Torsional rigidity of single actin filaments and actin–actin bond breaking force under torsion measured directly by in vitro micromanipulation

Knowledge of the elastic properties of actin filaments is crucial for considering its role in muscle contraction, cellular motile events, and formation of cell shape. The stiffness of actin filaments in the directions of stretching and bending has been determined. In this study, we have directly determined the torsional rigidity and breaking force of single actin filaments by measuring the rotational Brownian motion and tensile strength using optical tweezers and microneedles, respectively. Rotational angular fluctuations of filaments supplied the torsional rigidity as (8.0 ± 1.2) × 10−26 Nm2. This value is similar to that deduced from the longitudinal rigidity, assuming the actin filament to be a homogeneous rod. The breaking force of the actin–actin bond was measured while twisting a filament through various angles using microneedles. The breaking force decreased greatly under twist, e.g., from 600–320 pN when filaments were turned through 90°, independent of the rotational direction. Our results indicate that an actin filament exhibits comparable flexibility in the rotational and longitudinal directions, but breaks more easily under torsional load.

Enhancing the catalytic repertoire of nucleic acids: a systematic study of linker length and rigidity

The incorporation of potentially catalytic groups in DNA is of interest for the in vitro selection of novel deoxyribozymes. A series of 10 C5-modified analogues of 2′-deoxyuridine triphosphate have been synthesised that possess side chains of differing flexibility and bearing a primary amino or imidazole functionality. For each series of nucleotide analogues differing degrees of flexibility of the C5 side chain was achieved through the use of alkynyl, alkenyl and alkyl moieties. The imidazole function was conjugated to these C5-amino-modified nucleotides using either imidazole 4-acetic acid or imidazole 4-acrylic acid (urocanic acid). The substrate properties of the nucleotides (fully replacing dTTP) with Taq polymerase during PCR have been investigated in order to evaluate their potential applications for in vitro selection experiments. 5-(3-Aminopropynyl)dUTP and 5-(E-3-aminopropenyl)dUTP and their imidazole 4-acetic acid- and urocanic acid-modified conjugates were found to be substrates. In contrast, C5-amino-modified dUTPs with alkane or Z-alkene linkers and their corresponding conjugates were not substrates. The incorporation of these analogues during PCR has been confirmed by inhibition of restriction enzyme digestion using XbaI and by mass spectrometry of the PCR products.

Adjustment of conformational flexibility is a key event in the thermal adaptation of proteins

3-Isopropylmalate dehydrogenase (IPMDH, E.C. 1.1.1.85) from the thermophilic bacterium Thermus thermophilus HB8 is homologous to IPMDH from the mesophilic Escherichia coli, but has an approximately 17°C higher melting temperature. Its temperature optimum is 22–25°C higher than that of the E. coli enzyme; however, it is hardly active at room temperature. The increased conformational rigidity required to stabilize the thermophilic enzyme against heat denaturation might explain its different temperature-activity profile. Hydrogen/deuterium exchange studies were performed on this thermophilic-mesophilic enzyme pair to compare their conformational flexibilities. It was found that Th. thermophilus IPMDH is significantly more rigid at room temperature than E. coli IPMDH, whereas the enzymes have nearly identical flexibilities under their respective optimal working conditions, suggesting that evolutionary adaptation tends to maintain a “corresponding state” regarding conformational flexibility. These observations confirm that conformational fluctuations necessary for catalytic function are restricted at room temperature in the thermophilic enzyme, suggesting a close relationship between conformational flexibility and enzyme function.

