Fragment-based screening by protein-detected NMR spectroscopy.

Fragment-based drug discovery (FBDD) identifies low molecular weight compounds that can be developed into ligands with high affinity and selectivity for therapeutic targets. Screening fragment libraries (<10,000 molecules) with biophysical techniques against macromolecules provides information about novel chemical spaces that bind the macromolecule and scaffolds that can be modified to increase potency. A fragment-screening pipeline requires a standardized protocol for target selection, library assembly and maintenance, library screening, and hit validation to ensure hit integrity. Herein, the fundamental aspects of a fragment screening pipeline-focusing on protein-detected NMR data collection and analysis-are discussed in detail for researchers to use as a resource in their FBDD projects. Selected screening targets must undergo rigorous stability and buffer testing by NMR spectroscopy to ensure the protein structure is stable for the entire screen. Biophysical instrumentation that rapidly measures protein thermostability is helpful in buffer screening. Molecules in fragment libraries are analyzed computationally and physically, stored at appropriate temperatures, and multiplexed in well plates for library conservation. The screening protocol is streamlined using liquid handling robotics for sample preparation and customized Python scripts for protein-detected NMR data analysis. Molecules identified from the screen are titrated to determine their binding site(s) and Kd values and confirmed with an orthogonal biophysical assay. This detailed FBDD screening pipeline developed by the Program in Chemical Biology at the Medical College of Wisconsin has successfully screened many unrelated target proteins to identified novel molecules that selectively bind to these target proteins.

Preparation and Investigation of Crucial Oligomers in the Early Stages of Aß40 and Aß42 Aggregation.

Amyloid aggregation is a hallmark in many neuropathologies and other diseases of tremendous impact. It is increasingly evident that neuronal death associated with Alzheimer's disease (AD) is mainly produced by oligomers of the amyloid-ß (Aß) peptide. Yet little is known about the detailed structural and biophysical mechanisms of their formation. This lack of complete understanding comes from the labile nature and handling complexity of the oligomers. Consequently, providing reproducible and robust protocols for oligomer preparation is of particular importance.In this study, we describe detailed methods for the preparation and isolation of micellar oligomers of Aß that evolve towards larger and more stable oligomers enriched in beta-sheet structure and able to acquire a higher capacity to fibrillate. We also describe briefly some biophysical experiments allowing oligomer characterization.

Exiting the tunnel of uncertainty: crystal soak to validated hit.

Crystallographic fragment screens provide an efficient and effective way to identify small-molecule ligands of a crystallized protein. Due to their low molecular weight, such hits tend to have low, often unquantifiable, affinity for their target, complicating the twin challenges of validating the hits as authentic solution-phase ligands of the target and identifying the `best' hit(s) for further elaboration. In this article, approaches that address these challenges are assessed. Using retrospective analysis of a recent ATAD2 hit-identification campaign, alongside other examples of successful fragment-screening campaigns, it is suggested that hit validation and prioritization are best achieved by a `triangulation' approach in which the results of multiple available biochemical and biophysical techniques are correlated to develop qualitative structure-activity relationships (SARs). Such qualitative SARs may indeed be the only means by which to navigate a project through the tunnel of uncertainty that prevails before on-scale biophysical, biochemical and/or biological measurements become possible.

G-Quadruplex Aptamer-Ligand Characterization.

In this work we explore the structure of a G-rich DNA aptamer termed AT11-L2 (TGGTGGTGGTTGTTGTTGGTGGTGGTGGT; derivative of AT11) by evaluating the formation and stability of G-quadruplex (G4) conformation under different experimental conditions such as KCl concentration, temperature, and upon binding with a variety of G4 ligands (360A, BRACO-19, PDS, PhenDC3, TMPyP4). We also determined whether nucleolin (NCL) can be a target of AT11-L2 G4. Firstly, we assessed by circular dichroism, UV and NMR spectroscopies the formation of G4 by AT11-L2. We observed that, for KCl concentrations of 65 mM or less, AT11-L2 adopts hybrid or multiple topologies. In contrast, a parallel topology predominates for buffer containing 100 mM of KCl. The Tm of AT11-L2 in 100 mM of KCl is 38.9 °C, proving the weak stability of this sequence. We also found that upon titration with two molar equivalents of 360A, BRACO-19 and PhenDC3, the G4 is strongly stabilized and its topology is maintained, while the addition of 3.5 molar equivalents of TMPyP4 promotes the disruption of G4. The KD values between AT11-L2 G4, ligands and NCL were obtained by fluorescence titrations and are in the range of µM for ligand complexes and nM when adding NCL. In silico studies suggest that four ligands bind to the AT11-L2 G4 structure by stacking interactions, while the RBD1,2 domains of NCL interact preferentially with the thymines of AT11-L2 G4. Finally, AT11-L2 G4 co-localized with NCL in NCL-positive tongue squamous cell carcinoma cell line.

