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Sample records for adsorbed protein molecules

  1. The entropies of adsorbed molecules.

    PubMed

    Campbell, Charles T; Sellers, Jason R V

    2012-10-31

    Adsorbed molecules are involved in many reactions on solid surface that are of great technological importance. As such, there has been tremendous effort worldwide to learn how to predict reaction rates and equilibrium constants for reactions involving adsorbed molecules. Theoretical calculation of both the rate and equilibrium constants for such reactions requires knowing the entropy and enthalpy of the adsorbed molecule. While much effort has been devoted to measuring and calculating the enthalpies of well-defined adsorbates, few measurements of the entropies of adsorbates have been reported. We present here a new way to determine the standard entropies of adsorbed molecules (S(ad)(0)) on single crystal surfaces from temperature programmed desorption data, prove its accuracy by comparison to entropies measured by equilibrium methods, and apply it to published data to extract new entropies. Most importantly, when combined with reported entropies, we find that at high coverage, they linearly track the entropy of the gas-phase molecule at the same temperature (T), such that S(ad)(0)(T) = 0.70 S(gas)(0)(T) - 3.3R (R = the gas constant), with a standard deviation of only 2R over a range of 50R. These entropies, which are ~2/3 of the gas, are huge compared to most theoretical predictions. This result can be extended to reliably predict prefactors in the Arrhenius rate constant for surface reactions involving such species, as proven here for desorption. PMID:23033909

  2. Hydrophobic Porous Material Adsorbs Small Organic Molecules

    NASA Technical Reports Server (NTRS)

    Sharma, Pramod K.; Hickey, Gregory S.

    1994-01-01

    Composite molecular-sieve material has pore structure designed specifically for preferential adsorption of organic molecules for sizes ranging from 3 to 6 angstrom. Design based on principle that contaminant molecules become strongly bound to surface of adsorbent when size of contaminant molecules is nearly same as that of pores in adsorbent. Material used to remove small organic contaminant molecules from vacuum systems or from enclosed gaseous environments like closed-loop life-support systems.

  3. Size selective hydrophobic adsorbent for organic molecules

    NASA Technical Reports Server (NTRS)

    Sharma, Pramod K. (Inventor); Hickey, Gregory S. (Inventor)

    1997-01-01

    The present invention relates to an adsorbent formed by the pyrolysis of a hydrophobic silica with a pore size greater than 5 .ANG., such as SILICALITE.TM., with a molecular sieving polymer precursor such as polyfurfuryl alcohol, polyacrylonitrile, polyvinylidene chloride, phenol-formaldehyde resin, polyvinylidene difluoride and mixtures thereof. Polyfurfuryl alcohol is the most preferred. The adsorbent produced by the pyrolysis has a silicon to carbon mole ratio of between about 10:1 and 1:3, and preferably about 2:1 to 1:2, most preferably 1:1. The pyrolysis is performed as a ramped temperature program between about 100.degree. and 800.degree. C., and preferably between about 100.degree. and 600.degree. C. The present invention also relates to a method for selectively adsorbing organic molecules having a molecular size (mean molecular diameter) of between about 3 and 6 .ANG. comprising contacting a vapor containing the small organic molecules to be adsorbed with the adsorbent composition of the present invention.

  4. Isomerization reactions on single adsorbed molecules.

    PubMed

    Morgenstern, Karina

    2009-02-17

    Molecular switches occur throughout nature. In one prominent example, light induces the isomerization of retinal from the compact 11-cis form to the elongated all-trans form, a conversion that triggers the transformation of light into a neural impulse in the eye. Applying these natural principles to synthetic systems offers a promising way to construct smaller and faster nanoelectronic devices. In such systems, electronic switches are essential components for storage and logical operations. The development of molecular switches on the single-molecule level would represent a major step toward incorporating molecules as building units into nanoelectronic circuits. Molecular switches must be both reversible and bistable. To meet these requirements, a molecule must have at least two different thermally stable forms and a way to repeatedly interconvert between those forms based on changes in light, heat, pressure, magnetic or electric fields, pH, mechanical forces, or electric currents. The conversion should be connected to a measurable change in electronic, optical, magnetic, or mechanical properties. Because isomers can differ significantly in physical and chemical properties, isomerization could serve as a molecular switching mechanism. Integration of molecular switches into larger circuits will probably require arranging them on surfaces, which will require a better understanding of isomerization reactions in these environments. In this Account, we describe our scanning tunneling microscopy studies of the isomerization of individual molecules adsorbed on metal surfaces. Investigating chlorobenzene and azobenzene derivatives on the fcc(111) faces of Ag, Cu, and Au, we explored the influence of substituents and the substrate on the excitation mechanism of the isomerization reaction induced by inelastically tunneling electrons. We achieved an irreversible configurational (cis-trans) isomerization of individual 4-dimethyl-amino-azobenzene-4-sulfonic acid molecules on Au

  5. Adsorbed molecules in external fields: Effect of confining potential.

    PubMed

    Tyagi, Ashish; Silotia, Poonam; Maan, Anjali; Prasad, Vinod

    2016-12-01

    We study the rotational excitation of a molecule adsorbed on a surface. As is well known the interaction potential between the surface and the molecule can be modeled in number of ways, depending on the molecular structure and the geometry under which the molecule is being adsorbed by the surface. We explore the effect of change of confining potential on the excitation, which is largely controlled by the static electric fields and continuous wave laser fields. We focus on dipolar molecules and hence we restrict ourselves to the first order interaction in field-molecule interaction potential either through permanent dipole moment or/and the molecular polarizability parameter. It is shown that confining potential shapes, strength of the confinement, strongly affect the excitation. We compare our results for different confining potentials. PMID:27387127

  6. Inhomogeneous distribution of organic molecules adsorbed in sol gel glasses

    NASA Astrophysics Data System (ADS)

    Meneses-Nava, M. A.; Chávez-Cerda, S.; Sánchez-Villicaña, V.; Sánchez-Mondragón, J. J.; King, T. A.

    1999-09-01

    The effects of the porous matrix upon the radiative characteristics of quinine sulphate doped sol-gel glasses are investigated. The broadenings of the absorption and fluorescence spectra are explained by the attachment of the molecules on distorted sites or in a non-planar fashion, creating an inhomogeneous distribution of adsorbed molecules. For this reason, each emitting center relaxes with its own characteristics. This inhomogeneous distribution is also supported by the non-exponential and the wavelength dependence of the fluorescence decay.

  7. Hydrogen molecule on lithium adsorbed graphene: A DFT study

    NASA Astrophysics Data System (ADS)

    Kaur, Gagandeep; Gupta, Shuchi; Gaganpreet, Dharamvir, Keya

    2016-05-01

    Electronic structure calculations for the adsorption of molecular hydrogen on lithium (Li) decorated and pristine graphene have been studied systematically using SIESTA code [1] within the framework of the first-principle DFT under the Perdew-Burke-Ernzerhof (PBE) form of the generalized gradient approximation (GGA)[2], including spin polarization. The energy of adsorption of hydrogen molecule on graphene is always enhanced by the presence of co-adsorbed lithium. The most efficient adsorption configuration is when H2 is lying parallel to lithium adsorbed graphene which is in contrast to its adsorption on pristine graphene (PG) where it prefers perpendicular orientation.

  8. High capacity cryogel-type adsorbents for protein purification.

    PubMed

    Singh, Naveen Kumar; Dsouza, Roy N; Grasselli, Mariano; Fernández-Lahore, Marcelo

    2014-08-15

    Cryogel bodies were modified to obtain epoxy groups by graft-copolymerization using both chemical and gamma irradiation initiation techniques. The free epoxy adsorbents were reacted further to introduce diethylaminoethanol (DEAE) functionalities. The resulting weak anion-exchange cryogel adsorbents showed dynamic binding capacities of ca. 27±3mg/mL, which was significantly higher than previously reported for this type of adsorbent material. Gamma irradiated grafting initiation showed a 4-fold higher capacity for proteins than chemical grafting initiation procedures. The phosphate capacity for these DEAE cryogels was 119mmol/L and also showed similar column efficiency as compared to commercial adsorbents. The large pores in the cryogel structure ensure convective transport of the molecules to active binding sites located on the polymer-grafted surface of cryogels. However, as cryogels have relatively large pores (10-100μm), the BET area available for surface activation is low, and consequently, the capacity of the cryogels is relatively low for biomolecules, especially when compared to commercial beaded adsorbents. Nevertheless, we have shown that gamma ray mediated surface grafting of cryogel matrices greatly enhance their functional and adsorptive properties.

  9. High capacity cryogel-type adsorbents for protein purification.

    PubMed

    Singh, Naveen Kumar; Dsouza, Roy N; Grasselli, Mariano; Fernández-Lahore, Marcelo

    2014-08-15

    Cryogel bodies were modified to obtain epoxy groups by graft-copolymerization using both chemical and gamma irradiation initiation techniques. The free epoxy adsorbents were reacted further to introduce diethylaminoethanol (DEAE) functionalities. The resulting weak anion-exchange cryogel adsorbents showed dynamic binding capacities of ca. 27±3mg/mL, which was significantly higher than previously reported for this type of adsorbent material. Gamma irradiated grafting initiation showed a 4-fold higher capacity for proteins than chemical grafting initiation procedures. The phosphate capacity for these DEAE cryogels was 119mmol/L and also showed similar column efficiency as compared to commercial adsorbents. The large pores in the cryogel structure ensure convective transport of the molecules to active binding sites located on the polymer-grafted surface of cryogels. However, as cryogels have relatively large pores (10-100μm), the BET area available for surface activation is low, and consequently, the capacity of the cryogels is relatively low for biomolecules, especially when compared to commercial beaded adsorbents. Nevertheless, we have shown that gamma ray mediated surface grafting of cryogel matrices greatly enhance their functional and adsorptive properties. PMID:24980092

  10. Volumetric Interpretation of Protein Adsorption: Capacity Scaling with Adsorbate Molecular Weight and Adsorbent Surface Energy

    PubMed Central

    Parhi, Purnendu; Golas, Avantika; Barnthip, Naris; Noh, Hyeran; Vogler, Erwin A.

    2009-01-01

    Silanized-glass-particle adsorbent capacities are extracted from adsorption isotherms of human serum albumin (HSA, 66 kDa), immunoglobulin G (IgG, 160 kDa), fibrinogen (Fib, 341 kDa), and immunoglobulin M (IgM, 1000 kDa) for adsorbent surface energies sampling the observable range of water wettability. Adsorbent capacity expressed as either mass-or-moles per-unit-adsorbent-area increases with protein molecular weight (MW) in a manner that is quantitatively inconsistent with the idea that proteins adsorb as a monolayer at the solution-material interface in any physically-realizable configuration or state of denaturation. Capacity decreases monotonically with increasing adsorbent hydrophilicity to the limit-of-detection (LOD) near τo = 30 dyne/cm (θ~65o) for all protein/surface combinations studied (where τo≡γlvocosθ is the water adhesion tension, γlvo is the interfacial tension of pure-buffer solution, and θ is the buffer advancing contact angle). Experimental evidence thus shows that adsorbent capacity depends on both adsorbent surface energy and adsorbate size. Comparison of theory to experiment implies that proteins do not adsorb onto a two-dimensional (2D) interfacial plane as frequently depicted in the literature but rather partition from solution into a three-dimensional (3D) interphase region that separates the physical surface from bulk solution. This interphase has a finite volume related to the dimensions of hydrated protein in the adsorbed state (defining “layer” thickness). The interphase can be comprised of a number of adsorbed-protein layers depending on the solution concentration in which adsorbent is immersed, molecular volume of the adsorbing protein (proportional to MW), and adsorbent hydrophilicity. Multilayer adsorption accounts for adsorbent capacity over-and-above monolayer and is inconsistent with the idea that protein adsorbs to surfaces primarily through protein/surface interactions because proteins within second (or higher

  11. UV-induced protonation of molecules adsorbed on ice surfaces at low temperature.

    PubMed

    Moon, Eui-Seong; Lee, Chang-Woo; Kim, Joon-Ki; Park, Seong-Chan; Kang, Heon

    2008-05-21

    UV irradiation of ice films adsorbed with methylamine molecules induces protonation of the adsorbate molecules at low temperature (50-130 K). The observation indicates that long-lived protonic defects are created in the ice film by UV light, and they transfer protons to the adsorbate molecules via tunneling mechanism at low temperature. The methylammonium ion formed by proton transfer remains to be stable at the ice surface. It is suggested that this solid-phase protonation might play a significant role in the production of molecular ions in interstellar clouds.

