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ABSTRACT: The endosomal sorting complex required for transport (ESCRT) system traffics ubiquitinated cargo to lysosomes via an unusual membrane budding reaction that is directed away from the cytosol. Here, we show that human ESCRT-II self-assembles into clusters of 10-100 molecules on supported lipid bilayers. The ESCRT-II clusters are functional in that they bind to ubiquitin and the ESCRT-III subunit VPS20 at nanomolar concentrations on membranes with the same stoichiometries observed in solution and in crystals. The clusters only form when cholesterol is included in the lipid mixture at >10 mol %. The clusters induce the formation of ordered membrane domains that exclude the dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbo-cyanine perchlorate. These results show that ESCRT complexes are capable of inducing lateral lipid phase separation under conditions where the lipids themselves do not spontaneously phase-separate. This property could facilitate ESCRT-mediated membrane budding.
Journal of Biological Chemistry 06/2012; 287(33):28144-51. · 4.77 Impact Factor
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ABSTRACT: Integration of viral DNA into the host genome is an essential step in retroviral replication that is mediated by a stable nucleoprotein complex comprising a tetramer of integrase bridging the two ends of the viral DNA in a stable synaptic complex (SSC) or intasome. Assembly of HIV-1 intasomes requires several hundred base pairs of nonspecific internal DNA in addition to the terminal viral DNA sequence that is protected in footprinting experiments. We find that only one of the viral DNA ends in the intasome requires long-nonspecific internal DNA for intasome assembly. Although intasomes are unstable in solution when the nonspecific internal DNA is cut off after assembly, they are stable in agarose gels. These complexes are indistinguishable from SSCs with nonspecific internal DNA in Förster resonance energy transfer (FRET) experiments suggesting the interactions with the viral DNA and integrase tetramer are the same regardless of the presence of nonspecific internal DNA. We discuss models of how the internal DNA contributes to intasome assembly and stability. FRET is exquisitely sensitive to the distance between the fluorophores and given certain assumptions can be translated to distance measurements. We anticipated that a set of such distance constraints would provide a map of the DNA path within the intasome. In reality, the constraints we could impose from the FRET data were quite weak allowing a wide envelope for the possible path. We discuss the difficulties of converting the FRET signal to absolute distance within nucleoprotein complexes.
Protein Science 11/2011; 21(2):249-57. · 2.80 Impact Factor
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ABSTRACT: Min proteins of the Escherichia coli cell division system oscillate between the cell poles in vivo. In vitro on a solid-surface supported lipid bilayer, these proteins exhibit a number of interconverting modes of collective ATP-driven dynamic pattern formation including not only the previously described propagating waves, but also near uniformity in space surface concentration oscillation, propagating filament like structures with a leading head and decaying tail and moving and dividing amoeba-like structures with sharp edges. We demonstrate that the last behavior most closely resembles in vivo system behavior. The simple reaction-diffusion models previously proposed for the Min system fail to explain the results of the in vitro self-organization experiments. We propose the hypotheses that initiation of MinD binding to the surface is controlled by counteraction of initiation and dissociation complexes; the binding of MinD/E is stimulated by MinE and involves polymerization-depolymerization dynamics; polymerization of MinE over MinD oligomers triggers dynamic instability leading to detachment from the membrane. The physical properties of the lipid bilayer are likely to be one of the critical determinants of certain aspects of the dynamic patterns observed.
Proceedings of the National Academy of Sciences 03/2010; 107(18):8071-8. · 9.68 Impact Factor
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ABSTRACT: The objective of this report is to provide a practical and improved method for estimating Förster resonance energy transfer distance measurement error due to unknown angles in the dipole orientation factor based on emission anisotropy measurements. We improve on the method of Dale et al. (1979), which has minor mistakes and is frequently interpreted in overly optimistic ways in the literature. To facilitate proper fluorescence intensity measurements, we also evaluated instrument parameters that could impact the measurement. The apparent fluorescence intensity of isotropic samples depends on the sample emission anisotropy, fluorometer geometry, and optical apertures. We separate parameters of the sample, and those of the cylindrically symmetric illumination source and detector in the equations describing results of unpolarized and polarized fluorescence intensity measurements. This approach greatly simplifies calculations compared with the more universal method of Axelrod (1989). We provide a full computational method for calculating the Förster resonance energy transfer distance error and present a graph describing distance error in the simplest case.
