How Prion-like domains Drive Liquid-Liquid Phase Transitions in Cells

Single chain of the model (top), microscopy of liquid-liquid pase separation (middle), and images from phase separating simulations (bottom).

Liquid-liquid phase separation (LLPS) provides a way for cells to create membraneless micro-environments (“condensates”) that have been proposed to be involved in diverse cellular processes including stress responses, RNA splicing, mitosis, chromatin organization, and the clustering of receptors at membranes. Proteins driving LLPS often contain intrinsically disordered prion like domains (PLD’s) that appear to be necessary and sufficient to produce LLPS. PLDS’s have a high proportion of polar amino acids mixed in with aromatic residues. In a recent paper in the journal Science, researchers from St. Jude’s Children’s Research Hospital and Washington University at St. Louis used a combination of NMR, multiscale simulations and Size Exclusion Chromatography SAXS experiments at the BioCAT Beamline 18ID to discover sequence features that determine the phase behavior of PLD’s. They used their experimental findings to develop a “sticker-and-spacers” model that can predict the phase behavior of PLDs on the basis of their sequence. Phase condensation is driven by noncovalent, intra- and intermolecular cross-links between stickers (largely aromatic residues and other hydrophobic motifs), whereas spacers either facilitate or inhibit the formation …

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Sarcomere Structure and Nemaline Myopathy

NEM6 mutations cause changes in thin filament structure.

Nemaline myopathy (NM) is one of the most common congenital non-dystrophic myopathies and is characterized by severe hypotonia, muscle weakness, feeding difficulties, respiratory failure, and the presence of nemaline bodies (rods) in skeletal muscle biopsies. One form of nemaline myopathy is caused by mutations in the KBTBD13 (NEM6) gene. In addition to weakness, NEM6 patients have slow muscle relaxation, compromising contractility and daily-life activities. The role of KBTBD13 in muscle is unknown, and the p athomechanism underlying NEM6 is undetermined. A combination of transcranial magnetic stimulation-induced muscle relaxation, muscle fiber- and sarcomere-contractility assays, super-resolution microscopy, and low angle X-ray diffraction on the BioCAT Beamline 18ID revealed that the impaired muscle relaxation kinetics in NEM6 patients are caused by structural changes in the thin filament, a sarcomeric microstructure. Using homology modeling, binding- and contractility assays with recombinant KBTBD13, novel Kbtbd13-knockout and Kbtbd13R408C-knockin mouse models and a transgenic zebrafish model the authors discovered that KBTBD13 binds to actin – a major constituent of the thin filament - and that mutations in KBTBD13 cause structural changes impairing muscle relaxation kinetics. The authors propose that this actin-based impaired relaxation is central to NEM6 pathology.

See: Josine M. de …

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Cold temperatures put myosin in a refractory state unable to bind to actin

Low temperatures shift myosin heads from an ordered relaxed state to a disordered state that cannot bind actin in response to stimulation.

For many years, contraction in skeletal muscle was assumed to be primarily regulated by the binding of calcium to troponin on the thin filaments allowing tropomyosin to move unblocking the binding sites for myosin heads on the thin filament. More recently, it has been recognized that an additional thick filament base regulatory system coexists with thin filament regulation. The myosin heads in relaxed muscle are in an ordered quasi-helical arrangement around the thick filament backbone where they are unable to bind to actin. Current models propose that strain developed in the thick filament backbone generated by a small number of disordered, constitutively active myosin heads, once the strain surpasses some threshold, releases myosin heads form the ordered inactive heads to become disordered active heads. At this point, it is commonly assumed that ordered heads are in the OFF state, unable to bind to actin, while disordered heads are in the ON state, able to bind to actin and generate force. In a recent paper in J. General Physiology, researchers from the University Florence used the BioCAT Beamline 18ID …

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Frustration and Folding of a TIM Barrel Protein

Multiple folding pathways discovered by simulations, validated against the time resolved SAXS and FRET data.

