NMR View is a computer program designed for the visualization and analysis of NMR data. It allows the user to interact with a practically unlimited number of 2D, 3D and 4D NMR data files. Any number of spectral windows can be displayed on the screen in any size and location. Automatic peak picking and facilitated peak analysis features are included to aid in the assignment of complex NMR spectra. NMR View provides structure analysis features and data transfer to and from structure generation programs, allowing for a tight coupling between spectral analysis and structure generation. Visual correlation between structures and spectra can be done with the Molecular Data Viewer, a molecular graphics program with bidirectional communication to NMR View. The user interface can be customized and a command language is provided to allow for the automation of various tasks.

NMR View: A computer program for the visualization and analysis of NMR data.

https://zenodo.org/record/45375/files/article.pdf

Natural abundance nitrogen-15 NMR by enhanced heteronuclear spectroscopy

Communication between cells assumes particular importance in multicellular organisms. The growth, migration and differentiation of cells in the embryo, and their organization into specific tissues, depend on signals transmitted from one cell to another. In the adult, cell signalling orchestrates normal cellular behaviour and responses to wounding and infection. The consequences of breakdowns in this signalling underlie cancer, diabetes and disorders of the immune and cardiovascular systems. Conserved protein domains that act as key regulatory participants in many of these different signalling pathways are highlighted.

Protein modules and signalling networks

Fast transverse relaxation of 1H, 15N, and 13C by dipole-dipole coupling (DD) and chemical shift anisotropy (CSA) modulated by rotational molecular motions has a dominant impact on the size limit for biomacromolecular structures that can be studied by NMR spectroscopy in solution. Transverse relaxation-optimized spectroscopy (TROSY) is an approach for suppression of transverse relaxation in multidimensional NMR experiments, which is based on constructive use of interference between DD coupling and CSA. For example, a TROSY-type two-dimensional 1H,15N-correlation experiment with a uniformly 15N-labeled protein in a DNA complex of molecular mass 17 kDa at a 1H frequency of 750 MHz showed that 15N relaxation during 15N chemical shift evolution and 1HN relaxation during signal acquisition both are significantly reduced by mutual compensation of the DD and CSA interactions. The reduction of the linewidths when compared with a conventional two-dimensional 1H,15N-correlation experiment was 60% and 40%, respectively, and the residual linewidths were 5 Hz for 15N and 15 Hz for 1HN at 4°C. Because the ratio of the DD and CSA relaxation rates is nearly independent of the molecular size, a similar percentagewise reduction of the overall transverse relaxation rates is expected for larger proteins. For a 15N-labeled protein of 150 kDa at 750 MHz and 20°C one predicts residual linewidths of 10 Hz for 15N and 45 Hz for 1HN, and for the corresponding uniformly 15N,2H-labeled protein the residual linewidths are predicted to be smaller than 5 Hz and 15 Hz, respectively. The TROSY principle should benefit a variety of multidimensional solution NMR experiments, especially with future use of yet somewhat higher polarizing magnetic fields than are presently available, and thus largely eliminate one of the key factors that limit work with larger molecules.

https://www.pnas.org/content/pnas/94/23/12366.full.pdf

Attenuated T2 relaxation by mutual cancellation of dipole–dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution

The backbone dynamics of the C-terminal SH2 domain of phospholipase C gamma 1 have been investigated. Two forms of the domain were studied, one in complex with a high-affinity binding peptide derived from the platelet-derived growth factor receptor and the other in the absence of this peptide. 2-D 1H-15N NMR methods, employing pulsed field gradients, were used to determine steady-state 1H-15N NOE values and T1 and T2 15N relaxation times. Backbone dynamics were characterized by the overall correlation time (tau m), order parameters (S2), effective correlation times for internal motions (tau e), and, if required, terms to account for motions on a microsecond-to-millisecond-time scale. An extended two-time-scale formalism was used for residues having relaxation data and that could not be fit adequately using a single-time-scale formalism. The overall correlation times of the uncomplexed and complexed forms of SH2 were found to be 9.2 and 6.5 ns, respectively, suggesting that the uncomplexed form is in a monomer-dimer equilibrium. This was subsequently confirmed by hydrodynamic measurements. Analysis of order parameters reveals that residues in the so-called phosphotyrosine-binding loop exhibited higher than average disorder in both forms of SH2. Although localized differences in order parameters were observed between the uncomplexed and complexed forms of SH2, overall, higher order parameters were not found in the peptide-bound form, indicating that on average, picosecond-time-scale disorder is not reduced upon binding peptide. The relaxation data of the SH2-phosphopeptide complex were fit with fewer exchange terms than the uncomplexed form. This may reflect the monomer-dimer equilibrium that exists in the uncomplexed form or may indicate that the complexed form has lower conformational flexibility on a microsecond-to-millisecond-time scale.