Recent Advances in Fluorescence Recovery after Photobleaching for Decoupling Transport and Kinetics of Biomacromolecules in Cellular Physiology.

Among the new molecular tools available to scientists and engineers, some of the most useful include fluorescently tagged biomolecules. Tools, such as green fluorescence protein (GFP), have been applied to perform semi-quantitative studies on biological signal transduction and cellular structural dynamics involved in the physiology of healthy and disease states. Such studies focus on drug pharmacokinetics, receptor-mediated endocytosis, nuclear mechanobiology, viral infections, and cancer metastasis. In 1976, fluorescence recovery after photobleaching (FRAP), which involves the monitoring of fluorescence emission recovery within a photobleached spot, was developed. FRAP allowed investigators to probe two-dimensional (2D) diffusion of fluorescently-labelled biomolecules. Since then, FRAP has been refined through the advancements of optics, charged-coupled-device (CCD) cameras, confocal microscopes, and molecular probes. FRAP is now a highly quantitative tool used for transport and kinetic studies in the cytosol, organelles, and membrane of a cell. In this work, the authors intend to provide a review of recent advances in FRAP. The authors include epifluorescence spot FRAP, total internal reflection (TIR)/FRAP, and confocal microscope-based FRAP. The underlying mathematical models are also described. Finally, our understanding of coupled transport and kinetics as determined by FRAP will be discussed and the potential for future advances suggested.

Viscoelasticity, Like Forces, Plays a Role in Mechanotransduction.

Viscoelasticity and its alteration in time and space has turned out to act as a key element in fundamental biological processes in living systems, such as morphogenesis and motility. Based on experimental and theoretical findings it can be proposed that viscoelasticity of cells, spheroids and tissues seems to be a collective characteristic that demands macromolecular, intracellular component and intercellular interactions. A major challenge is to couple the alterations in the macroscopic structural or material characteristics of cells, spheroids and tissues, such as cell and tissue phase transitions, to the microscopic interferences of their elements. Therefore, the biophysical technologies need to be improved, advanced and connected to classical biological assays. In this review, the viscoelastic nature of cytoskeletal, extracellular and cellular networks is presented and discussed. Viscoelasticity is conceptualized as a major contributor to cell migration and invasion and it is discussed whether it can serve as a biomarker for the cells' migratory capacity in several biological contexts. It can be hypothesized that the statistical mechanics of intra- and extracellular networks may be applied in the future as a powerful tool to explore quantitatively the biomechanical foundation of viscoelasticity over a broad range of time and length scales. Finally, the importance of the cellular viscoelasticity is illustrated in identifying and characterizing multiple disorders, such as cancer, tissue injuries, acute or chronic inflammations or fibrotic diseases.

Hydrolipidic Characteristics and Clinical Efficacy of a Dermocosmetic Formulation for the Improvement of Homeostasis on Oily Mature Skin.

BACKGROUND: Although the scientific literature associates mature skin with dry skin and the secretion of sebum on the face decreases over the years, in tropical countries, such as Brazil, mature skin can still present oily characteristics. Thus, the knowledge of the hydrophilic characteristics of mature skin is fundamental to help the development of more effective treatments for this skin type. In this context, the study aimed to evaluate the hydrophilic characteristics and the clinical efficacy of a cosmetic formulation for mature skin added with alfalfa and lentil extracts by using biophysical and skin imaging techniques. METHODS: Twenty-eight healthy females aged between 45 and 59 years were enrolled. Measurements of the stratum corneum water content, sebum content, transepidermal water loss, skin microrelief, and pores count were performed before and after the 28-day formulation application. RESULTS: The mature skin presented as oily with wrinkles and pores. The proposed formulation significantly reduced the sebum content and the number of fine and large pores and improved skin microrelief and hydration after a 28-day period of the application when compared to the vehicle. CONCLUSIONS: The proposed formulation was effective in oily mature skin treatment, improving its general skin aging and oiliness conditions, and reducing pores count in just 28 days.

The concept of protein folding/unfolding and its impacts on human health.

