Importance of hydrophobic cavities in allosteric regulation of formylglycinamide synthetase: Insight from xenon trapping and statistical coupling analysis

No grant i

Using thermal scanning assays to test protein-protein interactions of inner-ear cadherins

<div><p>Protein-protein interactions play a crucial role in biological processes such as cell-cell adhesion, immune system-pathogen interactions, and sensory perception. Understanding the structural determinants of protein-protein complex formation and obtaining quantitative estimates of their dissociation constant (<i>K</i>_D) are essential for the study of these interactions and for the discovery of new therapeutics. At the same time, it is equally important to characterize protein-protein interactions in a high-throughput fashion. Here, we use a modified thermal scanning assay to test interactions of wild type (WT) and mutant variants of N-terminal fragments (EC1+2) of cadherin-23 and protocadherin-15, two proteins essential for inner-ear mechanotransduction. An environmentally sensitive fluorescent dye (SYPRO orange) is used to monitor melting temperature (<i>T</i>_m) shifts of protocadherin-15 EC1+2 (pcdh15) in the presence of increasing concentrations of cadherin-23 EC1+2 (cdh23). These <i>T</i>_m shifts are absent when we use proteins containing deafness-related missense mutations known to disrupt cdh23 binding to pcdh15, and are increased for some rationally designed mutants expected to enhance binding. In addition, surface plasmon resonance binding experiments were used to test if the <i>T</i>_m shifts correlated with changes in binding affinity. We used this approach to find a double mutation (cdh23(T15E)- pcdh15(G16D)) that enhances binding affinity of the cadherin complex by 1.98 kJ/mol, roughly two-fold that of the WT complex. We suggest that the thermal scanning methodology can be used in high-throughput format to quickly compare binding affinities (<i>K</i>_D from nM up to 100 μM) for some heterodimeric protein complexes and to screen small molecule libraries to find protein-protein interaction inhibitors and enhancers.</p></div

Affinity (<i>K</i>_D) and kinetic parameters for complexes of cdh23 bound to pcdh15.

<p>Affinity (<i>K</i>_D) and kinetic parameters for complexes of cdh23 bound to pcdh15.</p

Quantitative SPR measurements of cdh23 molecules binding to pcdh15.

<p>(A) Left, association and dissociation of the cdh23(WT)-pcdh15(WT) complex. Experimental data (sensorgrams) are represented in a gradient of green to blue colors for different concentrations of cdh23(WT) as labeled. Red lines indicate fitted model parameters (RMSD = 2.89). Injection peaks were removed and not fitted. Top three traces correspond to 5, 10, and 15 μM, respectively, but the 10 μM trace is not labeled for clarity. Black arrow indicates the position of equilibrium SPR signal (R_eq). Middle panel shows the fitting of R_eq to a Langmuir binding isotherm at different concentrations of analyte. Measurements for selected concentrations were done in duplicates. Right panel shows a heat map of the <i>k</i>_off and <i>K</i>_D distribution from the global fit of all traces in corresponding leftmost panel. The signal density of the peaks in the <i>k</i>_off and <i>K</i>_D distribution plot can directly be discerned from their color, which is scaled according to the color bar on the right side of the distribution plot [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189546#pone.0189546.ref048" target="_blank">48</a>]. (B) Association and dissociation curves for the T15E-G16D complex shown as in (A) (RMSD = 11.08). Top three traces correspond to 15, 20, and 25 μM, respectively, but the 15 and 20 μM traces are not labeled for clarity. The data were analyzed with the EVILFIT algorithm and the Biacore evaluation software.</p

Integrated photonic waveguides for on-chip SBS with OAM modes

Stimulated Brillouin Scattering (SBS) is a nonlinear optical process that can provide significant on-chip gain in various materials and platforms. The utilization of chalcogenide and silicon (Si) waveguides has facilitated the advancement of Brillouin lasers and diverse microwave photonic functionalities. The predominant approach has been centered on interactions between conventional guided modes, specifically TE/TM. This study introduces a novel approach that explores diverse guided-wave configurations capable of sustaining SBS with orbital angular momentum modes (OAM). These configurations include a cross-Si waveguide and a silica/chalcogenide waveguide. The simulations examine the forward and backward SBS utilizing OAM modes up to the 4th order in planar on-chip waveguides. A maximum on-chip SBS gain of up to ∼540 W-1 m-1 has been observed for pump and Stokes OAM mode numbers satisfying the topological charge conservation law. This OAM multiplexing approach can open new avenues for on-chip integrated nonlinear photonics for various applications, especially where pump and signal beam filtering or separation are critical

Heterotetrameric tip link made of CDH23 bound to PCDH15.

<p>(A) The tip link is formed by a CDH23 parallel dimer interacting tip-to-tip with a PCDH15 parallel dimer [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189546#pone.0189546.ref039" target="_blank">39</a>]. These proteins feature 27 and 11 extracellular cadherin (EC) repeats, respectively. (B) Ribbon diagram of mouse cdh23 (blue) bound to pcdh15 (magenta) with Ca²⁺ ions as green spheres (PDB ID: 4APX). Sites of deafness-causing mutations R113 and I108 in PCDH15 are shown in stick representation and circled. (C & D) Detail of I108 (C) and R113 (D) with surrounding residues in the cdh23 and pcdh15 interface.</p

Variation of <i>ΔT</i>_m at increasing ratios of cdh23 and pcdh15.

<p>(A) Variation of <i>ΔT</i>_m for WT and two deafness-related complexes at increasing concentration ratios. The WT <i>ΔT</i>_m increases from ~2 to ~5.5°C from 1:1 to 5:1 ratios. The deafness mutants have <i>ΔT</i>_m < ~1°C at all ratios. (B) <i>ΔT</i>_m variation of the most stabilizing mutant G16D-T15E (green), the WT complex (blue), and the less stabilizing mutant Q165L (pink). The double mutant has higher <i>ΔT</i>_m than the WT complex at all ratios. (C) Bar graph of <i>ΔT</i>_m variation of selected mutants at different cdh23:pcdh15 ratios. Asterisks indicate statistically significant differences with WT complex (p ≤ 0.05). Data for all mutants including pcdh15- A20V, D160W, L187W, Q165L, S92V and cdh23-T190W are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189546#pone.0189546.t001" target="_blank">Table 1</a>.</p

Mapping of rationally designed mutation sites on the structure of the cdh23 and pcdh15 complex.

<p>(A) Surface representation of cdh23 (blue and cyan) bound to pcdh15 (purple and pink; PDB ID: 4APX). (B) Cdh23 and pcdh15 interaction surfaces exposed with mutation sites labeled. Residues labeled in red are mutated in inherited deafness. Underlined labels indicate sites that belong to paired mutant complexes cdh23(H11K)-pcdh15(Q218E) and cdh23(T79E)-pcdh15(H91R).</p