Introduced more than fifty years ago, dynamic light scattering is routinely used to determine the size distribution of colloidal suspensions, as well as of macromolecules in solution, such as proteins, nucleic acids, and their complexes. More recently, differential dynamic microscopy has been proposed as a way to perform dynamic light scattering experiments with a microscope, with much less stringent constraints in terms of cleanliness of the optical surfaces, but a potentially lower sensitivity due to the use of camera-based detectors. In this work, we push bright-field differential dynamic microscopy beyond known limits and show it to be sufficiently sensitive to size small macromolecules in diluted solutions. By considering solutions of three different proteins (Bovine Serum Albumin, Lysozyme, and Pepsin), we accurately determine the diffusion coefficient and hydrodynamic radius of both single proteins and small protein aggregates down to concentrations of a few milligrams per milliliter. In addition, we present preliminary results showing unexplored potential for the determination of virial coefficients. Our results are in excellent agreement with the ones obtained in parallel with a state-of-the-art commercial dynamic light scattering setup, showing that differential dynamic microscopy represents a valuable alternative for rapid, label-free protein sizing with an optical microscope.

There are many fluorescence-based applications that can be used to characterize molecular interactions. However, available methods often depend on site-specific labeling techniques or binding-induced changes in conformation or size of the probed target molecule. To overcome these limitations, we applied a ratiometric dual-emission approach that quantifies ligand-induced spectral shifts with sub-nanometer sensitivity. The use of environment-sensitive near-infrared dyes with the method we describe enables affinity measurements and thermodynamic characterization without the explicit need for site-specific labeling or ligand-induced conformational changes. We demonstrate that in-solution spectral shift measurements enable precise characterization of molecular interactions for a variety of biomolecules, including proteins, antibodies, and nucleic acids. Thereby, the described method is not limited to a subset of molecules since even the most challenging samples of research and drug discovery projects like membrane proteins and intrinsically disordered proteins can be analyzed.

Introduction: Biologics are an important class of targets in drug discovery that are proven to be effective therapeutics.However, characterization of biologics and the associated workflows from early discovery to final formulation can often be very complex, time-consuming and lack accuracy and precision needed to appropriately monitor drug candidates.Objective: Here we demonstrate how the new Prometheus PANTA by NanoTemper Technologies can be used to completely characterize the behavior of proteins in solution and predict the long-term stability of biologics using a combination of thermal unfolding, turbidity measurement, and colloidal stability.This new developed instrument has been design with 3 detectors to giving the capability to measure, simultaneously, the unfolding, turbidity, particle size and polydispersity index (PDI), without affecting the data quality. Methodology:The new DLS detector developed by NanoTemper Technologies allows the instrument to measure particle size and polydispersity index through a thermal ramp, allowing to understand the changes on structure and aggregation in the folded to unfolded transition, or as standalone DLS system.The DLS detector have being designed to allow the fast measurement of particles from 0.5 nm to 2 µM with a very accurate and reproducible results.Results: All these characteristics allows the Prometheus PANTA to work in formulation development focused in optimize stability through direct ligand interaction as well as increasing colloidal stability or, it can be even used as a control quality or in force degradation studies to check the functionality of your biologic in each step, from development to product filling. Conclusion:This presentation will show how these 3 detectors work together to completely characterize the stability and functionality of biologics.

Aberrant WNT pathway activation, leading to nuclear accumulation of β-catenin, is a key oncogenic driver event. Mutations in the tumor suppressor gene APC lead to impaired proteasomal degradation of β-catenin and subsequent nuclear translocation. Restoring cellular degradation of β-catenin represents a potential therapeutic strategy. Here, we report the fragment-based discovery of a small molecule binder to β-catenin, including the structural elucidation of the binding mode by X-ray crystallography. The difficulty in drugging β-catenin was confirmed as the primary screening campaigns identified only few and very weak hits. Iterative virtual and NMR screening techniques were required to discover a compound with sufficient potency to be able to obtain an X-ray co-crystal structure. The binding site is located between armadillo repeats two and three, adjacent to the BCL9 and TCF4 binding sites. Genetic studies show that it is unlikely to be useful for the development of protein-protein interaction inhibitors but structural information and established assays provide a solid basis for a prospective optimization towards β-catenin proteolysis targeting chimeras (PROTACs) as alternative modality.

