Direct measure of cooperativity in DM-mediated peptide association.

<p>(a) Association rate of peptides to DR1 in the presence of DM was measured as described in Results. Data is plotted as the fraction of either peptide or DR1 forming complex. Reactions were performed in triplicate, and data points represent one of three independent experiments. Error bars are omitted for graphic clarity. Lines fit the data to a four parameter double exponential function. (b) Natural log (ln) plot of cooperativity (expected/observed <i>K</i>_eq) vs. association rate for each peptide tested. Error bars are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003722#pone-0003722-g001" target="_blank">figure 1b</a>. The line indicates the fit of the data to a linear regression.</p

DM mediated peptide exchange as function of reactant concentration.

<p>(a) Requirement of equimolar exchange peptide for initiating exchange. DM-mediated dissociation of the peptide HAS from DR1 measured in the presence of different concentration of unlabeled HA in excess as described in Results. The exchange peptide to complex ratio for each reaction is identified in the legend. Data points represent the mean and SD of three independent experiments, and lines represent the fit of the data to a five parameter double exponential decay function. (b) FP analysis of DM-catalyzed peptide binding to and release from DR. CLIP/DR complex at different concentrations (100, 300, 900 nM) was incubated with 3 fold DM and allowed to dissociate in absence of any free peptide. Simultaneously, loading of FAM-CLIP to an equimolar amount of DR at the same concentrations was measured. Reactions were set up in triplicates, and the average ±SDs are shown. Lines represent the fit of the data to either a five parameter double exponential decay or four parameters double exponential raise function. (c) Peptide release in the absence of exchange peptide is not a function of complex concentration. FP analysis of DM-catalyzed release of HAD from DR at four different concentrations in absence of any free peptide. At <i>t</i> = 1000 after steady state was reached, unlabeled peptide was added at an equimolar concentration to the complex at <i>t</i> = 0. Reactions were set up in triplicates, and the average values for each time point are shown. (d) DM-mediated binding is a function of reactant concentration. FP analysis of DM-catalyzed association of HAD to equimolar empty DR at the same four different concentrations as in panel B. Lines represent the fit of the data to a four parameter double exponential function. For (c) and (d), due to the small SD, error bars are hidden below data points.</p

Free peptide is a co-factor in DM-mediated peptide release.

<p>(a) Competitive binding analysis of P1, P3 and P7 MTSL-Cys substituted HA peptide variants to DR1. Data represent the mean and SD of three independent experiments. Lines indicate the fit of the data to a logistic equation. <i>K</i>_D values for the peptides as listed in the legend are respectively 83.3 nM, 28.8 μM, 237.9 nM and 404.7 nM. (b) Top view of the HA peptide P7 Leu→Cys substituted, labeled at this position with MTSL (HAsp7) and complexed with HLA-DR. The peptide is in orange, the α-chain is in green and the β-chain in yellow. The most energetically favored orientation of the probe (white) is shown. The model was generated using PyMol <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003722#pone.0003722-DeLano1" target="_blank">[45]</a>. Coordinates taken from ref. 41. (c) Spectra of HAsp7 peptide as recorded when free in solution (top), completely bound to HLA-DR (center) or as a composite of the two states (bottom). The broadening of the spectral lines and a slight shift in their positions in the spectrum appear evident. (d) Dissociation of HAsp7 peptide from DR1 was measured as described in Results. Data is plotted as the % of free (unbound) peptide detected at each time point as quantitated by spectral subtraction methods. Data points represent one of three independent experiments. Data points referring to the C sample at <i>t</i> = 0 and <i>t</i> = 24 are hidden below other data points.</p

Cooperative effect on peptide dissociation from HLA-DR1 in presence of DM is evidenced at the level of the exchange peptide.

