Forcing Switch from Short- to Intermediate- and Long-lived States of the αA Domain Generates LFA-1/ICAM-1 Catch Bonds

Binding of lymphocyte function-associated antigen-1 (LFA-1) to intercellular adhesion molecule-1 (ICAM-1) mediates leukocyte adhesion under force. Using a biomembrane force probe capable of measuring single bond interactions, we showed ICAM-1 binding to LFA-1 at different conformations, including the bent conformation with the lowest affinity. We quantify how force and conformations of LFA-1 regulate its kinetics with ICAM-1. At zero-force, on-rates were substantially changed by conditions that differentially favor a bent or extended LFA-1 with a closed or open headpiece; but off-rates were identical. With increasing force, LFA-1/ICAM-1 bond lifetimes (reciprocal off-rates) first increased (catch bonds) and then decreased (slip bonds). Three states with distinct off-rates were identified from lifetime distributions. Force shifted the associated fractions from the short- to intermediate- and long-lived states, producing catch bonds at low forces, but increased their off-rates exponentially, converting catch to slip bonds at high forces. An internal ligand antagonist that blocks pulling of the α₇-helix suppressed the intermediate-/long-lived states and eliminated catch bonds, revealing an internal catch bond between the αA and βA domains. These results elucidate an allosteric mechanism for the mechanochemistry of LFA-1/ICAM-1 binding.     

Demonstration of catch bonds between an integrin and its ligand

Binding of integrins to ligands provides anchorage and signals for the cell, making them prime candidates for mechanosensing molecules. How force regulates integrin–ligand dissociation is unclear. We used atomic force microscopy to measure the force-dependent lifetimes of single bonds between a fibronectin fragment and an integrin α₅β₁-Fc fusion protein or membrane α₅β₁. Force prolonged bond lifetimes in the 10–30-pN range, a counterintuitive behavior called catch bonds. Changing cations from Ca²⁺/Mg²⁺ to Mg²⁺/EGTA and to Mn²⁺ caused longer lifetime in the same 10–30-pN catch bond region. A truncated α₅β₁ construct containing the headpiece but not the legs formed longer-lived catch bonds that were not affected by cation changes at forces <30 pN. Binding of monoclonal antibodies that induce the active conformation of the integrin headpiece shifted catch bonds to a lower force range. Thus, catch bond formation appears to involve force-assisted activation of the headpiece but not integrin extension.     

分子黏附中的逆锁键研究进展

逆锁键是受体和配体蛋白质相互作用过程中的一种"反常"现象,属于受体-配体动态键的一种,在pN量级外力的影响下,其键寿命在特定范围内会随着所施加外力的增加而增加.逆锁键主要参与细胞黏附、细胞活化等过程,并与滑移键组成分子开关来调节各项与分子黏附相关的生命活动.其中,致病性大肠杆菌引发尿路感染、T细胞受体抗原识别、微丝解聚、Notch信号通路激活等重要生命活动已被证实与逆锁键密切相关.近期已有发现证实TCR-pMHC复合物中的逆锁键与癌症的引发也存在密切联系,这一发现提供了改进TCR-T (T细胞受体基因工程改造的T细胞)免疫疗法的新方向.本文概述了逆锁键在几类重要生命活动中的最新研究进展,并讨论了其潜在的医学研究前景.     

The N-terminal Flanking Region of the A1 Domain Regulates the Force-dependent Binding of von Willebrand Factor to Platelet Glycoprotein Ibα

Binding of platelet glycoprotein Ibα (GPIbα) to von Willebrand factor (VWF) initiates platelet adhesion to disrupted vascular surface under arterial blood flow. Flow exerts forces on the platelet that are transmitted to VWF-GPIbα bonds, which regulate their dissociation. Mutations in VWF and/or GPIbα may alter the mechanical regulation of platelet adhesion to cause hemostatic defects as found in patients with von Willebrand disease (VWD). Using a biomembrane force probe, we observed biphasic force-decelerated (catch) and force-accelerated (slip) dissociation of GPIbα from VWF. The VWF A1 domain that contains the N-terminal flanking sequence Gln¹²³⁸–Glu¹²⁶⁰ (1238-A1) formed triphasic slip-catch-slip bonds with GPIbα. By comparison, using a short form of A1 that deletes this sequence (1261-A1) abolished the catch bond, destabilizing its binding to GPIbα at high forces. Importantly, shear-dependent platelet rolling velocities on these VWF ligands in a flow chamber system mirrored the force-dependent single-bond lifetimes. Adding the Gln¹²³⁸–Glu¹²⁶⁰ peptide, which interacted with GPIbα and 1261-A1 but not 1238-A1, to whole blood decreased platelet attachment under shear stress. Soluble Gln¹²³⁸–Glu¹²⁶⁰ reduced the lifetimes of GPIbα bonds with VWF and 1238-A1 but rescued the catch bond of GPIbα with 1261-A1. A type 2B VWD 1238-A1 mutation eliminated the catch bond by prolonging lifetimes at low forces, a type 2M VWD 1238-A1 mutation shifted the respective slip-catch and catch-slip transition points to higher forces, whereas a platelet type VWD GPIbα mutation enhanced the bond lifetime in the entire force regime. These data reveal the structural determinants of VWF activation by hemodynamic force of the circulation.     

