The location of the carboxy-terminal region of γ chains in fibrinogen and fibrin D domains

Elongated fibrinogen molecules are comprised of two outer “D” domains, each connected through a “coiled-coil” region to the central “E” domain. Fibrin forms following thrombin cleavage in the E domain and then undergoes intermolecular end-to-middle D:E domain associations that result in double-stranded fibrils. Factor XIIIa mediates crosslinking of the C-terminal regions of γ chains in each D domain (the γ_XL site) by incorporating intermolecular -(γ-glutamyl)lysine bonds between amine donor γ406 lysine of one γ chain and a glutamine acceptor at γ398 or γ399 of another. Several lines of evidence show that crosslinked γ chains extend “transversely” between the strands of each fibril, but other data suggest instead that crosslinked γ chains can only traverse end-to-end-aligned D domains within each strand. To examine this issue and determine the location of the γ_XL site in fibrinogen and assembled fibrin fibrils, we incorporated an amine donor, thioacetyl cadaverine, into glutamine acceptor sites in fibrinogen in the presence of XIIIa, and then labeled the thiol with a relatively small (0.8 nm diameter) electron dense gold cluster compound, undecagold monoaminopropyl maleimide (Au₁₁). Fibrinogen was examined by scanning transmission electron microscopy to locate Au₁₁-cadaverine-labeled γ398/399 D domain sites. Seventy-nine percent of D domain Au₁₁ clusters were situated in middle to proximal positions relative to the end of the molecule, with the remaining Au₁₁ clusters in a distal position. In fibrin fibrils, D domain Au₁₁ clusters were located in middle to proximal positions. These findings show that most C-terminal γ chains in fibrinogen or fibrin are oriented toward the central domain and indicate that γ_XL sites in fibrils are situated predominantly between strands, suitably aligned for transverse crosslinking.     

Lateral diffusion of phospholipids in the plasma membrane of soybean protoplasts: Evidence for membrane lipid domains

Fluorescent lipid and phospholipid probes were incorporated at 4°C into soybean protoplasts prepared from cultured soybean (SB-1) cells. Fluorescence microscopy showed that the plasma membrane as well as the nucleus were labeled. Fluorescence redistribution after photobleaching (FRAP) analysis was performed on these cells at 18°C to monitor the lateral mobility of the incorporated probes. After labeling at low concentrations (40 μg/ml) of phosphatidyl-N-(4-nitrobenzo-2-oxa-1,3-diazolyl)ethanolamine (NBD-PtdEtn), a single mobile component was observed with a diffusion coefficient (D) of ≈3 × 10^-9 cm²/sec. After labeling at higher probe concentrations (≥100 μg/ml), two diffusing species were observed, with diffusion coefficients of ≈3 × 10^-9 cm²/sec (“fast”) and ≈5 × 10^-10 cm²/sec (“slow”). Similar results were observed with fluorescent derivatives of phosphatidylcholine and fatty acids. In contrast to these results, parallel analysis of 3T3 fibroblasts, using the same probes and conditions, yielded only a single diffusion component. These results suggest that the soybean plasma membrane may contain two distinct lipid domains in terms of lipid mobility. Consistent with this idea, experiments with soybean protoplasts yielded a single diffusion component under the following conditions: (i) labeling with NBD-PtdEtn (100 μg/ml), FRAP analysis at 37°C (D = 1.1 × 10^-8 cm²/sec); (ii) labeling with NBD-PtdEtn (100 μg/ml), FRAP analysis at 18°C in the presence of 2 mM EGTA (D = 4.2 × 10^-9 cm²/sec); (iii) labeling with 5-(N-dodecanoyl)aminofluorescein (a short-chain lipid probe), FRAP analysis at 18°C or 37°C (D = 2.5 × 10^-8 cm²/sec). These results suggest that the plasma membrane of soybean cells may contain stable immiscible domains of fluid and gel-like lipids.     

