Membrane topologies of the PGLa antimicrobial peptide and a transmembrane anchor sequence by Dynamic Nuclear Polarization/ solid-state NMR spectroscopy OPEN

International audienceDynamic Nuclear Polarization (DNP) has been introduced to overcome the sensitivity limitations of nuclear magnetic resonance (NMR) spectroscopy also of supported lipid bilayers. When investigated by solid-state NMR techniques the approach typically involves doping the samples with biradicals and their investigation at cryo-temperatures. Here we investigated the effects of temperature and membrane hydration on the topology of amphipathic and hydrophobic membrane polypeptides. Although the antimicrobial PGLa peptide in dimyristoyl phospholipids is particularly sensitive to topological alterations, the DNP conditions represent well its membrane alignment also found in bacterial lipids at ambient temperature. With a novel membrane-anchored biradical and purpose-built hardware a 17-fold enhancement in NMR signal intensity is obtained by DNP which is one of the best obtained for a truly static matrix-free system. Furthermore, a membrane anchor sequence encompassing 19 hydrophobic amino acid residues was investigated. Although at cryotemperatures the transmembrane domain adjusts it membrane tilt angle by about 10 degrees, the temperature dependence of two-dimensional separated field spectra show that freezing the motions can have beneficial effects for the structural analysis of this sequence

Phylogeny of the Sepia pharaonis species complex (Cephalopoda: Sepiida) based on analyses of mitochondrial and nuclear DNA sequence data

The pharaoh cuttlefish, Sepia pharaonis Ehrenberg, 1831, is a commercially fished species found from Japan to East Africa. Previous morphological and genetic work (the latter based on the 16S rRNA mitochondrial gene) suggested that S. pharaonis is a species complex, but relationships within the complex remained unresolved. To clarify these relationships, we have sequenced an additional mitochondrial gene region (cytochrome oxidase subunit I) and a nuclear gene region (rhodopsin) from over 50 specimens from throughout the range of S. pharaonis. We have also added sequence data from two specimens of Sepia ramani Neethiselvan, 2001, collected in southeastern India. Sepia ramani is a species that is morphologically very similar to S. pharaonis, and there is some question regarding its status as a distinct species. Phylogenetic analyses of a dataset comprising all three-gene regions revealed a monophyletic S. pharaonis complex consisting of a western Indian Ocean clade, a northeastern Australia clade, a Persian Gulf/Arabian Sea (‘Iranian’) clade, a western Pacific clade and a central Indian Ocean clade. Relationships among these clades remain somewhat poorly supported except for a clade comprising the Iranian clade, the western Pacific clade and the central Indian Ocean clade. One S. pharaonis specimen was collected in the Arabian Sea, but was found to be a member of the western Indian Ocean clade, suggesting that gene flow between these regions has either occurred recently or is ongoing. Both specimens of S. ramani are members of the S. pharaonis complex, but their mtDNA haplotypes are not closely related – one is a member of the central Indian Ocean clade, while the other is rather distantly related to the northeastern Australia clade. We suggest that ‘S. pharaonis’ may consist of several species, but morphological work is needed to clarify species-level taxonomy within this complex

Ultra low temperature microturbine for magic angle spinning system

We investigate the fluid dynamics of a microturbine system that is applied in a device for chemical and biological analysis—a so-called magic angle spinning (MAS) nuclear magnetic resonance (NMR) probe. The present system is utilized in a wide temperature range from 45K to 293 K. Pressurized air, nitrogen, or helium are used to drive a Pelton type microturbine. This turbine is mounted on a MAS rotor with a diameter between 0.7mm and 3.2 mm. The rotor system is equipped with a pressurized gas bearing that is operated by the same gas species as the turbine. Computational fluid dynamics (CFD) simulations have been performed and compared with fluid dynamics measurements of the MAS system for different diameters, temperatures, and spinning rates between 23 kHz and 120 kHz. To our knowledge, this work is the first comprehensive CFD and experimental study of such a wide temperature range that has been carried out for microturbines with pressurized gas bearings. The results show good agreement between measurements and CFD simulations with appropriate (real) gas models, i.e., the ideal gas model for air at room temperature, Peng–Robinson model for nitrogen at 105 K, and ideal gas model for helium at 45 K

Refined NMR solution structures of proteins using homo-and heteronuclear couplings, relaxation time measurements and relaxation matrix analysis