A Force-Activated Trip Switch Triggers Rapid Dissociation of a Colicin from Its Immunity Protein

Colicins are protein antibiotics synthesised by Escherichia coli strains to target and kill related bacteria. To prevent host suicide, colicins are inactivated by binding to immunity proteins. Despite their high avidity (K_d≈fM, lifetime ≈4 days), immunity protein release is a pre-requisite of colicin intoxication, which occurs on a timescale of minutes. Here, by measuring the dynamic force spectrum of the dissociation of the DNase domain of colicin E9 (E9) and immunity protein 9 (Im9) complex using an atomic force microscope we show that application of low forces (<20 pN) increases the rate of complex dissociation 10⁶-fold, to a timescale (lifetime ≈10 ms) compatible with intoxication. We term this catastrophic force-triggered increase in off-rate a trip bond. Using mutational analysis, we elucidate the mechanism of this switch in affinity. We show that the N-terminal region of E9, which has sparse contacts with the hydrophobic core, is linked to an allosteric activator region in E9 (residues 21–30) whose remodelling triggers immunity protein release. Diversion of the force transduction pathway by the introduction of appropriately positioned disulfide bridges yields a force resistant complex with a lifetime identical to that measured by ensemble techniques. A trip switch within E9 is ideal for its function as it allows bipartite complex affinity, whereby the stable colicin:immunity protein complex required for host protection can be readily converted to a kinetically unstable complex whose dissociation is necessary for cellular invasion and competitor death. More generally, the observation of two force phenotypes for the E9:Im9 complex demonstrates that force can re-sculpt the underlying energy landscape, providing new opportunities to modulate biological reactions in vivo; this rationalises the commonly observed discrepancy between off-rates measured by dynamic force spectroscopy and ensemble methods.     

Single-molecule force spectroscopy of cartilage aggrecan self-adhesion

We investigated self-adhesion between highly negatively charged aggrecan macromolecules extracted from bovine cartilage extracellular matrix by performing atomic force microscopy (AFM) imaging and single-molecule force spectroscopy (SMFS) in saline solutions. By controlling the density of aggrecan molecules on both the gold substrate and the gold-coated tip surface at submonolayer densities, we were able to detect and quantify the Ca²⁺-dependent homodimeric interaction between individual aggrecan molecules at the single-molecule level. We found a typical nonlinear sawtooth profile in the AFM force-versus-distance curves with a molecular persistence length of l_p = 0.31 ± 0.04 nm. This is attributed to the stepwise dissociation of individual glycosaminoglycan (GAG) side chains in aggrecans, which is very similar to the known force fingerprints of other cell adhesion proteoglycan systems. After studying the GAG-GAG dissociation in a dynamic, loading-rate-dependent manner (dynamic SMFS) and analyzing the data according to the stochastic Bell-Evans model for a thermally activated decay of a metastable state under an external force, we estimated for the single glycan interaction a mean lifetime of τ = 7.9 ± 4.9 s and a reaction bond length of x_β = 0.31 ± 0.08 nm. Whereas the x_β-value compares well with values from other cell adhesion carbohydrate recognition motifs in evolutionary distant marine sponge proteoglycans, the rather short GAG interaction lifetime reflects high intermolecular dynamics within aggrecan complexes, which may be relevant for the viscoelastic properties of cartilage tissue.     

