Nuclear Import Receptor Inhibits Phase Separation of FUS through Binding to Multiple Sites.

Liquid-liquid phase separation (LLPS) is believed to underlie formation of biomolecular condensates, cellular compartments that concentrate macromolecules without surrounding membranes. Physical mechanisms that control condensate formation/dissolution are poorly understood. The RNA-binding protein fused in sarcoma (FUS) undergoes LLPS in vitro and associates with condensates in cells. We show that the importin karyopherin-ß2/transportin-1 inhibits LLPS of FUS. This activity depends on tight binding of karyopherin-ß2 to the C-terminal proline-tyrosine nuclear localization signal (PY-NLS) of FUS. Nuclear magnetic resonance (NMR) analyses reveal weak interactions of karyopherin-ß2 with sequence elements and structural domains distributed throughout the entirety of FUS. Biochemical analyses demonstrate that most of these same regions also contribute to LLPS of FUS. The data lead to a model where high-affinity binding of karyopherin-ß2 to the FUS PY-NLS tethers the proteins together, allowing multiple, distributed weak intermolecular contacts to disrupt FUS self-association, blocking LLPS. Karyopherin-ß2 may act analogously to control condensates in diverse cellular contexts.

Sequence Determinants of Intracellular Phase Separation by Complex Coacervation of a Disordered Protein.

Liquid-liquid phase separation, driven by collective interactions among multivalent and intrinsically disordered proteins, is thought to mediate the formation of membrane-less organelles in cells. Using parallel cellular and in vitro assays, we show that the Nephrin intracellular domain (NICD), a disordered protein, drives intracellular phase separation via complex coacervation, whereby the negatively charged NICD co-assembles with positively charged partners to form protein-rich dense liquid droplets. Mutagenesis reveals that the driving force for phase separation depends on the overall amino acid composition and not the precise sequence of NICD. Instead, phase separation is promoted by one or more regions of high negative charge density and aromatic/hydrophobic residues that are distributed across the protein. Many disordered proteins share similar sequence characteristics with NICD, suggesting that complex coacervation may be a widely used mechanism to promote intracellular phase separation.

Average conservative chaos in quantum dusty plasmas.

We consider a hydrodynamic model of a quantum dusty plasma. We prove mathematically that the resulting dust ion-acoustic plasma waves present the property of being conservative on average. Furthermore, we test this property numerically, confirming its validity. Using standard techniques from the study of dynamical systems, as, for example, the Lyapunov characteristic exponents, we investigate the chaotic dynamics of the plasma and show numerically its existence for a wide range of parameter values. Finally, we illustrate how chaotic dynamics organizes in the parameter space for fixed values of the initial conditions, as the Mach number and the quantum diffraction parameter are continuously varied.

Improved strategy for isoleucine 1H/13C methyl labeling in Pichia pastoris.

Site specific methyl labeling combined with methyl TROSY offers a powerful NMR approach to study structure and dynamics of proteins and protein complexes of high molecular weight. Robust and cost-effective methods have been developed for site specific protein 1H/13C methyl labeling in an otherwise deuterated background in bacteria. However, bacterial systems are not suitable for expression and isotope labeling of many eukaryotic and membrane proteins. The yeast Pichia pastoris (P. pastoris) is a commonly used host for expression of eukaryotic proteins, and site-specific methyl labeling of perdeuterated eukaryotic proteins has recently been achieved with this system. However, the practical utility of methyl labeling and deuteration in P. pastoris is limited by high costs. Here, we describe an improved method for 1H/13C-labeling of the δ-methyl group of isoleucine residues in a perdeuterated background, which reduces the cost by ≥ 50% without compromising the efficiency of isotope enrichment. We have successfully implemented this method to label actin and a G-protein coupled receptor. Our approach will facilitate studies of the structure and dynamics of eukaryotic proteins by NMR spectroscopy.

Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote Pichia pastoris.

(13)C Methyl TROSY NMR spectroscopy has emerged as a powerful method for studying the dynamics of large systems such as macromolecular assemblies and membrane proteins. Specific (13)C labeling of aliphatic methyl groups and perdeuteration has been limited primarily to proteins expressed in E. coli, preventing studies of many eukaryotic proteins of physiological and biomedical significance. We demonstrate the feasibility of efficient (13)C isoleucine δ1-methyl labeling in a deuterated background in an established eukaryotic expression host, Pichia pastoris, and show that this method can be used to label the eukaryotic protein actin, which cannot be expressed in bacteria. This approach will enable NMR studies of previously intractable targets.