The complementary aggregation sites of fibrin investigated through examination of polymers of fibrinogen with fragment E

Fibrin polymerizes through the interaction of sites exposed by the thrombin-mediated cleavage of fibrinopeptides in the central E region of the protein and complementary sites near the ends of the molecules, open in the D regions of both fibrinogen and fibrin. A preparation of fragment E, containing the central domain and part of the coiled-coil regions of fibrin, was used in mixtures with fibrinogen in this electron microscopy study to investigate the formation of fibrillar structures. At short times, linearly ordered oligomers of fibrinogen were observed with an additional mass of E fragments at the end-to-end junctions. At later times, long flexible polymers made up of 30 or more fibrinogen and fragment E units, with a tendency for lateral aggregation and tangle formation, were seen. These single-stranded assemblies could be readily dissociated in dilute acetic acid into their fibrinogen and fragment E components. However, if the aggregates were treated with factor XIIIa so that all γ chains became ligated by N(γ-glutamyl)lysine linkages, the polymers could no longer be taken apart. Because the only γ chains in the preparation are present in the fibrinogen molecules interacting end-to-end, the findings show that the factor XIIIa-induced cross-linking of γ chains in the clotting of fibrinogen or fibrin must occur between molecules that are longitudinal (or end-to-end) rather than transverse (or half-staggered).     

Coated platelets function in platelet-dependent fibrin formation via integrin αIIbβ3 and transglutaminase factor XIII

Coated platelets, formed by collagen and thrombin activation, have been characterized in different ways: i) by the formation of a protein coat of α-granular proteins; ii) by exposure of procoagulant phosphatidylserine; or iii) by high fibrinogen binding. Yet, their functional role has remained unclear. Here we used a novel transglutaminase probe, Rhod-A14, to identify a subpopulation of platelets with a cross-linked protein coat, and compared this with other platelet subpopulations using a panel of functional assays. Platelet stimulation with convulxin/thrombin resulted in initial integrin α_IIbβ₃ activation, the appearance of a platelet population with high fibrinogen binding, (independently of active integrins, but dependent on the presence of thrombin) followed by phosphatidylserine exposure and binding of coagulation factors Va and Xa. A subpopulation of phosphatidylserine-exposing platelets bound Rhod-A14 both in suspension and in thrombi generated on a collagen surface. In suspension, high fibrinogen and Rhod-A14 binding were antagonized by combined inhibition of transglutaminase activity and integrin α_IIbβ₃. Markedly, in thrombi from mice deficient in transglutaminase factor XIII, platelet-driven fibrin formation and Rhod-A14 binding were abolished by blockage of integrin α_IIbβ₃. Vice versa, star-like fibrin formation from platelets of a patient with deficiency in α_IIbβ₃ (Glanzmann thrombasthenia) was abolished upon blockage of transglutaminase activity. We conclude that coated platelets, with initial α_IIbβ₃ activation and high fibrinogen binding, form a subpopulation of phosphatidylserine-exposing platelets, and function in platelet-dependent star-like fibrin fiber formation via transglutaminase factor XIII and integrin α_IIbβ₃.     

Supramolecular structure of stacked and unstacked regions of the photosynthetic membranes of Prochloron sp., a prokaryote

Freeze-fracture replicas of the photosynthetic prokaryote Prochloron sp., collected at Coconut Island, Hawaii, show that the thylakoids are differentiated into stacked and unstacked regions much like the thylakoids of green algae and higher plants. On the exoplasmic (E) fracture face, the particle density is greater in stacked regions (≈3100 particles/μm²) than in unstacked regions (≈925 particles/μm²). On the complementary protoplasmic (P) fracture face, the particle density is lower in stacked regions (≈2265 particles/μm² than in unstacked regions (≈4980 particles/μm²). The histogram of the diameters of E face particles in unstacked regions shows a single broad peak centered at 80 Å. In stacked regions, four distinct peaks, at 75, 105, 130, and 160 Å, are observed. These size classes are virtually identical to those found on E faces of thylakoids of the green alga Chlamydomonas and of greening pea chloroplasts. In the latter systems, the different size classes of E face particles are believed to represent photosystem II units surrounded by variable amounts of chlorophyll a/b light-harvesting complex. We propose that the same interpretation applies to the thylakoids of Prochloron, which contain a similar chlorophyll a/b complex. Our results add to the evidence supporting the view of the chlorophyll a/b complex as both a light-harvesting complex and a membrane adhesion factor. The similarity of the architecture of the thylakoids of Prochloron to that of green algal and plant chloroplasts also provides additional evidence of an evolutionary relationship between Prochloron and the chloroplasts of green plants.     

