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Find video protocols related to scientific articles indexed in Pubmed.
Intrinsically unstructured proteins by design-electrostatic interactions can control binding, folding, and function of a helix-loop-helix heterodimer.
J. Pept. Sci.
PUBLISHED: 02-18-2013
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Intrinsically disordered proteins that exist as unordered monomeric structures in aqueous solution at pH?7 but fold into four-helix bundles upon binding to recognized polypeptide targets have been designed. NMR and CD spectra of the monomeric polypeptides show the hallmarks of unordered structures, whereas in the bound state they are highly helical. Analytical ultracentrifugation data shows that the polypeptides bind to their targets to form exclusively heterodimers at neutral pH. To demonstrate the relationship between binding, folding, and function, a catalytic site for ester hydrolysis was introduced into an unordered and largely inactive monomer, but that was structured and catalytically active in the presence of a specific polypeptide target. Electrostatic interactions between surface-exposed residues inhibited the binding and folding of the monomers at pH?7. Charge-charge repulsion between ionizable amino acids was thus found to be sufficient to disrupt binding between polypeptide chains despite their inherent propensities for structure formation and may be involved in the folding and function of inherently disordered proteins in biology.
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Improving the diagnosis and management of neuroendocrine tumors: utilizing new advances in biomarker and molecular imaging science.
Neuroendocrinology
PUBLISHED: 02-11-2013
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Neuroendocrine tumors (NET) are malignant solid tumors that arise in hormone-secreting tissue of the diffuse neuroendocrine system or endocrine glands. Although traditionally understood to be a rare disease, the incidence and prevalence of NET have increased greatly in the past 3 decades. However, during this time, progress in diagnosis and outcome of NET has generally been modest. In order to achieve improved outcome in NET, a better understanding of NET biology combined with more reliable serum markers and better techniques to identify tumor localization and small lesions are needed. Although some NET biomarkers exist, sensitive and specific markers that predict tumor growth and behavior are generally lacking. In addition, the integration of new molecular imaging technologies in patient diagnosis and follow-up has the potential to enhance care. To discuss developments and issues required to improve diagnostics and management of NET patients, with specific focus on the latest advances in molecular imaging and biomarker science, 17 global leaders in the fields of NET, molecular imaging and biomarker technology gathered to participate in a 2-day meeting hosted by Prof. Kjell Öberg at the University of Uppsala in Sweden. During this time, findings were presented regarding methods with potential prognostic and treatment applications in NET or other types of cancers. This paper describes the symposium presentations and resulting discussions.
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Polypeptide folding-mediated tuning of the optical and structural properties of gold nanoparticle assemblies.
Nano Lett.
PUBLISHED: 11-08-2011
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Responsive hybrid nanomaterials with well-defined properties are of significant interest for the development of biosensors with additional applications in tissue engineering and drug delivery. Here, we present a detailed characterization using UV-vis spectroscopy and small angle X-ray scattering of a hybrid material comprised of polypeptide-decorated gold nanoparticles with highly controllable assembly properties. The assembly is triggered by a folding-dependent bridging of the particles mediated by the heteroassociation of immobilized helix-loop-helix polypeptides and a complementary nonlinear polypeptide present in solution. The polypeptides are de novo designed to associate and fold into a heterotrimeric complex comprised of two disulfide-linked four-helix bundles. The particles form structured assemblies with a highly defined interparticle gap (4.8±0.4 nm) that correlates to the size of the folded polypeptides. Transitions in particle aggregation dynamics, mass-fractal dimensions and ordering, as a function of particle size and the concentration of the bridging polypeptide, are observed; these have significant effects on the optical properties of the assemblies. The assembly and ordering of the particles are highly complex processes that are affected by a large number of variables including the number of polypeptides bridging the particles and the particle mobility within the aggregates. A fundamental understanding of these processes is of paramount interest for the development of novel hybrid nanomaterials with tunable structural and optical properties and for the optimization of nanoparticle-based colorimetric biodetection strategies.
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The molecular recognition of phosphorylated proteins by designed polypeptides conjugated to a small molecule that binds phosphate.
Org. Biomol. Chem.
