Engineered antigen-binding fragments for enhanced crystallization of antibody:antigen complexes.

The atomic-resolution structural information that X-ray crystallography can provide on the binding interface between a Fab and its cognate antigen is highly valuable for understanding the mechanism of interaction. However, many Fab:antigen complexes are recalcitrant to crystallization, making the endeavor a considerable effort with no guarantee of success. Consequently, there have been significant steps taken to increase the likelihood of Fab:antigen complex crystallization by altering the Fab framework.

Enzyme Evolution: An Epistatic Ratchet versus a Smooth Reversible Transition.

Evolutionary trajectories are deemed largely irreversible. In a newly diverged protein, reversion of mutations that led to the functional switch typically results in loss of both the new and the ancestral functions. Nonetheless, evolutionary transitions where reversions are viable have also been described.

Allosteric Modulation of Binding Specificity by Alternative Packing of Protein Cores.

Hydrophobic cores are often viewed as tightly packed and rigid, but they do show some plasticity and could thus be attractive targets for protein design. Here we explored the role of different functional pressures on the core packing and ligand recognition of the SH3 domain from human Fyn tyrosine kinase. We randomized the hydrophobic core and used phage display to select variants that bound to each of three distinct ligands.

Active Site Hydrophobicity and the Convergent Evolution of Paraoxonase Activity in Structurally Divergent Enzymes: The Case of Serum Paraoxonase 1.

Serum paraoxonase 1 (PON1) is a native lactonase capable of promiscuously hydrolyzing a broad range of substrates, including organophosphates, esters, and carbonates. Structurally, PON1 is a six-bladed β-propeller with a flexible loop (residues 70-81) covering the active site. This loop contains a functionally critical Tyr at position 71.

Engineering cell signaling modulators from native protein-protein interactions.

Recent studies on genome sequencing and genetic screens with RNAi and CRISPR technology have revolutionized our understanding of aberrant signaling networks in human diseases. A strategy combining both genetic and protein-based technologies should be at the heart of modern drug-development efforts, particularly in the era of precision medicine. Thus, there is an urgent need for efficient platforms to develop probes that can modulate protein function in cells to validate drug targets and to develop therapeutic leads.

Catalytic stimulation by restrained active-site floppiness--the case of high density lipoprotein-bound serum paraoxonase-1.

Despite the abundance of membrane-associated enzymes, the mechanism by which membrane binding stabilizes these enzymes and stimulates their catalysis remains largely unknown. Serum paraoxonase-1 (PON1) is a lipophilic lactonase whose stability and enzymatic activity are dramatically stimulated when associated with high-density lipoprotein (HDL) particles. Our mutational and structural analyses, combined with empirical valence bond simulations, reveal a network of hydrogen bonds that connect HDL binding residues with Asn168--a key catalytic residue residing >15Å from the HDL contacting interface.

Catalytic metal ion rearrangements underline promiscuity and evolvability of a metalloenzyme.

Although largely deemed as structurally conserved, catalytic metal ion sites can rearrange, thereby contributing to enzyme evolvability. Here, we show that in paraoxonase-1, a lipo-lactonase, catalytic promiscuity and divergence into an organophosphate hydrolase are correlated with an alternative mode of the catalytic Ca(2+). We describe the crystal structures of active-site mutants bearing mutations at position 115.

Evolved stereoselective hydrolases for broad-spectrum G-type nerve agent detoxification.

A preferred strategy for preventing nerve agents intoxication is catalytic scavenging by enzymes that hydrolyze them before they reach their targets. Using directed evolution, we simultaneously enhanced the activity of a previously described serum paraoxonase 1 (PON1) variant for hydrolysis of the toxic S(P) isomers of the most threatening G-type nerve agents. The evolved variants show ≤340-fold increased rates and catalytic efficiencies of 0.

Catalytic versatility and backups in enzyme active sites: the case of serum paraoxonase 1.