  12. Influence of particle size on the binding activity of proteins adsorbed onto gold nanoparticles.

    PubMed

    Kaur, Kanwarjeet; Forrest, James A

    2012-02-01

    We used optical extinction spectroscopy to study the structure of proteins adsorbed onto gold nanoparticles of sizes 5-60 nm and their resulting biological binding activity. For these studies, proteins differing in size and shape, with well-characterized and specific interactions-rabbit immunoglobulin G (IgG), goat anti-rabbit IgG (anti-IgG), Staphylococcal protein A, streptavidin, and biotin-were used as model systems. Protein interaction with gold nanoparticles was probed by optical extinction measurements of localized surface plasmon resonance (LSPR) of the gold nanoparticles. Binding of the ligands in solution to protein molecules already immobilized on the surface of gold causes a small but detectable shift in the LSPR peak of the gold nanoparticles. This shift can be used to probe the binding activity of the adsorbed protein. Within the context of Mie theory calculations, the thickness of the adsorbed protein layer as well as its apparent refractive index is shown to depend on the size of the gold nanoparticle. The results suggest that proteins can adopt different orientations that depend on the size of the gold nanospheres. These different orientations, in turn, can result in different levels of biological activity. For example, we find that IgG adsorbed on spheres with diameter ≥20 nm does not bind to protein A. This study illustrates the principle that the size of nanoparticles can strongly influence the binding activity of adsorbed proteins. In addition to the importance of this in cases of direct exposure of proteins to nanoparticles, the results have implications for proteins adsorbed to materials with nanometer scale surface roughness.

  13. Detection of Ordered Molecules Adsorbed on Graphene: a Theoretical Study

    NASA Astrophysics Data System (ADS)

    Wang, Yong; Zhang, Xue-Qing; Li, Hui

    2014-11-01

    Graphene has been demonstrated to be able to detect individual gas molecules [Schedin et al. Nat. Mater. 6 (2007) 652], which has attracted a lot of sensor research activities. Here we report for the first time that graphene is capable of detecting the ordering degree of absorbed water molecules. The efficiency of doping varies from the degrees of molecular ordering. The simulated results show that the highly ordered water molecules contribute more to the doping effect, which reduces the conductance of the water/graphene system.

  14. Experimental characterization of adsorbed protein orientation, conformation, and bioactivity

    PubMed Central

    Thyparambil, Aby A.; Wei, Yang; Latour, Robert A.

    2015-01-01

    Protein adsorption on material surfaces is a common phenomenon that is of critical importance in many biotechnological applications. The structure and function of adsorbed proteins are tightly interrelated and play a key role in the communication and interaction of the adsorbed proteins with the surrounding environment. Because the bioactive state of a protein on a surface is a function of the orientation, conformation, and accessibility of its bioactive site(s), the isolated determination of just one or two of these factors will typically not be sufficient to understand the structure–function relationships of the adsorbed layer. Rather a combination of methods is needed to address each of these factors in a synergistic manner to provide a complementary dataset to characterize and understand the bioactive state of adsorbed protein. Over the past several years, the authors have focused on the development of such a set of complementary methods to address this need. These methods include adsorbed-state circular dichroism spectropolarimetry to determine adsorption-induced changes in protein secondary structure, amino-acid labeling/mass spectrometry to assess adsorbed protein orientation and tertiary structure by monitoring adsorption-induced changes in residue solvent accessibility, and bioactivity assays to assess adsorption-induced changes in protein bioactivity. In this paper, the authors describe the methods that they have developed and/or adapted for each of these assays. The authors then provide an example of their application to characterize how adsorption-induced changes in protein structure influence the enzymatic activity of hen egg-white lysozyme on fused silica glass, high density polyethylene, and poly(methyl-methacrylate) as a set of model systems. PMID:25708632

  15. Experimental characterization of adsorbed protein orientation, conformation, and bioactivity.

    PubMed

    Thyparambil, Aby A; Wei, Yang; Latour, Robert A

    2015-01-01

    Protein adsorption on material surfaces is a common phenomenon that is of critical importance in many biotechnological applications. The structure and function of adsorbed proteins are tightly interrelated and play a key role in the communication and interaction of the adsorbed proteins with the surrounding environment. Because the bioactive state of a protein on a surface is a function of the orientation, conformation, and accessibility of its bioactive site(s), the isolated determination of just one or two of these factors will typically not be sufficient to understand the structure-function relationships of the adsorbed layer. Rather a combination of methods is needed to address each of these factors in a synergistic manner to provide a complementary dataset to characterize and understand the bioactive state of adsorbed protein. Over the past several years, the authors have focused on the development of such a set of complementary methods to address this need. These methods include adsorbed-state circular dichroism spectropolarimetry to determine adsorption-induced changes in protein secondary structure, amino-acid labeling/mass spectrometry to assess adsorbed protein orientation and tertiary structure by monitoring adsorption-induced changes in residue solvent accessibility, and bioactivity assays to assess adsorption-induced changes in protein bioactivity. In this paper, the authors describe the methods that they have developed and/or adapted for each of these assays. The authors then provide an example of their application to characterize how adsorption-induced changes in protein structure influence the enzymatic activity of hen egg-white lysozyme on fused silica glass, high density polyethylene, and poly(methyl-methacrylate) as a set of model systems. PMID:25708632

  16. Substrate-mediated interactions and intermolecular forces between molecules adsorbed on surfaces.

    PubMed

    Sykes, E Charles H; Han, Patrick; Kandel, S Alex; Kelly, Kevin F; McCarty, Gregory S; Weiss, Paul S

    2003-12-01

    Adsorbate interactions and reactions on metal surfaces have been investigated using scanning tunneling microscopy. The manners in which adsorbates perturb the surface electronic structure in their vicinity are discussed. The effects these perturbations have on other molecules are shown to be important in overlayer growth. Interactions of molecules with surface steps are addressed, and each molecule's electron affinity is shown to dictate its adsorption sites at step edges. Standing waves emanating from steps are demonstrated to effect transient molecular adsorption up to 40 A away from the step edge. Halobenzene derivatives are used to demonstrate how the surface is important in aligning reactive intermediates.

  17. Laser electrospray mass spectrometry of adsorbed molecules at atmospheric pressure

    NASA Astrophysics Data System (ADS)

    Brady, John J.; Judge, Elizabeth J.; Simon, Kuriakose; Levis, Robert J.

    2010-02-01

    Atmospheric pressure mass analysis of solid phase biomolecules is performed using laser electrospray mass spectrometry (LEMS). A non-resonant femtosecond duration laser pulse vaporizes native samples at atmospheric pressure for subsequent electrospray ionization and transfer into a mass spectrometer. LEMS was used to detect a complex molecule (irinotecan HCl), a complex mixture (cold medicine formulation with active ingredients: acetaminophen, dextromethorphan HBr and doxylamine succinate), and a biological building block (deoxyguanosine) deposited on steel surfaces without a matrix molecule.

  18. Auger electron spectroscopy as a tool for measuring intramolecular charges of adsorbed molecules

    NASA Astrophysics Data System (ADS)

    Magkoev, T. T.

    A way for the determination of the values of intramolecular charges of adsorbed molecules of some binary dielectrics, based on Auger electron spectroscopy (AES), is proposed. These values can be obtained from the coverage dependences of the ratios of intensities of anion KL 23L 23 and KL 1L 1 Auger transitions, which are sensitive to the amount of charge at the 2p-orbitals. As an example, MgO adsorbed on Mo(110) is presented.

  19. Auger electron spectroscopy as a tool for measuring intramolecular charges of adsorbed molecules

    NASA Astrophysics Data System (ADS)

    Magkoev, T. T.

    1993-10-01

    A way for the determination of the values of intramolecular charges of adsorbed molecules of some binary dielectrics, based on Auger electron spectroscopy (AES), is proposed. These values can be obtained from the coverage dependences of the ratios of intensities of anion KL 23L 23 and KL 1L 1 Auger transitions, which are sensitive to the amount of charge at the 2p-orbitals. As an example, MgO adsorbed on Mo(110) is presented.

  20. Activity of lactoperoxidase when adsorbed on protein layers.

    PubMed

    Haberska, Karolina; Svensson, Olof; Shleev, Sergey; Lindh, Liselott; Arnebrant, Thomas; Ruzgas, Tautgirdas

    2008-09-15

    Lactoperoxidase (LPO) is an enzyme, which is used as an antimicrobial agent in a number of applications, e.g., food technology. In the majority of applications LPO is added to a homogeneous product phase or immobilised on product surface. In the latter case, however, the measurements of LPO activity are seldom reported. In this paper we have assessed LPO enzymatic activity on bare and protein modified gold surfaces by means of electrochemistry. It was found that LPO rapidly adsorbs to bare gold surfaces resulting in an amount of LPO adsorbed of 2.9mg/m(2). A lower amount of adsorbed LPO is obtained if the gold surface is exposed to bovine serum albumin, bovine or human mucin prior to LPO adsorption. The enzymatic activity of the adsorbed enzyme is in general preserved at the experimental conditions and varies only moderately when comparing bare gold and gold surface pretreated with the selected proteins. The measurement of LPO specific activity, however, indicate that it is about 1.5 times higher if LPO is adsorbed on gold surfaces containing a small amount of preadsorbed mucin in comparison to the LPO directly adsorbed on bare gold.

  1. A molecule detector: Adsorbate induced conductance gap change of ultra-thin silicon nanowire

    NASA Astrophysics Data System (ADS)

    Zhang, Y. H.; Zhang, X. Q.; Li, H.; Taft, C. A.; Paiva, G.

    2009-03-01

    Inspired by the work of Lieber and co-workers [F. Patolsky, B.P. Timko, G. Zheng, C.M. Lieber, MRS Bull. 32 (2007) 142], we present a general discussion of the possibility of using atomic-chain scaled Si nanowires to detect molecules. Surface-modified Si nanowires were optimized by density functional theory (DFT) calculations. The electronic transport properties of the whole system, including Si nanowires and adsorbed molecules, sandwiched between two gold electrodes are investigated by means of non-equilibrium Green's function (NEGF) formalism. However, the overall transport properties, including current-voltage ( I- V) and conductance-voltage ( G- V) characteristics hardly show adsorbate sensitivity. Interestingly, our results show that the conductance gap clearly varies with the different adsorbates. Therefore different molecules can cause differences in the conductance gap compared with the bare Si nanowire. The results provide valuable information regarding the development of atomic-chain scaled molecular detectors.

