2006 - Abstracts
1. Christini, D. J., M. L. Riccio, C. A. Culianu, J. J. Fox, A. Karma and R. F. Gilmour (2006). "Control of electrical alternans in canine cardiac purkinje fibers." Physical Review Letters 96(10): 104101.
Alternation in the duration of consecutive cardiac action potentials (electrical alternans) may precipitate conduction block and the onset of arrhythmias. Consequently, suppression of alternans using properly timed premature stimuli may be antiarrhythmic. To determine the extent to which alternans control can be achieved in cardiac tissue, isolated canine Purkinje fibers were paced from one end using a feedback control method. Spatially uniform control of alternans was possible when alternans amplitude was small. However, control became attenuated spatially as alternans amplitude increased. The amplitude variation along the cable was well described by a theoretically expected standing wave profile that corresponds to the first quantized mode of the one-dimensional Helmholtz equation. These results confirm the wavelike nature of alternans and may have important implications for their control using electrical stimuli.
2. Cruceanu, M., R. J. Gorelick, K. Musier-Forsyth, I. Rouzina and M. C. Williams (2006). "Rapid kinetics of protein-nucleic acid interaction is a major component of HIV-1 nucleocapsid protein's nucleic acid chaperone function." Journal of Molecular Biology 363(5): 867-877.
The nucleic acid chaperone activity of the human immunodeficiency virus type-1 (HIV-1) nucleocapsid protein (NC) plays an important role in the retroviral life cycle, in part, by facilitating numerous nucleic acid rearrangements throughout the reverse transcription process. Recent studies have identified duplex destabilization and nucleic acid aggregation as the two major components of NC's chaperone activity. In order to better understand the contribution of the functional domains of NC to these two activities, we used optical tweezers to stretch single lambda DNA molecules through the helix-coil transition in the presence of wild-type or mutant HIV-1 NC. Protein-induced duplex destabilization was measured directly as an average decrease of the force-induced melting free energy, while NC's ability to facilitate strand annealing was determined by the amount of hysteresis in the DNA stretch-relax cycle. By studying zinc-free variants of full-length and truncated NC, the relative contributions of NC's zinc fingers and N-terminal basic domain to the two major components of chaperone activity were elucidated. In addition, examination of NC variants containing mutations affecting one or both zinc finger motifs showed that effective strand annealing activity is correlated with NC's ability to rapidly bind and dissociate from nucleic acids. NC variants with slow on/off rates are inefficient in strand annealing, even though they may still be capable of high affinity nucleic acid binding, duplex destabilization, and/or nucleic acid aggregation. Taken together, these observations establish the rapid kinetics of protein-nucleic acid interaction as another major component of NC's chaperone function. (c) 2006 Elsevier Ltd. All rights reserved.
3. Cruceanu, M., A. G. Stephen, P. J. Beuning, R. J. Gorelick, R. J. Fisher and M. C. Williams (2006). "Single DNA molecule stretching measures the activity of chemicals that target the HIV-1 nucleocapsid protein." Analytical Biochemistry 358(2): 159-170.
We develop a biophysical method for investigating chemical compounds that target the nucleic acid chaperone activity of HIV-1 nucleocapsid protein (NCp7). We used an optical tweezers instrument to stretch single ?,-DNA molecules through the helix-coil transition in the presence of NCp7 and various chemical compounds. The change in the helix-coil transition width induced by wild-type NCp7 and its zinc fingi.r variants correlates with in vitro nucleic acid chaperone activity measurements and in vivo assays. The compound-NC interaction measured here reduces NCpTs capability to alter the transition width. Purified compounds from the NO Diversity set, 119889, 119911, and 119913 reduce the chaperone activity of 5 nM NC in aqueous solution at 10, 25, and 100 nM concentrations respectively. Similarly,, gallein reduced the activity of 4 nM NC at 100 nM concentration. Further analysis allows us to dissect the impact of each compound on both sequence-specific and non-sequence-specific DNA binding of NC, two of the main components of NC's nucleic acid chaperone activity. These results suggest that DNA stretching experiments can be used to screen chemical compounds targeting NC proteins and to further explore the mechanisms by which these compounds interact with NC and alter its nucleic acid chaperone activity. (c) 2006 Elsevier Inc. All rights reserved.
