182-Pos Board B61 Mechanism Of DNA Translocation By SpoIIIE/Ftsk Marcelo Nollmann 1 , Jerod Ptacin 2 , Eric Becker 3 , Kit Pogliano 3 , Carlos Bustamante 4 . 1 CNRS U554, Montpellier, France, 2 Stanford University, Palo Alto, CA, USA, 3 University of California, San Diego, La Jolla, CA, USA, 4 University of California, Berkeley, Berkeley, CA, USA. During cell division, chromosomal DNA is segregated both rapidly and specif- ically. In bacteria, chromosomal segregation is conducted by the DNA translo- cases FtsK (Escherichia coli) and SpoIIIE (Bacillus subtilis).These enzymes assemble at the division septum and translocate the chromosome (~400 kb and ~3 Mbp, respectively) unidirectionally by using the energy of ATP hydrolysis by means of their AAAþ motor domains. Despite functional and sequence similarities, SpoIIIE and FtsK were proposed to use drastically different mech- anisms: SpoIIIE was suggested to be a unidirectional DNA transporter that exports DNA from the compartment in which it assembles, whereas FtsK was shown to establish translocation directionality by interacting with highly skewed chromosomal sequences. Here, we use a combination of single-mole- cule, bioinformatics and in vivo fluorescence methodologies to unveil the mechanism of DNA translocation by SpoIIIE, and to show that its wirestripping activity is largely responsible for compartment-specific gene expression during sporulation. We propose a sequence-directed DNA exporter model that recon- ciles previously proposed models for SpoIIIE and FtsK, and constitutes the first unified model for directional DNA transport by the SpoIIIE/FtsK/Tra family of AAAþ ring ATPases. 183-Pos Board B62 VirE2: A Unique ssDNA-Compacting Molecular Machine Martin Hegner 1 , Wilfried Grange 1 , Sudhir Husale 2 , Myriam Duckely 3 . 1 Trinity College Dublin, Dublin, Ireland, 2 Rowland Institute at Harvard, Boston, MA, USA, 3 Novartis AG, Basel, Switzerland. The translocation of single-stranded DNA (ssDNA) across membranes of two cells is a fundamental biological process occurring in both bacterial conjuga- tion and Agrobacterium pathogenesis. Whereas bacterial conjugation spreads antibiotic resistance, Agrobacterium facilitates efficient interkingdom transfer of ssDNA from its cytoplasm to the host plant cell nucleus. These processes rely on the Type IV secretion system (T4SS), an active multiprotein channel spanning the bacterial inner and outer membranes. T4SSs export specific pro- teins, among them relaxases, which covalently bind to the 5 0 end of the trans- located ssDNA and mediate ssDNA export. In Agrobacterium tumefaciens, another exported protein_VirE2_enhances ssDNA transfer efficiency 2000- fold. VirE2 binds cooperatively to the transferred ssDNA (T-DNA) and forms a compact helical structure, mediating T-DNA import into the host cell nucleus. We demonstrated_using single-molecule techniques_that by cooperatively binding to ssDNA, VirE2 proteins act as a powerful molecular machine. VirE2 actively pulls ssDNA and is capable of working against 50-pN loads with- out the need for external energy sources. Combining biochemical and cell biol- ogy data, we suggest that, in vivo, VirE2 binding to ssDNA allows an efficient import and pulling of ssDNA into the host. These findings provide a new insight into the ssDNA translocation mechanism from the recipient cell perspective. Ef- ficient translocation only relies on the presence of ssDNA binding proteins in the recipient cell that compacts ssDNA upon binding. This facilitated transfer could hence be a more general ssDNA import mechanism also occurring in bacterial conjugation and DNA uptake processes. 184-Pos Board B63 Experimental Evidence Of Polymer Hydrophobic Collapse Due To Water Density Fluctuation Isaac T.S. Li 1 , Nikhil Gunari 2 , Gilbert C. Walker 1,2 . 1 University of Toronto, Toronto, ON, Canada, 2 University of Pittsburgh, Pittsburgh, PA, USA. Hydrophobic collapse of protein chain is a major driving force for its folding in water. However, the detailed mechanism of hydrophobic collapse is not clear. One compelling theory explains the hydrophobic collapse due to the density fluctuation of water creating local voids in the vicinity of a hydrophobic poly- mer chain that drives its collapse. This density fluctuation happens rapidly in the time scale of picoseconds and is therefore difficult to detect directly. How- ever, a result of the fluctuation is the much longer lasting collapsed structure of hydrophobic polymer chains, which can be detected by single molecule force spectroscopy (SMFS). We performed experiments to investigate the effect of water density fluctuation in the collapse of single polystyrene (PS) chains using the atomic force microscope (AFM). In particular, we applied a constant ten- sion to single PS chain and detected end-to-end distance fluctuation, which directly probes the collapsed state of the PS chain and provides us insight to the hydrophobic collapse of the molecule. 