Proteins have evolved in specific 3D structures and play different functions in cells and determine various reactions and pathways. The newly synthesized amino acid chains once depart ribosome must crumple into three-dimensional structures so can be biologically active. This process of protein that makes a functional molecule is called protein folding. The protein folding is both a biological and a physicochemical process that depends on the sequence of it. In fact, this process occurs more complicated and in some cases and in exposure to some molecules like glucose (glycation), mistaken folding leads to amyloid structures and fatal disorders called conformational diseases. Such conditions are detected by the quality control system of the cell and these abnormal proteins undergo renovation or degradation. This scenario takes place by the chaperones, chaperonins, and Ubiquitin-proteasome complex. Understanding of protein folding mechanisms from different views including experimental and computational approaches has revealed some intermediate ensembles such as molten globule and has been subjected to biophysical and molecular biology attempts to know more about prevalent conformational diseases.

PDZ Sample Quality Assessment by Biochemical and Biophysical Characterizations.

PDZ domains are small globular domains involved in protein-protein interactions. They participate in a wide range of critical cellular processes. These domains, very abundant in the human proteome, are widely studied by high-throughput interactomics approaches and by biophysical and structural methods. However, the quality of the results is strongly related to the optimal folding and solubility of the domains. We provide here a detailed description of protocols for a strict quality assessment of the PDZ constructs. We describe appropriate experimental approaches that have been selected to overcome the small size of such domains to check the purity, identity, homogeneity, stability, and folding of samples.

Studying protein folding in health and disease using biophysical approaches.

Protein folding is crucial for normal physiology including development and healthy aging, and failure of this process is related to the pathology of diseases including neurodegeneration and cancer. Early thermodynamic and kinetic studies based on the unfolding and refolding equilibrium of individual proteins in the test tube have provided insight into the fundamental principles of protein folding, although the problem of predicting how any given protein will fold remains unsolved. Protein folding within cells is a more complex issue than folding of purified protein in isolation, due to the complex interactions within the cellular environment, including post-translational modifications of proteins, the presence of macromolecular crowding in cells, and variations in the cellular environment, for example in cancer versus normal cells. Development of biophysical approaches including fluorescence resonance energy transfer (FRET) and nuclear magnetic resonance (NMR) techniques and cellular manipulations including microinjection and insertion of noncanonical amino acids has allowed the study of protein folding in living cells. Furthermore, biophysical techniques such as single-molecule fluorescence spectroscopy and optical tweezers allows studies of simplified systems at the single molecular level. Combining in-cell techniques with the powerful detail that can be achieved from single-molecule studies allows the effects of different cellular components including molecular chaperones to be monitored, providing us with comprehensive understanding of the protein folding process. The application of biophysical techniques to the study of protein folding is arming us with knowledge that is fundamental to the battle against cancer and other diseases related to protein conformation or protein-protein interactions.

An Integrative Structural Biology Analysis of Von Willebrand Factor Binding and Processing by ADAMTS-13 in Solution.

Von Willebrand Factor (vWF), a 300-kDa plasma protein key to homeostasis, is cleaved at a single site by multi-domain metallopeptidase ADAMTS-13. vWF is the only known substrate of this peptidase, which circulates in a latent form and becomes allosterically activated by substrate binding. Herein, we characterised the complex formed by a competent peptidase construct (AD13-MDTCS) comprising metallopeptidase (M), disintegrin-like (D), thrombospondin (T), cysteine-rich (C), and spacer (S) domains, with a 73-residue functionally relevant vWF-peptide, using nine complementary techniques. Pull-down assays, gel electrophoresis, and surface plasmon resonance revealed tight binding with sub-micromolar affinity. Cross-linking mass spectrometry with four reagents showed that, within the peptidase, domain D approaches M, C, and S. S is positioned close to M and C, and the peptide contacts all domains. Hydrogen/deuterium exchange mass spectrometry revealed strong and weak protection for C/D and M/S, respectively. Structural analysis by multi-angle laser light scattering and small-angle X-ray scattering in solution revealed that the enzyme adopted highly flexible unbound, latent structures and peptide-bound, active structures that differed from the AD13-MDTCS crystal structure. Moreover, the peptide behaved like a self-avoiding random chain. We integrated the results with computational approaches, derived an ensemble of structures that collectively satisfied all experimental restraints, and discussed the functional implications. The interaction conforms to a 'fuzzy complex' that follows a 'dynamic zipper' mechanism involving numerous reversible, weak but additive interactions that result in strong binding and cleavage. Our findings contribute to illuminating the biochemistry of the vWF:ADAMTS-13 axis.

Estimation of pore dimensions in lipid membranes induced by peptides and other biomolecules: A review.