4. Cruceanu, M., M. A. Urbaneja, C. V. Hixson, D. G. Johnson, S. A. Datta, M. J. Fivash, A. G. Stephen, R. J. Fisher, R. J. Gorelick, J. R. Casas-Finet, A. Rein, I. Rouzina and M. C. Williams (2006). "Nucleic acid binding and chaperone properties of HIV-1 Gag and nucleocapsid proteins." Nucleic Acids Research 34(2): 593-605.
The Gag polyprotein of HIV-1 is essential for retroviral replication and packaging. The nucleocapsid (NC) protein is the primary region for the interaction of Gag with nucleic acids. In this study, we examine the interactions of Gag and its NC cleavage products (NCp15, NCp9 and NCp7) with nucleic acids using solution and single molecule experiments. The NC cleavage products bound DNA with comparable affinity and strongly destabilized the DNA duplex. In contrast, the binding constant of Gag to DNA was found to be similar to 10-fold higher than that of the NC proteins, and its destabilizing effect on dsDNA was negligible. These findings are consistent with the primary function of Gag as a nucleic acid binding and packaging protein and the primary function of the NC proteins as nucleic acid chaperones. Also, our results suggest that NCp7's capability for fast sequence-nonspecific nucleic acid duplex destabilization, as well as its ability to facilitate nucleic acid strand annealing by inducing electrostatic attraction between strands, likely optimize the fully processed NC protein to facilitate complex nucleic acid secondary structure rearrangements. In contrast, Gag's stronger DNA binding and aggregation capabilities likely make it an effective chaperone for processes that do not require significant duplex destabilization.
5. Haxhimali, T., A. Karma, F. Gonzales and M. Rappaz (2006). "Orientation selection in dendritic evolution." Nature Materials 5(8): 660-664.
Dendritic crystal growth patterns have fascinated scientists for several centuries. Much of the aesthetic appeal of these patterns stems from the hierarchical structure of primary-, secondary-, and higher-order branches, which typically grow along principal crystallographic axes. Atypical growth directions have also been observed. Here, we demonstrate both computationally and experimentally that the range of possible dendrite growth directions, and hence the morphological diversity of the resulting dendritic structures, is much richer than previously anticipated. In particular, we show that primary dendrite growth directions can vary continuously between different crystallographic directions as a function of the composition- dependent anisotropy parameters. The study combines phase-field simulations of equiaxed dendritic growth and directional freezing of Al-Zn alloys. Both simulations and experiments exhibit continuous changes of direction from < 100 > to < 110 > for an underlying cubic symmetry. These results have important implications for controlling the microstructure of a wide range of cast alloys that solidify dendritically.
6. Ionascu, D., F. Rosca, F. Gruia, A. Yu and P. M. Champion (2006). "Optical scanning instrument for ultrafast pump-probe spectroscopy of biomolecules at cryogenic temperatures." Review of Scientific Instruments 77(6): 064303.
We demonstrate novel optical scanning and detection instrumentation that is particularly useful for the interrogation of stationary cryogenic samples in pump-probe spectroscopy. The technique uses a spinning lens to scan multiple laser beams over a stationary sample while maintaining the focal properties of the beams. This significantly lengthens the time window for the sample reset to equilibrium and improves the photostability of stationary samples. In addition, we describe a signal processing methodology that discriminates against the strong background signal that can arise from leakage of the pump laser pulse train into the detector. These techniques are particularly useful in pump-probe studies of ultrafast processes in biological systems where sample deterioration, pump induced thermal lensing, and light scattering into the detection channel (e.g., induced by light scattering from a cryogenic matrix) are problematic. Generally, the optical scanning and detection instrumentation described here enable the study of a variety of biological systems, without the need for intricate spinning or flowing sample containers, making it possible to perform ultrafast pump-probe experiments on stationary samples such as a cryogenic matrix. (c) 2006 American Institute of Physics.
7. Martinez, R. A., A. Karma and M. C. Flemings (2006). "Spheroidal particle stability in semisolid processing." Metallurgical and Materials Transactions a-Physical Metallurgy and Materials Science 37A(9): 2807-2815.