185-Pos Board B64 Non-ideal Elasticity Of Single Stranded DNA At Low Forces Dustin McIntosh, Omar Saleh, Phil Pincus, Noah Ribeck. UC Santa Barbara, Santa Barbara, CA, USA. Single molecule force-extension experiments on dsDNA and ssDNA are usu- ally compared to the worm-like chain (WLC) or freely-jointed chain (FJC) models. The WLC and FJC are ideal polymer models: they account for local stiffness through the persistence or Kuhn length, but ignore long-range inter- actions between monomers, accounted for by, e.g., the excluded-volume parameter in the Flory scaling theory, that determine the self-avoiding ran- dom-walk structure of polymers not under tension. To attempt to bridge the gap between ideal force/extension models and the classic scaling picture of real polymers, we explore the low-force elasticity of chemically-denatured single-stranded DNA (ssDNA) using magnetic tweezers. We find a low-force regime where extension grows as a non-linear power-law with force, in con- tradiction with ideal predictions, but in agreement with scaling predictions made by P. Pincus (Macromolecules 9, 386 (1976)). By analyzing this power-law regime, we extract the dependence of the Kuhn length of ssDNA on monovalent salt concentration, and find that it is linearly proportional to the Debye screening length. This result is in contrast to the quadratic depen- dence predicted in the well-known OSF theory, but agrees with other theories and simulations. Finally, we identify a high-salt point ([NaCl] ~ 3 M) where ideal polymer behavior returns, and the data is well-fit by the WLC model at all forces. We identify this as a theta point of the polymer, and show that, be- yond 3M salt, the ssDNA aggregates in a manner consistent with a polymer in poor solution. 186-Pos Board B65 Single Molecule Force Spectroscopy of Guanine Quadruplex DNA Susanna Lynch 1 , Heather Baker 1 , Sarah Byker 1 , Dejian Zhou 2 , Kumar Sinniah 1 . 1 Calvin College, Grand Rapids, MI, USA, 2 University of Leeds, Leeds, United Kingdom. A single molecule force spectroscopy study was performed to examine the formation and stability of guanine quadruplexes using various G-rich oligo- mers. The study examined the differences in interactions of four-stranded and two-stranded G-quadruplexes. The two-stranded G-quadruplex system was used for dynamic force spectroscopy measurements. Rupture force depen- dency on the log loading rate was found to be non-linear. Using recently de- veloped microscopic models, the apparent kinetic and thermodynamic param- eters for the G-quadruplex system were estimated. The microscopic models predict barrier widths that are consistent with the bond length of the quadru- plex strand. 187-Pos Board B66 Kalman Filter Estimates of the Contour Length of an Unfolding Protein in Single-Molecule Force Spectroscopy Experiments Vicente I. Fernandez 1 , Pallav Kosuri 2 , Vicente Parot 3 , Julio M. Fernandez 2 . 1 Massachusetts Institute of Technology, Boston, MA, USA, 2 Columbia University, New York, NY, USA, 3 Pontificia Universidad Cato ´lica de Chile, Santiago, Chile. Force spectroscopy measurements of single molecules using AFM have enabled the study of a range of molecular properties not accessible with bulk methods. These properties of interest must typically be inferred by manually fitting models to selected portions of measured data. As manual intervention in the fitting process easily introduces a bias in the analysis, there is a need for more sophisticated analysis methods capable of interpreting data in an unbiased and repeatable ‘‘hands-off’’ manner. Here we apply an extended Kalman filter to the estimation of protein contour length (L c ) during mechanical unfolding, based on force and extension data from an AFM experiment. This filter provides an online and fully automated estimate of L c based on a system model, the exper- imental measurements, and noise statistics. The system model comprises a physical model of the cantilever and a nonlinear WLC approximation of the ex- tended protein. When manually fitting the WLC model to force-extension data from ubiquitin proteins, the estimate of the change in contour length during un- folding is distributed normally as N (22.7 nm, 6.59 nm 2 ). Testing the Kalman filter on the same protein yields DL c ~ N (24.54 nm, 0.24 nm 2 ). As the variance limits resolution in estimating the number of amino acids released by unfolding, it is clear that the Kalman filter presents a substantial improvement over the con- ventional method. We thereby demonstrate that the Kalman filter provides a powerful unbiased approach to interpreting force spectroscopy data, capable of increasing resolution beyond the traditional experimental limit. Due to the flexibility of this approach, it can be extended to monitoring other state variables of molecular systems observed by various forms of force spectroscopy, includ- ing optical and magnetic tweezers. 36a Sunday, March 1, 2009