The cytoplasmic membrane is one of the most frequent cell targets of antimicrobial peptides (AMPs) and other biomolecules. Understanding the mechanism of action of AMPs at the molecular level is of utmost importance for designing of new membrane-specific molecules. In particular, the formation of pores, the structure and size of these pores are of great interest and require nanoscale resolution approaches, therefore, biophysical strategies are essential to achieve an understanding of these processes at this scale. In the case of membrane active peptides, pore formation or general membrane disruption is usually the last step before cell death, and so, pore size is generally directly associated to pore structure and stability and loss of cellular homeostasis, implicated in overall peptide activity. Up to date, there has not been a critical review discussing the methods that can be used specifically for estimating the pore dimensions induced by membrane active peptides. In this review we discuss the scope, relevance and popularity of the different biophysical techniques such as liposome leakage experiments, advanced microscopy, neutron or X-ray scattering, electrophysiological techniques and molecular dynamics studies, all of them useful for determining pore structure and dimension.

Development of Photoprotective Formulations Containing Nanostructured Lipid Carriers: Sun Protection Factor, Physical-Mechanical and Sensorial Properties.

The effects of ultraviolet (UV) radiation emitted by the sun are cumulative and can result in chemical changes such as the generation of reactive oxygen species (ROS), leading to the regular use of sunscreen. As an alternative, the use of antioxidants, such as quercetin, into sunscreen can control these effects and provide additional skin photoprotection. However, quercetin presents low stability and poor permeation, alternatively, the encapsulation in nanoparticles can improve the stability and skin permeation. Thus, this study aimed to develop photoprotective formulations containing nanoencapsulated quercetin, characterize the physical-mechanical and sensorial properties, and evaluate the influence of nanocarriers on sun protection factor (SPF) and the immediate clinical effects. Sunscreen formulations with or without antioxidants in a free form or loaded in nanostructured lipid carriers (NLCs) were developed. After the stability, rheological behavior, texture profile, and in vivo SPF (sun protector factor) evaluation, sixty female participants, aged between 20 and 35 years, were enclosed to evaluate the sensorial properties and immediate clinical effects of the formulation in the skin hydration using biophysical and skin imaging techniques. The correlation of rheological behavior, texture profile, and sensory properties enabled the correct choice of formulation ingredients. In addition, the use of NLCs with quercetin significantly improved the SPF in vivo of the developed photoprotective formulation, without increasing the amount of UV filters. Finally, the association of NLCs in the photoprotective formulation showed synergistic effects in the SPF and an improvement in the skin barrier function and hydration.

The mitochondrial ADP/ATP carrier exists and functions as a monomer.

For more than 40 years, the oligomeric state of members of the mitochondrial carrier family (SLC25) has been the subject of debate. Initially, the consensus was that they were dimeric, based on the application of a large number of different techniques. However, the structures of the mitochondrial ADP/ATP carrier, a member of the family, clearly demonstrated that its structural fold is monomeric, lacking a conserved dimerisation interface. A re-evaluation of previously published data, with the advantage of hindsight, concluded that technical errors were at the basis of the earlier dimer claims. Here, we revisit this topic, as new claims for the existence of dimers of the bovine ADP/ATP carrier have emerged using native mass spectrometry of mitochondrial membrane vesicles. However, the measured mass does not agree with previously published values, and a large number of post-translational modifications are proposed to account for the difference. Contrarily, these modifications are not observed in electron density maps of the bovine carrier. If they were present, they would interfere with the structure and function of the carrier, including inhibitor and substrate binding. Furthermore, the reported mass does not account for three tightly bound cardiolipin molecules, which are consistently observed in other studies and are important stabilising factors for the transport mechanism. The monomeric carrier has all of the required properties for a functional transporter and undergoes large conformational changes that are incompatible with a stable dimerisation interface. Thus, our view that the native mitochondrial ADP/ATP carrier exists and functions as a monomer remains unaltered.

Residual film formation after emulsion application: Understanding the role and fate of excipients on skin surface.

This study focuses on the fate of excipients contained in topical emulsions once applied on the skin. The aim was thus to develop a methodology to characterize the residue left on the skin shortly after emulsion application. To this end, both the role and the impact of the different excipients on the formation and properties of the residue left on the skin surface once a product is applied were investigated. To that purpose, an O/W emulsion composed of an ester as oily phase, an emulsifier (alkylpolyglucoside-based vehicles), a polymer and a humectant (hydrophilic excipient) was first developed. Then, systems with fewer ingredients were prepared to understand their respective role in the residual film. This residual film was studied in vivo by means of biophysical instrumental methods, all being performed on the participants' forearm. Results highlighted the major role of the ester giving a bright and hydrophobic residue. While the surfactant structuration as the presence of glycerin and polymer provided a specific water distribution inside the residue on the skin surface. Finally, this work evidenced the ingredients organization in the residue depending on the systems composition, with a particular stratification on skin surface which could be considered in the formulation strategy for efficient active delivery and skin protection.