A model for diffusion-controlled spherical particle growth is presented and solved numerically, showing how, on cooling at a sufficient rate from a given fraction solid, growth velocity first increases and then decreases rapidly when solute fields of adjacent particles overlap. An approximate analytical solution for the spherical particle growth velocity is then developed and shown to be valid until the solute fields begin to overlap. A particle stability model is next presented, building on the preceding analytic solution. This model permits prediction of the maximum cooling rate at which a semisolid slurry or reheated semisolid billet can be cooled while still retaining the spherical growth morphology. The model shows that particle stability is favored by high particle density, high fraction solid, and low cooling rate. The predictions of the stability model are found to be in good quantitative agreement with experimental data collected for Al-4.5 wt pct Cu alloy. Engineering applications of the results obtained are discussed.
8. Mihailovic, A., L. Vladescu, M. McCauley, E. Ly, M. C. Williams, E. M. Spain and M. E. Nunez (2006). "Exploring the interaction of ruthenium(II) polypyridyl complexes with DNA using single-molecule techniques." Langmuir 22(10): 4699-4709.
Here we explore DNA binding by a family of ruthenium (II) polypyridyl complexes using anatomic force microscope (AFM) and optical tweezers. We demonstrate using AFM that Ru(bpy)(2)dppz(2+) intercalates into DNA (K-b = 1.5 x 10(5) M-1), as does its close relative Ru(bpy)(2)dppx(2+) (Kb = 1.5 x 10(5) M-1). However, intercalation by Ru(phen)(3)(2+) and other Ru(II) complexes with Kb values lower than that of Ru(bpy)(2)dppz(2+) is difficult to determine using AFM because of competing aggregation and surface-binding phenomena. At the high Ru(II) concentrations required to evaluate intercalation, most of the DNA strands acquire a twisted, curled conformation that is impossible to measure accurately. The condensation of DNA on mica in the presence of polycations is well known, but it clearly precludes the accurate assessment by AFM of DNA intercalation by most Ru(II) complexes, though not by ethidium bromide and other monovalent intercalators. When stretching individual DNA molecules using optical tweezers, the same limitation on high metal concentration does not exist. Using optical tweezers, we show that Ru(phen)(2)dppz(2+) intercalates avidly (K-b = 3.2 x 10(6) M-1) whereas Ru(bPY)(3)(2+) does not intercalate, even at micromolar ruthenium concentrations. Ru(phen)(3)(2+) is shown to intercalate weakly (i.e., at micromolar concentrations (Kb = 8.8 x 10(3) M-1)). The distinct differences in DNA stretching behavior between Ru(phen)(3)(2+) and Ru(bPY)(3)(2+) clearly illustrate that intercalation can be distinguished from groove binding by pulling the DNA with optical tweezers. Our results demonstrate both the benefits and challenges of two single-molecule methods of exploring DNA binding and help to elucidate the mode of binding of Ru(phen)(3)(2+).
9. Sato, D., Y. Shiferaw, A. Garfinkel, J. N. Weiss, Z. L. Qu and A. Karma (2006). "Spatially discordant alternans in cardiac tissue role of calcium cycling." Circulation Research 99(5): 520-527.
Spatially discordant alternans, where the action potential duration (APD) and intracellular calcium transient (Ca-i) alternate with opposite phase in different regions of tissue, is known to promote wave break and reentry. However, this phenomenon is not completely understood. It is known that alternans at the cellular level can be caused by dynamical instabilities arising from either membrane voltage (V-m) attributable to steep APD restitution or to calcium (Ca) cycling. Here, we used a mathematical model of intracellular Ca cycling, coupled with membrane ion currents, to investigate the dynamics of V-m and Ca-i transient alternans in an isolated cell, in two electrotonically coupled cells, and in 1D spatially homogeneous tissue. Our main finding is a novel instability mechanism in which the bidirectional coupling of V-m and Ca-i can drive the Ca-i transient of two neighboring cells to be out of phase. This instability is manifested in cardiac tissue by the dynamical formation of spatially discordant alternans. In this case, Ca-i transient alternans can reverse phase over a length scale of one cell, whereas APD alternans reverses phase over a much longer length scale set by the electrotonic coupling. We analyze this mechanism in detail and show that it is a robust consequence of experimentally established properties of the bidirectional coupling between Ca cycling and V-m dynamics. Finally, we address the experimental relevance of these findings and suggest physiological conditions under which these patterns can be observed.
