2008 - Abstracts
1. Escobar, G., T. Fares and A. Stepanyants (2008). "Structural plasticity of circuits in cortical neuropil." J Neurosci 28(34): 8477-88.
Learning and memory formation in the brain depend on the plasticity of neural circuits. In the adult and developing cerebral cortex, this plasticity can result from the formation and elimination of dendritic spines. New synaptic contacts appear in the neuropil where the gaps between axonal and dendritic branches can be bridged by dendritic spines. Such sites are termed potential synapses. Here, we describe a theoretical framework for the analysis of spine remodeling plasticity. We provide a quantitative description of two models of spine remodeling in which the presence of a bouton is either required or not for the formation of a new synapse. We derive expressions for the density of potential synapses in the neuropil, the connectivity fraction, which is the ratio of actual to potential synapses, and the number of structurally different circuits attainable with spine remodeling. We calculate these parameters in mouse occipital cortex, rat CA1, monkey V1, and human temporal cortex. We find that, on average, a dendritic spine can choose among 4-7 potential targets in rodents and 10-20 potential targets in primates. The potential of neuropil for structural circuit remodeling is highest in rat CA1 (7.1-8.6 bits/mum(3)) and lowest in monkey V1 (1.3-1.5 bits/mum(3)). We also evaluate the lower bound of neuron selectivity in the choice of synaptic partners. Postsynaptic excitatory neurons in rodents make synaptic contacts with >21-30% of presynaptic axons encountered with new spine growth. Primate neurons appear to be more selective, making synaptic connections with >7-15% of encountered axons.
2. Gruia, F., D. Ionascu, M. Kubo, X. Ye, J. Dawson, R. L. Osborne, S. G. Sligar, I. Denisov, A. Das, T. L. Poulos, J. Terner and P. M. Champion (2008). "Low-frequency dynamics of Caldariomyces fumago chloroperoxidase probed by femtosecond coherence Spectroscopy." Biochemistry 47(18): 5156-5167.
Ultrafast laser spectroscopy techniques are used to measure the low-frequency vibrational coherence spectra and nitric oxide rebinding kinetics of Caldariomyces fumago chloroperoxidase (CPO). Comparisons of the CPO coherence spectra with those of other heme species are made to gauge the protein-specific nature of the low-frequency spectra. The coherence spectrum of native CPO is dominated by a mode that appears near 32-33 cm(-1) at all excitation wavelengths,with a phase that is consistent with a ground-state Raman-excited vibrational wavepacket. On the basis of a normal coordinate structural decomposition (NSD) analysis, we assign this feature to the thiolate-bound heme doming mode. Spectral resolution of the probe pulse ("detuned" detection) reveals a mode at 349 cm-1, which has been previously assigned using Raman spectroscopy to the Fe-S stretching mode of native CPO. The ferrous species displays a larger degree of spectral inhomogeneity than the ferric species, as reflected by multiple shoulders in the optical absorption spectra. The inhomogeneities are revealed by changes in the coherence spectra at different excitation wavelengths. The appearance of a mode close to 220 cm(-1) in the coherence spectrum of reduced CPO excited at 440 nm suggests that a subpopulation of five coordinated histidine-ligated hemes is present in the ferrous state at a physiologically relevant pH. A significant increase in the amplitude of the coherence signal is observed for the resonance with the 440 nm subpopulation. Kinetics measurements reveal that nitric oxide binding to ferric and ferrous CPO can be described as a single-exponential process, with rebinding time constants of 29.4 +/- 1 and 9.3 +/- 1 ps, respectively. This is very similar to results previously reported for nitric oxide binding to horseradish peroxidase.
3. Gruia, F., M. Kubo, X. Ye and P. M. Champion (2008). "Investigations of vibrational coherence in the low-frequency region of ferric heme proteins." Biophysical Journal 94(6): 2252-2268.