  2. Pattern Recognition of Adsorbing HP Lattice Proteins

    NASA Astrophysics Data System (ADS)

    Wilson, Matthew S.; Shi, Guangjie; Wüst, Thomas; Landau, David P.; Schmid, Friederike

    2015-03-01

    Protein adsorption is relevant in fields ranging from medicine to industry, and the qualitative behavior exhibited by course-grained models could shed insight for further research in such fields. Our study on the selective adsorption of lattice proteins utilizes the Wang-Landau algorithm to simulate the Hydrophobic-Polar (H-P) model with an efficient set of Monte Carlo moves. Each substrate is modeled as a square pattern of 9 lattice sites which attract either H or P monomers, and are located on an otherwise neutral surface. The fully enumerated set of 102 unique surfaces is simulated with each protein sequence. A collection of 27-monomer sequences is used- each of which is non-degenerate and protein-like. Thermodynamic quantities such as the specific heat and free energy are calculated from the density of states, and are used to investigate the adsorption of lattice proteins on patterned substrates. Research supported by NSF.

  3. Volumetric Interpretation of Protein Adsorption: Interfacial Packing of Protein Adsorbed to Hydrophobic Surfaces from Surface-Saturating Solution Concentrations

    PubMed Central

    Kao, Ping; Parhi, Purnendu; Krishnan, Anandi; Noh, Hyeran; Haider, Waseem; Tadigadapa, Srinivas; Allara, David L.; Vogler, Erwin A.

    2010-01-01

    The maximum capacity of a hydrophobic adsorbent is interpreted in terms of square or hexagonal (cubic and face-centered-cubic, FCC) interfacial packing models of adsorbed blood proteins in a way that accommodates experimental measurements by the solution-depletion method and quartz-crystal-microbalance (QCM) for the human proteins serum albumin (HSA, 66 kDa), immunoglobulin G (IgG, 160 kDa), fibrinogen (Fib, 341 kDa), and immunoglobulin M (IgM, 1000 kDa). A simple analysis shows that adsorbent capacity is capped by a fixed mass/volume (e.g. mg/mL) surface-region (interphase) concentration and not molar concentration. Nearly analytical agreement between the packing models and experiment suggests that, at surface saturation, above-mentioned proteins assemble within the interphase in a manner that approximates a well-ordered array. HSA saturates a hydrophobic adsorbent with the equivalent of a single square-or-hexagonally-packed layer of hydrated molecules whereas the larger proteins occupy two-or-more layers, depending on the specific protein under consideration and analytical method used to measure adsorbate mass (solution depletion or QCM). Square-or-hexagonal (cubic and FCC) packing models cannot be clearly distinguished by comparison to experimental data. QCM measurement of adsorbent capacity is shown to be significantly different than that measured by solution depletion for similar hydrophobic adsorbents. The underlying reason is traced to the fact that QCM measures contribution of both core protein, water of hydration, and interphase water whereas solution depletion measures only the contribution of core protein. It is further shown that thickness of the interphase directly measured by QCM systematically exceeds that inferred from solution-depletion measurements, presumably because the static model used to interpret solution depletion does not accurately capture the complexities of the viscoelastic interfacial environment probed by QCM. PMID:21035180

  4. Photoacoustic spectroscopy of surface adsorbed molecules using a nanostructured coupled resonator array

    NASA Astrophysics Data System (ADS)

    Lee, Dongkyu; Kim, Seonghwan; Van Neste, C. W.; Lee, Moonchan; Jeon, Sangmin; Thundat, Thomas

    2014-01-01

    A rapid method of obtaining photoacoustic spectroscopic signals for trace amounts of surface adsorbed molecules using a nanostructured coupled resonator array is described. Explosive molecules adsorbed on a nanoporous anodic aluminum oxide cantilever, which has hexagonally ordered nanowells with diameters and well-to-well distances of 35 nm and 100 nm, respectively, are excited using pulsed infrared (IR) light with a frequency matching the common mode resonance frequency of the coupled resonator. The common mode resonance amplitudes of the coupled resonator as a function of illuminating IR wavelength present a photoacoustic IR absorption spectrum representing the chemical signatures of the adsorbed explosive molecules. In addition, the mass of the adsorbed molecules as an orthogonal signal for quantitative analysis is determined by measuring the variation of the localized, individual mode resonance frequency of a cantilever on the array. The limit of detection of the ternary mixture of explosive molecules (1:1:1 of trinitrotoluene (TNT), cyclotrimethylene trinitramine (RDX) and pentaerythritol tetranitrate (PETN)) is estimated to be ˜100 ng cm-2. These multi-modal signals enable us to perform quantitative and rapid chemical sensing and analysis in ambient conditions.

  5. Local electric field and configuration of CO molecules adsorbed on a nanostructured surface with nanocones

    NASA Astrophysics Data System (ADS)

    You, Rong-Yi; Huang, Xiao-Jing

    2009-09-01

    Based on the nanostructured surface model that the (platinum, Pt) nanocones grow out symmetrically from a plane substrate, the local electric field near the conical nanoparticle surface is computed and discussed. On the basis of these results, the adsorbed CO molecules are modelled as dipoles, and three kinds of interactions, i.e. interactions between dipoles and local electric field, between dipoles and dipoles, as well as between dipoles and nanostructured substrate, are taken into account. The spatial configuration of CO molecules adsorbed on the nanocone surface is then given by Monte-Carlo simulation. Our results show that the CO molecules adsorbed on the nanocone surface cause local agglomeration under the action of an external electric field, and this agglomeration becomes more compact with decreasing conical angle, which results in a stronger interaction among molecules. These results serve as a basis for explaining abnormal phenomena such as the abnormal infrared effect (AIRE), which was found when CO molecules were adsorbed on the nanostructured transit ion-metal surface.

  6. Calibrating the Bending of Molecule Adsorbed Nanoscale Si Cantilevers with a Modified Stoney Formula

    NASA Astrophysics Data System (ADS)

    Zang, Ji; Liu, Feng

    2007-03-01

    Fundamental understanding of mechanical bending of molecule adsorbed nanoscale thin films is of both scientific and technological importance. Our current understanding, however, is limited within macroscopic analysis that neglects the atomic details of film structure and surface effects. Here, we report atomistic simulation and theoretical analysis of bending of freestanding nanometer-thick silicon (Si) films induced by adsorption of hydrogen and acetylene molecules. It reveals the dominant role of atomic surface structure and surface stress in governing their bending behavior. We show that the bending curvature of molecule adsorbed Si nanofilm does not follow the classical Stoney formula, and we develop a modified Stoney formula by taking into account of the effects arising from atomic surface reconstruction and surface stress. Our findings suggest that re-calibration has to be made in detecting trace amount of molecules by nanoscale Si mechanochemical sensors.

  7. Spectroscopic observations of the displacement dynamics of physically adsorbed molecules-CO on C60

    NASA Astrophysics Data System (ADS)

    Yuan, Chunqing; Yates, John T.

    2016-10-01

    In this paper, we observed physically adsorbed CO molecules on C60 surface being displaced by impinging noble gas atoms (He, Ne, Ar, Kr), either through a dynamic displacement process or an exothermic replacement process, depending on their adsorption energies. This displacement mechanism could shift from one to the other depending on the surface coverage and temperature. Furthermore, rotational energy of the impinging molecules may also contribute to the dynamic displacement process by supplying additional energy.

  8. Giant Hysteresis of Single-Molecule Magnets Adsorbed on a Nonmagnetic Insulator.

    PubMed

    Wäckerlin, Christian; Donati, Fabio; Singha, Aparajita; Baltic, Romana; Rusponi, Stefano; Diller, Katharina; Patthey, François; Pivetta, Marina; Lan, Yanhua; Klyatskaya, Svetlana; Ruben, Mario; Brune, Harald; Dreiser, Jan

    2016-07-01

    TbPc2 single-molecule magnets adsorbed on a magnesium oxide tunnel barrier exhibit record magnetic remanence, record hysteresis opening, perfect out-of-plane alignment of the magnetic easy axes, and self-assembly into a well-ordered layer. PMID:27159732

  9. Giant Hysteresis of Single-Molecule Magnets Adsorbed on a Nonmagnetic Insulator.

    PubMed

    Wäckerlin, Christian; Donati, Fabio; Singha, Aparajita; Baltic, Romana; Rusponi, Stefano; Diller, Katharina; Patthey, François; Pivetta, Marina; Lan, Yanhua; Klyatskaya, Svetlana; Ruben, Mario; Brune, Harald; Dreiser, Jan

    2016-07-01

    TbPc2 single-molecule magnets adsorbed on a magnesium oxide tunnel barrier exhibit record magnetic remanence, record hysteresis opening, perfect out-of-plane alignment of the magnetic easy axes, and self-assembly into a well-ordered layer.

  10. Electrostatic model for treating long-range lateral interactions between polar molecules adsorbed on metal surfaces

    NASA Astrophysics Data System (ADS)

    Kokalj, Anton

    2011-07-01

    A classical polarizable point-dipole model for the adsorption of polar molecules on metal surfaces is presented. The main usefulness of the model is that lattice sums are represented by simple functions in closed form. This allows a simple extrapolation of adsorption energies of polar molecules—as calculated by first-principles calculations employing periodic boundary conditions—to the zero-coverage limit. Such an extrapolation is rather important for the proper evaluation of adsorption energy of highly polar molecules, because their long-range lateral interactions can extend beyond the nearest-neighbor distances of 50 bohrs. Moreover, the dependence of the adsorption energy on the orientation and configuration of molecular dipoles can be straightforwardly analyzed. It is demonstrated that an accumulation of polar molecules on the surface is favored provided that the molecular dipoles point parallel to the surface in the adsorbed state, whereas adsorbed molecules displaying dipoles oriented perpendicular to the surface would prefer to stay well separated due to repulsive lateral interactions. Further, the model is used to analyze the density-functional theory (DFT) calculated data of two polar molecules, triazole and benzotriazole, adsorbed onto Cu(111) surface. The DFT-calculated adsorption energies are about -0.5 eV, and it is shown that the main contribution to the molecule-surface bonding comes from the dipole-dipole electrostatic interactions.

  11. Vibrational dynamics of fullerene molecules adsorbed on metal surfaces studied with synchrotron infrared radiation

    SciTech Connect

    P. Rudolf; R. Raval; P. Dumas; Gwyn P. Williams

    2002-04-01

    Infrared (IR) spectroscopy of chemisorbed C{sub 60} on Ag (111), Au (110) and Cu (100) reveals that a non-IR-active mode becomes active upon adsorption, and that its frequency shifts proportionally with the charge transferred from the metal to the molecule by about 5 cm{sup -1} per electron. The temperature dependence of the frequency and the width of this IR feature have also been followed for C{sub 60>}/Cu (100) and were found to agree well with a weak anharmonic coupling (dephasing) to a low-frequency mode, which we suggest to be the frustrated translational mode of the adsorbed molecules.

    Additionally, the adsorption is accompanied by a broadband reflectance change, which is interpreted as due to the scattering of conduction electrons of the metal surface by the adsorbate. The reflectance change allows determination of the friction coefficient of the C{sub 60} molecules, which results in rather small values ({approx}2 x 10{sup 9}s{sup -1} for Ag and Au, and {approx}1.6 x 10{sup 9}s{sup -1} for Cu), consistent with a marked metallic character of the adsorbed molecules.

    Pre-dosing of alkali atoms onto the metal substrates drastically changes the IR spectra recorded during subsequent C{sub 60} deposition: anti-absorption bands, as well as an increase of the broadband reflectance, occur and are interpreted as due to strong electron-phonon coupling with induced surface states.

