Biophysical Techniques in Structural Biology.

Over the past six decades, steadily increasing progress in the application of the principles and techniques of the physical sciences to the study of biological systems has led to remarkable insights into the molecular basis of life. Of particular significance has been the way in which the determination of the structures and dynamical properties of proteins and nucleic acids has so often led directly to a profound understanding of the nature and mechanism of their functional roles. The increasing number and power of experimental and theoretical techniques that can be applied successfully to living systems is now ushering in a new era of structural biology that is leading to fundamentally new information about the maintenance of health, the origins of disease, and the development of effective strategies for therapeutic intervention. This article provides a brief overview of some of the most powerful biophysical methods in use today, along with references that provide more detailed information about recent applications of each of them. In addition, this article acts as an introduction to four authoritative reviews in this volume. The first shows the ways that a multiplicity of biophysical methods can be combined with computational techniques to define the architectures of complex biological systems, such as those involving weak interactions within ensembles of molecular components. The second illustrates one aspect of this general approach by describing how recent advances in mass spectrometry, particularly in combination with other techniques, can generate fundamentally new insights into the properties of membrane proteins and their functional interactions with lipid molecules. The third reviewdemonstrates the increasing power of rapidly evolving diffraction techniques, employing the very short bursts of X-rays of extremely high intensity that are now accessible as a result of the construction of free-electron lasers, in particular to carry out time-resolved studies of biochemical reactions. The fourth describes in detail the application of such approaches to probe the mechanism of the light-induced changes associated with bacteriorhodopsin's ability to convert light energy into chemical energy.

Rational design, synthesis, and biophysical characterization of a peptidic MDM2-MDM4 interaction inhibitor.

In recent years, the restoration of p53 physiological functions has become an attractive therapeutic approach to develop novel and efficacious cancer therapies. Among other mechanisms, the oncosuppressor protein p53 is functionally regulated by MDM2 through its E3 ligase function. MDM2 promotes p53 ubiquitination and degradation following homodimerization or heterodimerization with MDM4. Recently, we discovered Pep3 (1, Pellegrino et al., 2015), a novel peptidic inhibitor of MDM2 dimerization able to restore p53 oncosuppressive functions both in vitro and in vivo. In this work, we were able to identify the key interactions between peptide 1 and MDM2 RING domain and to design peptide 2, a truncated version of 1 that is still able to bind MDM2. Integrating both computational and biophysical techniques, we show that peptide 2 maintains the conserved peptide 1-MDM2 interactions and is still able to bind to full-length MDM2.

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.

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.

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.

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.

Application of biophysical and skin imaging techniques to evaluate the film-forming effect of cosmetic formulations.

OBJECTIVE: Products with film-forming effect, or 'second skin', which guarantees an immediate protective effect after application, is a highlight, especially when composed of natural ingredients. Thus, the objective of this study was to evaluate the immediate film-forming effect on skin of a gel and emulsion formulations added with Kappaphycus alvarezii and Caesalpinia spinosa extracts through biophysical and skin imaging techniques, especially with the Reflectance Confocal Microscopy (RCM). METHODS: The measurements were done in the forearm region before (baseline) and 1 h after of application of the developed formulation and its control. The parameters related to the stratum corneum water content, transepidermal water loss (TEWL), cutaneous microrelief and morphological and structural characteristics of the epidermis were analysed through the following biophysical and skin imaging techniques: Corneometer® CM 825, Tewameter® TM 300, Visioscan® VC98 and Vivascope® 1500, respectively. A sensorial analysis was also performed to study how the formulations were perceived on the skin. RESULTS: The obtained results showed that the active ingredient under study allows the film formation on the skin surface, leading to a reduction of TEWL and skin desquamation. The obtained images from RCM showed a reduction of furrows on the skin surface and a film formation after a single application of the formulations. However, these effects were more pronounced in the emulsion formulation, which suggests a synergistic effect of the active ingredient under study with the emollients of formulation composition. This result was also observed in the sensorial analysis, as both formulations added with the active substance were well evaluated. CONCLUSION: The presence of Kappaphycus alvarezii and Caesalpinia spinosa extracts in the studied cosmetic formulations, enabled a film formation on a skin surface, bringing benefits as a reduction of transepidermal water loss and skin desquamation, as well as a furrows reduction and an improvement of stratum corneum after 1 h of application. Finally, the skin imaging techniques can be suggested as an excellent tool to evaluate a film-forming effect of cosmetic formulations.