PUBLISHED: 10-06-2011
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The conjugation of polypeptides from a designed set to the small molecule ligand 3,5-bis[[bis(2-pyridylmethyl)amino]methyl]benzoic acid, which in the presence of Zn(2+) ions binds inorganic phosphate, has been shown to provide a polypeptide conjugate that binds ?-casein, a multiply phosphorylated protein, with a dissociation constant K(D) of 17 nM. The measured affinity is more than three orders of magnitude higher than that of the small molecule ligand for phosphate and the binding of 500 nM of ?-casein was not inhibited by 10 mM phosphate buffer, providing a 2000-fold excess of phosphate ion over protein. The selectivity for phosphoproteins was demonstrated by extraction of ?-casein from solutions of various complexity, including milk and human serum spiked with ?-casein. In addition to ?-casein, ?-casein was also recognized but not ovoalbumin. Conjugation of a polypeptide to the zinc chelating ligand was therefore shown to give rise to dramatically increased affinity and also increased selectivity. A set of polypeptide conjugates is expected to be able to capture a large number of phosphorylated proteins, perhaps all, and in combination with electrophoresis or mass spectrometry become a powerful tool for the monitoring of phosphorylation levels. The presented binder can easily be attached to various types of surfaces; here demonstrated for the case of polystyrene particles. The example of phosphoproteins was selected since posttranslational phosphorylation is of fundamental importance in cell biology due to its role in signaling and therefore of great interest in drug development. The reported concept for binder development is, however, quite general and high-affinity binders can conveniently be developed for a variety of proteins including those with posttranslational modifications for which small molecule recognition elements are available.
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Polypeptide conjugate binders that discriminate between two isoforms of human carbonic anhydrase in human blood.
Chembiochem
PUBLISHED: 01-24-2011
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Two binder candidates 4-C37L34-B and 3-C15L8-B from a 16-membered set of 42-residue polypeptide conjugates designed to bind human carbonic anhydrase II (HCAII), were shown to bind HCAII with high affinity in a fluorescence-based screening assay. Two carbonic anhydrase isoforms with 60?% homology exist in human blood with HCAI being present in five- to sevenfold excess over HCAII. The ability of the binders to discriminate between HCAI and HCAII was evaluated with regard to what selectivity could be achieved by the conjugation of polypeptides from a 16-membered set to a small organic molecule that binds both isoforms with similar affinities. The polypeptide conjugate 4-C37L34-B bound HCAII with a K(D) of 17 nM and HCAI with a K(D) of 470 nM, that is, with a 30-fold difference in affinity. The corresponding dissociation constants for the complexes formed from 3-C15L8-B and the two carbonic anhydrases were 60 and 390 nM, respectively. This demonstration of selectivity between two very similar proteins is striking in view of the fact that the molecular weight of each one of the conjugate molecules is little more than 5000, the fold is unordered, and the polypeptide sequences were designed de novo and have no prior relationship to carbonic anhydrases. The results suggest that synthetic polypeptide conjugates can be prepared from organic molecules that are considered to be weak binders with low selectivity, yielding conjugates with properties that make them attractive alternatives to biologically generated binders in biotechnology and biomedicine.
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Crossing borders to bind proteins--a new concept in protein recognition based on the conjugation of small organic molecules or short peptides to polypeptides from a designed set.
Anal Bioanal Chem
PUBLISHED: 01-18-2011
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A new concept for protein recognition and binding is highlighted. The conjugation of small organic molecules or short peptides to polypeptides from a designed set provides binder molecules that bind proteins with high affinities, and with selectivities that are equal to those of antibodies. The small organic molecules or peptides need to bind the protein targets but only with modest affinities and selectivities, because conjugation to the polypeptides results in molecules with dramatically improved binder performance. The polypeptides are selected from a set of only sixteen sequences designed to bind, in principle, any protein. The small number of polypeptides used to prepare high-affinity binders contrasts sharply with the huge libraries used in binder technologies based on selection or immunization. Also, unlike antibodies and engineered proteins, the polypeptides have unordered three-dimensional structures and adapt to the proteins to which they bind. Binder molecules for the C-reactive protein, human carbonic anhydrase II, acetylcholine esterase, thymidine kinase 1, phosphorylated proteins, the D-dimer, and a number of antibodies are used as examples to demonstrate that affinities are achieved that are higher than those of the small molecules or peptides by as much as four orders of magnitude. Evaluation by pull-down experiments and ELISA-based tests in human serum show selectivities to be equal to those of antibodies. Small organic molecules and peptides are readily available from pools of endogenous ligands, enzyme substrates, inhibitors or products, from screened small molecule libraries, from phage display, and from mRNA display. The technology is an alternative to established binder concepts for applications in drug development, diagnostics, medical imaging, and protein separation.