  12. A theoretical study of hydrogen diffraction following photodissociation of adsorbed molecules

    NASA Astrophysics Data System (ADS)

    Kosloff, Ronnie; Zeiri, Yehuda

    1992-08-01

    A new probe of surface structure is presented which is based on the photodissociation of hydrogen from an adsorbate molecule. The event creates an atomic hydrogen fragment, positioned between the adsorbate layer and the solid surface. Due to its light mass, the hydrogen dynamics is quantum mechanical in nature. A useful image is of the hydrogenic wave function behaving like a liquid able to fill all cracks. The coherent character of the hydrogenic wave function is crucial in the ability of the photodissociation experiment to act as a probe. A series of case studies has been carried out whose aim is to reveal the relation between the structure of the surface and the asymptotic energy resolved angular distribution of the hydrogen fragment. The dynamics of the hydrogen atom motion was modeled by the time dependent Schrödinger equation. The cases studied include the dissociation of a single HBr adsorbate on flat and corrugated surfaces. A broad specular peak was observed, in addition to diffraction peaks which can be correlated with the corrugation. Moreover, selective adsorption peaks, which can be correlated with the attractive part of the surface potential, have been identified. Systems in which the hydrogenic wave function scatters from several adsorbates were also investigated. It was found that the scattering is dominated by the trapping of the wave function by unstable periodic orbits. The quantization rules of these periodic orbits have been identified, creating a link between the structure of the adsorbates and the asymptotic angular distributions.

  13. On the interplay between chemical reactions and phase transitions for molecules adsorbed on solid surfaces

    NASA Astrophysics Data System (ADS)

    Stiles, M.; Metiu, H.

    1986-02-01

    There is a large body of experimental evidence suggesting that the rate of product formation in a reaction between adsorbed molecules is deeply modified when one of the reagents undergoes a phase transition or, more broadly speaking, an aggregation of some kind. Specific examples are: H2 and CO oxidation; methanol transformation into a methoxy radical on O/Cu; the water reaction with oxygen to form 2OH; the decomposition of N2O on Pt(111) which is affected by a phase transition in the outermost Pt layer; and the oxidation of Ni7. It is suspected that similar effects might operate on supported metal catalysts. We use Monte Carlo simulations to examine the manner in which reagent aggregation affects the reaction rate between molecules adsorbed on a solid surface. We discuss the temperature and concentration dependence of the rate of product formation.

  14. Orientation and heat capacity of horizontally adsorbed molecules in electric fields

    NASA Astrophysics Data System (ADS)

    Liao, Ying-Yen

    2014-02-01

    The orientation and the heat capacity of horizontally adsorbed molecules are investigated in static electric fields. We evaluate the energy spectrum and the wave function to probe the rotational characteristics of the molecule. Numerical results indicate that the electric field and the effect of quantum confinement lead to anticrossing behaviors in the energy levels. The orientation reveals a stepped feature due to the anticrossing in the ground state. Moreover, the heat capacity displays two peaks near the anticrossing. By means of comparison, each peak of the heat capacity corresponds to a particular degree of orientation.

  15. Evidence for photo-induced charge separation between dye molecules adsorbed to aluminium oxide surfaces

    NASA Astrophysics Data System (ADS)

    Cappel, Ute B.; Moia, Davide; Bruno, Annalisa; Vaissier, Valerie; Haque, Saif A.; Barnes, Piers R. F.

    2016-02-01

    Excited state dynamics and photo-induced charge transfer of dye molecules have been widely studied due to their relevance for organic and dye-sensitised solar cells. Herein, we present a femtosecond transient absorption spectroscopy study of the indolene dye D131 when adsorbed to inert Al2O3 substrates for different surface concentration of the dye. Surprisingly, we find that at high surface concentrations, the first singlet excited state of the dye is converted into a new state with an efficiency of about 80%. We assign the absorption features of this state to the oxidised dye and discuss the possibility of photo-induced charge separation between neighboring dye molecules. Our study is the first to show that this process can be highly efficient without the use of donor and acceptor molecules of different chemical structures.

  16. Homeotropic orientation of a nematic liquid crystal by bent-core molecules adsorbed on its surface

    NASA Astrophysics Data System (ADS)

    Hwang, Jiyong; Yang, Seungbin; Lee, Hyojin; Kim, Jongyoon; Lee, Ji-Hoon; Kang, Shin-Woong; Choi, E.-Joon

    2015-06-01

    We reported the promotion of a homeotropic alignment of a nematic liquid crystal (NLC) by bent-core liquid-crystal (BLC) Molecules adsorbed its surface. The BLC was mixed at various concentrations with the NLC, and the mixtures were injected into an empty cell with a cell gap of 13 μm. Although the pure NLC showed a heterogeneous orientation, the BLC-NLC mixture was gradually transformed to a homeotropic alignment with increasing concentration of the BLC. We investigated the surface topography of the samples by using an atomic force microscopy (AFM) and found that the BLC molecules were segregated into a polyimide (PI) surface and formed protrusion domains with diameters of 50-100 nm. The BLC protrusions might promote the homeotropic orientation of the NLC molecules.

  17. Evidence for photo-induced charge separation between dye molecules adsorbed to aluminium oxide surfaces

    PubMed Central

    Cappel, Ute B.; Moia, Davide; Bruno, Annalisa; Vaissier, Valerie; Haque, Saif A.; Barnes, Piers R. F.

    2016-01-01

    Excited state dynamics and photo-induced charge transfer of dye molecules have been widely studied due to their relevance for organic and dye-sensitised solar cells. Herein, we present a femtosecond transient absorption spectroscopy study of the indolene dye D131 when adsorbed to inert Al2O3 substrates for different surface concentration of the dye. Surprisingly, we find that at high surface concentrations, the first singlet excited state of the dye is converted into a new state with an efficiency of about 80%. We assign the absorption features of this state to the oxidised dye and discuss the possibility of photo-induced charge separation between neighboring dye molecules. Our study is the first to show that this process can be highly efficient without the use of donor and acceptor molecules of different chemical structures. PMID:26891851

  18. Exploring the interfacial structure of protein adsorbates and the kinetics of protein adsorption: an in situ high-energy X-ray reflectivity study.

    PubMed

    Evers, Florian; Shokuie, Kaveh; Paulus, Michael; Sternemann, Christian; Czeslik, Claus; Tolan, Metin

    2008-09-16

    The high energy X-ray reflectivity technique has been applied to study the interfacial structure of protein adsorbates and protein adsorption kinetics in situ. For this purpose, the adsorption of lysozyme at the hydrophilic silica-water interface has been chosen as a model system. The structure of adsorbed lysozyme layers was probed for various aqueous solution conditions. The effect of solution pH and lysozyme concentration on the interfacial structure was measured. Monolayer formation was observed for all cases except for the highest concentration. The adsorbed protein layers consist of adsorbed lysozyme molecules with side-on or end-on orientation. By means of time-dependent X-ray reflectivity scans, the time-evolution of adsorbed proteins was monitored as well. The results of this study demonstrate the capabilities of in situ X-ray reflectivity experiments on protein adsorbates. The great advantages of this method are the broad wave vector range available and the high time resolution.

  19. Exploring the interfacial structure of protein adsorbates and the kinetics of protein adsorption: an in situ high-energy X-ray reflectivity study.

    PubMed

    Evers, Florian; Shokuie, Kaveh; Paulus, Michael; Sternemann, Christian; Czeslik, Claus; Tolan, Metin

    2008-09-16

    The high energy X-ray reflectivity technique has been applied to study the interfacial structure of protein adsorbates and protein adsorption kinetics in situ. For this purpose, the adsorption of lysozyme at the hydrophilic silica-water interface has been chosen as a model system. The structure of adsorbed lysozyme layers was probed for various aqueous solution conditions. The effect of solution pH and lysozyme concentration on the interfacial structure was measured. Monolayer formation was observed for all cases except for the highest concentration. The adsorbed protein layers consist of adsorbed lysozyme molecules with side-on or end-on orientation. By means of time-dependent X-ray reflectivity scans, the time-evolution of adsorbed proteins was monitored as well. The results of this study demonstrate the capabilities of in situ X-ray reflectivity experiments on protein adsorbates. The great advantages of this method are the broad wave vector range available and the high time resolution. PMID:18715021

  20. Molecular resonant dissociation of surface-adsorbed molecules by plasmonic nanoscissors

    NASA Astrophysics Data System (ADS)

    Zhang, Zhenglong; Sheng, Shaoxiang; Zheng, Hairong; Xu, Hongxing; Sun, Mengtao

    2014-04-01

    The ability to break individual bonds or specific modes in chemical reactions is an ardently sought goal by chemists and physicists. While photochemistry based methodologies are very successful in controlling e.g. photocatalysis, photosynthesis and the degradation of plastic, it is hard to break individual molecular bonds for those molecules adsorbed on the surface because of the weak light-absorption in molecules and the redistribution of the resulting vibrational energy both inside the molecule and to its surrounding environment. Here we show how to overcome these obstacles with a plasmonic hot-electron mediated process and demonstrate a new method that allows the sensitive control of resonant dissociation of surface-adsorbed molecules by `plasmonic' scissors. To that end, we used a high-vacuum tip-enhanced Raman spectroscopy (HV-TERS) setup to dissociate resonantly excited NC2H6 fragments from Malachite green. The surface plasmons (SPs) excited at the sharp metal tip not only enhance the local electric field to harvest the light incident from the laser, but crucially supply `hot electrons' whose energy can be transferred to individual bonds. These processes are resonant Raman, which result in some active chemical bonds and then weaken these bonds, followed by dumping in lots of indiscriminant energy and breaking the weakest bond. The method allows for sensitive control of both the rate and probability of dissociation through their dependence on the density of hot electrons, which can be manipulated by tuning the laser intensity or tunneling current/bias voltage in the HV-TERS setup, respectively. The concepts of plasmonic scissors open up new versatile avenues for the deep understanding of in situ surface-catalyzed chemistry.The ability to break individual bonds or specific modes in chemical reactions is an ardently sought goal by chemists and physicists. While photochemistry based methodologies are very successful in controlling e.g. photocatalysis

  1. Evidence of conformational changes in adsorbed lysozyme molecule on silver colloids.

    PubMed

    Chandra, Goutam; Ghosh, Kalyan S; Dasgupta, Swagata; Roy, Anushree

    2010-10-01

    In this article, we discuss metal-protein interactions in the Ag-lysozyme complex by spectroscopic measurements. The analysis of the variation in relative intensities of SERS bands reveals the orientation and the change in conformation of the protein molecules on the Ag surface with time. The interaction kinetics of metal-protein complexes has been analyzed over a period of 3h via Raman measurements. Our analysis indicates that the Ag nanoparticles most likely interact with Trp123 which is in close proximity to Phe34 of the lysozyme molecule.

  2. Single-Molecule Magnets: Giant Hysteresis of Single-Molecule Magnets Adsorbed on a Nonmagnetic Insulator (Adv. Mater. 26/2016).

    PubMed

    Wäckerlin, Christian; Donati, Fabio; Singha, Aparajita; Baltic, Romana; Rusponi, Stefano; Diller, Katharina; Patthey, François; Pivetta, Marina; Lan, Yanhua; Klyatskaya, Svetlana; Ruben, Mario; Brune, Harald; Dreiser, Jan

    2016-07-01

    In Tb(Pc)2 single-molecule magnets, where Pc is phthalocyanine, adsorbed on magnesium oxide, the fluctuations of the terbium magnetic moment are strongly suppressed in contrast to the adsorption on silver. On page 5195, J. Dreiser and co-workers investigate that the molecules are perfectly organized by self-assembly, as seen in the scanning tunnelling microscopy image (top part of the design). The molecules are probed by circularly polarized X-rays depicted as green spirals. PMID:27383020

  3. Single-Molecule Magnets: Giant Hysteresis of Single-Molecule Magnets Adsorbed on a Nonmagnetic Insulator (Adv. Mater. 26/2016).