10. Shiferaw, Y. and A. Karma (2006). "Turing instability mediated by voltage diffusion in paced cardiac cells." Proceedings of the National Academy of Sciences of the United States of America 103(15): 5670-5675.
In cardiac cells, the coupling between the voltage across the cell membrane (V-m) and the release of calcium (Ca) from intracellular stores is a crucial ingredient of heart function. Under abnormal conditions and/or rapid pacing, both the action potential duration and the peak Ca concentration in the cell can exhibit well known period-doubling oscillations referred to as "alternans," which have been linked to sudden cardiac death. Fast diffusion of Vm keeps action potential duration alternans spatially synchronized over the approximate to 150-mu m-length scale of a cell, but slow diffusion of Ca ions allows Ca alternans within a cell to become spatially asynchronous, as observed in some experiments. This finding raises the question: When are Ca alternans spatially in-phase or out-of-phase on subcellular length scales? This question is investigated by using a spatially distributed model of Ca cycling coupled to Vm. Our main finding is the existence of a Turing-type symmetry breaking instability mediated by V-m and Ca diffusion that causes Ca alternans to become spontaneously out-of-phase at opposite ends of a cardiac cell. Pattern formation is governed by the interplay of short-range activation of Ca alternans, because of a dynamical instability of Ca cycling, and long-range inhibition of Ca alternans by V-m alternans through Ca-sensitive membrane ionic currents. These results provide a striking example of a Turing instability in a biological context where the morphogens can be clearly identified, as well as a potential link between dynamical instability on subcellular scales and life-threatening cardiac disorders.
11. Shiferaw, Y., Z. L. Qu, A. Garfinkel, A. Karma and J. N. Weiss (2006). "Nonlinear dynamics of paced cardiac cells." Interactive and Integrative Cardiology 1080: 376-394.
When a cardiac cell is rapidly paced it can exhibit a beat-to-beat alternation in the action potential duration (APD) and the intracellular calcium transient. This dynamical instability at the cellular level has been shown to correlate with the genesis of cardiac arrhythmias and has motivated the application of nonlinear dynamics in cardiology. In this article, we review mathematical approaches to describe the underlying mechanisms for alternans using beat-to-beat iterated maps. We explain the development and properties of these maps, and show that they provide a fruitful framework to understand dynamical instabilities of voltage and calcium in paced cardiac cells.
12. Shokri, L., B. Marintcheva, C. C. Richardson, I. Rouzina and M. C. Williams (2006). "Single molecule force spectroscopy of salt-dependent bacteriophage T7 gene 2.5 protein binding to single-stranded DNA." Journal of Biological Chemistry 281(50): 38689-38696.
The gene 2.5 protein (gp2.5) encoded by bacteriophage T7 binds preferentially to single-stranded DNA. This property is essential for its role in DNA replication and recombination in the phage-infected cell. gp2.5 lowers the phage lambda DNA melting force as measured by single molecule force spectroscopy. T7 gp2.5-Delta 26C, lacking 26 acidic C-terminal residues, also reduces the melting force but at considerably lower concentrations. The equilibrium binding constants of these proteins to single-stranded DNA (ssDNA) as a function of salt concentration have been determined, and we found for example that gp2.5 binds with an affinity of (3.5 +/- 0.6) x 10(5) M-1 in a 50mM Na+ solution, whereas the truncated protein binds to ssDNA with a much higher affinity of (7.8 +/- 0.9) x 10(7) M-1 under the same solution conditions. T7 gp2.5-Delta 26C binding to single-stranded DNA also exhibits a stronger salt dependence than the full-length protein. The data are consistent with a model in which a dimeric gp2.5 must dissociate prior to binding to ssDNA, a dissociation that consists of a weak non-electrostatic and a strong electrostatic component.
13. Sokoloff, J. B. (2006). "Theory of the effects of multiscale surface roughness and stiffness on static friction." Physical Review E 73(1): 016104.