OBJECTIF: Les produits ayant un effet filmogène, ou « deuxième peau ¼ qui garantit un effet protecteur immédiat après l'application, sont un plus, notamment lorsqu'ils sont composés d'ingrédients naturels. L'objectif de cette étude visait ainsi à évaluer l'effet filmogène immédiat sur la peau de formulations avec gel et émulsion enrichies d'extraits de Kappaphycus alvarezii et de Caesalpinia spinosa grâce à des techniques d'imagerie cutanée et biophysique, en particulier par microscopie confocale par réflectance (MCR). MÉTHODES: Les mesures ont été effectuées dans la région de l'avant-bras avant (référence) et 1 h après l'application de la formulation développée et de son contrôle. Les paramètres liés à la teneur en eau de la couche cornée, perte d'eau transépidermique (Transepidermal Water Loss, TEWL), au microrelief cutané et aux caractéristiques structurelles et morphologiques de l'épiderme ont été analysés à l'aide des techniques d'imagerie cutanée et biophysique suivantes : Corneometer® CM 825, Tewameter® TM 300, Visioscan® VC98 et Vivascope® 1500, respectivement. Une analyse sensorielle a également été réalisée pour étudier comment les formulations étaient perçues sur la peau. RÉSULTATS: Les résultats obtenus ont montré que le principe actif étudié permet la formation d'un film à la surface de la peau, entraînant une réduction de la TEWL et de la desquamation de la peau. Les images obtenues par MCR ont montré une réduction des sillons à la surface de la peau et la formation d'un film après une seule application des formulations. Ces effets étaient toutefois davantage prononcés avec la formulation de type émulsion, ce qui suggère un effet synergique du principe actif étudié avec les émollients contenus dans la formulation. Ce résultat a également été observé dans l'analyse sensorielle, car les deux formulations enrichies en substance active ont été bien évaluées. CONCLUSION: La présence d'extraits de Kappaphycus alvarezii et de Caesalpinia spinosa dans les préparations cosmétiques étudiées a permis la formation d'un film à la surface de la peau, procurant des bénéfices sous forme d'une réduction de la perte d'eau transépidermique et de la desquamation de la peau, de même qu'une réduction des sillons et une amélioration de la couche cornée 1 h après l'application. Enfin, les techniques d'imagerie cutanée peuvent être proposées comme excellent outil d'évaluation de l'effet filmogène des préparations cosmétiques.

Biophysical insight into the heparin-peptide interaction and its modulation by a small molecule.

The heparin-protein interaction plays a vital role in numerous physiological and pathological processes. Not only is the binding mechanism of these interactions poorly understood, studies concerning their therapeutic targeting are also limited. Here, we have studied the interaction of the heparin interacting peptide (HIP) from Tat (which plays important role in HIV infections) with heparin. Isothermal titration calorimetry binding exhibits distinct biphasic isotherm with two different affinities in the HIP-heparin complex formation. Overall, the binding was mainly driven by the nonionic interactions with a small contribution from ionic interactions. The stoichiometric analysis suggested that the minimal site for a single HIP molecule is a chain of 4 to 5 saccharide molecules, also supported by docking studies. The investigation was also focused on exploiting the possibility of using a small molecule as an inhibitor of the HIP-heparin complex. Quinacrine, because of its ability to mimic the HIP interactions with heparin, was shown to successfully modulate the HIP-heparin interactions. This result demonstrates the feasibility of inhibiting the disease relevant heparin-protein interactions by a small molecule, which could be an effective strategy for the development of future therapeutic agents.

Undesirable impact on structure and stability of insulin on addition of (+)-catechin hydrate with sugar.

Insulin (In) based formulation has been used over decades for the cure of In-dependent diabetic patients, however, more attempts are still required to improve the remedial use of In. In this regard, the use of green tea has become widespread nowadays. However, it is unknown that (+)-catechin hydrate (CAT), a major component of green tea which enhances anti-diabetic activity of In, will or will not enhance the structure and stability of In if ingested with sugars. Interestingly, by using biophysical techniques, present study reveals the fact that the use of sugar during the intake of green tea extract may produce unwanted effects on In which may further lead to some disorders associated with In stability and also create obstacle in successful implications of In formulations.

Exploration of ligand-induced protein conformational alteration, aggregate formation, and its inhibition: A biophysical insight.