    PubMed

    Wäckerlin, Christian; Donati, Fabio; Singha, Aparajita; Baltic, Romana; Rusponi, Stefano; Diller, Katharina; Patthey, François; Pivetta, Marina; Lan, Yanhua; Klyatskaya, Svetlana; Ruben, Mario; Brune, Harald; Dreiser, Jan

    2016-07-01

    In Tb(Pc)2 single-molecule magnets, where Pc is phthalocyanine, adsorbed on magnesium oxide, the fluctuations of the terbium magnetic moment are strongly suppressed in contrast to the adsorption on silver. On page 5195, J. Dreiser and co-workers investigate that the molecules are perfectly organized by self-assembly, as seen in the scanning tunnelling microscopy image (top part of the design). The molecules are probed by circularly polarized X-rays depicted as green spirals.

  4. Room temperature differential conductance measurements of triethylamine molecules adsorbed on Si(001).

    PubMed

    Naitabdi, Ahmed; Rochet, François; Carniato, Stéphane; Bournel, Fabrice; Gallet, Jean-Jacques

    2016-08-17

    We have measured the differential conductance of the triethylamine molecule (N(CH2CH3)3) adsorbed on Si(001)-2 × 1 at room temperature using scanning tunneling spectroscopy. Triethylamine can be engaged in a dative bonding with a silicon dimer, forming a Si-Si-N(CH2CH3)3 unit. We have examined the datively bonded adduct, either as an isolated molecule, or within an ordered molecular domain (reconstructed 4 × 2). The differential conductance curves, supported by DFT calculations, show that in the explored energy window (±2.5 near the Fermi level) the main features stem from the uncapped dangling bonds of the reacted dimer and of the adjacent unreacted ones that are electronically coupled The formation of a molecular domain, in which one dimer in two is left unreacted, is reflected in a shift of the up dimer atom occupied level away from the Fermi level, likely due to an increased π-bonding strength. In stark contrast with the preceding, pairs of dissociated molecule (a minority species) are electronically decoupled from the dimer dangling bond states. DFT calculation show that the lone-pair of the Si-N(CH2CH3)2 is a shallow level, that is clearly seen in the differential conductance curve. PMID:27499070

  5. Existence of hydration forces in the interaction between apoferritin molecules adsorbed on silica surfaces.

    PubMed

    Valle-Delgado, J J; Molina-Bolívar, J A; Galisteo-González, F; Gálvez-Ruiz, M J; Feiler, A; Rutland, M W

    2005-10-11

    The atomic force microscope, together with the colloid probe technique, has become a very useful instrument to measure interaction forces between two surfaces. Its potential has been exploited in this work to study the interaction between protein (apoferritin) layers adsorbed on silica surfaces and to analyze the effect of the medium conditions (pH, salt concentration, salt type) on such interactions. It has been observed that the interaction at low salt concentrations is dominated by electrical double layer (at large distances) and steric forces (at short distances), the latter being due to compression of the protein layers. The DLVO theory fits these experimental data quite well. However, a non-DLVO repulsive interaction, prior to contact of the protein layers, is observed at high salt concentration above the isoelectric point of the protein. This behavior could be explained if the presence of hydration forces in the system is assumed. The inclusion of a hydration term in the DLVO theory (extended DLVO theory) gives rise to a better agreement between the theoretical fits and the experimental results. These results seem to suggest that the hydration forces play a very important role in the stability of the proteins in the physiological media.

  6. Structure and dynamics of monolayer films of squalane molecules adsorbed on a solid surface

    NASA Astrophysics Data System (ADS)

    D. T Enevoldsen, A.; Hansen, F. Y.; Diama, A.; Taub, H.

    2003-03-01

    Squalane is a branched alkane (C_30H_62). It consists of a straight chain with 24 carbon atoms, as in tetracosane (C_24H_50), and has six methyl side groups. Branched polymers such as squalane are thought to be better lubricants than n-alkanes. At low temperature, our molecular dynamics (MD) simulations show that the molecules form an ordered monolayer which melts at approximately 325 K compared to the tetracosane monolayer melting point of ˜ 340 K. Our MD simulations indicate the same melting mechanism in the squalane monolayer that was found previously for tetracosane (F. Y. Hansen and H. Taub, Phys. Rev. Lett. 69, 652 (1992).) They also show that the adsorbed molecules are distorted from an all-trans carbon backbone in contrast to what was found for tetracosane. This may explain why the Bragg diffraction peaks were observed to be broader for the squalane monolayer than for tetracosane (D. Fuhrmann, A. P. Graham, L. Criswell, H. Mo, B. Matthies, K. W. Herwig, and H. Taub, Surf. Sci. 482-485, 77 (2001).). The diffusive motion in a squalane monolayer has been investigated by both quasielastic neutron scattering and MD simulations and compared to the dynamics in tetracosane monolayers. Focus will be on differences in the dynamics.

  7. Does Moisture Influence the Chemical Detection of Gas Molecules Adsorbed on Single-Wall Carbon Nanotubes?

    NASA Astrophysics Data System (ADS)

    Yu, Ming; Tian, W. Q.; Jayanthi, C. S.; Wu, S. Y.

    2009-03-01

    In this work, the role of water in the detection of hydrazine (N2H4) by a single-wall carbon nanotube (SWCNT) is investigated using first principles electronic structure calculations (DFT/GGA--USPP)[1]. This calculation is undertaken to interpret the experimental resistivity measurements for N2H4 adsorbed on SWCNT that reveal an n-type behavior [2]. Our preliminary theoretical studies of the adsorption of N2H4 on SWCNT revealed physisorption for N2H4 and an unaltered band structure for the SWCNT [3]. This prompted us to look into the role of water on the bonding of N2H4 to the SWCNT. We found that, by introducing a monolayer of water film on the (8,0) SWCNT, the adsorption of N2H4 can introduce occupied states near the Fermi level, exhibiting an n-type behavior. However, the introduction of just few water molecules was not sufficient to influence the electronic structure of N2H4/SWCNT. Presently, we are studying the influence of water films on the chemical detection of a variety of other gas molecules (N2, NH3, etc.) by SWCNTs, and the results from such studies will also be reported. [1]. G. Kresse et al. Phys. Rev. B 54, 11169 (1996). [2]. S. Desai, et al. (APS, March 2008). [3]. M. Yu, et al. (APS, March 2008).

  8. Brownian dynamics simulation of peeling a strongly-adsorbed polymer molecule from a frictionless substrate.

    PubMed

    Iliafar, Sara; Vezenov, Dmitri; Jagota, Anand

    2013-02-01

    We used brownian dynamics to study the peeling of a polymer molecule, represented by a freely jointed chain, from a frictionless surface in an implicit solvent with parameters representative of single-stranded DNA adsorbed on graphite. For slow peeling rates, simulations match the predictions of an equilibrium statistical thermodynamic model. We show that deviations from equilibrium peeling forces are dominated by a combination of Stokes (viscous) drag forces acting on the desorbed section of the chain and a finite rate of hopping over a desorption barrier. Characteristic velocities separating equilibrium and nonequilibrium regimes are many orders of magnitude higher than values accessible in force spectroscopy experiments. Finite probe stiffness resulted in disappearance of force spikes due to desorption of individual links predicted by the statistical thermodynamic model under displacement control. Probe fluctuations also masked sharp transitions in peeling force between blocks of distinct sequences, indicating limitation in the ability of single-molecule force spectroscopy to distinguish small differences in homologous molecular structures.

  9. Electrorheological crystallization of proteins and other molecules

    DOEpatents

    Craig, George D.; Rupp, Bernhard

    1996-01-01

    An electrorheological crystalline mass of a molecule is formed by dispersing the molecule in a dispersion fluid and subjecting the molecule dispersion to a uniform electrical field for a period of time during which time an electrorheological crystalline mass is formed. Molecules that may be used to form an electrorheological crystalline mass include any organic or inorganic molecule which has a permanent dipole and/or which is capable of becoming an induced dipole in the presence of an electric field. The molecules used to form the electrorheological crystalline mass are preferably macromolecules, such as biomolecules, such as proteins, nucleic acids, carbohydrates, lipoproteins and viruses. Molecules are crystallized by a method in which an electric field is maintained for a period of time after the electrorheological crystalline mass has formed during which time at least some of the molecules making up the electrorheological crystalline mass form a crystal lattice. The three dimensional structure of a molecule is determined by a method in which an electrorheological crystalline mass of the molecule is formed, an x-ray diffraction pattern of the electrorheological crystalline mass is obtained and the three dimensional structure of the molecule is calculated from the x-ray diffraction pattern.

  10. Electrorheological crystallization of proteins and other molecules

    DOEpatents

    Craig, G.D.; Rupp, B.

    1996-06-11

    An electrorheological crystalline mass of a molecule is formed by dispersing the molecule in a dispersion fluid and subjecting the molecule dispersion to a uniform electrical field for a period of time during which time an electrorheological crystalline mass is formed. Molecules that may be used to form an electrorheological crystalline mass include any organic or inorganic molecule which has a permanent dipole and/or which is capable of becoming an induced dipole in the presence of an electric field. The molecules used to form the electrorheological crystalline mass are preferably macromolecules, such as biomolecules, such as proteins, nucleic acids, carbohydrates, lipoproteins and viruses. Molecules are crystallized by a method in which an electric field is maintained for a period of time after the electrorheological crystalline mass has formed during which time at least some of the molecules making up the electrorheological crystalline mass form a crystal lattice. The three dimensional structure of a molecule is determined by a method in which an electrorheological crystalline mass of the molecule is formed, an X-ray diffraction pattern of the electrorheological crystalline mass is obtained and the three dimensional structure of the molecule is calculated from the X-ray diffraction pattern. 4 figs.

  11. Electronic structure and binding geometry of tetraphenylporphyrin-derived molecules adsorbed on metal and metal oxide surfaces

    NASA Astrophysics Data System (ADS)

    Coh, Senia

    Tetraphenylporphyrin (TPP)-derived molecules have been studied extensively as efficient photosensitizers when chemisorbed on the metal oxide substrates in dye-sensitized solar cells. Still, many fundamental electronic properties of the dye/oxide interface are not understood and need careful consideration. In this thesis we present a comprehensive study of the electronic structure, energy level alignment and the adsorption geometry of the TPP-derived dye molecules adsorbed on TiO2(110), ZnO(1120) and Ag(100) single crystal surfaces using ultra-high vacuum (UHV) based surface sensitive techniques. The alignment of the molecular energy levels with respect to the TiO 2 and ZnO band edges for all TPP-derived molecules we studied was found to be insensitive to either the nature of the functional groups located on the phenyl rings, presence of zinc as a central metal ion and different binding geometry of the molecules. Binding geometry, molecule-molecule interaction and the aggregation effects in the adsorbed layer, that were observed in the UV-visible spectra of the molecules adsorbed on ZnO substrate were not observed in the ultraviolet photoemission (UPS) and inverse photoemission (IPS) spectra of the occupied and unoccupied molecular states. Using near edge X-ray absorption fine structure (NEXAFS) and scanning tunneling microscopy (STM), binding geometry of the two representative TPP-derivatives was directly determined to be upright, with the porphyrin ring under large angle with respect to the surface for the p-ZnTCPP molecules and with the porphyrin ring parallel to the surface for the m-ZnTCPP molecules. We observe that the energies and the energy level alignment of the ZnTPP molecular levels measured in UPS and IPS depend on the substrate on which the molecules are adsorbed (Ag(100) or TiO2(110) single crystal surfaces). The differences are attributed to different charge screening properties of these two materials. Image charges created in the substrates during