Femtosecond coherence spectroscopy is applied to a series of ferric heme protein samples. The low-frequency vibrational spectra that are revealed show dominant oscillations near 40 cm(-1). MbCN is taken as a typical example of a histidine-ligated, six-coordinate, ferric heme and a comprehensive spectroscopic analysis is carried out. The results of this analysis reveal a new heme photoproduct species, absorbing near 418 nm, which is consistent with the photolysis of the His(93) axial ligand. The photoproduct undergoes subsequent rebinding/recovery with a time constant of similar to 4 ps. The photoproduct lineshapes are consistent with a photolysis quantum yield of 75-100%, although the observation of a relatively strong six-coordinate heme coherence near 252 cm(-1) (assigned to nu(9) in the MbCN Raman spectrum) suggests that the 75% lower limit is much more likely. The phase and amplitude excitation profiles of the low-frequency mode at 40 cm(-1) suggest that this mode is strongly coupled to the IMbCN photoproduct species and it is assigned to the doming mode of the transient penta-coordinated material. The absolute phase of the 40 cm(-1) mode is found to be pi/2 on the red side of 418 nm and it jumps to 3 pi/2 as excitation is tuned to the blue side of 418 nm. The absolute phase of the 40 cm(-1) signal is not explained by the standard theory for resonant impulsive stimulated Raman scattering. New mechanisms that give a dominant momentum impulse to the resonant wavepacket, rather than a coordinate displacement, are discussed. The possibilities of heme iron atom recoil after photolysis, as well as ultrafast nonradiative decay, are explored as potential ways to generate the strong momentum impulse needed to understand the phase properties of the 40 cm(-1) mode.
4. Gruia, F., M. Kubo, X. Ye, D. Ionascu, C. Lu, R. K. Poole, S. R. Yeh and P. M. Champion (2008). "Coherence spectroscopy investigations of the low-frequency vibrations of heme: Effects of protein-specific perturbations." Journal of the American Chemical Society 130(15): 5231-5244.
Femtosecond coherence spectroscopy is used to probe the low-frequency (20-200 cm(-1)) vibrational modes of heme proteins in solution. Horseradish peroxidase (HRP), myoglobin (Mb), and Campylobacter jejuni globin (Cgb) are compared and significant differences in the coherence spectra are revealed. It is concluded that hydrogen bonding and ligand charge do not strongly affect the low-frequency coherence spectra and that protein-specific deformations of the heme group lower its symmetry and control the relative spectral intensities. Such deformations potentially provide a means for proteins to tune heme reaction coordinates, so that they can perform a broad array of specific functions. Native HRP displays complex spectral behavior above similar to 50 cm(-1) and very weak activity below similar to 50 cm(-1). Binding of the substrate analog, benzhydroxamic acid, leads to distinct changes in the coherence and Raman spectra of HRP that are consistent with the stabilization of a heme water ligand. The CN derivatives of the three proteins are studied to make comparisons under conditions of uniform heme coordination and spin-state. MbCN is dominated by a doming mode near 40 cm(-1), while HRPCN displays a strong oscillation at higher frequency (96 cm(-1)) that can be correlated with the saddling distortion observed, in the X-ray structure. In contrast, CgbCN displays low-frequency coherence spectra that contain strong modes near 30 and 80 cm(-1), probably associated with a combination of heme doming and ruffling. HRPNO displays a strong doming mode near 40 cm(-1) that is activated by photolysis. The damping of the coherent motions is significantly reduced when the heme is shielded from solvent fluctuations by the protein material and reduced still further when T less than or similar to 50 K, as pure dephasing processes due to the protein-solvent phonon bath are frozen out.
5. Kubo, M., F. Gruia, A. Benabbas, A. Barabanschikov, W. R. Montfort, E. M. Maes and P. M. Champion (2008). "Low-frequency mode activity of heme: Femtosecond coherence spectroscopy of iron porphine halides and nitrophorin." Journal of the American Chemical Society 130(30): 9800-9811.