The origins of enzyme specificity are well established. However, the molecular details underlying the ability of a single active site to promiscuously bind different substrates and catalyze different reactions remain largely unknown. To better understand the molecular basis of enzyme promiscuity, we studied the mammalian serum paraoxonase 1 (PON1) whose native substrates are lipophilic lactones.

A new method for tumor detection using induced acoustic waves from tagged magnetic nanoparticles.

Unlabelled: Magnetoacoustic detection is a new method for the noninvasive, early detection of cancer. It uses specific superparamagnetic nanoparticles (NPs) that bind to tumor sites together with magnetic excitation and acoustic detection of the tumor-NPs complex. This work tests the feasibility of such method theoretically and experimentally.

Three dimension optical imaging in a high throughput layered expression system.

Layered peptide array (LPA) system enables multiplex screening of biomarkers [1-3]. One of the main problems of the LPA system is the screening of the layered-membranes stack. Nowadays, each membrane is imaged separately using conventional fluorescent imaging.

Directed evolution of hydrolases for prevention of G-type nerve agent intoxication.

Organophosphate nerve agents are extremely lethal compounds. Rapid in vivo organophosphate clearance requires bioscavenging enzymes with catalytic efficiencies of >10(7) (M(-1) min(-1)). Although serum paraoxonase (PON1) is a leading candidate for such a treatment, it hydrolyzes the toxic S(p) isomers of G-agents with very slow rates.

Coherent hollow-core waveguide bundles for thermal imaging.

There has been very little work done in the past to extend the wavelength range of fiber image bundles to the IR range. This is due, in part, to the lack of IR transmissive fibers with optical and mechanical properties analogous to the oxide glass fibers currently employed in the visible fiber bundles. Our research is aimed at developing high-resolution hollow-core coherent IR fiber bundles for transendoscopic infrared imaging.

Photothermal bundle measurement of phantoms and blood as a proof of concept for oxygenation saturation measurement.

This study's objective is to validate a method for the measurement of two compound phantoms as a proof of concept for oxygen saturation level measurement via a thermal imaging bundle. The method consists of a thermal imaging system and an algorithm which estimates the compound concentration according to temperature rise. A temperature rise is obtained by illuminating the tissue with a laser with different wavelengths in the NIR range and measured using a thermal camera.

A new thermography-based approach to early detection of cancer utilizing magnetic nanoparticles theory simulation and in vitro validation.

This work describes the utilization of tumor-specific magnetic nanoparticles together with an alternating magnetic field as a means to thermally mark a tumor so as to detect it using a thermal imaging system. Experiments were conducted using an in vitro tissue model, an inductive heating system, and an infrared camera. The thermal images, recorded by the infrared camera during the experiments, were analyzed using an algorithm that was developed as part of this work.

Minimal-invasive thermal imaging of a malignant tumor: a simple model and algorithm.

Purpose: This article deals with the development of a minimal-invasive, infrared (IR) (8-12 microm spectral range) imaging technique that would improve upon current methods by using superparamagnetic nanostructured core/shell particles for imaging as well as for therapy. This technique may function as a diagnostic tool, thanks to the ability of specific bioconjugation of these nanoparticles to a tumor's outer surface. Hence, by applying an alternating magnetic field, the authors could cause a selective elevation of temperature of the nanoparticles for +1 - +5 degrees C, enabling tumor's imaging.

Thermal imaging method for estimating oxygen saturation.

The objective of this study is to develop a minimal invasive thermal imaging method to determine the oxygenation level of an internal tissue. In this method, the tissue is illuminated using an optical fiber by several wavelengths in the visible and near-IR range. Each wavelength is absorbed by the tissue and thus causes increase in its temperature.

Assessment of CASP8 structure predictions for template free targets.

The biennial CASP experiment is a crucial way to evaluate, in an unbiased way, the progress in predicting novel 3D protein structures. In this article, we assess the quality of prediction of template free models, that is, ab initio prediction of 3D structures of proteins based solely on the amino acid sequences, that is, proteins that did not have significant sequence identity to any protein in the Protein Data Bank. There were 13 targets in this category and 102 groups submitted predictions.