Biological macromolecules

Magnetic actuation of hair cells
David Rowland, Yuttana Roongthumskul, Jae-Hyun Lee, Jinwoo Cheon, Dolores Bozovic
The bullfrog sacculus contains mechanically sensitive hair cells whose stereociliary bundles oscillate spontaneously when decoupled from the overlying membrane. Steady-state offsets on the resting position of a hair bundle can suppress or modulate this native motility. To probe the dynamics of spontaneous oscillation in the proximity of the critical point, we describe here a method for mechanical actuation that avoids loading the bundles or contributing to the viscous drag. Magnetite beads were attached to the tips of the stereocilia, and a magnetic probe was used to impose deflections. This technique allowed us to observe the transition from multi-mode to single-mode state in freely oscillating bundles, as well as the crossover from the oscillatory to the C quiescent state. V 2011 American Institute of Physics. [doi:10.1063/1.3659299]
JPEGs: Magnetic actuation of hair cells (333.97 KB)
PDFs: Rowland11.pdf (3.36 MB)
Research categories: Biological Macromolecules, Nonequilibrium physics

The folded protein as a viscoelastic solid
Y. Wang and G. Zocchi, “The folded protein as a visco-elastic solid”, Europhys. Lett. 96, 18003 (2011).
Yong Wang and Giovanni Zocchi
We apply a nanorheology technique to explore the mechanical properties of a globular protein in the frequency range 10 Hz–10 kHz and find that the folded state of the protein behaves like a viscoelastic solid. For increasing amplitude of the forcing, we observe three different regimes: linear elasticity, then a regime of viscoelastic but reversible deformations, and finally an irreversible regime. The second regime, which has the signature of a viscoelastic solid, gives access to the internal dissipation coefficient of the folded state, for which we find γ ≈ 4 × 10−5 kg/s, corresponding to an internal viscosity η ∼ 104 Pa · s for frequencies below ∼ 10 Hz. We propose that the large discrepancy between this value, which agrees with previous AFM indentation experiments, and the value of the internal viscosity extracted from refolding experiments is a consequence of the viscoelastic nature of the protein’s mechanics. Thus the present method yields detailed measurements of the mechanics of the folded state.
JPEGs: Nano_rheology.jpg (2.39 MB)
PDFs: Yong_4_reprint.pdf (4.74 MB)
Biological Macromolecules, Soft and fragile matter

Visualizing large RNA molecules in solution
A. Gopal, Z. H. Zhou, C. M. Knobler, and W. M. Gelbart, Visualizing large RNA molecules in solution, RNA 18, 284-299 (2012).
The intrinsic structures – size and shape – of large (many-thousands of nucleotides) RNA molecules in solution are imaged directly for the first time in this cryo-electron microscopy study of vitrified freely-suspended films having thicknesses of about 100nm. Any interactions of the RNA with solid substrate (as in AFM studies) or with staining agents (as in ordinary EM) would destroy the delicate secondary and tertiary structure of the molecules. By analyzing the projections of a large number of chemically identical viral RNA molecules, Gopal et al. are able to deduce the nature of the ensemble of structures associated with a given sequence, and to determine in this way the size and shape of large RNAs in solution. The cryo-EM results are compared against those obtained from small-angle synchrotron X-ray reconstructions and from coarse-grained molecular dynamics simulations.

PDFs: Visualizing RNA.pdf (10.88 MB)
JPEGs: Visualizing large RNA molecules in solution (330.13 KB)
Research categories: Biological Macromolecules