The low-frequency mode activity of metal loporphyrins has been studied for iron porphine-halides (Fe(P)(X), X = CI, Br) and nitrophorin 4 (NP4) using femtosecond coherence spectroscopy (FCS) in combination with polarized resonance Raman spectroscopy and density functional theory (DFT). It is confirmed that the mode symmetry selection rules for FCS are the same as for Raman scattering and that both Franck-Condon and Jahn-Teller mode activities are observed for Fe(P)(X) under Soret resonance conditions. The DFT-calculated low-frequency (20-400 cm(-1)) modes, and their frequency shifts upon halide substitution, are in good agreement with experimental Raman and coherence data, so that mode assignments can be made. The doming mode is located at similar to 80 cm(-1) for Fe(P)(CI) and at similar to 60 cm(-1) for Fe(P)(Br). NP4 is also studied with coherence techniques, and the NO-bound species of ferric and ferrous NP4 display a mode at similar to 30-40 cm(-1) that is associated with transient heme doming motion following NO photolysis. The coherence spectra of three ferric derivatives of NP4 with different degrees of heme ruffling distortion are also investigated. We find a mode at similar to 60 cm(-1) whose relative intensity in the coherence spectra depends quadratically on the magnitude of the ruffling distortion. To quantitatively account for this correlation, a new "distortion-induced" Raman enhancement mechanism is presented. This mechanism is unique to low-frequency "soft modes" of the molecular framework that can be distorted by environmental forces. These results demonstrate the potential of FCS as a sensitive probe of dynamic and functionally important nonplanar heme vibrational excitations that are induced by the protein environmental forces or by the chemical reactions in the aqueous phase.
6. Mahajan, A., Y. Shiferaw, D. Sato, A. Baher, R. Olcese, L. H. Xie, M. J. Yang, P. S. Chen, J. G. Restrepo, A. Karma, A. Garfinkel, Z. Qu and J. N. Weiss (2008). "A rabbit ventricular action potential model replicating cardiac dynamics at rapid heart rates." Biophysical Journal 94(2): 392-410.
Mathematical modeling of the cardiac action potential has proven to be a powerful tool for illuminating various aspects of cardiac function, including cardiac arrhythmias. However, no currently available detailed action potential model accurately reproduces the dynamics of the cardiac action potential and intracellular calcium (Ca-i) cycling at rapid heart rates relevant to ventricular tachycardia and fibrillation. The aim of this study was to develop such a model. Using an existing rabbit ventricular action potential model, we modified the L-type calcium (Ca) current (I-Ca,I-L) and Cai cycling formulations based on new experimental patch-clamp data obtained in isolated rabbit ventricular myocytes, using the perforated patch configuration at 35-37 degrees C. Incorporating a minimal seven-state Markovian model of I-Ca,I-L that reproduced Ca- and voltage-dependent kinetics in combination with our previously published dynamic Ca-i cycling model, the new model replicates experimentally observed action potential duration and Ca-i transient alternans at rapid heart rates, and accurately reproduces experimental action potential duration restitution curves obtained by either dynamic or S1S2 pacing.
7. McCauley, M. J., L. Shokri, J. Sefcikova, C. Venclovas, P. J. Beuning and M. C. Williams (2008). "Distinct Double- and Single-Stranded DNA Binding of E. coli Replicative DNA Polymerase III alpha Subunit." ACS Chem Biol.
The alpha subunit of the replicative DNA polymerase III of Escherichia coli is the active polymerase of the 10-subunit bacterial replicase. The C-terminal region of the alpha subunit is predicted to contain an oligonucleotide binding (OB-fold) domain. In a series of optical tweezers experiments, the alpha subunit is shown to have an affinity for both double- and single-stranded DNA, in distinct subdomains of the protein. The portion of the protein that binds to double-stranded DNA stabilizes the DNA helix, because protein binding must be at least partially disrupted with increasing force to melt DNA. Upon relaxation, the DNA fails to fully reanneal, because bound protein interferes with the reformation of the double helix. In addition, the single-stranded DNA binding component appears to be passive, as the protein does not facilitate melting but instead binds to single-stranded regions already separated by force. From DNA stretching measurements we determine equilibrium association constants for the binding of alpha and several fragments to dsDNA and ssDNA. The results demonstrate that ssDNA binding is localized to the C-terminal region that contains the OB-fold domain, while a tandem helix-hairpin-helix (HhH) 2 motif contributes significantly to dsDNA binding.
8. Pant, K., L. Shokri, R. L. Karpel, S. W. Morrical and M. C. Williams (2008). "Modulation of T4 gene 32 protein DNA binding activity by the recombination mediator protein UvsY." Journal of Molecular Biology 380(5): 799-811.
