The Use of Chlorobenzene as a Probe Molecule in Molecular Dynamics Simulations

We map ligand binding sites on protein surfaces in molecular dynamics simulations using chlorobenzene as a probe molecule. The method was validated on four proteins. Two types of affinity maps that identified halogen and hydrophobic binding sites on proteins were obtained. Our method could prove useful for the discovery and development of halogenated inhibitors

The Application of Ligand-Mapping Molecular Dynamics Simulations to the Rational Design of Peptidic Modulators of Protein–Protein Interactions

A computational ligand-mapping approach to detect protein surface pockets that interact with hydrophobic moieties is presented. In this method, we incorporated benzene molecules into explicit solvent molecular dynamics simulations of various protein targets. The benzene molecules successfully identified the binding locations of hydrophobic hot-spot residues and all-hydrocarbon cross-links from known peptidic ligands. They also unveiled cryptic binding sites that are occluded by side chains and the protein backbone. Our results demonstrate that ligand-mapping molecular dynamics simulations hold immense promise to guide the rational design of peptidic modulators of protein–protein interactions, including that of stapled peptides, which show promise as an exciting new class of cell-penetrating therapeutic molecules

Mass Spectrometry Reveals Protein Kinase CK2 High-Order Oligomerization <i>via</i> the Circular and Linear Assembly

CK2 is an intrinsically active protein kinase that is crucial for cellular viability. However, conventional kinase regulatory mechanisms do not apply to CK2, and its mode of regulation remains elusive. Interestingly, CK2 is known to undergo reversible ionic-strength-dependent oligomerization. Furthermore, a regulatory mechanism based on autoinhibitory oligomerization has been postulated on the basis of the observation of circular trimeric oligomers and linear CK2 assemblies in various crystal structures. Here, we employ native mass spectrometry to monitor the assembly of oligomeric CK2 species in an ionic-strength-dependent manner. A subsequent combination of ion mobility spectrometry–mass spectrometry and hydrogen–deuterium exchange mass spectrometry techniques was used to analyze the conformation of CK2 oligomers. Our findings support ionic-strength-dependent CK2 oligomerization, demonstrate the transient nature of the α/β interaction, and show that CK2 oligomerization proceeds <i>via</i> both the circular and linear assembly

Real Time Dual-Channel Multiplex SERS Ultradetection

Surface-enhanced Raman scattering (SERS) can be combined with microfluidics for rapid multiplex analyte screening. Through combination of the high intensity and complex signals provided by SERS with the flow characteristics of microfluidic channels, we engineered a microdevice that is capable of monitoring various analytes from different sources in real time. Detection limits down to the nM range may allow the generation of a new family of devices for remote, real time monitoring of environmental samples such as natural or waste waters and application to the high-throughput screening of multiple samples in healthcare diagnostics

Formation of Cucurbit[8]uril-Based Supramolecular Hydrogel Beads Using Droplet-Based Microfluidics

Herein we describe the use of microdroplets as templates for the fabrication of uniform-sized supramolecular hydrogel beads, assembled by supramolecular cross-linking of functional biopolymers with the macrocyclic host molecule, cucurbit[8]­uril (CB[8]). The microdroplets were formed containing diluted hydrogel precursors in solution, including the functional polymers and CB[8], in a microfluidic device. Subsequent evaporation of water from collected microdroplets concentrated the contents, driving the formation of the CB[8]-mediated host–guest ternary complex interactions and leading to the assembly of condensed three-dimensional polymeric scaffolds. Rehydration of the dried particles gave monodisperse hydrogel beads. Their equilibrium size was shown to be dependent on both the quantity of material loaded and the dimensions of the microfluidic flow focus. Fluorescein-labeled dextran was used to evaluate the efficacy of the hydrogel beads as a vector for controlled cargo release. Both passive, sustained release (hours) and triggered, fast release (minutes) of the FITC-dextran was observed, with the rate of sustained release dependent on the formulation. The kinetics of release was fitted to the Ritger-Peppas controlled release equation and shown to follow an anomalous (non-Fickian) transport mechanism

Nanoelectrospray Ionization Mass Spectrometric Study of <i>Mycobacterium tuberculosis</i> CYP121–Ligand Interactions

Nondenaturing nanoelectrospray ionization mass spectrometry (nanoESI MS) of intact protein complexes was used to study CYP121, one of the 20 cytochrome P450s in <i>Mycobacterium tuberculosis (Mtb</i>) and an enzyme that is essential for bacterial viability. The results shed new light on both ligand-free and ligand-bound states of CYP121. Isolated unbound CYP121 is a predominantly dimeric protein, with a minor monomeric form present. High affinity azoles cause the dissociation of dimeric CYP121 into monomer, whereas weaker azole binders induce partial dimer dissociation or do not significantly destabilize the dimer. Complexes of CYP121 with azoles were poorly detected by nanoESI MS, indicating kinetically labile complexes that are easily prone to gas-phase dissociation. Unlike with the azoles, CYP121 forms a stable complex with its natural substrate cYY that does not undergo gas-phase dissociation. In addition, a series of potential ligands from fragment-based studies were used as a test for nanoESI MS work against CYP121. Most of these ligands formed stable complexes with CYP121, and their binding did not promote dimer dissociation. On the basis of binding to the monomer and/or CYP121 dimer it was possible to determine the relative order of their CYP121 binding affinities. The top nanoESI MS screening hit was confirmed by heme absorbance shift assay to have a <i>K</i>_d of 40 μM

Sorting of <i>M</i>.<i>polymorpha</i> protoplasts.

<p>(A) Schematic representation of a platform for microfluidic sorting of encapsulated protoplasts. (B) Bright field and fluorescence micrographs of adjacent microdroplets containing protoplasts derived from wild-type and transgenic mpt0 <i>M</i>. <i>polymorpha</i>, respectively. (C) Bright field and fluorescence micrographs of microdroplets sorted into positive and negative channels based on their mVenus fluorescence intensity.</p

Chlorophyll fluorescence intensity of encapsulated protoplasts.

<p>(A) Experimental setup used for quantification of fluorescence intensity of encapsulated protoplasts. Long-pass filter-1: 495 nm; long-pass filter-2: 633 nm; dichroic filter-1: 495 nm; dichroic filter-2: 633 nm. (B) Bright field micrograph of an encapsulated protoplast passing through the excitation laser beam. (C) Representative PMT readout of chlorophyll fluorescence intensity recorded on chip over 120 s. Each line represents an individual encapsulated protoplast.</p

Reporter fluorescence quantification on encapsulated protoplasts.

<p>(A) Bright field and (B) mVenus fluorescence micrograph of an individual encapsulated protoplast derived from transgenic mpt0 <i>M</i>. <i>polymorpha</i> constitutively expressing mVenus. (C) Representative PMT readout of mVenus fluorescence intensity recorded on chip over 120 s. Each line represents an individual encapsulated protoplast.</p

Protoplast stability after encapsulation.

<p>(A) Bright field and (B) chlorophyll fluorescence micrographs of individual <i>M</i>. <i>polymorpha</i> protoplast encapsulated in microdroplets.</p