Unliganded gating of acetylcholine receptor channels

We estimated the unliganded opening and closing rate constants of neuromuscular acetylcholine receptor-channels (AChRs) having mutations that increased the gating equilibrium constant. For some mutant combinations, spontaneous openings occurred in clusters. For 25 different constructs, the unliganded gating equilibrium constant (E₀) was correlated with the product of the predicted fold-increase in the diliganded gating equilibrium constant caused by each mutation alone. We estimate that (i) E₀ for mouse, wild-type α₂βδε AChRs is ≈1.15 × 10⁻⁷; (ii) unliganded AChRs open for ≈80 μs, once every ≈15 min; (iii) the affinity for ACh of the O(pen) conformation is ≈10 nM, or ≈15,600 times greater than for the C(losed) conformation; (iv) the ACh-monoliganded gating equilibrium constant is ≈1.7 × 10⁻³; (v) the C→O isomerization reduces substantially ACh dissociation, but only slightly increases association; and (vi) ACh provides only ≈0.9 k_BT more binding energy per site than carbamylcholine but ≈3.1 k_BT more than choline, mainly because of a low O conformation affinity. Most mutations of binding site residue αW149 increase E₀. We estimate that the mutation αW149F reduces the ACh affinity of C only by 13-fold, but of O by 190-fold. Rate–equilibrium free-energy relationships for different regions of the protein show similar slopes (Φ values) for un- vs. diliganded gating, which suggests that the conformational pathway of the gating structural change is fundamentally the same with and without agonists. Agonist binding is a perturbation that (like most mutations) changes the energy, but not the mechanism, of the gating conformational change.     

A FERM domain autoregulates Drosophila myosin 7a activity

Full-length Drosophila myosin 7a (myosin 7a-FL) has a complex tail containing a short predicted coiled coil followed by a MyTH4-FERM domain, an SH3 domain, and a C-terminal MyTH4-FERM domain. Myosin 7a-FL expressed in Sf9 cells is monomeric despite the predicted coiled coil. We showed previously that Subfragment-1 (S1) from this myosin has MgATPase of V_max ≈ 1s⁻¹ and K_ATPase ≈ 1 μM actin. We find that myosin 7a-FL has V_max similar to S1 but K_ATPase ≈ 30 μM. Thus, at low actin concentrations (5 μM), the MgATPase of S1 is fully activated, whereas that of myosin 7a-FL is low, suggesting that the tail regulates activity. Electron microscopy of myosin 7a-FL with ATP shows the tail is tightly bent back against the motor domain. Myosin 7a-FL extends at either high ionic strength or without ATP, revealing the motor domain, lever, and tail. A series of C-terminal truncations show that deletion of 99 aa (the MyTH7 subdomain of the C-terminal FERM domain) is sufficient to abolish bending, and the K_ATPase is then similar to S1. This region is highly conserved in myosin 7a. We found that a double mutation in it, R2140A-K2143A, abolishes bending and reduces K_ATPase to S1 levels. In addition, the expressed C-terminal FERM domain binds actin with K_d ≈ 30 μM regardless of ATP, similar to the K_ATPase value for myosin 7a-FL. We propose that at low cellular actin concentrations, myosin 7a-FL is bent and inactive, but at high actin concentrations, it is unfolded and active because the C-terminal FERM domain binds to actin.     

Isolation and bacterial expression of a sesquiterpene synthase cDNA clone from peppermint (Mentha x piperita, L.) that produces the aphid alarm pheromone (E)-β-farnesene

(E)-β-Farnesene is a sesquiterpene semiochemical that is used extensively by both plants and insects for communication. This acyclic olefin is found in the essential oil of peppermint (Mentha x piperita) and can be synthesized from farnesyl diphosphate by a cell-free extract of peppermint secretory gland cells. A cDNA from peppermint encoding (E)-β-farnesene synthase was cloned by random sequencing of an oil gland library and was expressed in Escherichia coli. The corresponding synthase has a deduced size of 63.8 kDa and requires a divalent cation for catalysis (K_m for Mg²⁺ ≈ 150 μM; K_m for Mn²⁺ ≈ 7 μM). The sesquiterpenoids produced by the recombinant enzyme, as determined by radio-GC and GC-MS, are (E)-β-farnesene (85%), (Z)-β-farnesene (8%), and δ-cadinene (5%) with the native C15 substrate farnesyl diphosphate (K_m ≈ 0.6 μM; V_rel = 100) and Mg²⁺ as cofactor, and (E)-β-farnesene (98%) and (Z)-β-farnesene (2%) with Mn²⁺ as cofactor (V_rel = 80). With the C₁₀ analog, GDP, as substrate (K_m = 1.5 μM; V_rel = 3 with Mg²⁺ as cofactor), the monoterpenes limonene (48%), terpinolene (15%), and myrcene (15%) are produced.     