The Front Cover shows the discovered fragment in the process of binding to β-catenin bound to the transcription factor TCF4 and co-activator BCL9 (PDB ID: 2GL7). The electron density surrounding the ligand and the chemical shift perturbations observed in the fragment binding site represent the strong biophysical validation. The outreaching arm represents the prospective utility of this discovery to be used as a handle for proteolysis targeting chimeras (PROTACs) upon further optimization. More information can be found in the Communication by Dirk Kessler, Jark Böttcher et al. Cover art created by Mila Böttcher, Jark Böttcher, and Darryl B. McConnell.

The GRADFLEX experiment of ESA has shown that under microgravity conditions a stationary thermodiffusion process is accompanied by giant non-equilibrium fluctuations with size as large as the size of the sample. In the presence of small concentration gradients, the features of the non-equilibrium fluctuations can be described by means of linearized hydrodynamics. However, the linear models are not suitable to describe most cases of applicative interest, such as fluctuations induced by large gradients and under non-stationary conditions. Moreover, presently the investigation of non-equilibrium fluctuations has mainly involved single component fluids and binary mixtures, but recently transport processes in ternary mixtures have attracted increasing interest due to the experiments performed on the International Space Station in the framework of the DCMIX project of ESA. The Giant Fluctuations (NEUF-DIX) project of ESA will investigate non-equilibrium fluctuations during diffusive processes occurring in complex multi-component mixtures, where one of the components is a macromolecule, such as a polymer, a colloid or a protein. Important objectives will be the exploration of the features of the fluctuations under non-ideal conditions, such as large gradients, transient processes, and concentrated samples, and the understanding of how the fluctuations affect the interactions between macromolecules. The project involves the development of a dedicated facility, consisting of an array of shadowgraph optical instruments working in parallel, each one equipped with a thermal gradient cell. Here we outline the design concept of the facility and the results of performance tests performed on a breadboard to evaluate the suitability of the designed instrument to carry out scientific measurements of non-equilibrium concentration fluctuations in space.

This paper describes the European Space Agency (ESA) experiments devoted to study thermodiffusion of fluid mixtures in microgravity environment, where sedimentation and convection do not affect the mass flow induced by the Soret effect. First, the experiments performed on binary mixtures in the IVIDIL and GRADFLEX experiments are described. Then, further experiments on ternary mixtures and complex fluids performed in DCMIX and planned to be performed in the context of the NEUF-DIX project are presented. Finally, multi-component mixtures studied in the SCCO project are detailed.

Interactions between nucleic acids and proteins are driving gene expression programs and regulating the development of organisms. The binding affinities of transcription factors to their target sites are essential parameters to reveal their binding site occupancy and function in vivo. Microscale Thermophoresis (MST) is a rapid and precise method allowing for quantitative analysis of molecular interactions in solution on a microliter scale. The technique is based on the movement of molecules in temperature gradients, which is referred to as thermophoresis, and depends on molecule size, charge, and hydration shell. Since at least one of these parameters is typically affected upon binding of a ligand, the method can be used to analyze any kind of biomolecular interaction. This section provides a detailed protocol describing the analysis of DNA-protein interactions, using the transcription factor TTF-I as a model protein that recognizes a 10 bp long sequence motif.

The determination of protein unfolding and aggregation characteristics during preformulation is of major significance for the development of biopharmaceuticals. The aim of this study was to investigate the feasibility of a new immobilization-and label-free thermo-optical approach as an orthogonal method for material and time-saving early formulation and drugability screenings. In the experimental setup used, changes in the intrinsic tryptophan fluorescence of the protein were measured during IR laser-induced heating of the samples. This temperature increase leads to characteristic fluorescence changes over time, which can be attributed to separable effects of protein unfolding, aggregation, and precipitation, depending on the stability of the respective formulation. The obtained signals were compared with data from forced degradation and thermal stability measurements and correlated well both with the aggregation propensity and with the reversibility of unfolding in different formulations. These results, gathered with only 4-mL sample volume and 150 s measurement time per formulation, demonstrate potential for general applicability in rapid candidate and formulation selections. (C) 2017 American Pharmacists Association (R). Published by Elsevier Inc. All rights reserved.

