Binding Rate Constants Reveal Distinct Features of Disordered Protein Domains

Intrinsically disordered proteins (IDPs) are abundant in the proteome and involved in key cellular functions. However, experimental data about the binding kinetics of IDPs as a function of different environmental conditions are scarce. We have performed an extensive characterization of the ionic strength dependence of the interaction between the molten globular nuclear co-activator binding domain (NCBD) of CREB binding protein and five different protein ligands, including the intrinsically disordered activation domain of p160 transcriptional co-activators (SRC1, TIF2, ACTR), the p53 transactivation domain, and the folded pointed domain (PNT) of transcription factor ETS-2. Direct comparisons of the binding rate constants under identical conditions show that the association rate constant, <i>k</i>_on, for interactions between NCBD and disordered protein domains is high at low salt concentrations (90–350 × 10⁶ M^–1 s^–1 at 4 °C) but is reduced significantly (10–30-fold) with an increasing ionic strength and reaches a plateau around physiological ionic strength. In contrast, the <i>k</i>_on for the interaction between NCBD and the folded PNT domain is only 7 × 10⁶ M^–1 s^–1 (4 °C and low salt) and displays weak ionic strength dependence, which could reflect a distinctly different association that relies less on electrostatic interactions. Furthermore, the basal rate constant (in the absence of electrostatic interactions) is high for the NCBD interactions, exceeding those typically observed for folded proteins. One likely interpretation is that disordered proteins have a large number of possible collisions leading to a productive on-pathway encounter complex, while folded proteins are more restricted in terms of orientation. Our results highlight the importance of electrostatic interactions in binding involving IDPs and emphasize the significance of including ionic strength as a factor in studies that compare the binding properties of IDPs to those of ordered proteins

Stability of wild type and amide-to-ester variants.

<p>(<i>A</i>) Far-UV circular dichroism spectra and (<i>B–C</i>) urea-induced denaturation of wild type and amide-to-ester mutants of PSD-95 PDZ2. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095619#pone-0095619-t001" target="_blank">Table 1</a> for ΔΔ<i>G</i>_D-N values from the curve fitting in panel B.</p

Structural basis for the amide-to-ester mutations.

<p>(<i>A</i>) The X-ray crystal structure of PSD-95 PDZ2 from the PDZ1-2 tandem <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095619#pone.0095619-Sainlos1" target="_blank">[26]</a>, with the two engineered residues highlighted; Trp190 was used as a fluorescent probe and Cys178 for the semisynthesis of backbone amide-to-ester containing mutants. (<i>B</i>) and (<i>C</i>) close-ups showing the backbone hydrogen bonds perturbed by the amide-to-ester mutations (dashed lines).</p

Native and non-native bond formation in the transition states for folding of PSD-95 PDZ2.

<p>Φ-values are generally very low but tend to increase from TS1 (left panel) to TS2 (right panel). The exception is the F172φ mutation, which is going from a slightly negative value to a highly negative Φ-value in TS2. The ΔΔ<i>G</i>_D-N values of G171γ and G176γ were too low to allow calculation of Φ-values.</p

Equilibrium and kinetic parameters for wild type and amide-to-ester mutants of PSD-95 PDZ2.

1<p>The <i>m</i>_D-N value used in the curve fitting (1.0 kcal mol⁻¹ M⁻¹) was determined from the Y190W data set, which displayed well-defined native and denatured baselines.</p>2<p>Too low to calculate a reliable Φ value.</p>3<p>Φ_TS1 values were calculated in the absence of urea.</p>4<p>Φ_TS2 values were calculated at [urea] = 6 M.</p>5<p>Reports on the side chain mutation V178C.</p>6<p>Pseudo wild type.</p

Activation Barrier-Limited Folding and Conformational Sampling of a Dynamic Protein Domain

Folding reaction mechanisms of globular protein domains have been extensively studied by both experiment and simulation and found to be highly concerted chemical reactions in which numerous noncovalent bonds form in an apparent two-state fashion. However, less is known regarding intrinsically disordered proteins because their folding can usually be studied only in conjunction with binding to a ligand. We have investigated by kinetics the folding mechanism of such a disordered protein domain, the nuclear coactivator-binding domain (NCBD) from CREB-binding protein. While a previous computational study suggested that NCBD folds without an activation free energy barrier, our experimental data demonstrate that NCBD, despite its highly dynamic structure, displays relatively slow folding (∼10 ms at 277 K) consistent with a barrier-limited process. Furthermore, the folding kinetics corroborate previous nuclear magnetic resonance data showing that NCBD exists in two folded conformations and one more denatured conformation at equilibrium and, thus, that the folding mechanism is a three-state mechanism. The refolding kinetics is limited by unfolding of the less populated folded conformation, suggesting that the major route for interconversion between the two folded states is via the denatured state. Because the two folded conformations have been suggested to bind distinct ligands, our results have mechanistic implications for conformational sampling in protein–protein interactions

Development of a Standardised Readout System for Active Pixel Sensors in HV/HR-CMOS Technologies for ATLAS Inner Detector Upgrades

The LHC Phase-II Upgrade results in new challenges for tracking detectors for example in terms of cost effectiveness, resolution and radiation hardness. Active Pixel Sensors in HV/HR-CMOS technologies show promising results coping with these challenges. In order to demonstrate the feasibility of hybrid modules with active CMOS sensors and readout chips for the future ATLAS Inner Tracker, ATLAS R&D; activities have started. After introducing the basic concepts and the demonstrator program, the development of an ATLAS compatible readout system will be presented as well as tuning procedures and measurements with demonstrator modules to test the readout system

Additional file 7: Figure S5. of Evolution of the p53-MDM2 pathway

Sequence Logos based on the multiple sequence alignment of MDM p53/p63/p73BD. The color-coding is according to the eBioX alignment tool. (PDF 25 kb

Additional file 6: Figure S4. of Evolution of the p53-MDM2 pathway

Alignment of the p53/p63/p73BD in the MDM protein family. This alignment together with the alignment of the rest of the protein (not shown) was used to generate the phylogenetic tree. The color-coding is according to the eBioX alignment tool. (PDF 3715 kb

Additional file 2: Figure S1. of Evolution of the p53-MDM2 pathway

Alignment of the TAD in the p53/p63/p73 protein family. This alignment together with the alignment of the rest of the protein (not shown) was used to generate the phylogenetic tree. The color-coding is according to the eBioX alignment tool. (PDF 106 kb