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A new assay design for clinical diagnostics based on alternative recognition elements.
Biosens Bioelectron
PUBLISHED: 01-08-2010
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Herein, we present a new sandwich assay design containing a high affinity polypeptide scaffold as immobilized capture element and an antibody for detection. These polypeptide scaffolds provide a good affinity towards one antigen and can be linked to biosensor surfaces without affecting their binding capabilities. Furthermore, the small peptides are very stable, which allows for regenerating the surface several hundreds of times and thus for reuse of the biosensor. Moreover, these receptors can be synthesized with different affinities towards one antigen, which has been proven by characterizing them using a label-free detection method RIfS (reflectometric interference spectroscopy) for collecting kinetic data. Polypeptide scaffolds with different affinities have been chosen and characterized. Upon these results, sandwich-type assays have been set-up using a fluorescently labelled antibody as detection element. Thereby could be shown, that the working range of the assay can be shifted according to the affinity of the used capturing polypeptide scaffold. The scaffolds with a higher affinity towards the antigen can detect lower concentration, and in contrary, scaffolds with lower affinities can detect higher concentrations. In consequence, using this new sandwich-type assay, we avoid the complex procedure to immobilize antibodies in correct orientation, but simultaneously keep this well-known recognition element in the assay for detection. Furthermore, in addition to all the acknowledged properties of immunoassays, we add the possibility of tuning the working range of assays in distinct manner according to request.
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Colorimetric protein sensing by controlled assembly of gold nanoparticles functionalized with synthetic receptors.
Small
PUBLISHED: 07-10-2009
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A novel strategy is described for the colorimetric sensing of proteins, based on polypeptide-functionalized gold nanoparticles. Recognition is accomplished using a polypeptide sensor scaffold designed to specifically bind to the model analyte, human carbonic anhydrase II (HCAII). The extent of particle aggregation, induced by the Zn(2+)-triggered dimerization and folding of a second polypeptide also present on the surface of the gold nanoparticle, gives a readily detectable colorimetric shift that is dependent on the concentration of the target protein. In the absence of HCAII, particle aggregation results in a major redshift of the plasmon peak, whereas analyte binding prevented the formation of dense aggregates, significantly reducing the magnitude of the redshift. The versatility of the technique is demonstrated using a second model system based on the recognition of a peptide sequence from the tobacco mosaic virus coat protein (TMVP) by a recombinant antibody fragment (Fab57P). Concentrations down to approximately 10 nM and approximately 25 nM are detected for HCAII and Fab57P, respectively. This strategy is proposed as a generic platform for robust and specific protein analysis that can be further developed to monitor a wide range of target proteins.
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Powerful binders for the D-dimer by conjugation of the GPRP peptide to polypeptides from a designed set--illustrating a general route to new binders for proteins.
Bioconjug. Chem.
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The synthetic tetrapeptide GPRP based on the amino-terminal GPR sequence of the fibrin ?-chain binds the D-dimer protein with a dissociation constant K(D) of 25 ?M. The D-dimer protein, a well-known biomarker for thrombosis, contains two cross-linked D fragments from the fibrinogen protein formed upon degradation of the fibrin gel, the core component of blood clots. In order to develop a specific high-affinity binder for the D-dimer protein, GPRP was conjugated via an aliphatic spacer to each member of a set of sixteen polypeptides designed for the development of binder molecules for proteins in general. The binders were individually characterized and ranked using surface plasmon resonance (SPR) analysis. The dissociation constant of the complex formed from the D-dimer and 4-D15L8-GPRP labeled with fluorescein was determined by fluorescense titration and found to be 3 nM, an affinity 4 orders of magnitude higher than that of free GPRP. According to SPR analysis, binding was completely inhibited by free GPRP at mM concentrations and the polypeptide conjugate was therefore shown to bind specifically to the binding site of GPRP. Affinities were further enhanced by dimerization of the polypeptide conjugates via a bifunctional linker resulting in dissociation constants that were further decreased (affinities increased) by factors of 2-4. The results suggest an efficient route to specific binders for proteins based on short peptides with affinities that need only to be modest, thus shortening the time of binder development dramatically.