Isolation of a multispecific organic anion and cardiac glycoside transporter from rat brain

A novel multispecific organic anion transporting polypeptide (oatp2) has been isolated from rat brain. The cloned cDNA contains 3,640 bp. The coding region extends over 1,983 nucleotides, thus encoding a polypeptide of 661 amino acids. Oatp2 is homologous to other members of the oatp gene family of membrane transporters with 12 predicted transmembrane domains, five potential glycosylation, and six potential protein kinase C phosphorylation sites. In functional expression studies in Xenopus laevis oocytes, oatp2 mediated uptake of the bile acids taurocholate (K_m ≈ 35 μM) and cholate (K_m ≈ 46 μM), the estrogen conjugates 17β-estradiol-glucuronide (K_m ≈ 3 μM) and estrone-3-sulfate (K_m ≈ 11 μM), and the cardiac gylcosides ouabain (K_m ≈ 470 μM) and digoxin (K_m ≈ 0.24 μM). Although most of the tested compounds are common substrates of several oatp-related transporters, high-affinity uptake of digoxin is a unique feature of the newly cloned oatp2. On the basis of Northern blot analysis under high-stringency conditions, oatp2 is highly expressed in brain, liver, and kidney but not in heart, spleen, lung, skeletal muscle, and testes. These results provide further support for the overall significance of oatps as a new family of multispecific organic anion transporters. They indicate that oatp2 may play an especially important role in the brain accumulation and toxicity of digoxin and in the hepatobiliary and renal excretion of cardiac glycosides from the body.     

Opioids regulate cGMP formation in cloned neuroblastoma cells

Opioid agonists caused a rapid dose-related elevation of the cGMP content of N4TG1 murine neuroblastoma cells. An excellent correlation was found between the rank order of potency of agonists in stimulating cGMP accumulation and in displacing [³H]etorphine ([³H]ETP) bound to intact cells. The narcotic antagonists naloxone and diprenorphine failed to increase cGMP content; moreover, in the presence of 5 μM naloxone, the EC₅₀ of ETP increased from ≈9 nM to > 1 μM. N4TG1 cells that had been incubated for 20 min with 0.32 μM ETP and thoroughly washed displayed a marked loss in sensitivity to subsequent ETP challenge. This desensitization was characterized by a 40-50% decrease in maximal response and an increase in the apparent K_a of ETP from 4 to 50 nM. Desensitization was complete after a 7-min incubation with 0.32 μM ETP (t_½ ≈ 1 min) and was only slowly reversible (t_½ > 60 min). Naloxone (5 μM) and diprenorphine (0.1 μM) failed to elicit desensitization, but they blocked ETP-induced desensitization. Dextrophan and (+)-ethylketazocine were <1% as effective as levorphanol and (-)-ethylketazocine, respectively, in both stimulating cGMP accumulation and inducing desensitization. When the binding of [³H]ETP (0.2-20 nM) was examined under identical experimental conditions, cells that were completely desensitized by incubation with ETP (7 min with 0.32 μM or 20 min with 15 nM) showed no loss of high-affinity recognition sites. After longer incubation with ETP (0.32 μM for 20-60 min), the maximal binding of [³H]ETP was reduced 17-41%. The specific short-term desensitization of cGMP accumulation is not mediated or accompanied by a decrement in the number of agonist binding sites.     