In their continuing endeavor to understand misfolding proteins as part of the etiology of a variety of diseases, the Matthews lab particularly focuses on the different factors that impede a protein’s path from the unfolded state to the global free energy minimum. The complexity of the folding trajectory understandably depends on the size of the protein mostly because of the formation of intermediates many of which often stall the formation of an optimal native conformation.

The triosphosphate isomerase (TIM) barrel family of proteins have very well characterized and conserved folding path despite large variations in sequence which make it an ideal group of proteins to obtain widely applicable insights into the folding process. Like other proteins studied before, S.solfataricus indole-3-glycerol phosphate synthase (SsIGPS) a TIM barrel protein goes through a burst phase followed by a relaxation phase and then eventually folds into the native conformation. Mutational and hydrogen exchange experiments have helped characterize the species found in the few millisecond time range of the folding process. In this study, Halloran et al., have used a combination of continuous flow FRET, SAXS and simulations …

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Structure of BS Ric8A, a regulator of G-protein Biology

A model of the Ric8a protein structure showing the best scoring flexible tail (right), and the validation of the model against the SAXS data (left).

G-protein signaling has been the dominant theme in the Artemyev lab and their recent work specifically addresses Ric8A (Resistance to inhibitors of cholinesterase 8A) structure and function. Ric8A is a well-known regulator of G-protein biology and belongs to a class of proteins different from the G protein-coupled receptors (GPCRs), which act via interactions with monomeric Gα subunits as opposed to heterotrimeric Gαβγ proteins. SAXS was used in combination with crystallography and biochemical studies to show that the flexible C-terminal tail is important for the overall stability of Ric8A and the function as a guanine nucleotide exchange factor (GEF). The crystal structure revealed that Ric8A belongs to a functionally diverse class of proteins with what is known as an armadillo-fold (ARM) characterized by two layers of alpha helices arranged in a right handed superhelix. Ric8A diverged from the class in terms of the number of ARM repeats (8 as opposed to 10) and is further followed by a flexible region spanning ~ 70 residues.

Differential scanning fluorimetry (DSF) was used to determine that a construct without the …

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Protein-Folding Mechanisms Elucidated Using Chaotic-Flow SAXS

The unfolded-state ensemble (USE) undergoes a continuous reduction in chain dimensions as a function of decreased denaturant concentration.

While there is emerging consensus in the protein folding community concerning the behavior of proteins under unfolding conditions, the occurrence of unfolded states under physiological (native) conditions and their propensity to aggregate are the basis of several human pathologies. Valuable insights into these transient species were obtained by taking advantage of the temporal resolution afforded by combining time-resolved fluorescence and continuous (in this case chaotic) flow SAXS (CF-SAXS) with all atom simulations and polymer theory. A group of researchers led by the Raleigh lab (Stony Brook University) used the 59 amino acid N-terminal domain of the ribosomal protein L9 (NTL9), which has a well-studied two state folding mechanism. By introducing FRET pairs several pairwise distance distributions were measured in the unfolded and native conditions in equilibrium and also the unfolded states in native conditions using a continuous flow mixer Interestingly chain contraction as indicated by fluorescence decay was observed well within the dead time of the mixer (~40 µs) showing that chain collapse happens considerably faster than the time-scale required for completion of the folding process (2.5 ms for NTL9 …

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Probing the Powering of Contractions in Heart Failure

Sample x-ray diffraction patterns of resting cardiac muscle.

Current treatments can slow progression of heart failure, but do not address the underlying issues, including specific problems that cause systolic heart failure. In this condition, the heart doesn’t contract vigorously enough in pushing blood into the body’s circulation. But findings at nanometer and millisecond scales, based upon experimental data collected at BioCAT may help improve design of therapies directed at motor proteins to rescue failing hearts.

The heart muscle contractions that pump blood are generated by interactions between actin and myosin. These motor proteins power movement at the molecular level by converting the molecule ATP into energy. Earlier research in the lab of Michael Regnier, University of Washington (UW) professor of bioengineering, had shown that dATP, a natural variant of ATP, can be used to promote stronger heart function.

