Combining Single-Molecule Optical Trapping and Small-Angle X-Ray Scattering Measurements to Compute the Persistence Length of a Protein ER/K α-Helix

A relatively unknown protein structure motif forms stable isolated single α-helices, termed ER/K α-helices, in a wide variety of proteins and has been shown to be essential for the function of some molecular motors. The flexibility of the ER/K α-helix determines whether it behaves as a force transducer, rigid spacer, or flexible linker in proteins. In this study, we quantify this flexibility in terms of persistence length, namely the length scale over which it is rigid. We use single-molecule optical trapping and small-angle x-ray scattering, combined with Monte Carlo simulations to demonstrate that the Kelch ER/K α-helix behaves as a wormlike chain with a persistence length of ∼15 nm or ∼28 turns of α-helix. The ER/K α-helix length in proteins varies from 3 to 60 nm, with a median length of ∼5 nm. Knowledge of its persistence length enables us to define its function as a rigid spacer in a translation initiation factor, as a force transducer in the mechanoenzyme myosin VI, and as a flexible spacer in the Kelch-motif-containing protein.     

Ab initio structure solution of a dimeric cytochrome c 3 from Desulfovibrio gigas containing disulfide bridges

The 1.2?Å resolution crystal structure of the 29?kDa di-tetrahaem cytochrome c ₃ from the sulfate reducing bacterium Desulfovibrio gigas was solved by ab initio methods, making this the largest molecule to be solved by this procedure. The actual refined model of the cysteine-linked dimeric molecule reveals that this molecule is very similar to the non-covalently linked symmetrical dimer of the di-tetrahaem cytochrome c ₃ from Desulfomicrobium norvegicum. Each monomer has the typical polypeptide fold, haem arrangement and iron coordination found for the tetrahaem cytochrome c ₃ molecules. The interface between the covalently linked monomers in the asymmetric unit of the crystal shows a pseudo two-fold arrangement, disturbed from symmetry by crystal packing forces. The fact that D. gigas contains a dimeric tetrahaem cytochrome with solvent accessible disulfide bridges and that this cytochrome specifically couples hydrogen oxidation to thiosulfate reduction in bacterial extracts provides an interesting aspect related to disulfide exchange reactions in this microorganism.     

Torque-limited RecA polymerization on dsDNA

The assembly of RecA onto a torsionally constrained double-stranded DNA molecule was followed in real time using magnetic tweezers. Formation of a RecA–DNA filament on the DNA tether was stalled owing to different physical processes depending on the applied stretching force. For forces up to 3.6 pN, the reaction stalled owing to the formation of positive plectonemes in the remaining DNA molecule. Release of these plectonemes by rotation of the magnets led to full coverage of the DNA molecule by RecA. At stretching forces larger than 3.6 pN, the twist induced during filament formation caused the reaction to stall before positive supercoils were generated. We deduce a maximum built-up torsion of 10.1 ± 0.7 k_bT. In vivo this built-up torsion may be used to favor regression of a stalled replication fork or to free the chromosomal DNA in E.coli from its condensing proteins.     

Predicting maximum eccentric strength from surface EMG measurements

The origin of the well-documented discrepancy between maximum voluntary and in vitro tetanic eccentric strength has yet to be fully understood. This study aimed to determine whether surface EMG measurements can be used to reproduce the in vitro tetanic force–velocity relationship from maximum voluntary contractions. Five subjects performed maximal knee extensions over a range of eccentric and concentric velocities on an isovelocity dynamometer whilst EMG from the quadriceps were recorded. Maximum voluntary (MVC) force–length–velocity data were estimated from the dynamometer measurements and a muscle model. Normalised amplitude–length–velocity data were obtained from the EMG signals. Dividing the MVC forces by the normalised amplitudes generated EMG corrected force–length–velocity data. The goodness of fit of the in vitro tetanic force–velocity function to the MVC and EMG corrected forces was assessed. Based on a number of comparative scores the in vitro tetanic force–velocity function provided a significantly better fit to the EMG corrected forces compared to the MVC forces (p?0.05), Furthermore, the EMG corrected forces generated realistic in vitro tetanic force–velocity profiles. A 58±19% increase in maximum eccentric strength is theoretically achievable through eliminating neural factors. In conclusion, EMG amplitude can be used to estimate in vitro tetanic forces from maximal in vivo force measurements, supporting neural factors as the major contributor to the difference between in vitro and in vivo maximal force.     

