Characterizing the Monomer-Dimer Equilibrium of UbcH8/Ube2L6: A Combined SAXS and NMR Study.

Interferon-stimulated gene-15 (ISG15) is an interferon-induced protein with two ubiquitin-like (Ubl) domains linked by a short peptide chain and is a conjugated protein of the ISGylation system. Similar to ubiquitin and other Ubls, ISG15 is ligated to its target proteins through a series of E1, E2, and E3 enzymes known as Uba7, Ube2L6/UbcH8, and HERC5, respectively. Ube2L6/UbcH8 plays a central role in ISGylation, underscoring it as an important drug target for boosting innate antiviral immunity.

Protein NMR assignment by isotope pattern recognition.

The current standard method for amino acid signal identification in protein NMR spectra is sequential assignment using triple-resonance experiments. Good software and elaborate heuristics exist, but the process remains laboriously manual. Machine learning does help, but its training databases need millions of samples that cover all relevant physics and every kind of instrumental artifact.

A multi-iron enzyme installs copper-binding oxazolone/thioamide pairs on a nontypeable virulence factor.

The multinuclear nonheme iron-dependent oxidases (MNIOs) are a rapidly growing family of enzymes involved in the biosynthesis of ribosomally synthesized, posttranslationally modified peptide natural products (RiPPs). Recently, a secreted virulence factor from nontypeable (NTHi) was found to be expressed from an operon, which we designate the operon, that also encodes an MNIO. Here, we show by Mössbauer spectroscopy that the MNIO HvfB contains a triiron cofactor.

Insights on the G protein-coupled receptor helix 8 solution structure and orientation using a neurotensin receptor 1 peptide.

G-protein coupled receptors (GPCRs) are the largest class of membrane proteins encoded in the human genome with high pharmaceutical relevance and implications to human health. These receptors share a prevalent architecture of seven transmembrane helices followed by an intracellular, amphipathic helix 8 (H8) and a disordered C-terminal tail (Ctail). Technological advancements have led to over 1000 receptor structures in the last two decades, yet frequently H8 and the Ctail are conformationally heterogeneous or altogether absent.

Stabilization of pre-existing neurotensin receptor conformational states by β-arrestin-1 and the biased allosteric modulator ML314.

The neurotensin receptor 1 (NTS) is a G protein-coupled receptor (GPCR) with promise as a drug target for the treatment of pain, schizophrenia, obesity, addiction, and various cancers. A detailed picture of the NTS structural landscape has been established by X-ray crystallography and cryo-EM and yet, the molecular determinants for why a receptor couples to G protein versus arrestin transducers remain poorly defined. We used CH-methionine NMR spectroscopy to show that binding of phosphatidylinositol-4,5-bisphosphate (PIP2) to the receptor's intracellular surface allosterically tunes the timescale of motions at the orthosteric pocket and conserved activation motifs - without dramatically altering the structural ensemble.

Characterizing the monomer-dimer equilibrium of UbcH8/Ube2L6: A combined SAXS and NMR study.

Interferon-stimulated gene-15 (ISG15) is an interferon-induced protein with two ubiquitin-like (Ubl) domains linked by a short peptide chain, and the conjugated protein of the ISGylation system. Similar to ubiquitin and other Ubls, ISG15 is ligated to its target proteins through a series of E1, E2, and E3 enzymes known as Uba7, Ube2L6/UbcH8, and HERC5, respectively. Ube2L6/UbcH8 plays a literal central role in ISGylation, underscoring it as an important drug target for boosting innate antiviral immunity.

Ligands selectively tune the local and global motions of neurotensin receptor 1 (NTS).

Nuclear magnetic resonance (NMR) studies have revealed that fast methyl sidechain dynamics can report on entropically-driven allostery. Yet, NMR applications have been largely limited to the super-microsecond motional regimes of G protein-coupled receptors (GPCRs). We use C-methionine chemical shift-based global order parameters to test if ligands affect the fast dynamics of a thermostabilized GPCR, neurotensin receptor 1 (NTS).

A method for selective F-labeling absent of probe sequestration (SLAPS).