It is shown on the basis of simple scaling arguments that an interface between two three-dimensional elastic solids, consisting of completely flat disordered surfaces, which interact with interatomic hard core interactions, will be in a weak pinning regime, and hence exhibit negligibly small static friction. It is argued, however, that the presence of roughness on multiple length scales can lead to much larger friction (i.e., static friction coefficients not too much smaller than 1), as is characteristic of most solid surfaces. This approach suggests a possible way of understanding why coatings of materials with high elastic constants are often excellent lubricants.
14. Sun, D. Y., M. I. Mendelev, C. A. Becker, K. Kudin, T. Haxhimali, M. Asta, J. J. Hoyt, A. Karma and D. J. Srolovitz (2006). "Crystal-melt interfacial free energies in hcp metals: A molecular dynamics study of Mg." Physical Review B 73(2): 024116.
Crystal-melt interfacial free energies (gamma) are computed for hcp Mg by employing equilibrium molecular-dynamics (MD) simulations and the capillary-fluctuation method (CFM). This work makes use of a newly developed embedded-atom-method (EAM) interatomic potential for Mg fit to crystal, liquid, and melting properties. We describe how the CFM, which has previously been applied to cubic systems only, can be generalized for studies of hcp metals by employing a parametrization for the orientation dependence of gamma in terms of hexagonal harmonics. The method is applied in the calculation of the Turnbull coefficient (alpha) and crystalline anisotropies of gamma. We obtain a value of alpha=0.48, with interfacial free energies for different high-symmetry orientations differing by approximately 1%. These results are compared to those obtained in previous MD-CFM studies for cubic EAM metals as well as experimental studies of solid-liquid interfaces in hcp alloys. In addition, the implications of our results for the prediction of dendrite growth directions in hcp metals are discussed.
15. Trautt, Z. T., M. Upmanyu and A. Karma (2006). "Interface mobility from interface random walk." Science 314(5799): 632-635.
Computational studies aimed at extracting interface mobilities require driving forces orders of magnitude higher than those occurring experimentally. We present a computational methodology that extracts the absolute interface mobility in the zero driving force limit by monitoring the one-dimensional random walk of the mean interface position along the interface normal. The method exploits a fluctuation-dissipation relation similar to the Stokes-Einstein relation, which relates the diffusion coefficient of this Brownian-like random walk to the interface mobility. Atomic-scale simulations of grain boundaries in model crystalline systems validate the theoretical predictions and highlight the profound effect of impurities. The generality of this technique, combined with its inherent spatiotemporal efficiency, should allow computational studies to effectively complement experiments in understanding interface kinetics in diverse material systems.
16. van Thor, J. J. and J. T. Sage (2006). "Charge transfer in green fluorescent protein." Photochemical & Photobiological Sciences 5(6): 597-602.
Charge transfer reactions that contribute to the photoreactions of the wild type green fluorescent protein (GFP) do not occur in the isolated p-hydroxybenzylidene-imidazolidinone chromophore, demonstrating the role of the protein environment. The high quantum efficiency of the fluorescence photocycle that includes excited state proton transfer and the suppression of non-radiative pathways by the protein environment have been correlated with structural dynamics in the chromophore environment. A low quantum efficiency competing phototransformation reaction of GFP is accompanied by both proton and electron transfer, and closely mimics the charge redistribution that is occurring in the fluorescence photocycle. The protein response to this destabilising event has been demonstrated by cryo-trapping of early products in the reaction pathway and is found to be strong even at 100 K, including displacements of chromophore, protein, solvent and a photogenerated CO2 molecule derived from the decarboxylated Glu 222 side chain. We discuss the rami. cations of the observation of strong conformational perturbations below the protein dynamical transition at similar to 200 K, in view of low temperature work on other light sensitive proteins such as myoglobin and bacteriorhodopsin. The proton and electron transfer in the phototransformation pathway mimics the proton and charge transfer which occurs during the fluorescence cycle, which leads to common structural responses in both photoreactions as shown by ultrafast spectroscopy. We review and discuss literature on light-induced and thermal charge transfer events, focusing on recent findings addressing conformational dynamics and implications for thermodynamic properties.
17. Weiss, J. N., A. Karma, Y. Shiferaw, P. S. Chen, A. Garfinkel and Z. Qu (2006). "From pulsus to Pulseless the saga of cardiac alternans." Circulation Research 98(10): 1244-1253.