A highly branched storage polyglucan in the thermoacidophilic red microalga <Emphasis Type="Italic">Galdieria maxima</Emphasis> cells

The thermoacidophilic red alga Galdieria maxima is capable of heterotrophic growth. The content of carbohydrates in G. maxima grown heterotrophically increases by a factor of 4, reaching as much as 60% of cell dry weight. The increase in the level of carbohydrates in cells is due to accumulation of a storage α-glycan. According to a specific cleavage to glucose catalyzed by amyloglucosidase and the high positive specific optical rotation characteristic of polyglucans, this polysaccharide can be classified as a floridean starch. The data of ¹H NMR spectroscopy and the results of methylation showed that the average length of the unbranched regions of the polysaccharide molecule is six to seven glucose residues. The degree of branching of the starch molecule of G. maxima is greater than that of storage polysaccharides of other red algae, glycogens of yeast, and phytoglycogens of cyanobacteria.     

Unfolding individual nucleosomes by stretching single chromatin fibers with optical tweezers.

Single chromatin fibers were assembled directly in the flow cell of an optical tweezers setup. A single lambda phage DNA molecule, suspended between two polystyrene beads, was exposed to a Xenopus laevis egg extract, leading to chromatin assembly with concomitant apparent shortening of the DNA molecule. Assembly was force-dependent and could not take place at forces exceeding 10 pN. The assembled single chromatin fiber was subjected to stretching by controlled movement of one of the beads with the force generated in the molecule continuously monitored with the second bead trapped in the optical trap. The force displayed discrete, sudden drops upon fiber stretching, reflecting discrete opening events in fiber structure. These opening events were quantized at increments in fiber length of approximately 65 nm and are attributed to unwrapping of the DNA from around individual histone octamers. Repeated stretching and relaxing of the fiber in the absence of egg extract showed that the loss of histone octamers was irreversible. The forces measured for individual nucleosome disruptions are in the range of 20-40 pN, comparable to forces reported for RNA- and DNA-polymerases.     

DNA condensation in bacteria: Interplay between macromolecular crowding and nucleoid proteins

The volume of a typical Eschericia coli nucleoid is roughly 10⁴ times smaller than the volume of a freely coiling linear DNA molecule with the same length as the E. coli genome. We review the main forces that have been suggested to contribute to this compaction factor: macromolecular crowding (that “pushes” the DNA together), DNA charge neutralization by various polycationic species (that “glues” the DNA together), and finally, DNA deformations due to DNA supercoiling and nucleoid proteins. The direct contributions of DNA supercoiling and nucleoid proteins to the total compaction factor are probably small. Instead, we argue that the formation of the bacterial nucleoid can be described as a consequence of the influence of macromolecular crowding on thick, supercoiled protein-DNA fibers, that have been partly charge neutralized by small multivalent cations.     

In vivo outer hair cell length changes expose the active process in the cochlea

Background

Mammalian hearing is refined by amplification of the sound-evoked vibration of the cochlear partition. This amplification is at least partly due to forces produced by protein motors residing in the cylindrical body of the outer hair cell. To transmit power to the cochlear partition, it is required that the outer hair cells dynamically change their length, in addition to generating force. These length changes, which have not previously been measured in vivo, must be correctly timed with the acoustic stimulus to produce amplification.

Methodology/Principal Findings

Using in vivo optical coherence tomography, we demonstrate that outer hair cells in living guinea pigs have length changes with unexpected timing and magnitudes that depend on the stimulus level in the sensitive cochlea.

Conclusions/Significance

The level-dependent length change is a necessary condition for directly validating that power is expended by the active process presumed to underlie normal hearing.     

Two conformations of crystalline adenylate kinase.

Pig muscle adenylate kinase (EC2.7.4.3) can exist in three crystal forms, which are interconvertible. For crystal form A the enzyme structure is known in atomic detail. We report the X-ray diffraction analysis of crystal form B at 4.7 Å resolution and a comparison with the A form. During the transition from A to B the packing arrangement of the molecules changes slightly. Moreover, the individual molecule undergoes an appreciable conformational change: by displacing a chain segment of seven residues and two adjacent α-helices a hydrophobic pocket is opened deep in the cleft near the centre of the molecule. Concomitantly the β-pleated sheet is enlarged by about four hydrogen bonds in the B form. Several lines of evidence indicate that the observed conformational change is an intrinsic property of the molecule and is not induced by crystal packing forces.     