Molecular analysis of CaMnt1p, a mannosyl transferase important for adhesion and virulence of Candida albicans

There is an immediate need for identification of new antifungal targets in opportunistic pathogenic fungi like Candida albicans. In the past, efforts have focused on synthesis of chitin and glucan, which confer mechanical strength and rigidity upon the cell wall. This paper describes the molecular analysis of CaMNT1, a gene involved in synthesis of mannoproteins, the third major class of macromolecule found in the cell wall. CaMNT1 encodes an α-1,2-mannosyl transferase, which adds the second mannose residue in a tri-mannose oligosaccharide structure which represents O-linked mannan in C. albicans. The deduced amino acid sequence suggests that CaMnt1p is a type II membrane protein residing in a medial Golgi compartment. The absence of CaMnt1p reduced the ability of C. albicans cells to adhere to each other, to human buccal epithelial cells, and to rat vaginal epithelial cells. Both heterozygous and homozygous Camnt1 null mutants of C. albicans showed strong attenuation of virulence in guinea pig and mouse models of systemic candidosis, which, in guinea pigs, could be attributed to a decreased ability to reach and/or adhere internal organs. Therefore, correct CaMnt1p-mediated O-linked mannosylation of proteins is critical for adhesion and virulence of C. albicans.

Ionophores and receptors using cation-π interactions: Collarenes

Cation-π interactions are important forces in molecular recognition by biological receptors, enzyme catalysis, and crystal engineering. We have harnessed these interactions in designing molecular systems with circular arrangement of benzene units that are capable of acting as ionophores and models for biological receptors. [n]Collarenes are promising candidates with high selectivity for a specific cation, depending on n, because of their structural rigidity and well-defined cavity size. The interaction energies of [n]collarenes with cations have been evaluated by using ab initio calculations. The selectivity of these [n]collarenes in aqueous solution was revealed by using statistical perturbation theory in conjunction with Monte Carlo and molecular dynamics simulations. It has been observed that in [n]collarenes the ratio of the interaction energies of a cation with it and the cation with the basic building unit (benzene) can be correlated to its ion selectivity. We find that collarenes are excellent and efficient ionophores that bind cations through cation-π interactions. [6]Collarene is found to be a selective host for Li+ and Mg2+, [8]collarene for K+ and Sr2+, and [10]collarene for Cs+ and Ba2+. This finding indicates that [10]collarene and [8]collarene could be used for effective separation of highly radioactive isotopes, 137Cs and 90Sr, which are major constituents of nuclear wastes. More interestingly, collarenes of larger cavity size can be useful in capturing organic cations. [12]Collarene exhibits a pronounced affinity for tetramethylammonium cation and acetylcholine, which implies that it could serve as a model for acetylcholinestrase. Thus, collarenes can prove to be novel and effective ionophores/model-receptors capable of heralding a new direction in molecular recognition and host-guest chemistry.

Structural changes in hemoglobin during adsorption to solid surfaces: Effects of pH, ionic strength, and ligand binding

We have studied the adsorption of two structurally similar forms of hemoglobin (met-Hb and HbCO) to a hydrophobic self-assembled methyl-terminated thiol monolayer on a gold surface, by using a Quartz Crystal Microbalance (QCM) technique. This technique allows time-resolved simultaneous measurements of changes in frequency (f) (c.f. mass) and energy dissipation (D) (c.f. rigidity/viscoelastic properties) of the QCM during the adsorption process, which makes it possible to investigate the viscoelastic properties of the different protein layers during the adsorption process. Below the isoelectric points of both met-Hb and HbCO, the ΔD vs. Δf graphs displayed two phases with significantly different slopes, which indicates two states of the adsorbed proteins with different visco-elastic properties. The slope of the first phase was smaller than that of the second phase, which indicates that the first phase was associated with binding of a more rigidly attached, presumably denatured protein layer, whereas the second phase was associated with formation of a second layer of more loosely bound proteins. This second layer desorbed, e.g., upon reduction of Fe3+ of adsorbed met-Hb and subsequent binding of carbon monoxide (CO) forming HbCO. Thus, the results suggest that the adsorbed proteins in the second layer were in a native-like state. This information could only be obtained from simultaneous, time-resolved measurements of changes in both D and f, demonstrating that the QCM technique provides unique information about the mechanisms of protein adsorption to solid surfaces.