Diffusion and thermal diffusion processes in a liquid mixture are accompanied by long-range non-equilibrium fluctuations, whose amplitude is orders of magnitude larger than that of equilibrium fluctuations. The mean-square amplitude of the non-equilibrium fluctuations presents a scale-free power law behavior q-4 as a function of the wave vector q, but the divergence of the amplitude of the fluctuations at small wave vectors is prevented by the presence of gravity. In microgravity conditions the non-equilibrium fluctuations are fully developed and span all the available length scales up to the macroscopic size of the systems in the direction parallel to the applied gradient. Available theoretical models are based on linearized hydrodynamics and provide an adequate description of the statics and dynamics of the fluctuations in the presence of small temperature/concentration gradients and under stationary or quasi-stationary conditions. We describe a project aimed at the investigation of Non-EquilibriUm Fluctuations during DIffusion in compleX liquids (NEUF-DIX). The focus of the project is on the investigation in micro-gravity conditions of the non-equilibrium fluctuations in complex liquids, trying to tackle several challenging problems that emerged during the latest years, such as the theoretical predictions of Casimir-like forces induced by non-equilibrium fluctuations; the understanding of the non-equilibrium fluctuations in multi-component mixtures including a polymer, both in relation to the transport coefficients and to their behavior close to a glass transition; the understanding of the non-equilibrium fluctuations in concentrated colloidal suspensions, a problem closely related with the detection of Casimir forces; and the investigation of the development of fluctuations during transient diffusion. We envision to parallel these experiments with state-of-the-art multi-scale simulations.

Laforin is a human dual-specificity phosphatase (DSP) involved in glycogen metabolism regulation containing a carbohydrate-binding module (CBM). Mutations in the gene coding for laforin are responsible for the development of Lafora disease, a progressive fatal myoclonus epilepsy with early onset, characterized by the intracellular deposition of abnormally branched, hyperphosphorylated insoluble glycogen-like polymers, called Lafora bodies. Despite the known importance of the CBM domain of laforin in the regulation of glycogen metabolism, the molecular mechanism of laforin-glycogen interaction is still poorly understood. Recently, the structure of laforin with bound maltohexaose was determined and despite the importance of such breakthrough, some molecular interaction details remained missing. We herein report a thorough biophysical characterization of laforin-carbohydrate interaction using soluble glycans. We demonstrated an increased preference of laforin for the interaction with glycans with higher order of polymerization and confirmed the importance of tryptophan residues for glycan interaction. Moreover, and in line with what has been described for other CBMs and lectins, our results confirmed that laforin-glycan interactions occur with a favourable enthalpic contribution counter-balanced by an unfavourable entropic contribution. The analysis of laforin-glycan interaction through the glycan side by saturation transfer difference (STD)-NMR has shown that the CBM-binding site can accommodate between 5 and 6 sugar units, which is in line with the recently obtained crystal structure of laforin. Overall, the work in the present study complements the structural characterization of laforin and sheds light on the molecular mechanism of laforin-glycan interaction, which is a pivotal requisite to understand the physiological and pathological roles of laforin.

Fragment-based lead discovery has proved to be an effective alternative to high-throughput screenings in identifying chemical matter that can be developed into robust lead compounds. The search for optimal combinations of biophysical techniques that can correctly and efficiently identify and quantify binding can be challenging due to the physicochemical properties of fragments. In order to minimize the time and costs of screening, optimal combinations of biophysical techniques with maximal information content, sensitivity, and robustness are needed. Here we describe an approach utilizing automated microscale thermophoresis (MST) affinity screening to identify fragments active against MEK1 kinase. MST identified multiple hits that were confirmed by X-ray crystallography but not detected by orthogonal methods. Furthermore, MST also provided information about ligand-induced aggregation and protein denaturation. The technique delivered a large number of binders while reducing experimentation time and sample consumption, demonstrating the potential of MST to execute and maximize the efficacy of fragment screening campaigns.

The characterization and development of highly specific aptamers requires the analysis of the interaction strength between aptamer and target. MicroScale Thermophoresis (MST) is a rapid and precise method to quantify biomolecular interactions in solution at microliter scale. The basis of this technology is a physical effect referred to as thermophoresis, which describes the directed movement of molecules through temperature gradients. The thermophoretic properties of a molecule depend on its size, charge, and hydration shell. Since at least one of these parameters is altered upon binding of a ligand, this method can be used to analyze virtually any biomolecular interaction in any buffer or complex bioliquid. This section provides a detailed protocol describing how MST is used to obtain quantitative binding parameters for aptamer-target interactions. The two DNA-aptamers HD1 and HD22, which are targeted against human thrombin, are used as model systems to demonstrate a rapid and straightforward screening approach to determine optimal buffer conditions.