Biophysical Techniques for Target Validation and Drug Discovery in Transcription-Targeted Therapy.

In the post-genome era, pathologies become associated with specific gene expression profiles and defined molecular lesions can be identified. The traditional therapeutic strategy is to block the identified aberrant biochemical activity. However, an attractive alternative could aim at antagonizing key transcriptional events underlying the pathogenesis, thereby blocking the consequences of a disorder, irrespective of the original biochemical nature. This approach, called transcription therapy, is now rendered possible by major advances in biophysical technologies. In the last two decades, techniques have evolved to become key components of drug discovery platforms, within pharmaceutical companies as well as academic laboratories. This review outlines the current biophysical strategies for transcription manipulation and provides examples of successful applications. It also provides insights into the future development of biophysical methods in drug discovery and personalized medicine.

How does bovine serum albumin sustain in saccharomate® derived from pine tree biomass?

Nowadays, research on renewable raw materials and bioresources is a new concern towards the promotion of sustainable process and product development. The use of various plant biomasses such as starch, lignocellulosic and saccharide can be considered as an alternative for using cheaper and less polluting raw materials. In this regard, pine tree biomass, a lignocellulosic forest residue that has various value-added importance and it acts as a model of economic value to the agro-industrial fields. On the other hand, in order to meet and address the challenges of ever-increasing demands of bioresources, there has been significant research interest in deciphering the molecular interactions between proteins and biomass derived substances. No study reports the significance of saccharomate® derived from pine tree biomass on the structural and thermal stability of proteins. There is a sizable interest in the interactions between proteins and biomass derived substances, owing to their utilization and applications. Herein, we used various biophysical techniques such as absorption spectroscopy, fluorescence spectroscopy, circular dichroism (CD) and dynamic light scattering (DLS) to study the impact of pine tree biomass derived saccharomate® (PBDS) on bovine serum albumin (BSA). Further for better understanding of morphological changes of BSA in presence of biomass, Transmission electron microscopy (TEM) was also studied. The present study revealed that the increasing concentration of saccharomate® perturbs structural stability however; the thermal stability of BSA remained unchanged. The transition temperature of BSA remained approximately same in presence of different concentrations of PBDS. Furthermore, the size of BSA increases from 9.22 nm to 135.58 nm in presence of higher concentration of PBDS as revealed by DLS studies. To the best of our knowledge, the results represent first detailed proof of the unusual effect of PBDS on the model protein BSA.

Directive Effect of Chain Length in Modulating Peptide Nano-assemblies.

BACKGROUND: RADA-4 (Ac-RADARADARADARADA-NH2) is the most extensively studied and marketed self-assembling peptide, forming hydrogel, used to create defined threedimensional microenvironments for cell culture applications. OBJECTIVES: In this work, we use various biophysical techniques to investigate the length dependency of RADA aggregation and assembly. METHODS: We synthesized a series of RADA-N peptides, N ranging from 1 to 4, resulting in four peptides having 4, 8, 12, and 16 amino acids in their sequence. Through a combination of various biophysical methods including thioflavin T fluorescence assay, static right angle light scattering assay, Dynamic Light Scattering (DLS), electron microscopy, CD, and IR spectroscopy, we have examined the role of chain-length on the self-assembly of RADA peptide. RESULTS: Our observations show that the aggregation of ionic, charge-complementary RADA motifcontaining peptides is length-dependent, with N less than 3 are not forming spontaneous selfassemblies. CONCLUSION: The six biophysical experiments discussed in this paper validate the significance of chain-length on the epitaxial growth of RADA peptide self-assembly.

Biophysical approaches for exploring lipopeptide-lipid interactions.

In recent years, lipopeptides (LPs) have attracted a lot of attention in the pharmaceutical industry due to their broad-spectrum of antimicrobial activity against a variety of pathogens and their unique mode of action. This class of compounds has enormous potential for application as an alternative to conventional antibiotics and for pest control. Understanding how LPs work from a structural and biophysical standpoint through investigating their interaction with cell membranes is crucial for the rational design of these biomolecules. Various analytical techniques have been developed for studying intramolecular interactions with high resolution. However, these tools have been barely exploited in lipopeptide-lipid interactions studies. These biophysical approaches would give precise insight on these interactions. Here, we reviewed these state-of-the-art analytical techniques. Knowledge at this level is indispensable for understanding LPs activity and particularly their potential specificity, which is relevant information for safe application. Additionally, the principle of each analytical technique is presented and the information acquired is discussed. The key challenges, such as the selection of the membrane model are also been briefly reviewed.