(5 µM) on the pre-treated surface.

<p>The curves, (generated as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044287#pone-0044287-g006" target="_blank">Figure 6</a>) were fit to: <b>A</b>: a single exponential dissociation model (red line) or <b>B</b>: a single association model (red line). Calculated rate constants from these fits gave the following values: k_a = 3.7 10⁶ M⁻¹s⁻¹; k_d = 1.8 10⁻³s⁻¹; K_D = 4.9 10⁻¹⁰ M.</p

SPRi kinetic curves of PFV-1 IN (200 nM) interacting with immobilized dsDNA on the pre-treated surface.

<p>The values for % reflectivity were obtained from direct CCD camera measurements averaged across each spot shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044287#pone-0044287-g005" target="_blank">Figure 5</a> and as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044287#pone.0044287-Bouffartigues2" target="_blank">[27]</a>. <b>A</b>: Changes in % reflectivity at selected spots on the SPRi surface as a function of time as PFV-1 IN passes over the prism surface. The curves show binding to spots containing different concentrations of DNA (1, 2.5, 5 and 10 µM) or to reference spots (containing no DNA) outside of the zone to which DNA was applied (designated as background). <b>B</b>: The kinetic curve after subtraction of the background for reaction taking place on a spot where 10 µM DNA solution was deposited. The red line is a fit carried out by applying a single exponential model where is the % reflectivity at time <i>t</i>; is the amplitude of the phase, and the observed rate constant , is the association rate constant, is the dissociation rate constant calculated from a simple exponential fit of the dissociation phase using and [<i>C</i>] is the concentration of PFV-1 IN (200 nM). C: same curve as shown in <b>B</b> but fitted (red line) with a double exponential model for both association and dissociation. The model for association is and dissociation is obtained from where is the % reflectivity at time <i>t</i>; and and are the respective dissociation rate constants for the two phases and ; and .</p

SPRi difference images of the biochip surface at different times during the course of the experiment.

<p>The EG4 adsorption time was 30 s. 4 different SH-5T-dsDNA solution concentrations were spotted in rows from right to left: 10, 5, 2.5 and 1 µM respectively. Each SH-5T-dsDNA concentration was spotted 3 times in the same column. <b>A</b>: t = 0 s, no PFV-1 IN protein injected. <b>B</b>, <b>C</b>, and <b>D</b> are images taken at t = 18 s, t = 3 min and t = 5 min respectively after 200 nM PFV-1 IN injection. <b>E</b> is the image taken 12 min after the injection was stopped. <b>F</b>, <b>G</b> and <b>H</b> are images taken at t = 9 s, t = 2 min, t = 6 min and t = 10 min after the start of the 0.1% SDS injection.</p

Calculated rate constants from fits of the type shown in Figure 5C.

<p>Values are shown based on binding to the four DNA concentrations spotted on the surface; errors refer to standard error for the rate constants calculated for each DNA concentration. The equilibrium dissociation constants K_D1 and K_D2 were calculated from the ratio of the respective kd/ka values.</p

Cartoon describing the model for PFV-1 IN binding to DNA and subsequent accretion of proteins.

<p><b>A</b>: PFV-1 IN binds to the DNA; <b>B</b>: the nucleoprotein complex then binds more protein; <b>C</b>: which in turn continues to oligomerise until, <b>D</b>: the protein completely covers the DNA. The molecules are not to scale nor do they depict actual known orientations. The rate and equilibrium constants associated with B and C may be interchanged, as the model does not allow attribution of either to the structural forms suggested. The PFV-1 IN is represented in the monomeric form for simplicity.</p

Densities of DNA immobilized on pre-treated surfaces.

<p>The amount of DNA retained at the surfaces was calculated as described in the experimental section. <b>A</b>: Density of dsDNA with (OH-5T-dsDNA) and without (OH-dsDNA) the T₍₅₎ spacer on pre-treated surface. <b>B</b>: Density of the thiolated dsDNA with (SH-5T-dsDNA) and without (SH-dsDNA) the T₍₅₎ spacer on pre-treated surface. <b>C</b>: Density of immobilized DNA on the pre-treated gold surface as a function of the EG4 SAM immersion time ranging from 30 s to 2 hrs. SAM adsorption time = 0 corresponds to the bare gold surface. The SH*-5T-dsDNA and C*-dsDNA indicates which of the DNA strands were ³²P radiolabelled, namely the thiolated and the complementary strand respectively. OH-dsDNA refers to a ³²P radiolabelled non-thiolated dsDNA. All the dsDNA strands have the same sequence. <b>D</b>: Relative amount of double stranded DNA adsorbed on the surface after the pre-treated gold surface was immersed in 10 µM dsDNA solutions. The % dsDNA adsorbed is calculated from the ratio between C*-dsDNA and SH*-5T-dsDNA, 100% corresponding to the SH*-5T-dsDNA density. The error bars correspond to the dispersion after accumulating 8 experiments for each EG4 adsorption time.</p

Density of DNA adsorbed on a pretreatedEG4 SAM as a function of the applied SH-5T-dsDNA concentration.