Abstract In order to compare high resolution crystal structures of proteins with the corresponding solution structures a detailed analysis of NMR parameters obtained for various proteins was carried out. As many NOE values as possible were transformed into distances using a relaxation matrix analysis. In addition homo-and heteronuclear 3J couplings from I3C and I5N enriched protein species were determined. From these couplings the dihedral angles $, and x1 were evaluated. It was possible to interpret the various 3J values in terms of either distinct dihedral angles or with a certain variance of angles or with an equilibrium of different rotameric states. The refined solution structures were obtained using the distance constraints together with the dihedral angle constraints in a distance geometry algorithm (DIANA program package). The resulting DG structure was the starting conformation of a subsequent molecular dynamics simulation. From a determination of relaxation times TI, T, and NOE build-up rates of "N and I3C nuclei order parameters were obtained to describe the dynamic behaviour of protein molecular parts. Refined solution structures were obtained for ribonuclease TI, a flavodoxin from D.vulgaris, fatty acid binding protein from bovine heart and for a heat shock transcription factor from tomato. In most cases the high resolution crystal structures differ only slightly from the refined solution structures

Human PrimPol is a highly error-prone polymerase regulated by single-stranded DNA binding proteins

PrimPol is a recently identified polymerase involved in eukaryotic DNA damage tolerance, employed in both re-priming and translesion synthesis mechanisms to bypass nuclear and mitochondrial DNA lesions. In this report, we investigate how the enzymatic activities of human PrimPol are regulated. We show that, unlike other TLS polymerases, PrimPol is not stimulated by PCNA and does not interact with it in vivo. We identify that PrimPol interacts with both of the major single-strand binding proteins, RPA and mtSSB in vivo. Using NMR spectroscopy, we characterize the domains responsible for the PrimPol-RPA interaction, revealing that PrimPol binds directly to the N-terminal domain of RPA70. In contrast to the established role of SSBs in stimulating replicative polymerases, we find that SSBs significantly limit the primase and polymerase activities of PrimPol. To identify the requirement for this regulation, we employed two forward mutation assays to characterize PrimPol's replication fidelity. We find that PrimPol is a mutagenic polymerase, with a unique error specificity that is highly biased towards insertion-deletion errors. Given the error-prone disposition of PrimPol, we propose a mechanism whereby SSBs greatly restrict the contribution of this enzyme to DNA replication at stalled forks, thus reducing the mutagenic potential of PrimPol during genome replication

A dual-core NMR system for field-cycling singlet assisted diffusion NMR

Long-lived singlet spin order offers the possibility to extend the spin memory by more than an order of magnitude. This enhancement can be used, among other applications, to assist NMR diffusion experiments in porous media where the extended lifetime of singlet spin order can be used to gain information about structural features of the medium as well as the dynamics of the imbibed phase. Other than offering the possibility to explore longer diffusion times of the order of many minutes that, for example, gives unprecedented access to tortuosity in structures with interconnected pores, singlet order has the important advantage to be immune to the internal field gradients generated by magnetic susceptibility inhomogeneities. These inhomogeneities, however, are responsible for very short T2 decay constants in high magnetic field and this precludes access to the singlet order in the first instance. To overcome this difficulty and take advantage of singlet order in diffusion experiments in porous media, we have here developed a dual-core system with radiofrequency and 3-axis pulsed field gradients facilities in low magnetic field, for preparation and manipulation of singlet order and a probe, in high magnetic field, for polarisation and detection. The system operates in field-cycling and can be used for a variety of NMR experiments including diffusion tensor imaging (both singlet assisted and not). In this paper we present and discuss the new hardware and its calibration, and demonstrate its capabilities through a variety of examples

Hyperosmotic Stress Induces a Specific Pattern for Stress Granule Formation in Human-Induced Pluripotent Stem Cells

Stress granules (SGs) are assemblies of selective messenger RNAs (mRNAs), translation factors, and RNA-binding proteins in small untranslated messenger ribonucleoprotein (mRNP) complexes in the cytoplasm. Evidence indicates that different types of cells have shown different mechanisms to respond to stress and the formation of SGs. In the present work, we investigated how human-induced pluripotent stem cells (hiPSCs/IMR90-1) overcome hyperosmotic stress compared to a cell line that does not harbor pluripotent characteristics (SH-SY5Y cell line). Gradient concentrations of NaCl showed a different pattern of SG formation between hiPSCs/IMR90-1 and the nonpluripotent cell line SH-SY5Y. Other pluripotent stem cell lines (hiPSCs/CRTD5 and hESCs/H9 (human embryonic stem cell line)) as well as nonpluripotent cell lines (BHK-21 and MCF-7) were used to confirm this phenomenon. Moreover, the formation of hyperosmotic SGs in hiPSCs/IMR90-1 was independent of eIF2α phosphorylation and was associated with low apoptosis levels. In addition, a comprehensive proteomics analysis was performed to identify proteins involved in regulating this specific pattern of hyperosmotic SG formation in hiPSCs/IMR90-1. We found possible implications of microtubule organization on the response to hyperosmotic stress in hiPSCs/IMR90-1. We have also unveiled a reduced expression of tubulin that may protect cells against hyperosmolarity stress while inhibiting SG formation without affecting stem cell self-renewal and pluripotency. Our observations may provide a possible cellular mechanism to better understand SG dynamics in pluripotent stem cells