Alkylammonium chlorobenzoates are a new group of ester-containing reversible inhibitors of cholinesterases of different animals

Interaction with cholinesterases (ChEs) of nine specially synthesized derivatives of dimethylaminoalkyl esters of 2-chloro-and 2,4-dichlorobenzoic acids and their iodoalkylates is studied. Used as enzyme sources were partially purified preparations of acetylcholinesterase (AChE) from human erythrocytes and butyrylcholinesterase (BChE) from horse blood serum, as well as water homogenates of the frog Rana temporaria brain and of the Pacific squid Todarodes pacificus optical ganglia. The studied benzoates failed to be hydrolyzed by the studied ChEs at the enzyme concentrations exceeding 10 times those used for determination of the acetylthiocholine hydrolysis rate. These compounds have turned out to be reversible inhibitors of ChEs of the mixed-noncompetitive type of action. Effects on the anticholinesterase activity of such structural elements of the inhibitors as the acidic part of the benzoate molecule, length of polymethylene chain in the molecule alcoholic part, and the structure of ammonium group are studied. This study has allowed revealing some peculiarities of the reaction capability of vertebrate and invertebrate ChEs.     

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:     

The Structure of High Molecular Weight Ribonucleic Acid in Solution: A Small-Angle X-Ray Scattering Study

Small-angle x-ray scattering studies on an absolute scale have been carried out on isotropic solutions of high molecular weight RNA obtained from ascites tumor cells, E. coli, and yeast. It was found that in all three cases the RNA is composed of short rigid rods (50 to 150Å in length) joined by small flexible regions. The rods account for almost the entire structure (at least 90 per cent); their radius of gyration about the axis and their mass per unit length are similar to those of DNA, suggesting a double-stranded helical structure. The rods are joined in an array forming the compact RNA molecule. On thermal degradation, the molecular superstructure disappears while the rods persist.     

Mechanical forces regulate elastase activity and binding site availability in lung elastin

Many fundamental cellular and extracellular processes in the body are mediated by enzymes. At the single molecule level, enzyme activity is influenced by mechanical forces. However, the effects of mechanical forces on the kinetics of enzymatic reactions in complex tissues with intact extracellular matrix (ECM) have not been identified. Here we report that physiologically relevant macroscopic mechanical forces modify enzyme activity at the molecular level in the ECM of the lung parenchyma. Porcine pancreatic elastase (PPE), which binds to and digests elastin, was fluorescently conjugated (f-PPE) and fluorescent recovery after photobleach was used to evaluate the binding kinetics of f-PPE in the alveolar walls of normal mouse lungs. Fluorescent recovery after photobleach indicated that the dissociation rate constant (k_off) for f-PPE was significantly larger in stretched than in relaxed alveolar walls with a linear relation between k_off and macroscopic strain. Using a network model of the parenchyma, a linear relation was also found between k_off and microscopic strain on elastin fibers. Further, the binding pattern of f-PPE suggested that binding sites on elastin unfold with strain. The increased overall reaction rate also resulted in stronger structural breakdown at the level of alveolar walls, as well as accelerated decay of stiffness and decreased failure stress of the ECM at the macroscopic scale. These results suggest an important role for the coupling between mechanical forces and enzyme activity in ECM breakdown and remodeling in development, and during diseases such as pulmonary emphysema or vascular aneurysm. Our findings may also have broader implications because in vivo, enzyme activity in nearly all cellular and extracellular processes takes place in the presence of mechanical forces.     

Substrate Deformation Predicts Neuronal Growth Cone Advance

Although pulling forces have been observed in axonal growth for several decades, their underlying mechanisms, absolute magnitudes, and exact roles are not well understood. In this study, using two different experimental approaches, we quantified retrograde traction force in Aplysia californica neuronal growth cones as they develop over time in response to a new adhesion substrate. In the first approach, we developed a novel method, to our knowledge, for measuring traction forces using an atomic force microscope (AFM) with a cantilever that was modified with an Aplysia cell adhesion molecule (apCAM)-coated microbead. In the second approach, we used force-calibrated glass microneedles coated with apCAM ligands to guide growth cone advance. The traction force exerted by the growth cone was measured by monitoring the microneedle deflection using an optical microscope. Both approaches showed that Aplysia growth cones can develop traction forces in the 10⁰–10² nN range during adhesion-mediated advance. Moreover, our results suggest that the level of traction force is directly correlated to the stiffness of the microneedle, which is consistent with a reinforcement mechanism previously observed in other cell types. Interestingly, the absolute level of traction force did not correlate with growth cone advance toward the adhesion site, but the amount of microneedle deflection did. In cases of adhesion-mediated growth cone advance, the mean needle deflection was 1.05 ± 0.07 μm. By contrast, the mean deflection was significantly lower (0.48 ± 0.06 μm) when the growth cones did not advance. Our data support a hypothesis that adhesion complexes, which can undergo micron-scale elastic deformation, regulate the coupling between the retrogradely flowing actin cytoskeleton and apCAM substrates, stimulating growth cone advance if sufficiently abundant.     