Temperature-induced disruptive growth rate behavior due to streaming instability in semiconductor quantum plasma with nanoparticles.

The nature of the growth rate due to streaming instability in a semiconductor quantum plasma implanted with nanoparticles has been analyzed using the quantum hydrodynamic model. In this study, the intriguing effect of temperature, beam electron speed, and electron-hole density on growth rate and frequency is investigated. The results show that the growth rate demonstrates a nonlinear behavior, strongly linked to the boron implantation, beam electron streaming speed and quantum correction factor. A noteworthy finding in this work is the discontinuous nature of the growth rate of streaming instability in boron implanted semiconducting plasma system. The implantation leads to a gap in the growth rate which further gets enhanced upon increase in concentration of implantation. This behavior is apparent only for a specific range of the ratio of thermal speed of the electrons to that of the holes.

Solution nuclear magnetic resonance structure of the GATase subunit and structural basis of the interaction between GATase and ATPPase subunits in a two-subunit-type GMPS from Methanocaldococcus jannaschii.

The solution structure of the monomeric glutamine amidotransferase (GATase) subunit of the Methanocaldococcus janaschii (Mj) guanosine monophosphate synthetase (GMPS) has been determined using high-resolution nuclear magnetic resonance methods. Gel filtration chromatography and ¹5N backbone relaxation studies have shown that the Mj GATase subunit is present in solution as a 21 kDa (188-residue) monomer. The ensemble of 20 lowest-energy structures showed root-mean-square deviations of 0.35 ± 0.06 Å for backbone atoms and 0.8 ± 0.06 Å for all heavy atoms. Furthermore, 99.4% of the backbone dihedral angles are present in the allowed region of the Ramachandran map, indicating the stereochemical quality of the structure. The core of the tertiary structure of the GATase is composed of a seven-stranded mixed ß-sheet that is fenced by five α-helices. The Mj GATase is similar in structure to the Pyrococcus horikoshi (Ph) GATase subunit. Nuclear magnetic resonance (NMR) chemical shift perturbations and changes in line width were monitored to identify residues on GATase that were responsible for interaction with magnesium and the ATPPase subunit, respectively. These interaction studies showed that a common surface exists for the metal ion binding as well as for the protein-protein interaction. The dissociation constant for the GATase-Mg(2+) interaction has been found to be ∼1 mM, which implies that interaction is very weak and falls in the fast chemical exchange regime. The GATase-ATPPase interaction, on the other hand, falls in the intermediate chemical exchange regime on the NMR time scale. The implication of this interaction in terms of the regulation of the GATase activity of holo GMPS is discussed.

Bound nucleotide can control the dynamic architecture of monomeric actin.

Polymerization of actin into cytoskeletal filaments is coupled to its bound adenine nucleotides. The mechanism by which nucleotide modulates actin functions has not been evident from analyses of ATP- and ADP-bound crystal structures of the actin monomer. We report that NMR chemical shift differences between the two forms are globally distributed. Furthermore, microsecond-millisecond motions are spread throughout the molecule in the ATP form, but largely confined to subdomains 1 and 2, and the nucleotide binding site in the ADP form. Through these motions, the ATP- and ADP-bound forms sample different high-energy conformations. A deafness-causing, fast-nucleating actin mutant populates the high-energy conformer of ATP-actin more than the wild-type protein, suggesting that this conformer may be on the pathway to nucleation. Together, the data suggest a model in which differential sampling of a nucleation-compatible form of the actin monomer may contribute to control of actin filament dynamics by nucleotide.

1H, 13C, 15N assignment and secondary structure determination of glutamine amido transferase subunit of gaunosine monophosphate synthetase from Methanocaldococcus jannaschii.

Sequence specific resonance assignments have been obtained for (1)H, (13)C and (15)N nuclei of the 21 kDa (188 residues long) glutamine amido transferase subunit of guanosine monophosphate synthetase from Methanocaldococcus jannaschii. From an analysis of (1)H and (13)C(α), (13)C(ß) secondary chemical shifts, (3) JH(N)H(α) scalar coupling constants and sequential, short and medium range (1)H-(1)H NOEs, it was deduced that the glutamine amido transferase subunit has eleven strands and five helices as the major secondary structural elements in its tertiary structure.