Pre-Synaptic Release Deficits in a DYT1 Dystonia Mouse Model

DYT1 early-onset generalized torsion dystonia (DYT1 dystonia) is an inherited movement disorder caused by mutations in one allele of DYT1 (TOR1A), coding for torsinA. The most common mutation is a trinucleotide deletion (ΔGAG), which causes a deletion of a glutamic acid residue (ΔE) in the C-terminal region of torsinA. Although recent studies using cultured cells suggest that torsinA contributes to protein processing in the secretory pathway, endocytosis, and the stability of synaptic proteins, the nature of how this mutation affects synaptic transmission remains unclear. We previously reported that theta-burst-induced long-term potentiation (LTP) in the CA1 region of the hippocampal slice is not altered in Dyt1 ΔGAG heterozygous knock-in (KI) mice. Here, we examined short-term synaptic plasticity and synaptic transmission in the hippocampal slices. Field recordings in the hippocampal Schaffer collaterals (SC) pathway revealed significantly enhanced paired pulse ratios (PPRs) in Dyt1 ΔGAG heterozygous KI mice, suggesting an impaired synaptic vesicle release. Whole-cell recordings from the CA1 neurons showed that Dyt1 ΔGAG heterozygous KI mice exhibited normal miniature excitatory post-synaptic currents (mEPSC), suggesting that action-potential independent spontaneous pre-synaptic release was normal. On the other hand, there was a significant decrease in the frequency, but not amplitude or kinetics, of spontaneous excitatory post-synaptic currents (sEPSC) in Dyt1 ΔGAG heterozygous KI mice, suggesting that the action-potential dependent pre-synaptic release was impaired. Moreover, hippocampal torsinA was significantly reduced in Dyt1 ΔGAG heterozygous KI mice. Although the hippocampal slice model may not represent the neurons directly associated with dystonic symptoms, impaired release of neurotransmitters caused by partial dysfunction of torsinA in other brain regions may contribute to the pathophysiology of DYT1 dystonia.     

Uncoiling mechanics of Escherichia coli type I fimbriae are optimized for catch bonds

We determined whether the molecular structures through which force is applied to receptor–ligand pairs are tuned to optimize cell adhesion under flow. The adhesive tethers of our model system, Escherichia coli, are type I fimbriae, which are anchored to the outer membrane of most E. coli strains. They consist of a fimbrial rod (0.3–1.5 μm in length) built from a helically coiled structural subunit, FimA, and an adhesive subunit, FimH, incorporated at the fimbrial tip. Previously reported data suggest that FimH binds to mannosylated ligands on the surfaces of host cells via catch bonds that are enhanced by the shear-originated tensile force. To understand whether the mechanical properties of the fimbrial rod regulate the stability of the FimH–mannose bond, we pulled the fimbriae via a mannosylated tip of an atomic force microscope. Individual fimbriae rapidly elongate for up to 10 μm at forces above 60 pN and rapidly contract again at forces below 25 pN. At intermediate forces, fimbriae change length more slowly, and discrete 5.0 ± 0.3–nm changes in length can be observed, consistent with uncoiling and coiling of the helical quaternary structure of one FimA subunit at a time. The force range at which fimbriae are relatively stable in length is the same as the optimal force range at which FimH–mannose bonds are longest lived. Higher or lower forces, which cause shorter bond lifetimes, cause rapid length changes in the fimbria that help maintain force at the optimal range for sustaining the FimH–mannose interaction. The modulation of force and the rate at which it is transmitted from the bacterial cell to the adhesive catch bond present a novel physiological role for the fimbrial rod in bacterial host cell adhesion. This suggests that the mechanical properties of the fimbrial shaft have codeveloped to optimize the stability of the terminal adhesive under flow.     

Random Coil to Globular Thermal Response of a Protein (H3.1) with Three Knowledge-Based Coarse-Grained Potentials

