediate (I-state) has a higher propensity to oligomerize compared to the native state. Our data agree well with a simple five-state kinetic model that involves only monomeric and dimeric species. The dimers have an elongated shape with the dimerization interface located at the apical side of ß2-microglobulin close to Pro32, the residue that has a trans conformation in the I-state and a cis conformation in the native (N) state. Our experimental data suggest that partial unfolding in the apical half of the protein close to Pro32 leads to an excited state conformation with enhanced propensity for oligomerization. This excited state becomes more populated in the transient I-state due to the destabilization of the native conformation by the trans-Pro32 configuration.