Bacteriophage T4 UvsY is a recombination mediator protein that promotes assembly of the UvsX-ssDNA presynaptic filament. UvsY helps UvsX to displace T4 gene 32 protein (gp32) from ssDNA, a reaction necessary for proper formation of the presynaptic filament. Here we use DNA stretching to examine UvsY interactions with single DNA molecules in the presence and absence of gp32 and a gp32 C-terminal truncation (*I), and show that in both cases UvsY is able to destabilize gp32-ssDNA interactions. In these experiments UvsY binds more strongly to dsDNA than ssDNA due to its inability to wrap ssDNA at high forces. To support this hypothesis, we show that ssDNA created by exposure of stretched DNA to glyoxal is strongly wrapped by UvsY, but wrapping occurs only at low forces. Our results demonstrate that UvsY interacts strongly with stretched DNA in the absence of other proteins. In the presence of gp32 and *I, UvsY is capable of strongly destabilizing gp32-DNA complexes in order to facilitate ssDNA wrapping, which in turn prepares the ssDNA for presynaptic filament assembly in the presence of UvsX. Thus, UvsY mediates UvsX binding to ssDNA by converting rigid gp32-DNA filaments into a structure that can be strongly bound by UvsX. (C) 2008 Elsevier Ltd. All rights reserved.
9. Paramanathan, T., F. Westerlund, M. J. McCauley, I. Rouzina, P. Lincoln and M. C. Williams (2008). "Mechanically manipulating the DNA threading intercalation rate." Journal of the American Chemical Society 130(12): 3752-+.
The dumbbell shaped binuclear ruthenium complex Delta Delta-P requires transiently melted DNA in order to thread through the DNA bases and intercalate DNA. Because such fluctuations are rare at room temperature, the binding rates are extremely low in bulk experiments. Here, single DNA molecule stretching is used to lower the barrier to DNA melting, resulting in direct mechanical manipulation of the barrier to DNA binding by the ligand. The rate of DNA threading depends exponentially on force, consistent with theoretical predictions. From the observed force dependence of the binding rate, we demonstrate that only one base pair must be transiently melted for DNA threading to occur.
10. Shokri, L., M. J. McCauley, L. Rouzina and M. C. Williams (2008). "DNA overstretching in the presence of glyoxal: Structural evidence of force-induced DNA melting." Biophysical Journal 95(3): 1248-1255.
When a long DNA molecule is stretched beyond its B-form contour length, a transition occurs in which its length increases by a factor of 1.7, with very little force increase. A quantitative model was proposed to describe this transition as force-induced melting, where double-stranded DNA is converted into single-stranded DNA. The force-induced melting model accurately describes the thermodynamics of DNA overstretching as a function of solution conditions and in the presence of DNA binding ligands. An alternative explanation suggests a transformation into S-DNA, a double-stranded form which preserves the interstrand base pairing. To determine the extent to which DNA base pairs are exposed to solution during the transition, we held DNA overstretched to different lengths within the transition in the presence of glyoxal. If overstretching involved strand separation, then force-melted basepairs would be glyoxal-modified, thus essentially permanently sing le-stranded. Subsequent stretches confirm that a significant fraction of the DNA melted by force is permanently melted. This result demonstrates that DNA overstretching is accompanied by a disruption of the DNA helical structure, including a loss of hydrogen bonding.
11. Sokoloff, J. B. (2008). "Theory of the observed ultralow friction between sliding polyelectrolyte brushes." Journal of Chemical Physics 129(1): 014901.
It is shown using a method based on a modified version of the mean field theory of Miklavic and Marcelja [J. Phys. Chem. 92, 6718 (1988)] that it should be possible for osmotic pressure due to the counterions associated with the two polyelectrolyte polymer brush coated surfaces to support a reasonable load (i.e., about 10(6) Pa) with the brushes held sufficiently far apart to prevent entanglement of polymers belonging to the two brushes, thus avoiding what is believed to be the dominant mechanisms for static and dry friction. (C) 2008 American Institute of Physics.