The effect of temperature on the conformation of a histone (H3.1) is studied by a coarse-grained Monte Carlo simulation based on three knowledge-based contact potentials (MJ, BT, BFKV). Despite unique energy and mobility profiles of its residues, the histone H3.1 undergoes a systematic (possibly continuous) structural transition from a random coil to a globular conformation on reducing the temperature. The range over which such a systematic response in variation of the radius of gyration (R_g) with the temperature (T) occurs, however, depends on the potential, i.e. ΔT_MJ ≈ 0.013–0.020, ΔT_BT ≈ 0.018–0.026, and ΔT_BFKV ≈ 0.006–0.013 (in reduced unit). Unlike MJ and BT potentials, results from the BFKV potential show an anomaly where the magnitude of R_g decreases on raising the temperature in a range ΔT_A ≈ 0.015–0.018 before reaching its steady-state random coil configuration. Scaling of the structure factor, S(q) ∝ q^−1/ν, with the wave vector, q = 2π/λ, and the wavelength, λ, reveals a systematic change in the effective dimension (D_e∼1/ν) of the histone with all potentials (MJ, BT, BFKV): D_e∼3 in the globular structure with D_e∼2 for the random coil. Reproducibility of the general yet unique (monotonic) structural transition of the protein H3.1 with the temperature (in contrast to non-monotonic structural response of a similar but different protein H2AX) with three interaction sets shows that the knowledge-based contact potential is viable tool to investigate structural response of proteins. Caution should be exercise with the quantitative comparisons due to differences in transition regimes with these interactions.     

Binding of Imidazole to the Heme of Cytochrome c1 and Inhibition of the bc1 Complex from Rhodobacter sphaeroides: I. EQUILIBRIUM AND MODELING STUDIES*

We have used imidazole (Im) and N-methylimidazole (MeIm) as probes of the heme-binding cavity of membrane-bound cytochrome (cyt) c₁ in detergent-solubilized bc₁ complex from Rhodobacter sphaeroides. Imidazole binding to cyt c₁ substantially lowers the midpoint potential of the heme and fully inhibits bc₁ complex activity. Temperature dependences showed that binding of Im (K_d ≈ 330 μm, 25 °C, pH 8) is enthalpically driven (ΔH⁰ = −56 kJ/mol, ΔS⁰ = −121 J/mol/K), whereas binding of MeIm is 30 times weaker (K_d ≈ 9.3 mm) and is entropically driven (ΔH⁰ = 47 kJ/mol, ΔS⁰° = 197 J/mol/K). The large enthalpic and entropic contributions suggest significant structural and solvation changes in cyt c₁ triggered by ligand binding. Comparison of these results with those obtained previously for soluble cyts c and c₂ suggested that Im binding to cyt c₁ is assisted by formation of hydrogen bonds within the heme cleft. This was strongly supported by molecular dynamics simulations of Im adducts of cyts c, c₂, and c₁, which showed hydrogen bonds formed between the N_δH of Im and the cyt c₁ protein, or with a water molecule sequestered with the ligand in the heme cleft.     

The proton-driven rotor of ATP synthase: ohmic conductance (10 fS), and absence of voltage gating

The membrane portion of F₀F₁-ATP synthase, F₀, translocates protons by a rotary mechanism. Proton conduction by F₀ was studied in chromatophores of the photosynthetic bacterium Rhodobacter capsulatus. The discharge of a light-induced voltage jump was monitored by electrochromic absorption transients to yield the unitary conductance of F₀. The current-voltage relationship of F₀ was linear from 7 to 70 mV. The current was extremely proton-specific (>10⁷) and varied only slightly (≈threefold) from pH 6 to 10. The maximum conductance was ≈10 fS at pH 8, equivalent to 6240 H⁺ s⁻¹ at 100-mV driving force, which is an order-of-magnitude greater than of coupled F₀F₁. There was no voltage-gating of F₀ even at low voltage, and proton translocation could be driven by ΔpH alone, without voltage. The reported voltage gating in F₀F₁ is thus attributable to the interaction of F₀ with F₁ but not to F₀ proper. We simulated proton conduction by a minimal rotary model including the rotating c-ring and two relay groups mediating proton exchange between the ring and the respective membrane surface. The data fit attributed pK values of ≈6 and ≈10 to these relays, and placed them close to the membrane/electrolyte interface.     