Cleavage planes in frog eggs are altered by strong magnetic fields

Early cleavages of Xenopus embryos were oriented in strong, static magnetic fields. Third-cleavage planes, normally horizontal, were seen to orient to a vertical plane parallel with a vertical magnetic field. Second cleavages, normally vertical, could also be oriented by applying a horizontal magnetic field. We argue that these changes in cleavage-furrow geometries result from changes in the orientation of the mitotic apparatus. We hypothesize that the magnetic field acts directly on the microtubules of the mitotic apparatus. Considerations of the length of the astral microtubules, their diamagnetic anisotropy, and flexural rigidity predict the required field strength for an effect that agrees with the data. This observation provides a clear example of a static magnetic-field effect on a fundamental cellular process, cell division.

Polymer Models of Meiotic and Mitotic Chromosomes

Polymers tied together by constraints exhibit an internal pressure; this idea is used to analyze physical properties of the bottle-brush–like chromosomes of meiotic prophase that consist of polymer-like flexible chromatin loops, attached to a central axis. Using a minimal number of experimental parameters, semiquantitative predictions are made for the bending rigidity, radius, and axial tension of such brushes, and the repulsion acting between brushes whose bristles are forced to overlap. The retraction of lampbrush loops when the nascent transcripts are stripped away, the oval shape of diplotene bivalents between chiasmata, and the rigidity of pachytene chromosomes are all manifestations of chromatin pressure. This two-phase (chromatin plus buffer) picture that suffices for meiotic chromosomes has to be supplemented by a third constituent, a chromatin glue to understand mitotic chromosomes, and explain how condensation can drive the resolution of entanglements. This process resembles a thermal annealing in that a parameter (the affinity of the glue for chromatin and/or the affinity of the chromatin for buffer) has to be tuned to achieve optimal results. Mechanical measurements to characterize this protein–chromatin matrix are proposed. Finally, the propensity for even slightly chemically dissimilar polymers to phase separate (cluster like with like) can explain the apparent segregation of the chromatin into A+T- and G+C-rich regions revealed by chromosome banding.

Mapping the intrinsic curvature and flexibility along the DNA chain

The energy of DNA deformation plays a crucial and active role in its packaging and its function in the cell. Considerable effort has gone into developing methodologies capable of evaluating the local sequence-directed curvature and flexibility of a DNA chain. These studies thus far have focused on DNA constructs expressly tailored either with anomalous flexibility or curvature tracts. Here we demonstrate that these two structural properties can be mapped also along the chain of a “natural” DNA with any sequence on the basis of its scanning force microscope (SFM) images. To know the orientation of the sequence of the investigated DNA molecules in their SFM images, we prepared a palindromic dimer of the long DNA molecule under study. The palindromic symmetry also acted as an internal gauge of the statistical significance of the analysis carried out on the SFM images of the dimer molecules. It was found that although the curvature modulus is not efficient in separating static and dynamic contributions to the curvature of the population of molecules, the curvature taken with its direction (its sign in two dimensions) permits the direct separation of the intrinsic curvature from the flexibility contributions. The sequence-dependent flexibility seems to vary monotonically with the chain's intrinsic curvature; the chain rigidity was found to modulate as its local thermodynamic stability and does not correlate with the dinucleotide chain rigidities evaluation made from x-ray data by other authors.

A Cross-Polarization, Magic-Angle-Spinning, 13C-Nuclear-Magnetic-Resonance Study of Polysaccharides in Sugar Beet Cell Walls1

Solid-state nuclear magnetic resonance relaxation experiments were used to study the rigidity and spatial proximity of polymers in sugar beet (Beta vulgaris) cell walls. Proton T1ρ decay and cross-polarization patterns were consistent with the presence of rigid, crystalline cellulose microfibrils with a diameter of approximately 3 nm, mobile pectic galacturonans, and highly mobile arabinans. A direct-polarization, magic-angle-spinning spectrum recorded under conditions adapted to mobile polymers showed only the arabinans, which had a conformation similar to that of beet arabinans in solution. These cell walls contained very small amounts of hemicellulosic polymers such as xyloglucan, xylan, and mannan, and no arabinan or galacturonan fraction closely associated with cellulose microfibrils, as would be expected of hemicelluloses. Cellulose microfibrils in the beet cell walls were stable in the absence of any polysaccharide coating.