  12. Heavy meromyosin molecules extending more than 50 nm above adsorbing electronegative surfaces.

    PubMed

    Persson, Malin; Albet-Torres, Nuria; Ionov, Leonid; Sundberg, Mark; Höök, Fredrik; Diez, Stefan; Månsson, Alf; Balaz, Martina

    2010-06-15

    In the in vitro motility assay, actin filaments are propelled by surface-adsorbed myosin motors, or rather, myosin motor fragments such as heavy meromyosin (HMM). Recently, efforts have been made to develop actomyosin powered nanodevices on the basis of this assay but such developments are hampered by limited understanding of the HMM adsorption geometry. Therefore, we here investigate the HMM adsorption geometries on trimethylchlorosilane- [TMCS-] derivatized hydrophobic surfaces and on hydrophilic negatively charged surfaces (SiO(2)). The TMCS surface is of great relevance in fundamental studies of actomyosin and both surface substrates are important for the development of motor powered nanodevices. Whereas both the TMCS and SiO(2) surfaces were nearly saturated with HMM (incubation at 120 microg mL(-1)) there was little actin binding on SiO(2) in the absence of ATP and no filament sliding in the presence of ATP. This contrasts with excellent actin-binding and motility on TMCS. Quartz crystal microbalance with dissipation (QCM-D) studies demonstrate a HMM layer with substantial protein mass up to 40 nm above the TMCS surface, considerably more than observed for myosin subfragment 1 (S1; 6 nm). Together with the excellent actin transportation on TMCS, this strongly suggests that HMM adsorbs to TMCS mainly via its most C-terminal tail part. Consistent with this idea, fluorescence interference contrast (FLIC) microscopy showed that actin filaments are held by HMM 38 +/- 2 nm above the TMCS-surface with the catalytic site, on average, 20-30 nm above the surface. Viewed in a context with FLIC, QCM-D and TIRF results, the lack of actin motility and the limited actin binding on SiO(2) shows that HMM adsorbs largely via the actin-binding region on this surface with the C-terminal coiled-coil tails extending >50 nm into solution. The results and new insights from this study are of value, not only for the development of motor powered nanodevices but also for the

  13. Protein Scaffolding for Small Molecule Catalysts

    SciTech Connect

    Baker, David

    2014-09-14

    We aim to design hybrid catalysts for energy production and storage that combine the high specificity, affinity, and tunability of proteins with the potent chemical reactivities of small organometallic molecules. The widely used Rosetta and RosettaDesign methodologies will be extended to model novel protein / small molecule catalysts in which one or many small molecule active centers are supported and coordinated by protein scaffolding. The promise of such hybrid molecular systems will be demonstrated with the nickel-phosphine hydrogenase of DuBois et. al.We will enhance the hydrogenase activity of the catalyst by designing protein scaffolds that incorporate proton relays and systematically modulate the local environment of the catalyticcenter. In collaboration with DuBois and Shaw, the designs will be experimentally synthesized and characterized.

  14. Profiling protein function with small molecule microarrays

    PubMed Central

    Winssinger, Nicolas; Ficarro, Scott; Schultz, Peter G.; Harris, Jennifer L.

    2002-01-01

    The regulation of protein function through posttranslational modification, local environment, and protein–protein interaction is critical to cellular function. The ability to analyze on a genome-wide scale protein functional activity rather than changes in protein abundance or structure would provide important new insights into complex biological processes. Herein, we report the application of a spatially addressable small molecule microarray to an activity-based profile of proteases in crude cell lysates. The potential of this small molecule-based profiling technology is demonstrated by the detection of caspase activation upon induction of apoptosis, characterization of the activated caspase, and inhibition of the caspase-executed apoptotic phenotype using the small molecule inhibitor identified in the microarray-based profile. PMID:12167675

  15. Micro-differential thermal analysis detection of adsorbed explosive molecules using microfabricated bridges.

    PubMed

    Senesac, Larry R; Yi, Dechang; Greve, Anders; Hales, Jan H; Davis, Zachary J; Nicholson, Don M; Boisen, Anja; Thundat, Thomas

    2009-03-01

    Although micromechanical sensors enable chemical vapor sensing with unprecedented sensitivity using variations in mass and stress, obtaining chemical selectivity using the micromechanical response still remains as a crucial challenge. Chemoselectivity in vapor detection using immobilized selective layers that rely on weak chemical interactions provides only partial selectivity. Here we show that the very low thermal mass of micromechanical sensors can be used to produce unique responses that can be used for achieving chemical selectivity without losing sensitivity or reversibility. We demonstrate that this method is capable of differentiating explosive vapors from nonexplosives and is additionally capable of differentiating individual explosive vapors such as trinitrotoluene, pentaerythritol tetranitrate, and cyclotrimethylenetrinitromine. This method, based on a microfabricated bridge with a programmable heating rate, produces unique and reproducible thermal response patterns within 50 ms that are characteristic to classes of adsorbed explosive molecules. We demonstrate that this micro-differential thermal analysis technique can selectively detect explosives, providing a method for fast direct detection with a limit of detection of 600x10(-12) g.

  16. Micro differential thermal analysis detection of adsorbed explosive molecules using microfabricated bridges

    SciTech Connect

    Senesac, Larry R; Yi, Dechang; Greve, Anders; Hales, Jan; Davis, Zachary; Nicholson, Don M; Boisen, Anja; Thundat, Thomas George

    2009-01-01

    Although micromechanical sensors enable chemical vapor sensing with unprecedented sensitivity using variations in mass and stress, obtaining chemical selectivity using the micromechanical response still remains as a crucial challenge. Chemoselectivity in vapor detection using immobilized selective layers that rely on weak chemical interactions provides only partial selectivity. Here we show that the very low thermal mass of micromechanical sensors can be used to produce unique responses that can be used for achieving chemical selectivity without losing sensitivity or reversibility. We demonstrate that this method is capable of differentiating explosive vapors from nonexplosives and is additionally capable of differentiating individual explosive vapors such as trinitrotoluene, pentaerythritol tetranitrate, and cyclotrimethylenetrinitromine. This method, based on a microfabricated bridge with a programmable heating rate, produces unique and reproducible thermal response patterns within 50 ms that are characteristic to classes of adsorbed explosive molecules. We demonstrate that this micro-differential thermal analysis technique can selectively detect explosives, providing a method for fast direct detection with a limit of detection of 600 x 10{sup -12} g.

  17. Direct comparison of the electronic coupling efficiency of sulfur and selenium alligator clips for molecules adsorbed onto gold electrodes

    NASA Astrophysics Data System (ADS)

    Patrone, L.; Palacin, S.; Bourgoin, J. P.

    2003-05-01

    Scanning tunneling microscopy experiments have been performed to compare the electronic coupling provided by S and by Se used as alligator clips for bisthiol- and biselenol-terthiophene molecules adsorbed onto gold. The molecules were inserted in a dodecanethiol (DT) self-assembled monolayer. Their apparent height above the dodecanethiol matrix was used as a measure of the electronic coupling strength corresponding to S and Se, respectively. We show that the insertion behaviors of the two molecules are qualitatively the same, and that Se provides systematically a better coupling link than S, whatever the tunneling conditions.

  18. Direct comparison of the electronic coupling efficiency of sulfur and selenium anchoring groups for molecules adsorbed onto gold electrodes

    NASA Astrophysics Data System (ADS)

    Patrone, L.; Palacin, S.; Bourgoin, J. P.; Lagoute, J.; Zambelli, T.; Gauthier, S.

    2002-08-01

    We performed air and ultra-high vacuum scanning tunneling microscopy experiments in order to compare the electronic coupling provided by S and by Se used as alligator clips for bisthiol- and biselenol-terthiophene molecules adsorbed onto gold. The molecules were inserted in a dodecanethiol self-assembled monolayer. Their apparent height above the dodecanethiol matrix was used as a measure of the electronic coupling strength corresponding to S and Se, respectively. We show that the insertion behaviors of the two molecules are qualitatively the same, and that Se provides systematically a better coupling link than S whatever the tunneling conditions.

  19. Tunneling Spectroscopy Studies of Urea, Thiourea, and Selected Phosphonate Molecules Adsorbed on Aluminum Oxide

    NASA Astrophysics Data System (ADS)

    Crowder, Charles D.

    Experimental and calculated inelastic electron tunneling intensities were compared for several of the vibrational modes of thiourea adsorbed on aluminum oxide. The partial charge model of Kirtley, Scalapino, and Hansma was used to compute the theoretical intensities of each mode. The required partial charges were determined using a method developed by Momany. Essentially, the Coulomb potential resulting from point charges located at atom sites was fitted to the quantum mechanical electrostatic potential of a molecule calculated from Hartree-Fock theory. The effect of a vibrational mode pattern on the electrostatic potential of a molecule was investigated. This effect could not be acceptably modeled with a single point charge located on each atom, so one charge was used to represent the positive nucleus of each atom and a second charge was used to represent the valence cloud. The valence charge was allowed to move independently of the nuclear charge during a molecular vibration, and the motions of the two charges were found to be very different for hydrogen atoms. This model gave very reasonable agreement between the theoretical and observed relative intensities for the in plane vibrational modes of thiourea. An acceptable set of out of plane force constants could not be found. This caused problems in the interpretation of the out of plane relative intensities. Based on the in plane modes, it was concluded that thiourea bonded to aluminum oxide with the sulfur atom near the oxide and the sulfur-carbon bond perpendicular to the aluminum oxide surface. Quantum mechanical electrostatic potentials were also calculated for urea, phosphoric acid (PA), methylphosphonic acid (MPA), hydroxymethylphosphonic acid (HMP), and nitrotrismethylphosphonic acid (NTMP). Electron tunneling spectra were taken for PA, HMP and NTMP, and the observed frequencies were compared to values obtained from Fourier transform infrared, infrared and Raman spectroscopy. Upward shifts in the P=O and P

  20. Proteins Are the Body's Worker Molecules

    MedlinePlus

    ... Each "bead" is a small molecule called an amino acid. There are 20 standard amino acids, each with its own shape, size, and properties. Proteins typically contain from 50 to 2,000 amino acids hooked end-to-end in many combinations. Each ...

  1. Protein immobilization in hollow nanostructures and investigation of the adsorbed protein behavior.

    PubMed

    Qian, Xi; Levenstein, Alex; Gagner, Jennifer E; Dordick, Jonathan S; Siegel, Richard W

    2014-02-11

    Understanding nanomaterial-biomolecule interactions is critical to develop broad applications in sensors, devices, and therapeutics. During the past decade, in-depth studies have been performed on the effect of nanoscale surface topography on adsorbed protein structure and function. However, a fundamental understanding of nanobio interactions at concave surfaces is limited; the greatest challenge is to create a nanostructure that allows such interactions to occur and to be characterized. We have synthesized hollow nanocages (AuNG) through careful control of morphology and surface chemistry. Lysozyme was used as a model to probe interactions between a protein and these nanostructures. Solid Au nanoparticles with a similar morphology and surface chemistry were also used as a reference. Through a series of quantitative analyses of protein adsorption profiles and enzymatic activity assays of both nanobioconjugates, we discovered that a significant amount of protein could be delivered into the core of AuNG, while maintaining a substantial fraction of native activity.