12. Sokoloff, J. B. and I. Webman (2008). "Theory of depinning of monolayer films adsorbed on a quartz crystal microbalance." Physical Review E 77(4): 042601.
In quartz crystal microbalance studies of the friction between an adsorbed monolayer film and a metallic substrate, the films are observed to slide relative to the substrate under inertial forces of order 10(-14) dyn per film atom, a force much smaller than all existing theoretical estimates of the force that surface defects are capable of exerting on the film. We argue that defect potentials with a range comparable to an atomic spacing or more will produce a pinning force below the inertial force.
13. Stepanyants, A., J. A. Hirsch, L. M. Martinez, Z. F. Kisvaday, A. S. Ferecsko and D. B. Chklovskii (2008). "Local potential connectivity in cat primary visual cortex." Cerebral Cortex 18(1): 13-28.
Time invariant description of synaptic connectivity in cortical circuits may be precluded by the ongoing growth and retraction of dendritic spines accompanied by the formation and elimination of synapses. On the other hand, the spatial arrangement of axonal and dendritic branches appears stable. This suggests that an invariant description of connectivity can be cast in terms of potential synapses, which are locations in the neuropil where an axon branch of one neuron is proximal to a dendritic branch of another neuron. In this paper, we attempt to reconstruct the potential connectivity in local cortical circuits of the cat primary visual cortex (V1). Based on multiple single-neuron reconstructions of axonal and dendritic arbors in 3 dimensions, we evaluate the expected number of potential synapses and the probability of potential connectivity among excitatory (pyramidal and spiny stellate) neurons and inhibitory basket cells. The results provide a quantitative description of structural organization of local cortical circuits. For excitatory neurons from different cortical layers, we compute local domains, which contain their potentially pre- and postsynaptic excitatory partners. These domains have columnar shapes with laminar specific radii and are roughly of the size of the ocular dominance column. Therefore, connections between most excitatory neurons in the ocular dominance column can be implemented by local synaptogenesis. Structural connectivity involving inhibitory basket cells is generally weaker than excitatory connectivity. Here, only nearby neurons are capable of establishing more than one potential synapse, implying that within the ocular dominance column these connections have more limited potential for circuit remodeling.
14. Stewart-Maynard, K. M., M. Cruceanu, F. Wang, M. N. Vo, R. J. Gorelick, M. C. Williams, I. Rouzina and K. Musier-Forsyth (2008). "Retroviral Nucleocapsid Proteins Display Non-Equivalent Levels of Nucleic Acid Chaperone Activity." J Virol.
Human immunodeficiency virus type-1 (HIV-1) nucleocapsid protein (NC) is a nucleic acid chaperone that facilitates remodeling of nucleic acids during various steps of the viral life cycle. Two main features of NC's chaperone activity are its ability to aggregate and destabilize nucleic acids. These functions are associated with NC's highly basic character and with its zinc finger domains, respectively. While the chaperone activity of HIV-1 NC has been extensively studied, less is known about the chaperone activity of other retroviral NCs. In this work, complementary experimental approaches are used to characterize and compare the chaperone activity of NC proteins from four different retroviruses: HIV-1, Moloney murine leukemia virus (MLV), Rous sarcoma virus (RSV) and human T-cell lymphotropic virus type-1 (HTLV-1). The different NCs exhibit significant differences in their overall chaperone activity, as demonstrated by gel-shift annealing assays, decreasing in the order HIV-1 approximately RSV > MLV >> HTLV-1. In addition, whereas HIV-1, RSV and MLV NCs are effective aggregating agents, HTLV-1 NC, which exhibits poor overall chaperone activity, is unable to aggregate nucleic acids. Equilibrium binding measurements to single- and double-stranded oligonucleotides suggest that all four NC proteins have moderate duplex destabilization capability. Single-molecule DNA stretching studies reveal striking differences in the kinetics of nucleic acid dissociation between the NC proteins, revealing an excellent correlation between nucleic acid dissociation kinetics and overall chaperone activity.
15. van Thor, J. J., K. L. Ronayne, M. Towrie and J. T. Sage (2008). "Balance between ultrafast parallel reactions in the green fluorescent protein has a structural origin." Biophysical Journal 95(4): 1902-1912.