Predicting the sizes of large RNA molecules
A. M. Yoffe, P. Prinsen, A. Gopal, C.M. Knobler, W. M. Gelbart, and A. Ben-Shaul, Predicting the sizes of large RNA molecules, Proc. Natl. Acad. Sci. (USA) 105, 16153-8 (2008).
The genomes of many viruses are large single-stranded (ss) RNA molecules, often several thousand nucleotides long, which are packaged spontaneously into small rigid shells by one of their gene products, the capsid protein. Yoffe et al. argue that this fact is associated with evolutionary pressure for these genomes to be more compact – have a smaller radius of gyration – than non-viral RNA sequences of the same length. This compaction is arises from the special large-scale properties of the secondary structures – self-complementarity – arising from viral (vs nonviral) nucleotide sequences. To quantify this effect, Yoffe et al. introduce the notion of “maximum ladder distance” (MLD), quantifying the “extendedness” of any RNA secondary structure, and demonstrate for a large number of viral genomes that their MLDs (and hence radii of gyration) are indeed significantly smaller than those of nonviral and random sequences with the same lengths.
PDFs: RNA sizes.pdf (3.37 MB)
PNGs: Secondary structures for viral and nonviral (random) sequences of RNA molecules (299.45 KB)
Research categories: Biological Macromolecules

Nucleosome Switches
David Schwab, Robijn F. Bruinsma, and Joseph Rudnick
“Nucleosomes” are groups of eight proteins (“histones”) around which genomic DNA is wound (see figure) as a means of “condensing” DNA. Jonathan Widom had demonstrated that the positioning of these nucleosomes is encoded by the DNA itself. We applied an exactly soluble one-dimensional statistical mechanics model that could predict the locations of the the nucleosomes based on the DNA sequence. Based on this model, we found that nucleosome positioning can act as “switches” regulating gene expression.
PDFs: nihms-124886.pdf (3.95 MB)
PNGs: Nucleosome Switches (440.41 KB)

Mechano-chemistry of the enzyme Guanylate Kinase
C-Y. Tseng, A. Wang, and G. Zocchi, “Mechano-chemistry of the enzyme Guanylate Kinase”, Europhys. Lett. 91, 18005 (2010).
C.-Y. Tseng, A. Wang and G. Zocchi
We address the coupling of mechanics and chemistry in an enzyme through equilibrium experiments where we mechanically deform the enzyme and measure the effect on the chemical reaction it catalyzes. We use the DNA molecular spring method to exert stresses at three different specific locations on the enzyme Guanylate Kinase, and for each case determine the changes in substrates binding affinities and catalytic rate. We find that the enzyme kinetics parameters can be affected separately, depending on where the mechanical stress is applied. For one configuration the applied stress mainly affects the catalytic rate kcat , for another it mainly affects the binding affinity of the substrate GMP. These experiments show that a stress applied by pulling two residues on the surface of the protein generally results in a strain propagating into the structure.
JPEGs: GK_chimera.jpg (403.34 KB)
PDFs: Chiao-Yu2_reprint.pdf (6.52 MB)
Research categories: Biological Macromolecules

Real-time observation of bacteriophage T4 gp41 helicase reveals an unwinding mechanism
Timothee Lionnet, Michelle M. Spiering, Stephen J. Benkovic, David Bensimon, Vincent Croquette
Helicases are enzymes that couple ATP hydrolysis to the unwinding of double-stranded (ds) nucleic acids. The bacteriophage T4 helicase (gp41) is a hexameric helicase that promotes DNA replication within a highly coordinated protein complex termed the replisome. Despite recent progress, the gp41 unwinding mechanism and regulatory interactions within the replisome remain unclear. Here we use a single tethered DNA hairpin as a real-time reporter of gp41-mediated dsDNA unwinding and single-stranded (ss) DNA translocation with 3-base pair (bp) resolution. Although gp41 translocates on ssDNA as fast as the in vivo replication fork (400 bp/s), its unwinding rate extrapolated to zero force is much slower (30 bp/s). Together, our results have two implications: first, gp41 unwinds DNA through a passive mechanism; second, this weak helicase cannot efficiently unwind the T4 genome alone. Our results suggest that important regulations occur within the replisome to achieve rapid and processive replication.
JPEGs: Real-time observation of bacteriophage T4 gp41.jpg (390.16 KB)
PDFs: Real-time observation of bacteriophage T4 gp41.pdf (8.38 MB)
Research categories: Biological Macromolecules, Cellular mechanics, Nonequilibrium physics

