Fast multi-dimensional NMR acquisition and processing using the sparse FFT

Increasing the dimensionality of NMR experiments strongly enhances the spectral resolution and provides invaluable direct information about atomic interactions. However, the price tag is high: long measurement times and heavy requirements on the computation power and data storage. We introduce sparse fast Fourier transform as a new method of NMR signal collection and processing, which is capable of reconstructing high quality spectra of large size and dimensionality with short measurement times, faster computations than the fast Fourier transform, and minimal storage for processing and handling of sparse spectra. The new algorithm is described and demonstrated for a 4D BEST-HNCOCA spectrum.Swedish Research Council (Research Grant 2011-5994)Swedish National Infrastructure for Computing (Grant SNIC 001/12-271

A C2HC zinc finger is essential for the RING-E2 interaction of the ubiquitin ligase RNF125

The activity of RING ubiquitin ligases (E3s) depends on an interaction between the RING domain and ubiquitin conjugating enzymes (E2), but posttranslational events or additional structural elements, yet largely undefined, are frequently required to enhance or regulate activity. Here, we show for the ubiquitin ligase RNF125 that, in addition to the RING domain, a C2HC Zn finger (ZnF) is crucial for activity, and a short linker sequence (Li2(120-128)) enhances activity. The contribution of these regions was first shown with truncated proteins, and the essential role of the ZnF was confirmed with mutations at the Zn chelating Cys residues. Using NMR, we established that the C2HC ZnF/Li2(120-128) region is crucial for binding of the RING domain to the E2 UbcH5a. The partial X-ray structure of RNF125 revealed the presence of extensive intramolecular interactions between the RING and C2HC ZnF. A mutation at one of the contact residues in the C2HC ZnF, a highly conserved M112, resulted in the loss of ubiquitin ligase activity. Thus, we identified the structural basis for an essential role of the C2HC ZnF and conclude that this domain stabilizes the RING domain, and is therefore required for binding of RNF125 to an E2

An oxygen-sensitive toxin-antitoxin system

The Hha and TomB proteins from Escherichia coli form an oxygen-dependent toxin-antitoxin (TA) system. Here we show that YmoB, the Yersinia orthologue of TomB, and its single cysteine variant [C117S]YmoB can replace TomB as antitoxins in E. coli. In contrast to other TA systems, [C117S]YmoB transiently interacts with Hha (rather than forming a stable complex) and enhances the spontaneous oxidation of the Hha conserved cysteine residue to a -SOxH- containing species (sulfenic, sulfinic or sulfonic acid), which destabilizes the toxin. The nuclear magnetic resonance structure of [C117S]YmoB and the homology model of TomB show that the two proteins form a four-helix bundle with a conserved buried cysteine connected to the exterior by a channel with a diameter comparable to that of an oxygen molecule. The Hha interaction site is located on the opposite side of the helix bundle

Measurement of protein backbone (CO)-C-13 and N-15 relaxation dispersion at high resolution

Peak overlap in crowded regions of two-dimensional spectra prevents characterization of dynamics for many sites of interest in globular and intrinsically disordered proteins. We present new three-dimensional pulse sequences for measurement of Carr-Purcell-Meiboom-Gill relaxation dispersions at backbone nitrogen and carbonyl positions. To alleviate increase in the measurement time associated with the additional spectral dimension, we use non-uniform sampling in combination with two distinct methods of spectrum reconstruction: compressed sensing and co-processing with multi-dimensional decomposition. The new methodology was validated using disordered protein CD79A from B-cell receptor and an SH3 domain from Abp1p in exchange between its free form and bound to a peptide from the protein Ark1p. We show that, while providing much better resolution, the 3D NUS experiments give the similar accuracy and precision of the dynamic parameters to ones obtained using traditional 2D experiments. Furthermore, we show that jackknife resampling of the spectra yields robust estimates of peak intensities errors, eliminating the need for recording duplicate data points.Funding Agencies|Swedish Research Council [2015-04614]; Swedish National Infrastructure for Computing [SNIC 2016/5-61]</p

About structural changes of lignin during kraft cooking and the kinetics of delignification

Wood meal was submitted to kraft cooking in a small-scale flow-through reactor and the structural changes of lignin have been investigated. The rate determining steps in kraft cooking were in focus. Based on two-dimensional nuclear magnetic resonance (2D-NMR) measurements on lignin fractions extracted at different cooking times from the black liquor, it was observed that the main lignin reactions occur within 10-20 min and thus the kinetics of the chemical reaction cannot be the rate-determining step. On the other hand, the molecular weight (MW) of lignin is shifted towards larger fragments in the course of cooking time but the MW decreases with increasing ionic strength. Obviously, the kinetics of the delignification are strongly dependent on solubility and/or mass transport at the cell wall level. At chip size level, the mass transport of cooking chemicals into the wood chip may influence the overall kinetics in the initial part of the cooking. At longer cooking times the concentration of chemicals becomes sufficiently high in the wood chips, and the delignification is progressively governed by solubility and/or mass transport of lignin molecules occurring at the cell wall level

Tyrosine Phosphorylation within the Intrinsically Disordered Cytosolic Domains of the B-Cell Receptor: An NMR-Based Structural Analysis

<div><p>Intrinsically disordered proteins are found extensively in cell signaling pathways where they often are targets of posttranslational modifications e.g. phosphorylation. Such modifications can sometimes induce or disrupt secondary structure elements present in the modified protein. CD79a and CD79b are membrane-spanning, signal-transducing components of the B-cell receptor. The cytosolic domains of these proteins are intrinsically disordered and each has an immunoreceptor tyrosine-based activation motif (ITAM). When an antigen binds to the receptor, conserved tyrosines located in the ITAMs are phosphorylated which initiate further downstream signaling. Here we use NMR spectroscopy to examine the secondary structure propensity of the cytosolic domains of CD79a and CD79b <i>in vitro</i> before and after phosphorylation. The phosphorylation patterns are identified through analysis of changes of backbone chemical shifts found for the affected tyrosines and neighboring residues. The number of the phosphorylated sites is confirmed by mass spectrometry. The secondary structure propensities are calculated using the method of intrinsic referencing, where the reference random coil chemical shifts are measured for the same protein under denaturing conditions. Our analysis revealed that CD79a and CD79b both have an overall propensity for α-helical structure that is greatest in the C-terminal region of the ITAM. Phosphorylation of CD79a caused a decrease in helical propensity in the C-terminal ITAM region. For CD79b, the opposite was observed and phosphorylation resulted in an increase of helical propensity in the C-terminal part.</p></div

Backbone Assignment of the MALT1 Paracaspase by Solution NMR.

Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is a unique paracaspase protein whose protease activity mediates oncogenic NF-κB signalling in activated B cell-like diffuse large B cell lymphomas (ABC-DLBCLs). ABC-DLBCLs are aggressive lymphomas with high resistance to current chemotherapies. Low survival rate among patients emphasizes the urgent need for alternative treatment options. The characterization of the MALT1 will be an essential tool for developing new target-directed drugs against MALT1 dependent disorders. As the first step in the atomic-level NMR studies of the system, here we report, the (15)N/(13)C/(1)H backbone assignment of the apo form of the MALT1 paracaspase region together with the third immunoglobulin-like (Ig3) domain, 44 kDa, by high resolution NMR. In addition, the non-uniform sampling (NUS) based targeted acquisition procedure is evaluated as a mean of decreasing acquisition and analysis time for larger proteins