The fluorescence photocycle of the green fluorescent protein is functionally dependent on the specific structural protein environment. A direct relationship between equilibrium protein side-chain conformation of glutamate 222 and reactivity is established, particularly the rate of ultrafast proton transfer reactions in the fluorescence photocycle. We show that parallel transformations in the photocycle have a structural origin, and we report on the vibrational properties of responsive amino acids on an ultrafast timescale. Blue excitation of GFP drives two parallel, excited-state deuteron transfer reactions with 10 ps and 75 ps time constants to the buried carboxylic acid side chain of glutamate 222 via a hydrogen-bonding network. Assignment of 1456 cm(-1) and 1441 cm(-1) modes to nsym and assignment of 1564 cm(-1) and 1570 cm(-1) features to nu(asym) of E222 in the 10 ps and 75 ps components, respectively, was possible from the analysis of the transient absorption data of an E222D mutant and was consistent with photoselection measurements. In contrast to the wild-type, measurements of E222D can be described with only one difference spectrum, with the nsym mode at 1435 cm(-1) and the nasym mode at 1567 cm(-1), also correlating a large Delta nu(asym-sym) with slow excited-state proton transfer kinetics. Density Functional Theory calculations and published model compound and theoretical studies relate differences in Delta nu(asym-sym) to the strength and number of hydrogen-bonding interactions that are detected via equilibrium geometry and COO-stretching frequency differences of the carboxylate. The correlation of photocycle kinetics with side-chain conformation of the acceptor suggests that proton transfer from S205 to E222 controls the rate of the overall excited-state proton transfer process, which is consistent with recent theoretical predictions. Photoselection measurements show agreement for localized C = O vibrations of chromophore, Q69, and E222 with Density Functional Theory and ab initio calculations placed in the x-ray geometry and provide their vibrational response in the intermediates in the photocycle.
16. Williams, M. C., I. Rouzina and R. L. Karpel (2008). "Quantifying DNA-Protein interactions by single molecule stretching." Biophysical Tools for Biologists: Vol 1 in Vitro Techniques 84: 517-+.
In this chapter, we discuss a new method for quantifying DNA-protein interactions. A single double-stranded DNA (dsDNA) molecule is stretched beyond its contour length, causing the base pairs to break while increasing the length from that of dsDNA to that of ssDNA. When applied in a solution containing DNA binding ligands, this method of force-induced DNA melting can be used to quantify the free energy of ligand binding, including the free energy of protein binding. The dependence of melting force on protein concentration is used to obtain the equilibrium binding constant of the ligand to DNA. We have applied this method to a well-studied DNA-binding protein, bacteriophage T4 gene 32 protein (gp32), and have obtained binding constants for the protein to single-stranded DNA (ssDNA) under a wide range of solution conditions. Our analysis of measurements conducted at several salt concentrations near physiological conditions indicates that a salt-dependent conformational change regulates DNA binding by gp32.
17. Zeng, W., A. Barabanschikov, Y. Zhang, J. Zhao, W. Sturhahn, E. E. Alp and J. T. Sage (2008). "Synchrotron-derived vibrational data confirm unprotonated oxo ligand in myoglobin compound II." Journal of the American Chemical Society 130(6): 1816
Recent structural investigations have generated uncertainty regarding the protonation state of the exogenous oxo ligand in ferryl derivatives of several heme proteins. We used nuclear resonance vibrational spectroscopy (NRVS) to reveal the complete spectrum of Fe-ligand modes for compound II of myoglobin. Comparison with vibrational DFT predictions allows us to identify vibrations involving FeO tilting, coupled with stretching of the Fe-N bonds to the heme, and stretching of the proximal Fe-His bond, in addition to the previously observed Fe-O stretching vibration. Additional calculations, coupled with measurements on the hydroxyl derivative of metmyoglobin, reveal vibrational signatures for the putative protonated ferryl species. These include a 33 cirri splitting of the FeO tilling modes due to the asymmetrically placed proton, as well as a 250 cm(-1) decrease of the Fe-O stretching frequency. The vibrational data suggest a fully deprotonated oxo ligand in compound II.