Larval abundance in different pH treatments over time.

<p>The relationship between logarithmic larval abundance and time for four different pH treatments. Lines were plotted using coefficients from the GLMM. The first two sampling points were on days 3 and 6, after which the larval sampling was conducted every other day. Each replicate (n = 20) for each sampling point is shown.</p

Results of the GLMM (Poisson error distribution, log link function and replicate identity as a random factor).

<p>Effects of pH treatment and time on larval abundance.</p><p>All two-way interactions between time and pH treatment were significant and are denoted in bold.</p

Larval sizes at the end of the 20-day experiment in different pH treatments.

<p>Larval sizes are illustrated as size frequency distributions in pH treatments (A) 7.2, (B) 7.4, (C) 7.7 and (D) 8.1, and presented with mode values illustrating the increase of large size fractions in the control treatment.</p

Larval sizes at the start and end of the experiment in different pH treatments.

<p>Mean ±SD, n = 105.</p

Mean pH levels during the experiment.

<p>Measured from all replicate bottles (n = 5) in each treatment. For clarity SD are not shown.</p

Indication of shell dissolution in different pH treatments.

<p>The left picture shows the situation after the 20-day experiment in control conditions, picturing several empty shells of <i>M. balthica</i> larvae. The right picture is from the pH_7.2 at the same time point, with the majority of the dead shells absent, implying a high dissolution of the shell material in low pH conditions.</p

Distinguishing orthosteric and allosteric effects in Hsp90.

<p><b>(A)</b> The absolute difference in numbers of deuterons (y-axis) between the free and ligand bound state is plotted for each pepsin digest fragment listed from the N to C terminus (x-axis) of Hsp90 for each deuterium exchange time point (t = 0.5, 2, 5, 10 min) in a ‘difference plot’. Shifts in the positive scale represent decreases in deuterium exchange and shifts in the negative scale represent increases in deuterium exchange when compared to apo-Hsp90. Regions showing significant differences above a threshold of 0.5 Da (red dashed line) are compared with orthosteric sites (blue boxes) to predict allosteric regions. Peptides highlighted in red show regions showing differences in distal allosteric regions, not involved in orthosteric binding. Peptides spanning these regions are marked in red boxes and divided into four allosteric regions A1 to A4. Radicicol and 17-AAG shows differences in A1 and A2, while only radicicol showed changes in A3 and A4. Time points are colored according to key. <b>(B)</b> Predicted allosteric regions are mapped on to the structure of Hsp90 (red), together with the orthosteric regions, in blue. Radicicol bound at the ligand binding pocket is shown as sticks (PDB ID: 4EGK).</p

Fragments 1 and 2 differ in the nature of the allosteric effect in Hsp90.

<p><b>(A)</b> The absolute difference in numbers of deuterons (inferred from difference in mass in Daltons (Da) (y-axis) between the free and ligand bound state is plotted for each pepsin digest fragment listed from the N to C terminus (x-axis) of Hsp90 for each deuterium exchange time point (t = 0.5, 2, 5, 10 min) in a ‘difference plot’. Shifts in the positive scale represent decreases in deuterium exchange and shifts in the negative scale represent increases in deuterium exchange when compared to the apo-Hsp90. Regions showing significant differences above a threshold of 0.5 Da (red dashed line) are compared with orthosteric sites (blue boxes) to establish allosteric regions (red boxed). Fragment <b>2</b> does not show any changes in region A4, similar to 17-AAG, while fragment <b>1</b> shows differences, similar to Radicicol. In addition, fragment <b>1</b> shows an allosteric response at the regions A5 (residues 201–213 shown in orange box), which is not observed in the other three ligands. Time points are colored according to key. <b>(B,C)</b> The identified orthosteric (blue) and allosteric regions (red) for fragments are mapped on to the structure of Hsp90 in blue. <b>(C)</b> The allosteric site A5 in Hsp90, which is observed only fragment <b>2</b> is highlighted in orange. Radicicol bound at the ligand binding pocket is shown as sticks (PDB ID: 4EGK).</p

Mapping protein-ligand interactions by HDXMS.

<p>Protein-ligand interactions can be analyzed by HDXMS by comparing deuterium exchange of the unliganded state of the protein with that bound to ligand (shown in yellow sticks). An ensemble view entails that the target protein <b>(E)</b> would exist in multiple conformations in the absence of ligand. Here a representative target protein is shown containing two sites- an orthosteric <b>(O)</b> site forming the ligand binding pocket (sites 1–4 are represented) and an allosteric <b>(A)</b> site. Deuterium exchange at the orthosteric site (O-site) (blue) is then governed by ligand binding kinetic parameters: kon, koff, concentration of ligand as well as the observed rate of HDX exchange, kex, which varies across different regions of the protein. The HDXMS output encompasses changes at the orthosteric O-site and long range conformational changes (red) at the allosteric A-site. Binding of ligand at the O-site <b>(E:L)</b> would result in decreased exchange while changes at the A-site <b>(E*:L)</b> could be reflected as decreases or increases in deuterium exchange.</p

Hydrogen/Deuterium Exchange Mass Spectrometry Reveals Specific Changes in the Local Flexibility of Plasminogen Activator Inhibitor 1 upon Binding to the Somatomedin B Domain of Vitronectin

The native fold of plasminogen activator inhibitor 1 (PAI-1) represents an active metastable conformation that spontaneously converts to an inactive latent form. Binding of the somatomedin B domain (SMB) of the endogenous cofactor vitronectin to PAI-1 delays the transition to the latent state and increases the thermal stability of the protein dramatically. We have used hydrogen/deuterium exchange mass spectrometry to assess the inherent structural flexibility of PAI-1 and to monitor the changes induced by SMB binding. Our data show that the PAI-1 core consisting of β-sheet B is rather protected against exchange with the solvent, while the remainder of the molecule is more dynamic. SMB binding causes a pronounced and widespread stabilization of PAI-1 that is not confined to the binding interface with SMB. We further explored the local structural flexibility in a mutationally stabilized PAI-1 variant (14-1B) as well as the effect of stabilizing antibody Mab-1 on wild-type PAI-1. The three modes of stabilizing PAI-1 (SMB, Mab-1, and the mutations in 14-1B) all cause a delayed latency transition, and this effect was accompanied by unique signatures on the flexibility of PAI-1. Reduced flexibility in the region around helices B, C, and I was seen in all three cases, which suggests an involvement of this region in mediating structural flexibility necessary for the latency transition. These data therefore add considerable depth to our current understanding of the local structural flexibility in PAI-1 and provide novel indications of regions that may affect the functional stability of PAI-1