  2. Effects of molecule-insulator interaction on geometric property of a single phthalocyanine molecule adsorbed on an ultrathin NaCl film

    NASA Astrophysics Data System (ADS)

    Miwa, Kuniyuki; Imada, Hiroshi; Kawahara, Shota; Kim, Yousoo

    2016-04-01

    The adsorption structure and orientation of a metal-free phthalocyanine (H2Pc ) and a magnesium phthalocyanine (MgPc) on a bilayer of NaCl films were investigated both theoretically and experimentally by means of first-principles calculations based on density functional theory and by scanning tunneling microscopy. H2Pc is adsorbed with its center over the sodium cation, and H-N bonds in the molecule are aligned with the [100] or [010] surface direction of a bilayer (001)-terminated NaCl film. The most stable structures of MgPc on the NaCl film show two kinds of orientations corresponding to the molecule rotated by ±7∘ relative to the [110] surface direction, with the Mg cation positioned over the chlorine anion in both cases. The energetic barrier for switching between these orientations is as low as 9.0 meV, and during an STM measurement, an orientational change of MgPc can be observed. The interaction between the adsorbed molecule and the NaCl film were analyzed in terms of dispersion interaction, Mg-Cl chemical bonding, and electrostatic interaction. It is found that the small electrostatic interaction between the molecule and the film gives a dominant contribution to determining the molecular orientation. Our detailed and comprehensive studies of the molecule-insulator interaction will provide knowledge to understand and control the properties of molecules on an insulating material.

  3. Small molecules that target phosphorylation dependent protein-protein interaction.

    PubMed

    Watanabe, Nobumoto; Osada, Hiroyuki

    2016-08-01

    Protein-protein interaction is one of the key events in the signal transduction pathway. The interaction changes the conformations, activities, localization and stabilities of the proteins, and transduces the signal to the next step. Frequently, this interaction occurs upon the protein phosphorylation. When upstream signals are stimulated, protein kinase(s) is/are activated and phosphorylate(s) their substrates, and induce the phosphorylation dependent protein-protein interaction. For this interaction, several domains in proteins are known to specifically recognize the phosphorylated residues of target proteins. These specific domains for interaction are important in the progression of the diseases caused by disordered signal transduction such as cancer. Thus small molecules that modulate this interaction are attractive lead compounds for the treatment of such diseases. In this review, we focused on three examples of phosphorylation dependent protein-protein interaction modules (14-3-3, polo box domain of Plk1 and F-box proteins in SCF ubiquitin ligases) and summarize small molecules that modulate their interaction. We also introduce our original screening system to identify such small molecules.

  4. Id proteins: small molecules, mighty regulators.

    PubMed

    Ling, Flora; Kang, Bin; Sun, Xiao-Hong

    2014-01-01

    The family of inhibitor of differentiation (Id) proteins is a group of evolutionarily conserved molecules, which play important regulatory roles in organisms ranging from Drosophila to humans. Id proteins are small polypeptides harboring a helix-loop-helix (HLH) motif, which are best known to mediate dimerization with other basic HLH proteins, primarily E proteins. Because Id proteins do not possess the basic amino acids adjacent to the HLH motif necessary for DNA binding, Id proteins inhibit the function of E protein homodimers, as well as heterodimers between E proteins and tissue-specific bHLH proteins. However, Id proteins have also been shown to have E protein-independent functions. The Id genes are broadly but differentially expressed in a variety of cell types. Transcription of the Id genes is controlled by transcription factors such as C/EBPβ and Egr as well as by signaling pathways triggered by different stimuli, which include bone morphogenic proteins, cytokines, and ligands of T cell receptors. In general, Id proteins are capable of inhibiting the differentiation of progenitors of different cell types, promoting cell-cycle progression, delaying cellular senescence, and facilitating cell migration. These properties of Id proteins enable them to play significant roles in stem cell maintenance, vasculogenesis, tumorigenesis and metastasis, the development of the immune system, and energy metabolism. In this review, we intend to highlight the current understanding of the function of Id proteins and discuss gaps in our knowledge about the mechanisms whereby Id proteins exert their diverse effects in multiple cellular processes.

  5. [Analysis of FT-IR-ATR spectra of serum proteins adsorbed on carbonaceous materials].

    PubMed

    Li, Bogang; Na, Juanjuan; Yin, Jie; Yin, Guangfu

    2006-10-01

    To clarify the reason causing difference of serum proteins adsorbability on different carbonaceous materials, FT-IR-ATR spectra of human serum albumin (HSA) and human serum fibrinogen(HFG) before and after adsorbing on diamond like carbon film (DLC),diamond film (DF) and graphite were analyzed. It has been shown that there are hydrogen bond because of -NH at the interfaces of HSA-DLC, HFG-DF and HFG-graphite. Based on the results, earlier research conclusion that the adsorbability of HSA on DLC higher than that on DF and graphite, but on DF and graphite the adsorption of HFG takes precedence can be explained rationally.

  6. Small-molecule inhibitors of myosin proteins

    PubMed Central

    Bond, Lisa M; Tumbarello, David A; Kendrick-Jones, John; Buss, Folma

    2014-01-01

    Advances in screening and computational methods have enhanced recent efforts to discover/design small-molecule protein inhibitors. One attractive target for inhibition is the myosin family of motor proteins. Myosins function in a wide variety of cellular processes, from intracellular trafficking to cell motility, and are implicated in several human diseases (e.g., cancer, hypertrophic cardiomyopathy, deafness and many neurological disorders). Potent and selective myosin inhibitors are, therefore, not only a tool for understanding myosin function, but are also a resource for developing treatments for diseases involving myosin dysfunction or overactivity. This review will provide a brief overview of the characteristics and scientific/therapeutic applications of the presently identified small-molecule myosin inhibitors before discussing the future of myosin inhibitor and activator design. PMID:23256812

  7. Effect of the interplay between protein and surface on the properties of adsorbed protein layers.

    PubMed

    Ouberai, Myriam M; Xu, Kairuo; Welland, Mark E

    2014-08-01

    Although protein adsorption to surface is a common phenomenon, investigation of the process is challenging due to the complexity of the interplay between external factors, protein and surface properties. Therefore experimental approaches have to measure the properties of adsorbed protein layers with high accuracy in order to achieve a comprehensive description of the process. To this end, we used a combination of two biosensing techniques, dual polarization interferometry and quartz crystal microbalance with dissipation. From this, we are able to extract surface coverage values, layer structural parameters, water content and viscoelastic properties to examine the properties of protein layers formed at the liquid/solid interface. Layer parameters were examined upon adsorption of proteins of varying size and structural properties, on surfaces with opposite polarity. We show that "soft" proteins such as unfolded α-synuclein and high molecular weight albumin are highly influenced by the surface polarity, as they form a highly diffuse and hydrated layer on the hydrophilic silica surface as opposed to the denser, less hydrated layer formed on a hydrophobic methylated surface. These layer properties are a result of different orientations and packing of the proteins. By contrast, lysozyme is barely influenced by the surface polarity due to its intrinsic structural stability. Interestingly, we show that for a similar molecular weight, the unfolded α-synuclein forms a layer with the highest percentage of solvation not related to surface coverage but resulting from the highest water content trapped within the protein. Together, these data reveal a trend in layer properties highlighting the importance of the interplay between protein and surface for the design of biomaterials. PMID:24780165

  8. Heat capacity measurements of atoms and molecules adsorbed on evaporated metal films

    SciTech Connect

    Kenny, T.W.

    1989-05-01

    Investigations of the properties of absorbed monolayers have received great experimental and theoretical attention recently, both because of the importance of surface processes in practical applications such as catalysis, and the importance of such systems to the understanding of the fundamentals of thermodynamics in two dimensions. We have adapted the composite bolometer technology to the construction of microcalorimeters. For these calorimeters, the adsorption substrate is an evaporated film deposited on one surface of an optically polished sapphire wafer. This approach has allowed us to make the first measurements of the heat capacity of submonolayer films of /sup 4/He adsorbed on metallic films. In contrast to measurements of /sup 4/He adsorbed on all other insulating substrates, we have shown that /sup 4/He on silver films occupies a two-dimensional gas phase over a broad range of coverages and temperatures. Our apparatus has been used to study the heat capacity of Indium flakes. CO multilayers, /sup 4/He adsorbed on sapphire and on Ag films and H/sub 2/ adsorbed on Ag films. The results are compared with appropriate theories. 68 refs., 19 figs.

  9. Volumetric Interpretation of Protein Adsorption: Ion-Exchange Adsorbent Capacity, Protein pI, and Interaction Energetics

    PubMed Central

    Noh, Hyeran; Yohe, Stefan; Vogler, Erwin A.

    2008-01-01

    Adsorption of lysozyme (Lys), human serum albumin (HSA), and immunoglobulin G (IgG) to anion- and cation-exchange resins is dominated by electrostatic interactions between protein and adsorbent. The solution-depletion method of measuring adsorption shows, however, that these proteins do not irreversibly adsorb to ion-exchange surfaces, even when the charge disparity between adsorbent and protein inferred from protein pI is large. Net-positively-charged Lys (pI = 11) and net-negatively-charged HSA (pI = 5.5) adsorb so strongly to sulfopropyl sepharose (SP; a negatively-charged, strong cation exchange resin, −0.22 mmol/mL exchange capacity) that both resist displacement by net-neutral IgG (pI = 7.0) in simultaneous adsorption-competition experiments. By contrast, IgG readily displaces both Lys and HSA adsorbed either to quarternary-ammonium sepharose (Q; a positively-charged, strong anion exchanger, + 0.22 mmol/mL exchange capacity) or octadecyl sepharose (ODS, a neutral hydrophobic resin, 0 mmol/mL exchange capacity). Thus it is concluded that adsorption results do not sensibly correlate with protein pI and that pI is actually a rather poor predictor of affinity for ion-exchange surfaces. Adsorption of Lys, HSA, and IgG to ion-exchange resins from stagnant solution leads to adsorbed multi-layers, into-or-onto which IgG adsorbs in adsorption-competition experiments. Comparison of adsorption to ion-exchange resins and neutral ODS leads to the conclusion that the apparent standard free-energy-of-adsorption ΔGadso of Lys, HSA, and IgG is not large in comparison to thermal energy due to energy-compensating interactions between water, protein, and ion-exchange surfaces that leaves a small net ΔGadso. Thus water is found to control protein adsorption to a full range of substratum types spanning hydrophobic (poorly water wettable) surfaces, hydrophilic surfaces bearing relatively-weak Lewis acid/base functionalities that wet with (hydrogen bond to) water but do not

  10. Multiscale simulations of protein G B1 adsorbed on charged self-assembled monolayers.

    PubMed

    Liu, Jie; Liao, Chenyi; Zhou, Jian

    2013-09-10

    The orientation of an antibody plays an important role in the development of immunosensors. Protein G is an antibody binding protein, which specifically targets the Fc fragment of an antibody. In this work, the orientation of prototypical and mutated protein G B1 adsorbed on positively and negatively charged self-assembled monolayers was studied by parallel tempering Monte Carlo and all-atom molecular dynamics simulations. Both methods present generally similar orientation distributions of protein G B1 for each kind of surface. The root-mean-square deviation, DSSP, gyration radius, eccentricity, dipole moment, and superimposed structures of protein G B1 were analyzed. Moreover, the orientation of binding antibody was also predicted in this work. Simulation results show that with the same orientation trends, the mutant exhibits narrower orientation distributions than does the prototype, which was mainly caused by the stronger dipole of the mutant. Both kinds of proteins adsorbed on charged surfaces were induced by the competition of electrostatic interaction and vdW interaction; the electrostatic interaction energy dominated the adsorption behavior. The protein adsorption was also largely affected by the distribution of charged residues within the proteins. Thus, the prototype could adsorb on a negatively charged surface, although it keeps a net charge of -4 e. The mutant has imperfect opposite orientation when it adsorbed on oppositely charged surfaces. For the mutant on a carboxyl-functionalized self-assembled monolayer (COOH-SAM), the orientation was the same as that inferred by experiments. While for the mutant on amine-functionalized self-assembled monolayer (NH2-SAM), the orientation was induced by the competition between attractive interactions (led by ASP40 and GLU56) and repulsive interactions (led by LYS10); thus, the perfect opposite orientation could not be obtained. On both surfaces, the adsorbed protein could retain its native conformation. The desired

  11. Adsorbed Proteins Influence the Biological Activity and Molecular Targeting of Nanomaterials

    SciTech Connect

    Dutta, Debamitra; Sundaram, S. K.; Teeguarden, Justin G.; Riley, Brian J.; Fifield, Leonard S.; Jacobs, Jon M.; Addleman, Raymond S.; Kaysen, George A.; Moudgil, Brij M.; Weber, Thomas J.