Biophysical Journal 09/2009; 97(3):922-9. · 3.65 Impact Factor
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ABSTRACT: This paper presents a review of largely our own work on the DNA melting transition, and some new measurements of the elastic energy of sharp bends in single-stranded DNA and RNA. The purpose is to present the point of view that studying the transition of intermediate size oligomers leads to valuable tests of the models, in particular the ingredients most important for a reduced-degrees-of-freedom description, such as the different role of base pairing and base stacking. We make the case that, with intermediate size oligomers, one can actually measure the bubble length, which exhibits a more interesting behavior than the fraction of dissociated bases alone. Here is where more work seems necessary, both on the experimental and the modeling side, to understand the differences between theory and experiments. We summarize our previous results on the cooperativity parameters, which suggest that the transition is never exactly two-state no matter how short the molecule, or in other words the nucleation size for bubbles opening at the ends of the molecule is essentially 1 base pair. We briefly discuss our own modification of the nearest-neighbor model which treats pairing and stacking separately, as a way to fit the experimental melting profiles in this intermediate length regime. Finally we go on to present some new measurements on the stability of DNA and RNA hairpins with very short loops.
Journal of Physics Condensed Matter 01/2009; 21(3):034102. · 2.55 Impact Factor
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ABSTRACT: We propose a statistical mechanics model for the melting transition of DNA. Base pairing and stacking are treated as separate degrees of freedom, and the interplay between pairing and stacking is described by a set of local rules which mimic the geometrical constraints in the real molecule. This microscopic mechanism intrinsically accounts for the cooperativity related to the free energy penalty of bubble nucleation. The model describes both the unpairing and unstacking parts of the spectroscopically determined experimental melting curves. Furthermore, the model explains the observed temperature dependence of the effective thermodynamic parameters used in models of the nearest neighbor type. We compute the partition function for the model through the transfer matrix formalism, which we also generalize to include nonlocal chain entropy terms.
Physical Review E 05/2005; 71(4 Pt 1):041909. · 2.26 Impact Factor
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ABSTRACT: We propose a statistical mechanics model for DNA melting in which base stacking and pairing are explicitly introduced as distinct degrees of freedom. Unlike previous approaches, this model describes thermal denaturation of DNA secondary structure in the whole experimentally accessible temperature range. Base pairing is described through a zipper model, base stacking through an Ising model. We present experimental data on the unstacking transition, obtained exploiting the observation that at moderately low pH this transition is moved down to experimentally accessible temperatures. These measurements confirm that the Ising model approach is indeed a good description of base stacking. On the other hand, comparison with the experiments points to the limitations of the simple zipper model description of base pairing.
Physical Review E 12/2004; 70(5 Pt 1):051907. · 2.26 Impact Factor
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ABSTRACT: We report here the study of the transition of a B-form of d(CGCGCGCGCGCG) to the Z-form as a function of the ions strength of PBS buffer. We used circular dichroism (CD) spectroscopy. Decomposition CD spectra by linear superposition provides evidence for one or more intermediate states that are distinct from the B- and Z-forms of DNA. Linear decomposition is performed according to the locally linearized model (LLM) modified by the explicit introduction of the weight function essentially present in any quadratic integral of the spectra.
10/2003;
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ABSTRACT: We demonstrate the detection of nanometer-scale conformational changes of single DNA oligomers through a micromechanical technique. The quantity monitored is the displacement of a micrometer-size bead tethered to a surface by the probe molecule undergoing the conformational change. This technique allows probing of conformational changes within distances beyond the range of fluorescence resonance energy transfer. We apply the method to detect single hybridization events of label-free target oligomers. Hybridization of the target is detected through the conformational change of the probe.
Proceedings of the National Academy of Sciences 07/2003; 100(13):7605-10. · 9.68 Impact Factor