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A synthetic polypeptide conjugate from a 42-residue polypeptide and salicylhydroxamic acid binds human myeloperoxidase with high affinity.
J. Pept. Sci.
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Myeloperoxidase (MPO) is a 150?kD tetrameric heme protein consisting of two heavy chains and two light chains, which is present in neutrophils, white blood cells, at concentrations between 2% and 5% and plays an important role in the innate immune system. The MPO concentration in serum or plasma has been shown to be linked to the risk for cardiovascular diseases, and MPO is considered to be a high potential diagnostic biomarker. To develop a molecule that binds MPO, salicylhydroxamic acid (SHA), a substrate analog inhibitor of MPO with a K(D) ?=?2??M, was conjugated to a designed set of 42-residue polypeptide scaffolds via 9- and 11-carbon atom aliphatic spacers to form 20 different protein binder candidates, and their interactions with MPO were evaluated by surface plasmon resonance analysis. The polypeptide conjugate 4C37L34C11SHA was found to bind to MPO with an affinity that could be estimated to have a dissociation constant of around 400?pM, nearly four orders of magnitude higher than that of SHA. Inhibition of binding to MPO by free SHA was observed in competition experiments demonstrating that the binding of the polypeptide conjugate is dominated by the interactions of SHA with the heme cavity. Although still in the future, the discovery of these new synthetic binders for MPO suggests a route to clinical diagnostic tests in vivo or in vitro, independent of antibodies. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.
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Designed protein binders in combination with nanocrystalline diamond for use in high-sensitivity biosensors.
Anal Bioanal Chem
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A platform for diagnostic applications showing signal-to-noise ratios that by far surpass those of traditional bioanalytical test formats has been developed. It combines the properties of modified nanocrystalline diamond (NCD) surfaces and those of polyethylene oxide and polypropylene oxide based block copolymers for surface passivation and binder conjugation with a new class of synthetic binders for proteins. The NCD surfaces were fluorine-, hydrogen-, or oxygen-terminated prior to further biofunctionalization and the surface composition was characterized by X-ray photoelectron spectroscopy. In a proof of principle demonstration targeting the C-reactive protein, an ELISA carried out using an F-terminated diamond surface showed a signal-to-noise ratio of 3,900 which compares well to the signal-to-noise of 89 obtained in an antibody-based ELISA on a polystyrene microtiter plate, a standard test format used in most life science laboratories today. The increase in signal-to-noise ratio is to a large extent the result of extremely efficient passivation of the diamond surface. The results suggest that significant improvements can be obtained in standardized test formats using new materials in combination with new types of chemical coatings and receptor molecules.
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Efficient formation of heterodimers from peptides and proteins using unsymmetrical polyfluorophenyl esters of dicarboxylic acids.
J. Pept. Sci.
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An efficient method for the heteroconjugation of biomolecules carrying free amino groups was reported previously, where mixed polyfluorophenyl diesters of dicarboxylic acids with varied aliphatic chain length were shown to be efficient reagents for the conjugation of a variety of model biomolecules. The concept was based on the differential reactivity of the esters towards amines. The concept has now been further optimized, and a 2,6-difluorophenyl-pentafluorophenyl diester combination has been demonstrated to be the most efficient, both with respect to selectivity and to reaction rate. A pentafluorophenyl ester reacts faster with an amino group and requires a weaker base than a 2,6-difluorophenyl ester that requires a stronger base and longer reaction time. With the use of this combination of esters, we obtained considerably shortened reaction times compared with those reported previously, yet still retaining the desired selectivity in heteroconjugation. The increased reactivity of the bifunctional reagent allowed the construction of sophisticated peptide heteroconjugates from peptides, carbohydrates and proteins, showing a wide scope of applicability in the field of assembling functional bioconjugates.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.