Phe317 Is Essential for Rubber Oxygenase RoxA Activity

RoxA is an extracellular c-type diheme cytochrome secreted by Xanthomonas sp. strain 35Y during growth on rubber. RoxA cleaves poly(cis-1,4-isoprene) to 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (ODTD). Analysis of the RoxA structure revealed that Phe317 is located in close proximity (≈5 Å) to the N-terminal heme that presumably represents the active site. To find evidence of whether Phe317 is important for catalysis, we changed it to tyrosine, tryptophan, leucine, histidine, or alanine. All five RoxA muteins were expressed after integration of the respective gene into the chromosome of a Xanthomonas sp. ΔroxA strain. Residual clearing zone formation on opaque latex agar was found for Xanthomonas sp. strains expressing the Phe317Leu, Phe317Ala, or Phe317His variant (wild type > Leu > Ala > His). Strains in which Phe317 was changed to tyrosine or tryptophan were inactive. Phe317Ala and Phe312Leu RoxA muteins were purified, and polyisoprene cleavage activities were reduced to ≈3% and 10%, respectively. UV-visible spectroscopy of RoxA muteins confirmed that both heme groups were present in an oxidized form, but spectral responses to the addition of low-molecular-weight (inhibitory) ligand molecules such as imidazole and pyridine were different from those of wild-type RoxA. Our results show that residue 317 is involved in interaction with substrates. This is the first report on structure-function analysis of a polyisoprene-cleaving enzyme and on the identification of an amino acid that is essential for polyisoprene cleavage activity.     

Calcium Causes a Conformational Change in Lamin A Tail Domain that Promotes Farnesyl-Mediated Membrane Association

Lamin proteins contribute to nuclear structure and function, primarily at the inner nuclear membrane. The posttranslational processing pathway of lamin A includes farnesylation of the C-terminus, likely to increase membrane association, and subsequent proteolytic cleavage of the C-terminus. Hutchinson Gilford progeria syndrome is a premature aging disorder wherein a mutant version of lamin A, Δ50 lamin A, retains its farnesylation. We report here that membrane association of farnesylated Δ50 lamin A tail domains requires calcium. Experimental evidence and molecular dynamics simulations collectively suggest that the farnesyl group is sequestered within a hydrophobic region in the tail domain in the absence of calcium. Calcium binds to the tail domain with an affinity K_D ≈ 250 μM where it alters the structure of the Ig-fold and increases the solvent accessibility of the C-terminus. In 2 mM CaCl₂, the affinity of the farnesylated protein to a synthetic membrane is K_D ≈ 2 μM, as measured with surface plasmon resonance, but showed a combination of aggregation and binding. Membrane binding in the absence of calcium could not be detected. We suggest that a conformational change induced in Δ50 lamin A with divalent cations plays a regulatory role in the posttranslational processing of lamin A, which may be important in disease pathogenesis.     

Deletion of Vacuolar Proton-translocating ATPase Voa Isoforms Clarifies the Role of Vacuolar pH as a Determinant of Virulence-associated Traits in Candida albicans

Vacuolar proton-translocating ATPase (V-ATPase) is a central regulator of cellular pH homeostasis, and inactivation of all V-ATPase function has been shown to prevent infectivity in Candida albicans. V-ATPase subunit a of the V_o domain (V_oa) is present as two fungal isoforms: Stv1p (Golgi) and Vph1p (vacuole). To delineate the individual contribution of Stv1p and Vph1p to C. albicans physiology, we created stv1Δ/Δ and vph1Δ/Δ mutants and compared them to the corresponding reintegrant strains (stv1Δ/ΔR and vph1Δ/ΔR). V-ATPase activity, vacuolar physiology, and in vitro virulence-related phenotypes were unaffected in the stv1Δ/Δ mutant. The vph1Δ/Δ mutant exhibited defective V₁V_o assembly and a 90% reduction in concanamycin A-sensitive ATPase activity and proton transport in purified vacuolar membranes, suggesting that the Vph1p isoform is essential for vacuolar V-ATPase activity in C. albicans. The vph1Δ/Δ cells also had abnormal endocytosis and vacuolar morphology and an alkalinized vacuolar lumen (pH_vph1Δ_/Δ = 6.8 versus pH_vph1Δ_/Δ_R = 5.8) in both yeast cells and hyphae. Secreted protease and lipase activities were significantly reduced, and M199-induced filamentation was impaired in the vph1Δ/Δ mutant. However, the vph1Δ/Δ cells remained competent for filamentation induced by Spider media and YPD, 10% FCS, and biofilm formation and macrophage killing were unaffected in vitro. These studies suggest that different virulence mechanisms differentially rely on acidified vacuoles and that the loss of both vacuolar (Vph1p) and non-vacuolar (Stv1p) V-ATPase activity is necessary to affect in vitro virulence-related phenotypes. As a determinant of C. albicans pathogenesis, vacuolar pH alone may prove less critical than originally assumed.     

