Solution structure and backbone dynamics of the second PDZ domain of postsynaptic density-95

The second PDZ domain of postsynaptic density-95 (PSD-95 PDZ2) plays a critical role in coupling N-methyl-D-aspartate receptors to neuronal nitric oxide synthase (nNOS). In this work, the solution structure of PSD-95 PDZ2 was determined to high resolution by NMR spectroscopy. The structure of PSD-95 PDZ2 was compared in detail with that of a1-syntrophin PDZ domain, as the PDZ domains share similar target interaction properties. The interaction of the PSD-95 PDZ2 with a carboxyl-terminal peptide derived from a cytoplasmic protein CAPON was studied by NMR titration experiments. Complex formation between PSD-95 PDZ2 and the nNOS PDZ was modelled on the basis of the crystal structure of the a1-syntrophin PDZ/nNOS PDZ dimer. We found that the prolonged loop connecting the bB and bC strands of PSD-95 PDZ2 is likely to play a role in both the binding of the carboxyl-terminal peptide and the nNOS b-®nger. Finally, the backbone dynamics of the PSD-95 PDZ2 in the absence of bound peptide were studied using a model-free approach. Thè`G LGF''-loop and the loop connecting aB and bF of the protein display some degree of¯exibility in solution. The rest of the protein is rigid and lacks detectable slow time-scale (microseconds to milliseconds) motions. In particular, the loop connecting bB and bC loop adopts a well-de®ned, rigid structure in solution. It appears that the loop adopts a pre-aligned conformation for the PDZ domain to interact with its targets

A novel magnetic resonance-based method to measure gene expression in living cells

In unicellular and multicellular eukaryotes, elaborate gene regulatory mechanisms facilitate a broad range of biological processes from cell division to morphological differentiation. In order to fully understand the gene regulatory networks involved in these biological processes, the spatial and temporal patterns of expression of many thousands of genes will need to be determined in real time in living organisms. Currently available techniques are not sufficient to achieve this goal; however, novel methods based on magnetic resonance (MR) imaging may be particularly useful for sensitive detection of gene expression in opaque tissues. This report describes a novel reporter gene system that monitors gene expression dynamically and quantitatively, in yeast cells, by measuring the accumulation of inorganic polyphosphate (polyP) using MR spectroscopy (MRS) or MR spectroscopic imaging (MRI). Because this system is completely non-invasive and does not require exogenous substrates, it is a powerful tool for studying gene expression in multicellular organisms, as well

ALS mutations in the TIA-1 prion-like domain trigger highly condensed pathogenic structures

筋萎縮性側索硬化症（ALS）の発症機構の一端を解明 --タンパク質の高密度な凝縮構造が鍵--. 京都大学プレスリリース. 2022-09-13.T cell intracellular antigen-1 (TIA-1) plays a central role in stress granule (SG) formation by self-assembly via the prion-like domain (PLD). In the TIA-1 PLD, amino acid mutations associated with neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) or Welander distal myopathy (WDM), have been identified. However, how these mutations affect PLD self-assembly properties has remained elusive. In this study, we uncovered the implicit pathogenic structures caused by the mutations. NMR analysis indicated that the dynamic structures of the PLD are synergistically determined by the physicochemical properties of amino acids in units of five residues. Molecular dynamics simulations and three-dimensional electron crystallography, together with biochemical assays, revealed that the WDM mutation E384K attenuated the sticky properties, whereas the ALS mutations P362L and A381T enhanced the self-assembly by inducing β-sheet interactions and highly condensed assembly, respectively. These results suggest that the P362L and A381T mutations increase the likelihood of irreversible amyloid fibrillization after phase-separated droplet formation, and this process may lead to pathogenicity

Characterizing conformational ensembles of multi-domain proteins using anisotropic paramagnetic NMR restraints

It has been over two decades since paramagnetic NMR started to form part of the essential techniques for structural analysis of proteins under physiological conditions. Paramagnetic NMR has significantly expanded our understanding of the inherent flexibility of proteins, in particular, those that are formed by combinations of two or more domains. Here, we present a brief overview of techniques to characterize conformational ensembles of such multi-domain proteins using paramagnetic NMR restraints produced through anisotropic metals, with a focus on the basics of anisotropic paramagnetic effects, the general procedures of conformational ensemble reconstruction, and some representative reweighting approaches

An innate interaction between IL-18 and the propeptide that inactivates its precursor form

Uncontrolled secretion of mature interleukin (IL)-1β and IL-18 is responsible for severe autoinflammatory or autoimmune disorders and various allergic diseases. Here we report an intramolecular interaction between IL-18 and its propeptide, which is proteolytically removed from its precursor proIL-18 during maturation. The intramolecular interaction was recapitulated intermolecularly using recombinant propeptide. These results suggest the possibility of developing a novel class of peptide-based IL-18 inhibitors that could serve as therapeutic agents for IL-18-related inflammatory diseases

Effective production of fluorescent nanodiamonds containing negatively-charged nitrogen-vacancy centers by ion irradiation

Fluorescence from negatively-charged nitrogen-vacancy centers (NV[−]s) in diamonds has unique optical properties with none of the undesirable effects such as photo-bleaching and photo-blinking. In addition, the spin-dependent fluorescence intensity of NV[−]s allows us to perform optically detected magnetic resonance (ODMR) investigation for evaluating the presence of NV−s and for the electronic local environment. In this work, we irradiated H[+], He[+], Li[+] and N[+] ions to nanodiamonds with a median size of 26 nm at various irradiation energies and doses for improving the NV[−] concentration. ODMR observations of the nanodiamonds showed that ion irradiation increased the number of nanodiamonds containing NV[−]s up to 200 ppm, whereas without ion irradiation, only few NV[−]s were found. The number of nanodiamonds containing NV[−]s at various ion irradiation doses was not monotonous, but had maxima at certain irradiation doses. These results suggest a competition in two opponent roles of vacancies, effective for pairing with nitrogen atoms and as defects for developing damage in crystalline. We also found that sharp and strong ODMR signals were obtained from nanodiamonds irradiated at the optimal condition for the highest yield of NV−s. We concluded that He[+] ion irradiations with 60 or 80 keV at a dose of 1 × 10[13] ions cm[–2] are the conditions required for the most efficient production of a high quantity of nanodiamonds containing NV[−]s