<p>(a) Dissociation rate of peptides from HLA-DR1 in the presence of DM was measured as described in Results. Data is plotted as the fraction of DR1/labeled peptide complex remaining relative to <i>t</i> = 0. Reactions were performed in triplicate, and data points represent one of two independent experiments. Lines fit the data to a single exponential decay function. (b) Natural log (ln) plot of cooperativity (expected/observed <i>t</i>_1/2) vs. DM-mediated (solid line) and intrinsic (dashed line) dissociation rate for each DR1/peptide complex tested. To facilitate the comparison, data points were plotted on different scales for <i>t</i>_1/2 values. Top x-axis scale refers to intrinsic dissociation rate. Bottom x-axis scale refers to DM-mediated off rate. Since we defined cooperativity <i>C</i> as the ratio of the expected to observed values for Δ<i>t</i>_1/2, and <i>t</i>_1/2 is directly proportional to stability, the cooperative effect is positive if 0≤<i>C</i><1, while if C>1 the cooperative effect is negative. In the ln plot, positive cooperativity in stability is indicated on the y-axis by values <0 and negative cooperativity by values >0. Horizontal error bars represent the SD of the <i>t</i>_1/2 measurement. Vertical error bars represent the error of cooperativity as calculated through SE propagation. Lines indicate the fit of the data to a linear regression. (c) DM-mediated dissociation of the HAS peptide from DR1. The nature of the competing peptide present in excess during the reaction is identified in the legend. Data points represent the mean of two independent experiments, and lines represent the fit of the data to a two or three parameter single exponential decay function. (d) Natural log (ln) plot of cooperativity (expected/observed t_1/2) vs. dissociation rate of DR1/HAS complex for each multiple substituted exchange peptide tested. Error bars are as in panel B. The line indicates the fit of the data to a linear regression. (e) Intrinsic dissociation of the HAS peptide from DR1. The nature of the competing peptide present in excess during the reaction is identified in the legend. Data points represent the mean of two independent experiments, and lines represent the fit of the data to a two or three parameter single exponential decay function. (f) Natural log (ln) plot of cooperativity (expected/observed t_1/2) vs. dissociation rate of DR1/HAS complex for each multiple substituted exchange peptide tested. Error bars are as above. The line indicates the fit of the data to a linear regression. (g) The ratio of <i>t</i>_1/2 for the DR1/HAS complex measured in the presence of different exchange peptides and DM as compared to the <i>t</i>_1/2 measured in the presence of HAS is plotted as function of the natural log of exchange peptide <i>K</i>_D. The line indicate the fit of the data to an exponential function (r² = 0.98). (h) The ratio of <i>t</i>_1/2 for the DR1/HAS complex measured in the presence of different exchange peptides and in absence of DM as compared to the <i>t</i>_1/2 measured in the presence of HAS is plotted as function of the natural log of exchange peptide <i>K</i>_D. The line indicate the fit of the data to an exponential function (r² = 0.97).</p

Co-localization of prebound and free peptide during DM-mediated exchange.

<p>(a) DM-mediated and intrinsic dissociation of HAD from DR1 was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003722#s4" target="_blank">Methods</a>. The nature of the exchange peptide present in excess during the reaction is identified in the legend. Data points represent the mean of three independent experiments, and lines represent the fit of the data to a five parameter double exponential decay function. (b) Reduction of the overall fluorescence due to FRET between FAM-labeled HAD and QSY7-HA (shaded bars) either with (striped bars) or without (squared bars) DM as compared with the fluorescence detected in presence of unlabeled free HA peptide during the dissociation of HAD from DR1 (dark bars). Plain bars represent the fluorescence detected in the following reactions: HAD (dark bar), HAD+QSY7-HA (shaded bar) and HAD+QSY7-HA+DM.</p

FP analysis of the role of free peptide in DM-mediated peptide dissociation.

<p>(a) Real-time analysis of DR1/HAC stability as described in Results. (b) Real-time FP analysis of DR1/CLIP stability as described in Results. For (a) and (b) initial reaction conditions are identified in the legend. Data is plotted as the % of bound peptide detected at a certain time point. Reactions were performed in triplicate, and data points represent one of two independent experiments. (c) DM-mediated dissociation of the CLIP peptide from DR1. The nature of the exchange peptide present in excess during the reaction is identified in the legend. Data points represent the mean of three independent experiments, and lines represent the fit of the data to a five parameter double exponential decay function. Due to the small SD, error bars are hidden below data points.</p