Fluorine ( F) offers several distinct advantages for biomolecular nuclear magnetic resonance spectroscopy such as no background signal, 100% natural abundance, high sensitivity, and a large chemical shift range. Exogenous cysteine-reactive F-probes have proven especially indispensable for characterizing large, challenging systems that are less amenable to other isotopic labeling strategies such as G protein-coupled receptors. As fluorine linewidths are inherently broad, limiting reactions with offsite cysteines is critical for spectral simplification and accurate deconvolution of component peaks-especially when analyzing systems with intermediate to slow timescale conformational exchange.

Effect of Ligands and Transducers on the Neurotensin Receptor 1 Conformational Ensemble.

Using a discrete, intracellular F nuclear magnetic resonance (NMR) probe on transmembrane helix 6 of the neurotensin receptor 1 (NTS1), we aim to understand how ligands and transducers modulate the receptor's structural ensemble in a solution. For apo NTS1, F NMR spectra reveal an ensemble of at least three conformational substates (one inactive and two active-like) in equilibrium that exchange on the millisecond to second timescale. Dynamic NMR experiments reveal that these substates follow a linear three-site exchange process that is both thermodynamically and kinetically remodeled by orthosteric ligands.

NUScon: a community-driven platform for quantitative evaluation of nonuniform sampling in NMR.

Although the concepts of nonuniform sampling (NUS​​​​​​​) and non-Fourier spectral reconstruction in multidimensional NMR began to emerge 4 decades ago , it is only relatively recently that NUS has become more commonplace. Advantages of NUS include the ability to tailor experiments to reduce data collection time and to improve spectral quality, whether through detection of closely spaced peaks (i.e.

TRACT revisited: an algebraic solution for determining overall rotational correlation times from cross-correlated relaxation rates.

Accurate rotational correlation times ([Formula: see text]) are critical for quantitative analysis of fast timescale NMR dynamics. As molecular weights increase, the classic derivation of [Formula: see text] using transverse and longitudinal relaxation rates becomes increasingly unsuitable due to the non-trivial contribution of remote dipole-dipole interactions to longitudinal relaxation. Derivations using cross-correlated relaxation experiments, such as TRACT, overcome these limitations but are erroneously calculated in 65% of the citing literature.

Optimal control theory enables homonuclear decoupling without Bloch-Siegert shifts in NMR spectroscopy.

The Bloch-Siegert shift is a phenomenon in NMR spectroscopy and atomic physics in which the observed resonance frequency is changed by the presence of an off-resonance applied field. In NMR, it occurs especially in the context of homonuclear decoupling. Here we develop a practical method for homonuclear decoupling that avoids inducing Bloch-Siegert shifts.

Mixed pyruvate labeling enables backbone resonance assignment of large proteins using a single experiment.

Backbone resonance assignment is a critical first step in the investigation of proteins by NMR. This is traditionally achieved with a standard set of experiments, most of which are not optimal for large proteins. Of these, HNCA is the most sensitive experiment that provides sequential correlations.

Interpolating and extrapolating with hmsIST: seeking a t for optimal sensitivity, resolution and frequency accuracy.

Non-Uniform Sampling has the potential to exploit the optimal resolution of high-field NMR instruments. This is not possible in 3D and 4D NMR experiments when using traditional uniform sampling due to the long overall measurement time. Nominally, uniformly sampled time domain data acquired to a maximum evolution time t can be extended to high resolution via a virtual maximum evolution time t* while extrapolating with linear prediction or iterative soft thresholding (IST).

Backbone and side chain NMR assignments of Geobacillus stearothermophilus ZapA allow identification of residues that mediate the interaction of ZapA with FtsZ.

Bacterial division begins with the formation of a contractile protein ring at midcell, which constricts the bacterial envelope to generate two daughter cells. The central component of the division ring is FtsZ, a tubulin-like protein capable of self-assembling into filaments which further associate into a higher order structure known as the Z ring. Proteins that bind to FtsZ play a crucial role in the formation and regulation of the Z ring.

Perspectives in magnetic resonance: NMR in the post-FFT era.

Multi-dimensional NMR spectra have traditionally been processed with the fast Fourier transformation (FFT). The availability of high field instruments, the complexity of spectra of large proteins, the narrow signal dispersion of some unstructured proteins, and the time needed to record the necessary increments in the indirect dimensions to exploit the resolution of the highfield instruments make this traditional approach unsatisfactory. New procedures need to be developed beyond uniform sampling of the indirect dimensions and reconstruction methods other than the straight FFT are necessary.