Direct observation of ultrafast folding and denatured state dynamics in single protein molecules

Single-molecule fluorescence resonance energy transfer (smFRET) experiments are extremely useful in studying protein folding but are generally limited to time scales of greater than ≈100 μs and distances greater than ≈2 nm. We used single-molecule fluorescence quenching by photoinduced electron transfer, detecting short-range events, in combination with fluorescence correlation spectroscopy (PET-FCS) to investigate folding dynamics of the small binding domain BBL with nanosecond time resolution. The kinetics of folding appeared as a 10-μs decay in the autocorrelation function, resulting from stochastic fluctuations between denatured and native conformations of individual molecules. The observed rate constants were probe independent and in excellent agreement with values derived from conventional temperature-jump (T-jump) measurements. A submicrosecond relaxation was detected in PET-FCS data that reported on the kinetics of intrachain contact formation within the thermally denatured state. We engineered a mutant of BBL that was denatured under the reaction conditions that favored folding of the parent wild type (“D_phys”). D_phys had the same kinetic signature as the thermally denatured state and revealed segmental diffusion with a time constant of intrachain contact formation of 500 ns. This time constant was more than 10 times faster than folding and in the range estimated to be the “speed limit” of folding. D_phys exhibited significant deviations from a random coil. The solvent viscosity and temperature dependence of intrachain diffusion showed that chain motions were slaved by the presence of intramolecular interactions. PET-FCS in combination with protein engineering is a powerful approach to study the early events and mechanism of ultrafast protein folding.     

Effects of extracellular Ca2+ concentration on hair-bundle stiffness and gating-spring integrity in hair cells

When a hair cell is stimulated by positive deflection of its hair bundle, increased tension in gating springs opens transduction channels, permitting cations to enter stereocilia and depolarize the cell. Ca²⁺ is thought to be required in mechanoelectrical transduction, for exposure of hair bundles to Ca²⁺ chelators eliminates responsiveness by disrupting tip links, filamentous interstereociliary connections that probably are the gating springs. Ca²⁺ also participates in adaptation to stimuli by controlling the activity of a molecular motor that sets gating-spring tension. Using a flexible glass fiber to measure hair-bundle stiffness, we investigated the effect of Ca²⁺ concentration on stiffness before and after the disruption of gating springs. The stiffness of intact hair bundles depended nonmonotonically on the extracellular Ca²⁺ concentration; the maximal stiffness of ≈1200 μNm⁻¹ occurred when bundles were bathed in solutions containing 250 μM Ca²⁺, approximately the concentration found in frog endolymph. For cells exposed to solutions with sufficient chelator capacity to reduce the Ca²⁺ concentration below ≈100 nM, hair-bundle stiffness fell to ≈200 μNm⁻¹ and no longer exhibited Ca²⁺-dependent changes. Because cells so treated lost mechanoelectrical transduction, we attribute the reduction in bundle stiffness to tip-link disruption. The results indicate that gating springs are not linearly elastic; instead, they stiffen with increased strain, which rises with adaptation-motor activity at the physiological extracellular Ca²⁺ concentration.     

INTRAMOLECULAR LOCALIZATION OF THE ACCEPTOR CROSS-LINKING SITES IN FIBRIN

Using 1-¹⁴C-glycine ethyl ester to titrate and label the acceptor cross-linking sites in fibrin, it was possible to localize and characterize the reactivity of these sites. In terms of sulfitolyzed chain fragments, both α and γ chains were shown to act as amine-acceptors, the site in γ being more reactive. Identification and isolation of the acceptor loci were also achieved after cyanogen bromide fragmentation. It is interesting that the “N-terminal disulfide knot” portion of fibrin does not seem to contain acceptor functions.     

Strong reversible Fe3+-mediated bridging between dopa-containing protein films in water

Metal-containing polymer networks are widespread in biology, particularly for load-bearing exoskeletal biomaterials. Mytilus byssal cuticle is an especially interesting case containing moderate levels of Fe³⁺ and cuticle protein—mussel foot protein-1 (mfp-1), which has a peculiar combination of high hardness and high extensibility. Mfp-1, containing 13 mol % of dopa (3, 4-dihydroxyphenylalanine) side-chains, is highly positively charged polyelectrolyte (pI ∼ 10) and didn’t show any cohesive tendencies in previous surface forces apparatus (SFA) studies. Here, we show that Fe³⁺ ions can mediate unusually strong interactions between the positively charged proteins. Using an SFA, Fe³⁺ was observed to impart robust bridging (W_ad ≈ 4.3 mJ/m²) between two noninteracting mfp-1 films in aqueous buffer approaching the ionic strength of seawater. The Fe³⁺ bridging between the mfp-1-coated surfaces is fully reversible in water, increasing with contact time and iron concentration up to 10 μM; at 100 μM, Fe³⁺ bridging adhesion is abolished. Bridging is apparently due to the formation of multivalent dopa-iron complexes. Similar Fe-mediated bridging (W_ad ≈ 5.7 mJ/m²) by a smaller recombinant dopa-containing analogue indicates that bridging is largely independent of molecular weight and posttranslational modifications other than dopa. The results suggest that dopa-metal interactions may provide an energetic new paradigm for engineering strong, self-healing interactions between polymers under water.     