Backbone Dynamics of a Free and a Phosphopeptide-Complexed Src Homology 2 Domain Studied by 15N NMR Relaxation

Sensitivity enhanced detection of weak nuclei using heteronuclear multiple quantum coherence

The Dynamic Process of β2-Adrenergic Receptor Activation

Dipolar coupling interactions represent an extremely valuable source of long-range distance and angle information that was previously not available for solution structure determinations of macromolecules. This is because observation of these dipolar coupling data requires creating an anisotropic environment for the macromolecule. Here we introduce a new method for generating tunable degrees of alignment of macromolecules by addition of magnetically aligned Pf1 filamentous bacteriophage as a cosolute. This phage-induced alignment technique has been used to study 1H-1H, 1H-13C, and 1H-15N dipolar coupling interactions in a DNA duplex, an RNA hairpin and several proteins including thioredoxin and apo-calmodulin. The phage allow alignment of macromolecules over a wide range of temperature and solution conditions and thus represent a stable versatile method for generating partially aligned macromolecules in solution.

Tunable alignment of macromolecules by filamentous phage yields dipolar coupling interactions

G-protein-coupled receptors (GPCRs) mediate the majority of cellular responses to hormones and neurotransmitters, and these membrane proteins are the largest group of therapeutic targets for a broad range of diseases. It is very difficult to obtain high-resolution X-ray crystal structures of GPCRs; little is known about the functional role(s) of the extracellular surface in receptor activation or about the conformational coupling of the extracellular surface to the native ligand-binding pocket. In this study, Bokoch et al. used NMR spectroscopy to investigate ligand-specific conformational changes around a salt bridge linking extracellular loops 2 and 3 of the β2 adrenergic receptor. They found that drugs that bind within the transmembrane core (and exhibit different efficacies towards G-protein activation) stabilize distinct conformations of the extracellular surface. New therapeutic agents that target this diverse surface could function as allosteric modulators with high subtype selectivity. G-protein-coupled receptors (GPCRs) mediate the majority of cellular responses to hormones and neurotransmitters and are the largest group of therapeutic targets for a range of diseases. The extracellular surface (ECS) of GPCRs is diverse and therefore an ideal target for the discovery of subtype-selective drugs. Here, NMR spectroscopy is used to investigate ligand-specific conformational changes around a central structural feature in the ECS of a GPCR. G-protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters. They are the largest group of therapeutic targets for a broad spectrum of diseases. Recent crystal structures of GPCRs1,2,3,4,5 have revealed structural conservation extending from the orthosteric ligand-binding site in the transmembrane core to the cytoplasmic G-protein-coupling domains. In contrast, the extracellular surface (ECS) of GPCRs is remarkably diverse and is therefore an ideal target for the discovery of subtype-selective drugs. However, little is known about the functional role of the ECS in receptor activation, or about conformational coupling of this surface to the native ligand-binding pocket. Here we use NMR spectroscopy to investigate ligand-specific conformational changes around a central structural feature in the ECS of the β2 adrenergic receptor: a salt bridge linking extracellular loops 2 and 3. Small-molecule drugs that bind within the transmembrane core and exhibit different efficacies towards G-protein activation (agonist, neutral antagonist and inverse agonist) also stabilize distinct conformations of the ECS. We thereby demonstrate conformational coupling between the ECS and the orthosteric binding site, showing that drugs targeting this diverse surface could function as allosteric modulators with high subtype selectivity. Moreover, these studies provide a new insight into the dynamic behaviour of GPCRs not addressable by static, inactive-state crystal structures.

Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor

NMR resonances of backbone nuclei have been assigned for >95% of the residues in oxidized, substrate-free, perdeuterated 13C/15N-labelled MurB (347 residues). A novel approach utilizing minimum chemical shift changes was employed to localize the NADP+ binding site on MurB.

Luciano Mueller

Papers

Sensitivity enhanced detection of weak nuclei using heteronuclear multiple quantum coherence

The Dynamic Process of β2-Adrenergic Receptor Activation

Tunable alignment of macromolecules by filamentous phage yields dipolar coupling interactions

Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor

Localizing the NADP+ binding site on the MurB enzyme by NMR.