The complete bending energy function for nicked DNA
Hao Qu and Giovanni Zocchi, “The complete bending energy function for nicked DNA”, Europhys. Lett. 94, 18003 (2011).
We derive an analytic expression for the bending elastic energy of short DNA molecules, valid in the entire range from low to high energies. The elastic energy depends on three parameters: the length of the molecule (2L), the bending modulus B, and a critical torque τc at which the molecule develops a kink. In the kinked state, the elastic energy is linear in the kink angle, i.e. the torque at the kink is constant (= τc ). τc depends (weakly) on the sequence around the nick, but is about 27 pN × nm. We measure it for a specific sequence, through experiments where the elastic energy of constrained DNA molecules is directly measured.
JPEGs: DNA_stressed.jpg (733.84 KB)
PDFs: Hao_2_reprint.pdf (7.82 MB)
Research categories: Biological Macromolecules, Experimental probes

Mechanisms of chiral discrimination by topoisomerase IV
K. C. Neuman, G. Charvin, D. Bensimon, and V. Croquette
Topoisomerase IV (Topo IV), an essential ATP-dependent bacterial type II topoisomerase, transports one segment of DNA through a transient double-strand break in a second segment of DNA. In vivo, Topo IV unlinks catenated chromosomes before cell division and relaxes positive supercoils generated during DNA replication. In vitro, Topo IV relaxes positive supercoils at least 20-fold faster than negative supercoils. The mechanisms underlying this chiral discrimination by Topo IV and other type II topoisomerases remain speculative. We used magnetic tweezers to measure the relaxation rates of single and multiple DNA crossings by Topo IV. These measurements allowed us to determine unambiguously the relative importance of DNA crossing geometry and enzymatic processivity in chiral discrimination by Topo IV. Our results indicate that Topo IV binds and passes DNA strands juxtaposed in a nearly perpendicular orientation and that relaxation of negative supercoiled DNA is perfectly distributive. Together, these results suggest that chiral discrimination arises primarily from dramatic differences in the processivity of relaxing positive and negative supercoiled DNA: Topo IV is highly processive on positively supercoiled DNA, whereas it is perfectly distributive on negatively supercoiled DNA. These results provide fresh insight into topoisomerase mechanisms and lead to a model that reconciles contradictory aspects of previous findings while providing a framework to interpret future results.
JPEGs: Mechanisms of chiral discrimination.jpg (500.38 KB)
PDFs: Mechanisms of chiral discrimination.pdf (4.31 MB)
Research categories: Biological Macromolecules, Experimental probes

Measurement of the Torque on a Single Stretched and Twisted DNA Using Magnetic Tweezers
Francesco Mosconi, Jean Franc¸ois Allemand, David Bensimon, and Vincent Croquette
We deduced the torque applied on a single stretched and twisted DNA by integrating the change in the molecule’s extension with respect to force as it is coiled. While consistent with previous direct measurements of the torque at high forces (F > 1 pN), this method, which is simple and does not require a sophisticated setup, allows for lower force estimates. We used this approach to deduce the effective torsional modulus of DNA, which decreases with force, and to estimate the buckling torque of DNA as a function of force in various salt conditions.
JPEGs: Measurement of the Torque on a Single Stretched and Twisted DNA Using Magnetic Tweezers (527.58 KB)
PDFs: Measurement of the Torque on a Single Stretched and Twisted DNA Using Magnetic Tweezers.pdf (8.96 MB)
Research categoriesBiological Macromolecules, Cellular mechanics, Experimental probes