The primary fibrin polymerization pocket: Three-dimensional structure of a 30-kDa C-terminal γ chain fragment complexed with the peptide Gly-Pro-Arg-Pro

After vascular injury, a cascade of serine protease activations leads to the conversion of the soluble fibrinogen molecule into fibrin. The fibrin monomers then polymerize spontaneously and noncovalently to form a fibrin gel. The primary interaction of this polymerization reaction is between the newly exposed N-terminal Gly-Pro-Arg sequence of the α chain of one fibrin molecule and the C-terminal region of a γ chain of an adjacent fibrin(ogen) molecule. In this report, the polymerization pocket has been identified by determining the crystal structure of a 30-kDa C-terminal fragment of the fibrin(ogen) γ chain complexed with the peptide Gly-Pro-Arg-Pro. This peptide mimics the N terminus of the α chain of fibrin. The conformational change in the protein upon binding the peptide is subtle, with electrostatic interactions primarily mediating the association. This is consistent with biophysical experiments carried out over the last 50 years on this fundamental polymerization reaction.     

GABA(A)-current rundown of temporal lobe epilepsy is associated with repetitive activation of GABA(A) "phasic" receptors

A study was made of the “rundown” of GABA_A receptors, microtransplanted to Xenopus oocytes from surgically resected brain tissues of patients afflicted with drug-resistant human mesial temporal lobe epilepsy (mTLE). Cell membranes, isolated from mTLE neocortex specimens, were injected into frog oocytes that rapidly incorporated functional GABA_A receptors. Upon repetitive activation with GABA (1 mM), “epileptic” GABA_A receptors exhibited a GABA_A-current (I_GABA) rundown that was significantly enhanced by Zn²⁺ (≤250 μM), and practically abolished by the high-affinity GABA_A receptor inverse agonist SR95531 (gabazine; 2.5–25 μM). Conversely, I_GABA generated by “control” GABA_A receptors microtransplanted from nonepileptic temporal lobe, lesional TLE, or authoptic disease-free tissues remained stable during repetitive stimulation, even in oocytes treated with Zn²⁺. We conclude that rundown of mTLE epileptic receptors depends on the presence of “phasic GABA_A receptors” that have low sensitivity to antagonism by Zn²⁺. Additionally, we found that GABA_A receptors, microtransplanted from the cerebral cortex of adult rats exhibiting recurrent seizures, caused by pilocarpine-induced status epilepticus, showed greater rundown than control tissue, an event also occurring in patch-clamped rat pyramidal neurons. Rundown of epileptic rat receptors resembled that of human mTLE receptors, being enhanced by Zn²⁺ (40 μM) and sensitive to the antiepileptic agent levetiracetam, the neurotrophin brain-derived neurotrophic factor, and the phosphatase blocker okadaic acid. Our findings point to the rundown of GABA_A receptors as a hallmark of TLE and suggest that modulating tonic and phasic mTLE GABA_A receptor activity may represent a useful therapeutic approach to the disease.     