Computer simulations and nonlinear dynamics have provided invaluable tools for illuminating the underlying mechanisms of cardiac arrhythmias. Here, we review how this approach has led to major insights into the mechanisms of spatially discordant alternans, a key arrhythmogenic factor predisposing the heart to re-entry and lethal arrhythmias. During spatially discordant alternans, the action potential duration (APD) alternates out of phase in different regions of the heart, markedly enhancing dispersion of refractoriness so that ectopic beats have a high probability of inducing reentry. We show how, at the cellular level, instabilities in membrane voltage (ie, steep APD restitution slope) and intracellular Ca (Ca-i) cycling dynamics cause APD and the Cai transient to alternate and how the characteristics of alternans are affected by different "modes" of the bidirectional coupling between voltage and Cai. We illustrate how, at the tissue level, additional factors, such as conduction velocity restitution and ectopic beats, promote spatially discordant alternans. These insights have illuminated the mechanistic basis underlying the clinical association of cardiac alternans (eg, T wave alternans) with arrhythmia risk, which may lead to novel therapeutic approaches to avert sudden cardiac death.
18. Williams, M. C., L. Rouzina and R. L. Karpel (2006). "Thermodynamics and kinetics of DNA-protein interactions from single molecule force spectroscopy measurements." Current Organic Chemistry 10(4): 419-432.
When single DNA molecules are stretched, the measurement of the resulting force as a function of extension has yielded new information about the physical, chemical, and biological properties of these important molecules. It has been shown that double-stranded DNA molecules undergo a force-induced melting transition at high forces. Force-extension measurements of single DNA molecules using optical tweezers allow us to measure the stability of DNA under a variety of solution conditions and in the presence of DNA binding proteins. Here we review our studies of DNA force-induced melting in the presence of the classical single-stranded DNA binding protein, gene 32 protein. Bacteriophage T4 gene 32 protein (gp32) is a well studied representative of a large class of single-stranded DNA binding proteins, which are essential for the replication, recombination and repair of DNA. We have developed several new single molecule methods, which when applied to gp32, have led to significant new insights about this protein's structure-function relationships. We discuss a technique for measuring K-ss, the association constant of these proteins to ssDNA, which we can determine over a large range of salt concentrations not available to bulk binding studies. In addition, we have measured the noncooperative association constants (K-ds) of the weak but biologically-significant interaction with double-stranded DNA as a function of salt concentration for full-length protein and *I, a truncation of gp32 lacking the 48-residue C-terminal domain. Our results have led to a quantitative model for the salt dependence of protein binding, which we postulate to be regulated by a salt-dependent conformational change within the-protein involving the C-terminal domain. With this new force spectroscopy technique, we have obtained binding rates and binding free energies for these interactions Under a broad range of conditions. Our methodologies should have useful applications in many areas of DNA-protein interactions.
19. Wu, K. A., A. Karma, J. J. Hoyt and M. Asta (2006). "Ginzburg-Landau theory of crystalline anisotropy for bcc-liquid interfaces." Physical Review B 73(9): 094101.
The weak anisotropy of the interfacial free energy gamma is a crucial parameter influencing dendritic crystal growth morphologies in systems with atomically rough solid-liquid interfaces. The physical origin and quantitative prediction of this anisotropy are investigated for body-centered-cubic (bcc) forming systems using a Ginzburg-Landau theory where the order parameters are the amplitudes of density waves corresponding to principal reciprocal lattice vectors. We find that this theory predicts the correct sign gamma(100 >)gamma(110) and magnitude (gamma(100)-gamma(110))/(gamma(100)+gamma(110))approximate to 1% of this anisotropy in good agreement with the results of molecular dynamics (MD) simulations for Fe. The results show that the directional dependence of the rate of spatial decay of solid density waves into the liquid, imposed by the crystal structure, is a main determinant of anisotropy. This directional dependence is validated by MD computations of density wave profiles for different reciprocal lattice vectors for {110} crystal faces. Our results are contrasted with the prediction of the reverse ordering gamma(100)<gamma(110) from an earlier formulation of Ginzburg-Landau theory.
20. Ye, X., A. C. Yu and P. M. Champion (2006). "Dynamics of nitric oxide rebinding and escape in horseradish peroxidase." Journal of the American Chemical Society 128(5): 1444-1445.