Kinetic stability as a mechanism for protease longevity

The folding of the extracellular serine protease, α-lytic protease (αLP; EC 3.4.21.12) reveals a novel mechanism for stability that appears to lead to a longer functional lifetime for the protease. For αLP, stability is based not on thermodynamics, but on kinetics. Whereas this has required the coevolution of a pro region to facilitate folding, the result has been the optimization of native-state properties independent of their consequences on thermodynamic stability. Structural and mutational data lead to a model for catalysis of folding in which the pro region binds to a conserved β-hairpin in the αLP C-terminal domain, stabilizing the folding transition state and the native state. The pro region is then proteolytically degraded, leaving the active αLP trapped in a metastable conformation. This metastability appears to be a consequence of pressure to evolve properties of the native state, including a large, highly cooperative barrier to unfolding, and extreme rigidity, that reduce susceptibility to proteolytic degradation. In a test of survival under highly proteolytic conditions, homologous mammalian proteases that have not evolved kinetic stability are much more rapidly degraded than αLP. Kinetic stability as a means to longevity is likely to be a mechanism conserved among the majority of extracellular bacterial pro-proteases and may emerge as a general strategy for intracellular eukaryotic proteases subject to harsh conditions as well.

Force and kinetic barriers to unzipping of the DNA double helix

A theory of the unzipping of double-stranded DNA is presented and is compared to recent micromanipulation experiments. It is shown that the interactions that stabilize the double helix and the elastic rigidity of single strands simply determine the sequence-dependent ≈12-pN force threshold for DNA strand separation. Using a semimicroscopic model of the binding between nucleotide strands, we show that the greater rigidity of the strands when formed into double-stranded DNA, relative to that of isolated strands, gives rise to a potential barrier to unzipping. The effects of this barrier are derived analytically. The force to keep the extremities of the molecule at a fixed distance, the kinetic rates for strand unpairing at fixed applied force, and the rupture force as a function of loading rate are calculated. The dependence of the kinetics and of the rupture force on molecule length is also analyzed.

A rigid trans-spanning dinitrile ligand.

A rigid dinitrile ligand was synthesized from two xanthene units condensed to a naphthalene-1,4,5,8-diimide spacer. The rigidity and C shape of the ligand gave exclusively trans complexes with Pd(II), Ag(I), and Au(I). Evidence for complexation, coordination geometry, and stoichiometry was provided by a combination of 1H NMR, 19F NMR, and IR spectroscopy. The AuBF4 and PdCl2 complexes were shown to have a 1:1 (metal-to-ligand) stoichiometry and the AgBF4 complex was shown to have a 1:2 stoichiometry in solution. The preorganization of the dinitrile ligand resulted in complexes much more stable than their monodentate counterparts.

Role of aromatic residues in stabilization of the [Fe4S4] cluster in high-potential iron proteins (HiPIPs): physical characterization and stability studies of Tyr-19 mutants of Chromatium vinosum HiPIP.

The functional role of residue Tyr-19 of Chromatium vinosum HiPIP has been evaluated by site-directed mutagenesis experiments. The stability of the [Fe4S4] cluster prosthetic center is sensitive to side-chain replacements. Polar residues result in significant instability, while nonpolar residues (especially with aromatic side chains) maintain cluster stability. Two-dimensional NMR data of native and mutant HiPIPs are consistent with a model where Tyr-19 serves to preserve the structural rigidity of the polypeptide backbone, thereby maintaining a hydrophobic barrier for exclusion of water from the cluster cavity. Solvent accessibility results in more facile oxidation of the cluster by atmospheric oxygen, with subsequent rapid hydrolysis of the [Fe4S4]3+ core.