Collaborative coupling between polymerase and helicase for leading-strand synthesis
Maria Manosas, Michelle M. Spiering, Fangyuan Ding, Vincent Croquette and Stephen J. Benkovic
Rapid and processive leading-strand DNA synthesis in the bacteriophage T4 system requires functional coupling between the helicase and the holoenzyme, consisting of the polymerase and trimeric clamp loaded by the clamp loader. We investigated the mechanism of this coupling on a DNA hairpin substrate manipulated by a magnetic trap. In stark contrast to the isolated enzymes, the coupled system synthesized DNA at the maximum rate without exhibiting fork regression or pauses. DNA synthesis and unwinding activities were coupled at low forces, but became uncoupled displaying separate activities at high forces or low dNTP concentration. We propose a collaborative model in which the helicase releases the fork regression pressure on the holoenzyme allowing it to adopt a processive polymerization conformation and the holoenzyme destabilizes the first few base pairs of the fork thereby increasing the efficiency of helicase unwinding. The model implies that both enzymes are localized at the fork, but does not require a specific interaction between them. The model quantitatively reproduces homologous and heterologous coupling results under various experimental conditions.
JPEGs: Collaborative coupling between polymerase and.jpg (1.06 MB)
PDFs: Collaborative coupling between polymerase.pdf (7.69 MB)
Research categories: Biological Macromolecules, Cellular mechanics, Experimental probes

Criterion for Amino Acid Composition of Defensins and Antimicrobial Peptides Based on Geometry of Membrane Destabilization
N. W. Schmidt, A. Mishra, G. H. Lai, M. Davis, L. K. Sanders, D. Tran, A. Garcia, K. P. Tai, P. B. McCray Jr., A. J. Ouellette, M. E. Selsted, G. C. L. Wong, “Criterion for amino acid composition of defensins and antimicrobial peptides based on geometry
Nathan W. Schmidt, Abhijit Mishra, Ghee Hwee Lai, Matthew Davis, Lori K. Sanders, Dat Tran, Angie Garcia, Kenneth P. Tai, Paul B. McCray, Jr., Andre J. Ouellette, Michael E. Selsted, Gerard C. L. Wong
Defensins comprise a potent class of membrane disruptive antimicrobial peptides (AMPs) with well-characterized broad spectrum and selective microbicidal effects. By using high-resolution synchrotron small-angle X-ray scattering to investigate
interactions between heterogeneous membranes and members of the defensin subfamilies, R-defensins (Crp-4), β-defensins (HBD-2, HBD-3), and θ-defensins (RTD-1, BTD-7), we show how these peptides all permeabilize model bacterial membranes but not model eukaryotic membranes: defensins selectively generate saddle-splay (“negative Gaussian”) membrane curvature in model membranes rich in negative curvature lipids such as those with phosphoethanolamine (PE) headgroups. These results are shown to be consistent with vesicle leakage assays. A mechanism of action based on saddle-splay membrane curvature generation is broadly enabling, because it is a necessary condition for processes such as pore formation, blebbing, budding, and vesicularization, all of which destabilize the barrier function of cell membranes. Importantly, saddle-splay membrane curvature generation places constraints on the amino acid composition of membrane disruptive peptides. For example, we show that the requirement for generating saddle-splay curvature implies that a decrease in arginine content in an AMP can be offset by an increase in both lysine and hydrophobic content. This “design rule” is consistent with the amino acid compositions of 1080 known cationic AMPs.
JPEGs: Wong group discovers selection rule for antimicrobial peptide sequences.jpg (126.45 KB)
PDFs: JACS Schmidt Wong 2011.pdf (23.51 MB)
Research categories: Biological Macromolecules, Cellular mechanics

High energy deformation of filaments with internal structure and localized torque-induced melting of DNA
We develop a continuum elastic approach to examining the bending mechanics of semiflexible filaments with a local internal degree of freedom that couples to the bending modulus. We apply this model to study the nonlinear mechanics of a double stranded DNA oligomer (shorter than its thermal persistence length) whose free ends are linked by a single standed DNA chain. This construct, studied by Qu et al. [Europhys. Lett., 94, 18003, 2011], displays nonlinear strain softening associated with the local melting of the double stranded DNA under applied torque and serves as a model system with which to study the nonlinear elasticity of DNA under large energy deformations. We show that one can account quantitatively for the observed bending mechanics using an augmented worm-like chain model, the helix coil worm-like chain. We also predict that the highly bent and partially molten dsDNA should exhibit particularly large end-to-end fluctuations associated with the fluctuation of the length of the molten region, and propose appropriate experimental tests. We suggest that the augmented worm-like chain model discussed here is a useful analytic approach to the nonlinear mechanics of DNA or other biopolymer systems. 
PDFs: MeltingElastica_v4.pdf (11.68 MB)
Research categories: Biological Macromolecules