Metastability transfer spectroscopy with two like ions in the same trap

High-resolution optical spectroscopy of an individual trapped ion is hampered by lack of sharp lasers. This suggests the use of a second metastable excited ion as an ultrasharp light source. To this end, laser-cool two barium ions to an equilibrium distance of ≈8 μm on the z (symmetry) axis of the trap and, in this (earth)(Ba⁺)₂-molecule, visually or photoelectrically identify them as A and B by their location. Briefly turn on a 455-nm spectral lamp until one of the ions, say the A ion, is pumped into the metastable D_5/2 level and turns invisible. Focus on the visible, spatially well-resolved B ion and turn off the blue and red illumination lasers for ≈15 s. Then turn them back on again and check on whether the excitation by chance has been transferred to the B ion and is now in the D_5/2 level and dark while the A ion is bright. The cross section for absorption of the λ(D_5/2 → S_1/2) λ₀ = 1.76 μm radiation by a stationary ion can be >λ₀²/2π. Thus, by pushing the two ions together to ≈λ₀/4 by turning on a much stronger trapping field during the excitation exchange period, one might be able to detect excitation transfer in >10% of the attempts. The ions are tuned relative to each other by a 0- to 10-mV/cm variable dc field in the z direction, which displaces them axially and causes them to see different rf fields, which Stark-shifts their frequencies. In this way, a resonant transfer response as sharp as twice the natural width of the D_5/2 level, 11 mHz or a Q ≈ 0.4 × 10¹⁷, might be demonstrated.     

Targeted rescue of a destabilized mutant of p53 by an in silico screened drug

The tumor suppressor p53 is mutationally inactivated in ≈50% of human cancers. Approximately one-third of the mutations lower the melting temperature of the protein, leading to its rapid denaturation. Small molecules that bind to those mutants and stabilize them could be effective anticancer drugs. The mutation Y220C, which occurs in ≈75,000 new cancer cases per annum, creates a surface cavity that destabilizes the protein by 4 kcal/mol, at a site that is not functional. We have designed a series of binding molecules from an in silico analysis of the crystal structure using virtual screening and rational drug design. One of them, a carbazole derivative (PhiKan083), binds to the cavity with a dissociation constant of ≈150 μM. It raises the melting temperature of the mutant and slows down its rate of denaturation. We have solved the crystal structure of the protein–PhiKan083 complex at 1.5-Å resolution. The structure implicates key interactions between the protein and ligand and conformational changes that occur on binding, which will provide a basis for lead optimization. The Y220C mutant is an excellent “druggable” target for developing and testing novel anticancer drugs based on protein stabilization. We point out some general principles in relationships between binding constants, raising of melting temperatures, and increase of protein half-lives by stabilizing ligands.     

Fatty acids suppress voltage-gated Na+ currents in HEK293t cells transfected with the α-subunit of the human cardiac Na+ channel

Studies have shown that fish oils, containing n-3 fatty acids, have protective effects against ischemia-induced, fatal cardiac arrhythmias in animals and perhaps in humans. In this study we used the whole-cell voltage-clamp technique to assess the effects of dietary, free long-chain fatty acids on the Na⁺ current (I_Na,α) in human embryonic kidney (HEK293t) cells transfected with the α-subunit of the human cardiac Na⁺ channel (hH1_α). Extracellular application of 0.01 to 30 μM eicosapentaenoic acid (EPA, C20:5n-3) significantly reduced I_Na,α with an IC₅₀ of 0.51 ± 0.06 μM. The EPA-induced suppression of I_Na,α was concentration- and voltage-dependent. EPA at 5 μM significantly shifted the steady-state inactivation relationship by −27.8 ± 1.2 mV (n = 6, P < 0.0001) at the V_1/2 point. In addition, EPA blocked I_Na,α with a higher “binding affinity” to hH1_α channels in the inactivated state than in the resting state. The transition from the resting state to the inactivated state was markedly accelerated in the presence of 5 μM EPA. The time for 50% recovery from the inactivation state was significantly slower in the presence of 5 μM EPA, from 2.1 ± 0.8 ms for control to 34.8 ± 2.1 ms (n = 5, P < 0.001). The effects of EPA on I_Na,α were reversible. Furthermore, docosahexaenoic acid (C22:6n-3), α-linolenic acid (C18:3n-3), conjugated linoleic acid (C18:2n-7), and oleic acid (C18:1n-9) at 5 μM and all-trans-retinoic acid at 10 μM had similar effects on I_Na,α as EPA. Even 5 μM of stearic acid (C18:0) or palmitic acid (C16:0) also significantly inhibited I_Na,α. In contrast, 5 μM EPA ethyl ester did not alter I_Na,α (8 ± 4%, n = 8, P > 0.05). The present data demonstrate that free fatty acids suppress I_Na,α with high “binding affinity” to hH1_α channels in the inactivated state and prolong the duration of recovery from inactivation.