Unraveling the Nanoscale Surface Properties of Chitin Synthase Mutants of?Aspergillus fumigatus and Their Biological Implications

Understanding the surface properties of the human opportunistic pathogen Aspergillus fumigatus conidia is essential given the important role they play during the fungal interactions with the human host. Although chitin synthases with myosin motor-like domain (CSM) play a major role in cell wall biosynthesis, the extent to which deletion of the CSM genes alter the surface structural and biophysical-biological properties of conidia is not fully characterized. We used three complementary atomic force microscopy techniques—i.e., structural imaging, chemical force microscopy with hydrophobic tips, and single-molecule force spectroscopy with lectin tips—to gain detailed insights into the nanoscale surface properties (ultrastructure, hydrophobicity) and polysaccharide composition of the wild-type and the chitin synthase mutant (ΔcsmA, ΔcsmB, and ΔcsmA/csmB) conidia of A. fumigatus. Wild-type conidia were covered with a highly hydrophobic layer of rodlet nanostructures. By contrast, the surface of the ΔcsmA mutant was almost completely devoid of rodlets, leading to loss of hydrophobicity and exposure of mannan and chitin polysaccharides. The ΔcsmB and ΔcsmA/csmB mutants showed a different behavior, i.e., the surfaces featured poorly organized rodlet layers, yet with a low hydrophobicity and substantial amounts of exposed mannan and chitin at the surface. As the rodlet layer is important for masking recognition of immunogenic fungal cell wall components by innate immune cells, disappearance of rodlet layers in all three chitin synthase mutant conidia was associated with an activation of human dendritic cells. These nanoscale analyses emphasize the important and distinct roles that the CSMA and CSMB genes play in modulating the surface properties and immune interactions of A. fumigatus and demonstrate the power of atomic force microscopy in fungal genetic studies for assessing the phenotypic characteristics of mutants altered in cell surface organization.     

Behavioral and Electrophysiological Characterization of Dyt1 Heterozygous Knockout Mice

DYT1 dystonia is an inherited movement disorder caused by mutations in DYT1 (TOR1A), which codes for torsinA. Most of the patients have a trinucleotide deletion (ΔGAG) corresponding to a glutamic acid in the C-terminal region (torsinA^ΔE). Dyt1 ΔGAG heterozygous knock-in (KI) mice, which mimic ΔGAG mutation in the endogenous gene, exhibit motor deficits and deceased frequency of spontaneous excitatory post-synaptic currents (sEPSCs) and normal theta-burst-induced long-term potentiation (LTP) in the hippocampal CA1 region. Although Dyt1 KI mice show decreased hippocampal torsinA levels, it is not clear whether the decreased torsinA level itself affects the synaptic plasticity or torsinA^ΔE does it. To analyze the effect of partial torsinA loss on motor behaviors and synaptic transmission, Dyt1 heterozygous knock-out (KO) mice were examined as a model of a frame-shift DYT1 mutation in patients. Consistent with Dyt1 KI mice, Dyt1 heterozygous KO mice showed motor deficits in the beam-walking test. Dyt1 heterozygous KO mice showed decreased hippocampal torsinA levels lower than those in Dyt1 KI mice. Reduced sEPSCs and normal miniature excitatory post-synaptic currents (mEPSCs) were also observed in the acute hippocampal brain slices from Dyt1 heterozygous KO mice, suggesting that the partial loss of torsinA function in Dyt1 KI mice causes action potential-dependent neurotransmitter release deficits. On the other hand, Dyt1 heterozygous KO mice showed enhanced hippocampal LTP, normal input-output relations and paired pulse ratios in the extracellular field recordings. The results suggest that maintaining an appropriate torsinA level is important to sustain normal motor performance, synaptic transmission and plasticity. Developing therapeutics to restore a normal torsinA level may help to prevent and treat the symptoms in DYT1 dystonia.     