However, there remains a pressing need for data to explain why dATP helps to increase contractile force in heart disease. A new study headed by Regnier, who is a researcher at the UW Medicine Institute for Stem Cell and Regenerative Medicine and director of the Center for Translational Muscle Research, offers new insights, with unprecedented precision, about the nature of …

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New Insights into Traumatic Brain Injury

X-ray diffraction patterns (a) and integrated data (intensity versus d-spacings) (b) of control and impact-loaded rat optic nerve samples.

Traumatic brain injury, or TBI, is often referred to as the “invisible injury” — while on the surface everything seems normal with brain structure, symptoms may present themselves in the behavior of the injured and cannot be explained. According to the Centers for Disease Control and Prevention, about 2.8 million TBI-related emergency department (ED) visits, hospitalizations and deaths occurred in the United States in 2013 alone. Every day, 153 people in the United States die from injuries that include TBI.

As reported in the Journal of Synchrotron Radiation, investigators from Joseph Orgel at the Illinois Institute of Technology, Argonne National Laboratory and the Army Research Lab studied the effect of controlled amounts of compressive force on rat optic nerves to attempt to identify the changes that occur in otherwise normal looking brain neurons due to the specific impact forces experienced during head trauma. This first-of-its-kind study used x-ray diffraction data obtained at the Biophysics Collaborative Access Team (Bio-CAT) 18ID beamline at the APS to examine the changes to myelin, the fatty material that wraps around nerve cell projections in …

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A Target Mutation that Renders a Cancer Drug Ineffective

The wild-type enzyme SHP2 contains a protein tyrosine phosphatase catalytic domain (PTP domain) and two SH2 domains (N-SH2 and C-SH2). This version of SHP2 assumes a closed conformation in which the enzyme is autoinhibited. An allosteric inhibitor called SHP099 stabilizes SHP2. A specific mutation (E76K) to the gene, PTPN11, that encodes SHP2, results in a 120-degree pivot of the C-SH2 domain and relocalization of the N-SH2 domain. In this more open conformation of SHP2, the binding pocket for SHP099 is eliminated, thereby making SHP2 100 times more resistant to the allosteric inhibitor.

Mutations in the gene PTPN11, which encodes a common enzyme called SHP2, can result in developmental disorders, such as Noonan Syndrome, and act as oncogenic drivers in patients with certain blood cancers. Due to the well understood role of the enzyme SHP2 in Noonan Syndrome and in tumorigenesis, many companies are currently trying to develop drugs that inhibit the enzyme. However, it is unclear what impact mutations to SHP2 may have on the potential efficacy of drugs targeting this enzyme. Using the U.S. Department of Energy’s Advanced Photon Source (APS), researchers investigated the effect that the most frequently observed mutation of PTPN11 …

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Unraveling the role of a “nebulous” protein

Force in muscle is generated by interactions between myosin motor proteins arranged in thick filaments and their binding sites on thin filaments made mostly of the protein actin. Nebulin is a giant actin-binding protein in skeletal muscle that is found along the length of the thin filaments but up to now, not much was known about what it does to affect muscle function. Mutations in the nebulin protein cause the muscles in patients with nemaline myopathy disease to be weak in patients, suggesting that properly functioning nebulin is important to generate force. This condition can be recapitulated in genetically engineered mice who have mutated nebulin or entirely lack nebulin so that the muscles in these animals also show weakness. Investigators from the University of Arizona and the Illinois Institute of Technology used the BioCAT beamline 18ID to study mice entirely lacking nebulin to discover the effects of nebulin on the nanoscale structure of muscle.

Using X-ray diffraction, they found that the thin filaments were found to be 3-fold less stiff in nebulin-deficient muscles and that the action of other proteins that normally function to turn on and off the thick filament were impaired. As a consequence, fewer myosin motors are …

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