The association of protein aggregates with plentiful human diseases has fascinated studies regarding the biophysical characterization of protein misfolding and ultimately their aggregate formation mechanism. Protein-ligand interaction, their mechanism, conformational changes by ligands, and protein aggregate formation have been studied upon exploiting experimental techniques and computational methodologies. Such studies for the exploration of ligand-induced conformational changes in protein, misfolding and aggregation, has confirmed drastic progresses in the study of aggregate formation pathways. This review comprises of an inclusive description of contemporary experimental techniques as well as theoretical improvements in the interpretation of the conformational properties of protein. We have also discussed various factors responsible for the microenvironment change around protein that sequentially causes amyloidoses. Biophysical techniques and cell-based assays to gain comprehensive understandings of protein-ligand interaction, protein folding, and aggregation pathways have also been described. The promising therapeutic methods used to inhibit the protein fibrillogenesis have also been discussed.

Structural and component mining of nails using bioengineering techniques.

The human nail is one of the challenging membranes for the scientists to target and to improve the clinical efficacy of ungual formulations. The understanding of nail physiology, impact of hydration on its properties and presence of trace elements in nails as biomarkers has been explored by various researchers in clinical studies. Despite the importance of biophysical techniques for the assessment of structure and physiology of nail, minimum literature analyses biophysical, biochemical and bioanalytical approaches. However, nowadays scientists in bioengineering field are keen in developing non-invasive, reliable and reproducible techniques for the assessment of different anatomical and functional parameters of nails for testing of ungual products.

A growing toolbox of techniques for studying ß-barrel outer membrane protein folding and biogenesis.

Great strides into understanding protein folding have been made since the seminal work of Anfinsen over 40 years ago, but progress in the study of membrane protein folding has lagged behind that of their water soluble counterparts. Researchers in these fields continue to turn to more advanced techniques such as NMR, mass spectrometry, molecular dynamics (MD) and single molecule methods to interrogate how proteins fold. Our understanding of ß-barrel outer membrane protein (OMP) folding has benefited from these advances in the last decade. This class of proteins must traverse the periplasm and then insert into an asymmetric lipid membrane in the absence of a chemical energy source. In this review we discuss old, new and emerging techniques used to examine the process of OMP folding and biogenesis in vitro and describe some of the insights and new questions these techniques have revealed.

Physical basis for the ofloxacin-induced acceleration of lysozyme aggregation and polymorphism in amyloid fibrils.

Aggregation of globular proteins is an intractable problem which generally originates from partially folded structures. The partially folded structures first collapse non-specifically and then reorganize into amyloid-like fibrils via one or more oligomeric intermediates. The fibrils and their on/off pathway intermediates may be toxic to cells and form toxic deposits in different human organs. To understand the basis of origins of the aggregation diseases, it is vital to study in details the conformational properties of the amyloidogenic partially folded structures of the protein. In this work, we examined the effects of ofloxacin, a synthetic fluoroquinolone compound on the fibrillar aggregation of hen egg-white lysozyme. Using two aggregation conditions (4M GuHCl at pH 7.0 and 37 °C; and pH 1.7 at 65 °C) and a number of biophysical techniques, we illustrate that ofloxacin accelerates fibril formation of lysozyme by binding to partially folded structures and modulating their secondary, tertiary structures and surface hydrophobicity. We also demonstrate that Ofloxacin-induced fibrils show polymorphism of morphology, tinctorial properties and hydrophobic surface exposure. This study will assist in understanding the determinant of fibril formation and it also indicates that caution should be exercised in the use of ofloxacin in patients susceptible to various aggregation diseases.

Expression, purification and characterization of Esx-1 secretion-associated protein EspL from Mycobacterium tuberculosis.

The Esx-1 cluster encodes a special secretion system that is important for granuloma formation and virulence when Mycobacterium tuberculosis infects the host. As one of the 'core' genes in the cluster, Rv3880c gene codes an Esx-1 secretion-associated protein EspL from Mycobacterium tuberculosis (MtEspL). It has been reported that EspL had a strong influence on the secretion of other two virulence factors, EsxA and EspE. However, so far little is known about the tertiary structure and specific function of MtEspL due to the difficulty in preparing the high-quality protein. In this study, we tried several fusion tags and various expression conditions to recombinantly express MtEspL. Through a four-step purification procedure, ultimately, we successfully prepared the full-length MtEspL in Escherichia coli BL21 (DE3) with a purity of 98%. The yields of the purified MtEspL protein were 14 mg/L in Luria Bertani medium and 5.6 mg/L in M9 minimal medium, respectively. Biophysical experiments showed that MtEspL existed in a dimeric form. Moreover, the (1)H-(15)N HSQC spectrum recorded on MtEspL illustrates a favorable dispersion of the resonance peaks, indicating that the symmetric dimeric MtEspL adopted a well-folded structure and might be feasible to determine its solution structure by NMR spectroscopy. Moreover, we identified a strong DNA-binding ability of MtEspL with fluorescence quenching experiments. Our work lays the basis for further structural determination and functional exploration of MtEspL.