<p>The incubation time of the EG4 SAM is 30 s. The error bars correspond to the dispersion of the results over 4 separate measurements.</p

SPRi difference images of PFV-1 IN interacting with the biochip surface.

<p>ssDNA solutions at 50, 25, 10, 6, 3, 1, 0.5 and 0.1 µM were spotted in replicates on the pre-treated surface. The entire spot image was between 0.8 and 0.9 cm in diameter and each individual spot was between 400 and 450 µm in diameter. The spotting, of decreasing ssDNA concentration, starts from left to right, and downward. <b>A</b>: ssDNA spotted on EG4 pre-treated surfaces, PFV-1 IN at 200 nM was flowed across the SPRi surfaces containing the spotted ssDNA molecules. Difference images during the injection are shown in panels <b>a</b> and <b>b</b>, while panel <b>c</b> is a difference image after the injection of the protein was stopped. Panel <b>d</b> and <b>e</b> shows difference images during the injection of a solution of 0.1% SDS across the surface during the dissociation phase of PFV-1 IN. Panel <b>f</b> shows difference images of PFV-1 IN retained at the surface after the SDS injection. <b>B</b>: Surfaces were prepared as previously by spotting ssDNA at various concentrations on the pre-treated surface. Then the complementary ssDNA oligomers was flowed across the surface in order to hybridize and form dsDNA before injecting PFV-1 IN at 200 nM across the SPRi surface. Panels <b>a</b> to <b>c</b> are difference images of PFV-1 IN flowing across the SPRi surface and panel d is a difference image after the injection of the protein was stopped. Panel <b>e</b> and <b>f</b> are difference images during and after the injection of a 0.1% SDS solution.</p

Surface plasmon resonance measurements for H-NS binding to DNA fragments immobilized on a surface

<p><b>Copyright information:</b></p><p>Taken from "Rapid coupling of Surface Plasmon Resonance (SPR and SPRi) and ProteinChip™ based mass spectrometry for the identification of proteins in nucleoprotein interactions"</p><p></p><p>Nucleic Acids Research 2007;35(6):e39-e39.</p><p>Published online 7 Feb 2007</p><p>PMCID:PMC1874600.</p><p>© 2007 The Author(s)</p> () SPR measurements on surfaces containing immobilized 5A6A DNA fragments were carried out on a BIAcore 2000™ as described in Materials and Methods section. Sensorgrams are shown of H-NS at various concentrations (20–600 nM) flowing across the 5A6A surface. () SPRi measurements on surfaces containing immobilized 5A6A DNA fragments. (i) Images of the prism surface of the ™ SPR device (GenOptics) containing immobilized DNA fragments. The spots have been circled in this representation with the name of the respective DNA fragment in each circle. The DNA was applied at a concentration of 10 µg/ml (∼50 nM) for all fragments. Each spot has a surface area of ∼0.78 mm. H-NS (500 µl of 500 nM) was flowed across the surface at 50 µl/min (thus contact time = 10 min) in binding buffer. (ii) Kinetic curve of the binding of H-NS (500 nM) to the prism surface. Images were taken at 1 s intervals and the relative change in resonance response plotted as a function of time of injection. The arrows show the image associated with a specific time point on the curve

Schematic representation of the JCV NCCRs.

<p>The NCCR is delimited by the translation start sites of early and late regions. A highly conserved region which includes ORI is followed by sections <i>a</i>, <i>b</i>, <i>c</i>, <i>d</i>, <i>e</i> and <i>f</i> in the archetype <i>at</i>-NCCR. These regions have been defined because of their deletion and/or repeat in variant NCCRs. Accordingly, NCCRs have been classified into four groups [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199171#pone.0199171.ref025" target="_blank">25</a>]: I-S, I-R (like Mad-1 and Mad4), II-S and II-R (like Mad-7 and Mad-8). NCCRs of type I do not contain insert whereas NCCRs of type II present an insertion of at least a portion of the sequence from one of the sections <i>b</i> or <i>d</i>. The sub-types “S” (for singular) do not present a repeat whereas the subtypes “R” (for repeat) do. NCCRs of type II-S are also named archetype or archetype-like if deletions occur. NCCRs of type II-R have generally been termed rearranged NCCRs (“<i>rr</i>-NCCR”) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199171#pone.0199171.ref025" target="_blank">25</a>]. Numbers indicate the base-pair length of each section within CY <i>at</i>-NCCR [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199171#pone.0199171.ref012" target="_blank">12</a>].</p