Translocation of TAT peptide and analogs induced by multiplexed membrane and cytoskeletal interactions
A. Mishra, G. H. Lai, N. W. Schmidt, V. Z. Sun, A. Rodriguez, R. Tong, L. Tang, J. J. Cheng, T. J. Deming, D. T. Kamei, G. C. L. Wong, “Translocation of TAT peptide and analogs induced by multiplexed membrane and cytoskeletal interactions”, Proc. Nat.
Cell-penetrating peptides (CPPs), such as the HIV TAT peptide, are able to translocate across cellular membranes efficiently. A number of mechanisms, from direct entry to various endocytotic mechanisms (both receptor independent and receptor dependent), have been observed but how these specific amino acid sequences accomplish these effects is unknown. We show how CPP sequences can multiplex interactions with the membrane, the actin cytoskeleton, and cell-surface receptors to facilitate different translocation pathways under different conditions. Using “nunchuck” CPPs, we demonstrate that CPPs permeabilize membranes by generating topologically active saddle-splay (“negative Gaussian”) membrane curvature through multidentate hydrogen bonding of lipid head groups. This requirement for negative Gaussian curvature constrains but underdetermines the amino acid content of CPPs. We observe that in most CPP sequences decreasing arginine content is offset by a simultaneous increase in lysine and hydrophobic content. Moreover, by densely organizing cationic residues while satisfying the above constraint, TAT peptide is able to combine cytoskeletal remodeling activity with membrane translocation activity. We show that the TAT peptide can induce structural changes reminiscent of macropinocytosis in actin-encapsulated giant vesicles without receptors.
JPEGs: Wong group finds molecular mechanisms for cell penetrating peptides.jpg (700.20 KB)
PDFs: PNAS-2011-Mishra-16883-8.pdf (6.61 MB)
Research categories: Biological Macromolecules, Cellular mechanics

Squalamine as a broad-spectrum systemic antiviral agent with therapeutic potential
M. Zasloff, A. Adams, B. Beckerman, A. Campbell, Z. Han, E. Luijten, I. Meza, J. Julander, A. Mishra, W. Qu, J. Taylor, S. Weaver, G. C. L. Wong, “Squalamine as a broad spectrum antiviral with therapeutic potential”, Proc. Nat. Acad. Sci. USA, 108 159
Michael Zasloffa, A. Paige Adams, Bernard Beckerman, Ann Campbell, Ziying Han, Erik Luijten, Isaura Meza, Justin Julander, Abhijit Mishra, Wei Qu, John M. Taylor, Scott C. Weaver, and Gerard C. L. Wong
Antiviral compounds that increase the resistance of host tissues represent an attractive class of therapeutic. Here, we show that squalamine, a compound previously isolated from the tissues of the dogfish shark (Squalus acanthias) and the sea lamprey (Petromyzon marinus), exhibits broad-spectrum antiviral activity against human pathogens, which were studied in vitro as well as in vivo. Both RNA- and DNA-enveloped viruses are shown to be susceptible. The proposed mechanism involves the capacity of squalamine, a cationic amphipathic sterol, to neutralize the negative electrostatic surface charge of intracellular membranes in a way that renders the cell less effective in supporting viral replication. Because squalamine can be readily synthesized and has a known safety profile in man, we believe its potential as a broad-spectrum human antiviral agent should be explored.
JPEGs: Wong group elucidates antiviral immune system of shark.jpg (188.24 KB)
PDFs: PNAS Zasloff Mishra Wong 2011.pdf (6.61 MB)
Research categories: Viruses, Biological Macromolecules