Equilibrium and Kinetics of DNA Overstretching Modeled with a Quartic Energy Landscape

It is well known that the dsDNA molecule undergoes a phase transition from B-DNA into an overstretched state at high forces. For some time, the structure of the overstretched state remained unknown and highly debated, but recent advances in experimental techniques have presented evidence of more than one possible phase (or even a mixed phase) depending on ionic conditions, temperature, and basepair sequence. Here, we present a theoretical model to study the overstretching transition with the possibility that the overstretched state is a mixture of two phases: a structure with portions of inner strand separation (melted or M-DNA), and an extended phase that retains the basepair structure (S-DNA). We model the double-stranded DNA as a chain composed of n segments of length l, where the transition is studied by means of a Landau quartic potential with statistical fluctuations. The length l is a measure of cooperativity of the transition and is key to characterizing the overstretched phase. By analyzing the different values of l corresponding to a wide spectrum of experiments, we find that for a range of temperatures and ionic conditions, the overstretched form is likely to be a mix of M-DNA and S-DNA. For a transition close to a pure S-DNA state, where the change in extension is close to 1.7 times the original B-DNA length, we find l ≈ 25 basepairs regardless of temperature and ionic concentration. Our model is fully analytical, yet it accurately reproduces the force-extension curves, as well as the transient kinetic behavior, seen in DNA overstretching experiments.     

Equilibrium and Kinetics of DNA Overstretching Modeled with a Quartic Energy Landscape

It is well known that the dsDNA molecule undergoes a phase transition from B-DNA into an overstretched state at high forces. For some time, the structure of the overstretched state remained unknown and highly debated, but recent advances in experimental techniques have presented evidence of more than one possible phase (or even a mixed phase) depending on ionic conditions, temperature, and basepair sequence. Here, we present a theoretical model to study the overstretching transition with the possibility that the overstretched state is a mixture of two phases: a structure with portions of inner strand separation (melted or M-DNA), and an extended phase that retains the basepair structure (S-DNA). We model the double-stranded DNA as a chain composed of n segments of length l, where the transition is studied by means of a Landau quartic potential with statistical fluctuations. The length l is a measure of cooperativity of the transition and is key to characterizing the overstretched phase. By analyzing the different values of l corresponding to a wide spectrum of experiments, we find that for a range of temperatures and ionic conditions, the overstretched form is likely to be a mix of M-DNA and S-DNA. For a transition close to a pure S-DNA state, where the change in extension is close to 1.7 times the original B-DNA length, we find l ≈ 25 basepairs regardless of temperature and ionic concentration. Our model is fully analytical, yet it accurately reproduces the force-extension curves, as well as the transient kinetic behavior, seen in DNA overstretching experiments.     

Mechanical Forces Impeding Exocytotic Surfactant Release Revealed by Optical Tweezers

The release of surfactant from alveolar type II cells is essential to lower the surface tension in the lung and to facilitate inspiration. However, the factors controlling dispersal and diffusion of this hydrophobic material are still poorly understood. Here we report that release of surfactant from the fused vesicle, termed lamellar body (LB), resisted mechanical forces applied by optical tweezers: At constant trapping force, the probability to expand LB contents, i.e., to “pull” surfactant into the extracellular fluid, increased with time after LB fusion with the plasma membrane, consistent with slow fusion pore expansion in these cells. Elevations of the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_c) had a similar effect. Inasmuch as surfactant did not disintegrate in the extracellular space, this method permitted for the first time the determination of elastic and recoil properties of the macromolecular complex, yielding a spring constant of ~12.5 pN/μm. This is the first functional evidence that release of hydrophobic material is mechanically impeded and occurs in an “all-or-none” fashion. This mode of release is most probably the result of cohesive forces of surfactant, combined with adhesive forces and/or retaining forces exerted by a constrictive fusion pore acting as a regulated mechanical barrier, withstanding forces up to 160 pN. In independent experiments equiaxial strain was exerted on cells without optical tweezers. Strain facilitated surfactant release from preexisting fused vesicles, consistent with the view of mechanical impediments during the release process, which can be overcome by cell strain.     

DNA Motion Capture Reveals the Mechanical Properties of DNA at the Mesoscale

Single-molecule studies probing the end-to-end extension of long DNAs have established that the mechanical properties of DNA are well described by a wormlike chain force law, a polymer model where persistence length is the only adjustable parameter. We present a DNA motion-capture technique in which DNA molecules are labeled with fluorescent quantum dots at specific sites along the DNA contour and their positions are imaged. Tracking these positions in time allows us to characterize how segments within a long DNA are extended by flow and how fluctuations within the molecule are correlated. Utilizing a linear response theory of small fluctuations, we extract elastic forces for the different, ∼2-μm-long segments along the DNA backbone. We find that the average force-extension behavior of the segments can be well described by a wormlike chain force law with an anomalously small persistence length.