Entropy,Energy, and Bending of DNA in Viral Capsids

Inspired by novel single-molecule and bulk solution measurements, the physics underlying the forces and pressures involved in DNA packaging into bacteriophage capsids became the focus of numerous recent theoretical models. These fall into two general categories: Continuum-elastic theories (CT), and simulation studies—mostly of the molecular dynamics (MD) genre. Both types of models account for the dependence of the force, and hence the packaging free energy (ΔF), on the loaded DNA length, but differ markedly in interpreting their origin. While DNA confinement entropy is a dominant contribution to ΔF in the MD simulations, in the CT theories this role is fulfilled by interstrand repulsion, and there is no explicit entropy term. The goal of this letter is to resolve this apparent contradiction, elucidate the origin of the entropic term in the MD simulations, and point out its tacit presence in the CT treatments.The genomic double-stranded (ds) DNA inside bacteriophage heads is highly stressed, leading to internal pressures of up to ∼50 atmospheres, reflecting the tight packing and extreme bending of this highly charged and rigid molecule (1). The interaxial distance (d) between neighboring (nonbonded) dsDNA segments in the fully packaged virus is typically ≈2.5 nm (2,3), just slightly larger than the hardcore diameter of dsDNA (b = 2.0 nm) and well into the repulsive regime (d ≤ 2.8 nm) of DNA-DNA interaction in ionic solutions (4–6). Moreover, free dsDNA in (physiological) solution is a fluctuating, semiflexible, wormlike chain (WLC), with persistence length ξ ≈ 50 nm, larger than the radius of most viral capsids. Thus, on a molecular scale, packaging the long (e.g., the 330-ξ long λ-phage genome) viral DNA into its tiny capsid requires enormous mechanical work.The force needed to package the DNA is provided by an ATP-driven motor protein situated at the capsid portal. Recent single molecule measurements reveal that this force, f(L_int), increases sharply with the loaded genome length, L_int, rising to ∼30–100 pN, depending on the virus in question (7,8). These studies inspired the formulation of many theoretical models of DNA packaging in viral capsids, which fall roughly into two categories:     

Triphasic Force Dependence of E-Selectin/Ligand Dissociation Governs Cell Rolling under Flow

During inflammation, flowing leukocytes tether to and roll on vascular surfaces through the association and dissociation of selectin/ligand bonds. The interactions of P- and L- selectins with their respective ligands exhibit catch-slip bonds, such that increasing force initially prolongs and then shortens bond lifetimes. In addition, catch-slip bonds have been shown to govern L-selectin-mediated cell rolling. Using a flow chamber and biomembrane force probe, we show a triphasic force dependence of E-selectin/ligand dissociation that initially behaves as slip bonds, then transitions to catch bonds, and finally transitions again to slip bonds as the force increases. These transitions govern the velocities of neutrophils, HL-60 cells, and Colo-205 cells rolling on E-selectin, as evidenced by the fact that their velocities exhibited a triphasic force dependence that inversely matched the triphasic lifetime-force relationship. At low forces, slip bonds may also precede catch bonds for interactions of P- and L-selectin with their ligands.     

Proton motive force during growth of Streptococcus lactis cells

Experiments with the aerotolerant anaerobe Streptococcus lactis provide the opportunity for determining the proton motive force (Δp) in dividing cells. The two components of Δp, ΔΨ (the transmembrane potential) and ΔpH (the chemical gradient of H⁺), were determined by the accumulation of radiolabeled tetraphenylphosphonium (TPP⁺) and benzoate ions. The ΔΨ was calibrated with the K⁺ diffusion potential in starved, valinomycin-treated cells. With resting, glycolyzing cells, the Δp was measured also by the accumulation of the non-metabolizable sugar thiomethyl-β-galactoside (TMG). In resting cells the Δp, calculated either by adding ΔΨ and ZΔpH or from the levels of TMG, was relatively constant between pH 5 to 7, decreasing from 160 to 150 mV and decreasing further to 100 mV at pH 8.0. With the TPP⁺ probe for ΔΨ, we confirmed our previous finding that the K⁺ ions dissipate ΔΨ and increase ΔpH, whereas Na⁺ ions have little effect on ΔΨ and no effect on ΔpH. [³H]TPP⁺ and [¹⁴C]benzoate were added during exponential phase to S. lactis cells growing at pH 5 to 7 at 28°C in a defined medium with glucose as energy source. As with resting cells, the ΔpH and ΔΨ were dependent on the pH of the medium. At pH 5.1, the ΔpH was equivalent to 60 mV (alkaline inside) and decreased to 25 mV at pH 6.8. The ΔΨ increased from 83 mV (negative inside) at pH 5.1 to 108 mV at pH 6.8. The Δp, therefore, was fairly constant between pH 5 and 7, decreasing from 143 to 133 mV. The values for Δp in growing cells, just as in resting cells, are consistent with a system in which the net efflux of H⁺ ions is effected by a membrane-bound adenosine triphosphatase and glycolytically generated adenosine triphosphate. The data suggest that in both growing and resting cells the pH of the medium and its K⁺ concentration are the two principal factors that determine the relative contribution of ΔpH and ΔΨ to the proton motive force.