Exploring signal-to-noise ratio and sensitivity in non-uniformly sampled multi-dimensional NMR spectra.

It is well established that non-uniform sampling (NUS) allows acquisition of multi-dimensional NMR spectra at a resolution that cannot be obtained with traditional uniform acquisition through the indirect dimensions. However, the impact of NUS on the signal-to-noise ratio (SNR) and sensitivity are less well documented. SNR and sensitivity are essential aspects of NMR experiments as they define the quality and extent of data that can be obtained.

NMR solution structure and condition-dependent oligomerization of the antimicrobial peptide human defensin 5.

Human defensin 5 (HD5) is a 32-residue host-defense peptide expressed in the gastrointestinal, reproductive, and urinary tracts that has antimicrobial activity. It exhibits six cysteine residues that are regiospecifically oxidized to form three disulfide bonds (Cys(3)-Cys(31), Cys(5)-Cys(20), and Cys(10)-Cys(30)) in the oxidized form (HD5(ox)). To probe the solution structure and oligomerization properties of HD5(ox), and select mutant peptides lacking one or more disulfide bonds, NMR solution studies and analytical ultracentrifugation experiments are reported in addition to in vitro peptide stability assays.

Staphylococcus aureus uses a novel multidomain receptor to break apart human hemoglobin and steal its heme.

Staphylococcus aureus is a leading cause of life-threatening infections in the United States. It requires iron to grow, which must be actively procured from its host to successfully mount an infection. Heme-iron within hemoglobin (Hb) is the most abundant source of iron in the human body and is captured by S.

Solution structure of the sortase required for efficient production of infectious Bacillus anthracis spores.

Bacillus anthracis forms metabolically dormant endospores that upon germination can cause lethal anthrax disease in humans. Efficient sporulation requires the activity of the SrtC sortase (BaSrtC), a cysteine transpeptidase that covalently attaches the BasH and BasI proteins to the peptidoglycan of the forespore and predivisional cell, respectively. To gain insight into the molecular basis of protein display, we used nuclear magnetic resonance to determine the structure and backbone dynamics of the catalytic domain of BaSrtC (residues Ser(56)-Lys(198)).

Transient weak protein-protein complexes transfer heme across the cell wall of Staphylococcus aureus.

Iron is an essential nutrient for the bacterial pathogen Staphylococcus aureus . Heme in hemoglobin (Hb) is the most abundant source of iron in the human body and during infections is captured by S. aureus using iron-regulated surface determinant (Isd) proteins.

Assembly of minicellulosomes on the surface of Bacillus subtilis.

To cost-efficiently produce biofuels, new methods are needed to convert lignocellulosic biomass into fermentable sugars. One promising approach is to degrade biomass using cellulosomes, which are surface-displayed multicellulase-containing complexes present in cellulolytic Clostridium and Ruminococcus species. In this study we created cellulolytic strains of Bacillus subtilis that display one or more cellulase enzymes.

Evidence from artificial septal targeting and site-directed mutagenesis that residues in the extracytoplasmic β domain of DivIB mediate its interaction with the divisomal transpeptidase PBP 2B.

Bacterial cytokinesis is achieved through the coordinated action of a multiprotein complex known as the divisome. The Escherichia coli divisome is comprised of at least 10 essential proteins whose individual functions are mostly unknown. Most divisomal proteins have multiple binding partners, making it difficult to pinpoint epitopes that mediate pairwise interactions between these proteins.

A heteronuclear zero quantum coherence Nz-exchange experiment that resolves resonance overlap and its application to measure the rates of heme binding to the IsdC protein.

Chemical exchange phenomena in NMR spectra can be quantitatively interpreted to measure the rates of ligand binding, as well as conformational and chemical rearrangements. In macromolecules, processes that occur slowly on the chemical shift time scale are frequently studied using 2D heteronuclear ZZ or N(z)-exchange spectroscopy. However, to successfully apply this method, peaks arising from each exchanging species must have unique chemical shifts in both dimensions, a condition that is often not satisfied in protein-ligand binding equilibria for (15)N nuclei.