Exploring the antioxidant potentiality of two food by-products into a topical cream: stability, in vitro and in vivo evaluation.

CONTEXT: Coffee silverskin (CS), a food by-product of the coffee roasting industry, has been studied as an active ingredient for skin care products due to its high potential of antioxidant activity and low cytotoxicity. Another food waste used as ingredient with promising characteristics is obtained from Medicago sativa (MS), which antioxidants and isoflavones content is high. OBJECTIVE: The aim of this study is to evaluate and characterize a new body formulation containing two food by-products extracts. MATERIALS AND METHODS: Different parameters (such as pH, rheological behavior, color, antioxidant content and microbiological analysis) of a body cream formulation containing by-products (CSMS) and a formulation without extracts (F) were evaluated under a stability study during 180 days at different temperatures. Moreover, the in vitro cell toxicity and the in vivo skin safety and protective effects were also assessed. RESULTS: Formulation showed stable physical properties and antioxidant activity during 180 days of storage. In vitro toxicity was screened in two skin cell lines (fibroblasts and keratinocytes) and any toxicity was reported. The in vivo test carried out showed that, with respect to irritant effects, CSMS formulation can be regarded as safe for topical application and the skin hydratation improved after 30 days of its use. Also, considering the consumer acceptance, more than 90% of volunteers classified it as very pleasant. CONCLUSIONS: CSMS formulation is stable and safe for topical use as no adverse and/or side effects were observed during the application period of testing, improving skin protective properties.

Protein-Directed Dynamic Combinatorial Chemistry: A Guide to Protein Ligand and Inhibitor Discovery.

Protein-directed dynamic combinatorial chemistry is an emerging technique for efficient discovery of novel chemical structures for binding to a target protein. Typically, this method relies on a library of small molecules that react reversibly with each other to generate a combinatorial library. The components in the combinatorial library are at equilibrium with each other under thermodynamic control. When a protein is added to the equilibrium mixture, and if the protein interacts with any components of the combinatorial library, the position of the equilibrium will shift and those components that interact with the protein will be amplified, which can then be identified by a suitable biophysical technique. Such information is useful as a starting point to guide further organic synthesis of novel protein ligands and enzyme inhibitors. This review uses literature examples to discuss the practicalities of applying this method to inhibitor discovery, in particular, the set-up of the combinatorial library, the reversible reactions that may be employed, and the choice of detection methods to screen protein ligands from a mixture of reversibly forming molecules.

Integrated approach in the assessment of skin compatibility of cosmetic formulations with green coffee oil.

OBJECTIVE: Green coffee oil (GCO) has been used in cosmetic formulations due to its emollient and anti-ageing properties. However, there are insufficient studies about its safety when applied in cosmetic formulations. METHODS: Cytotoxicity of GCO and of formulations containing 2.5-15% of GCO was evaluated by the MTT reduction assay, in human keratinocytes. Formulations containing 15% of GCO and the vehicle were applied under in use conditions in the volar forearm of human volunteers during 3 days. Transepidermal water loss, stratum corneum water content and erythema index were evaluated each 24 h using biophysical techniques. The same formulations were probed for skin tolerance through a patch test. RESULTS: Neither pure GCO nor its formulations showed cytotoxic effects in concentrations up to 100 µg mL(-1) . Transepidermal water loss values showed a slight reduction when the formulation containing GCO was applied. Stratum corneum water content and erythema index did not show significant differences, as the results observed in the first day of the study were maintained throughout 3 days. None of the volunteers display any reaction after using an occlusive patch. CONCLUSION: The results obtained in the study indicate that GCO seems to be safe for topical applications and showed good skin compatibility under the experimental conditions of the study.

Surface plasmon resonance using the catalytic domain of soluble guanylate cyclase allows the detection of enzyme activators.

Soluble Guanylate Cyclase (sGC) is the receptor for the signalling agent nitric oxide (NO) and catalyses the production of the second messenger cyclic guanosine monophosphate (cGMP) from guanosine triphosphate (GTP). The enzyme is an attractive drug target for small molecules that act in the cardiovascular and pulmonary systems, and has also shown to be a potential target in neurological disorders. We have discovered that 5-(indazol-3-yl)-1,2,4-oxadiazoles activate the enzyme in the absence of added NO and shown they bind to the catalytic domain of the enzyme after development of a surface plasmon resonance assay that allows the biophysical detection of intrinsic binding of ligands to the full length sGC and to a construct of the catalytic domain.

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.