    2007-11-01

    The possible combination of unique physicochemical properties operating at unique sites of action within cells and tissues has led to considerable uncertainty surrounding nanomaterial toxic potential. Here we have investigated the relative importance of proteins adsorbed onto nanomaterial surfaces in guiding uptake and toxicity to determine whether a priori identification of adsorbed proteins will contribute to nanomaterial toxicity assessment. Albumin was identified as the major protein adsorbed onto single walled carbon nanotubes (SWCNTs) following incubation with fetal bovine or human serum/plasma, but not when plasma from the Nagase Analbuminemic Rat (NAR) was used, and precoating SWCNTs with a non-ionic surfactant (Pluronic F127) inhibited albumin adsorption. Damaged or structurally altered albumin is rapidly cleared by scavenger receptors. In the RAW 264.7 macrophage-like model, we observed that SWCNTs inhibited the induction of cyclooxygenase-2 (Cox-2) by lipopolysaccharide (LPS; 1 ng/ml, 6 hr) and this anti-inflammatory response was inhibited by fucoidan (scavenger receptor antagonist) and by precoating SWCNTs with Pluronic F127. Fucoidan also reduced the uptake of fluorescent SWCNTs (Alexa647) in RAW 264.7 cells. Albumin-coated SWCNTs reduced LPS-mediated Cox-2 induction. SWCNTs did not appear to reduce binding of a fluorescent LPS (Alexa488) to RAW 264.7 cells. The profile of proteins adsorbed onto amorphous silica (50 – 1000 nm) was qualitatively different, relative to SWCNTs, and coating amorphous silica with Pluronic F127 dramatically reduced protein binding and toxicity. Collectively, these observations are consistent with an important role for adsorbed proteins in guiding nanomaterial disposition and toxicity.

  12. The special features of protein adsorption isotherms on silica adsorbents

    NASA Astrophysics Data System (ADS)

    Chukhrai, E. S.; Atyaksheva, L. F.; Pilipenko, O. S.

    2011-05-01

    The adsorption isotherms of hemoglobin, peroxidase, and β-galactosidase on silochrome and mesoporous and biporous silicas were comparatively studied. Adsorption developed in two stages, including fast "reversible" protein adsorption (equilibrium was reached in t ≤ 1-2 h) and a "slow stage" of irreversible binding in t ≫ 24 h (multipoint adsorption). The corresponding equilibrium constants were determined. The mechanism of unlimited linear association of peroxidase in the adsorption layer on the surface of silochrome was established.

  13. Charge-transfer photodissociation of adsorbed molecules via electron image states

    SciTech Connect

    Jensen, E. T.

    2008-01-28

    The 248 and 193 nm photodissociations of submonolayer quantities of CH{sub 3}Br and CH{sub 3}I adsorbed on thin layers of n-hexane indicate that the dissociation is caused by dissociative electron attachment from subvacuum level photoelectrons created in the copper substrate. The characteristics of this photodissociation-translation energy distributions and coverage dependences show that the dissociation is mediated by an image potential state which temporarily traps the photoelectrons near the n-hexane-vacuum interface, and then the charge transfers from this image state to the affinity level of a coadsorbed halomethane which then dissociates.

  14. Adsorbed states of chlorophenol on Cu(110) and controlled switching of single-molecule junctions

    NASA Astrophysics Data System (ADS)

    Okuyama, H.; Kitaguchi, Y.; Hattori, T.; Ueda, Y.; Ferrer, N. G.; Hatta, S.; Aruga, T.

    2016-06-01

    A molecular junction of substituted benzene (chlorophenol) is fabricated and controlled by using a scanning tunneling microscope (STM). Prior to the junction formation, the bonding geometry of the molecule on the surface is characterized by STM and electron energy loss spectroscopy (EELS). EELS shows that the OH group of chlorophenol is dissociated on Cu(110) and that the molecule is bonded nearly flat to the surface via an O atom, with the Cl group intact. We demonstrate controlled contact of an STM tip to the "available" Cl group and lift-up of the molecule while it is anchored to the surface via an O atom. The asymmetric bonding motifs of the molecule to the electrodes allow for reversible control of the junction.

  15. Role of Structure and Glycosylation of Adsorbed Protein Films in Biolubrication

    PubMed Central

    Veeregowda, Deepak H.; Busscher, Henk J.; Vissink, Arjan; Jager, Derk-Jan; Sharma, Prashant K.; van der Mei, Henny C.

    2012-01-01

    Water forms the basis of lubrication in the human body, but is unable to provide sufficient lubrication without additives. The importance of biolubrication becomes evident upon aging and disease, particularly under conditions that affect secretion or composition of body fluids. Insufficient biolubrication, may impede proper speech, mastication and swallowing, underlie excessive friction and wear of articulating cartilage surfaces in hips and knees, cause vaginal dryness, and result in dry, irritated eyes. Currently, our understanding of biolubrication is insufficient to design effective therapeutics to restore biolubrication. Aim of this study was to establish the role of structure and glycosylation of adsorbed protein films in biolubrication, taking the oral cavity as a model and making use of its dynamics with daily perturbations due to different glandular secretions, speech, drinking and eating, and tooth brushing. Using different surface analytical techniques (a quartz crystal microbalance with dissipation monitoring, colloidal probe atomic force microscopy, contact angle measurements and X-ray photo-electron spectroscopy), we demonstrated that adsorbed salivary conditioning films in vitro are more lubricious when their hydrophilicity and degree of glycosylation increase, meanwhile decreasing their structural softness. High-molecular-weight, glycosylated proteins adsorbing in loops and trains, are described as necessary scaffolds impeding removal of water during loading of articulating surfaces. Comparing in vitro and in vivo water contact angles measured intra-orally, these findings were extrapolated to the in vivo situation. Accordingly, lubricating properties of teeth, as perceived in 20 volunteers comprising of equal numbers of male and female subjects, could be related with structural softness and glycosylation of adsorbed protein films on tooth surfaces. Summarizing, biolubrication is due to a combination of structure and glycosylation of adsorbed protein

  16. Simulation Studies of Protein and Small Molecule Interactions and Reaction.

    PubMed

    Yang, L; Zhang, J; Che, X; Gao, Y Q

    2016-01-01

    Computational studies of protein and small molecule (protein-ligand/enzyme-substrate) interactions become more and more important in biological science and drug discovery. Computer modeling can provide molecular details of the processes such as conformational change, binding, and transportation of small molecules/proteins, which are not easily to be captured in experiments. In this chapter, we discussed simulation studies of both protein and small molecules from three aspects: conformation sampling, transportations of small molecules in enzymes, and enzymatic reactions involving small molecules. Both methodology developments and examples of simulation studies in this field were presented.

  17. Simulation Studies of Protein and Small Molecule Interactions and Reaction.

    PubMed

    Yang, L; Zhang, J; Che, X; Gao, Y Q

    2016-01-01

    Computational studies of protein and small molecule (protein-ligand/enzyme-substrate) interactions become more and more important in biological science and drug discovery. Computer modeling can provide molecular details of the processes such as conformational change, binding, and transportation of small molecules/proteins, which are not easily to be captured in experiments. In this chapter, we discussed simulation studies of both protein and small molecules from three aspects: conformation sampling, transportations of small molecules in enzymes, and enzymatic reactions involving small molecules. Both methodology developments and examples of simulation studies in this field were presented. PMID:27497167

  18. Identification of polymer surface adsorbed proteins implicated in pluripotent human embryonic stem cell expansion.

    PubMed

    Hammad, Moamen; Rao, Wei; Smith, James G W; Anderson, Daniel G; Langer, Robert; Young, Lorraine E; Barrett, David A; Davies, Martyn C; Denning, Chris; Alexander, Morgan R

    2016-08-16

    Improved biomaterials are required for application in regenerative medicine, biosensing, and as medical devices. The response of cells to the chemistry of polymers cultured in media is generally regarded as being dominated by proteins adsorbed to the surface. Here we use mass spectrometry to identify proteins adsorbed from a complex mouse embryonic fibroblast (MEF) conditioned medium found to support pluripotent human embryonic stem cell (hESC) expansion on a plasma etched tissue culture polystyrene surface. A total of 71 proteins were identified, of which 14 uniquely correlated with the surface on which pluripotent stem cell expansion was achieved. We have developed a microarray combinatorial protein spotting approach to test the potential of these 14 proteins to support expansion of a hESC cell line (HUES-7) and a human induced pluripotent stem cell line (ReBl-PAT) on a novel polymer (N-(4-Hydroxyphenyl) methacrylamide). These proteins were spotted to form a primary array yielding several protein mixture 'hits' that enhanced cell attachment to the polymer. A second array was generated to test the function of a refined set of protein mixtures. We found that a combination of heat shock protein 90 and heat shock protein-1 encourage elevated adherence of pluripotent stem cells at a level comparable to fibronectin pre-treatment. PMID:27466628

  19. Mechanism of charge transfer and its impacts on Fermi-level pinning for gas molecules adsorbed on monolayer WS{sub 2}

    SciTech Connect

    Zhou, Changjie; Zhu, Huili; Yang, Weihuang

    2015-06-07

    Density functional theory calculations were performed to assess changes in the geometric and electronic structures of monolayer WS{sub 2} upon adsorption of various gas molecules (H{sub 2}, O{sub 2}, H{sub 2}O, NH{sub 3}, NO, NO{sub 2}, and CO). The most stable configuration of the adsorbed molecules, the adsorption energy, and the degree of charge transfer between adsorbate and substrate were determined. All evaluated molecules were physisorbed on monolayer WS{sub 2} with a low degree of charge transfer and accept charge from the monolayer, except for NH{sub 3}, which is a charge donor. Band structure calculations showed that the valence and conduction bands of monolayer WS{sub 2} are not significantly altered upon adsorption of H{sub 2}, H{sub 2}O, NH{sub 3}, and CO, whereas the lowest unoccupied molecular orbitals of O{sub 2}, NO, and NO{sub 2} are pinned around the Fermi-level when these molecules are adsorbed on monolayer WS{sub 2}. The phenomenon of Fermi-level pinning was discussed in light of the traditional and orbital mixing charge transfer theories. The impacts of the charge transfer mechanism on Fermi-level pinning were confirmed for the gas molecules adsorbed on monolayer WS{sub 2}. The proposed mechanism governing Fermi-level pinning is applicable to the systems of adsorbates on recently developed two-dimensional materials, such as graphene and transition metal dichalcogenides.

  20. The effect of gold nanoparticle structure on the conformation and function of adsorbed proteins

    NASA Astrophysics Data System (ADS)

    Gagner, Jennifer E.

    Many applications of nanobiomaterials rely on or are enhanced by specific, protein-mediated interactions with biological systems. These interactions can be engineered by chemically modifying the surface of the material to affect protein adsorption, or by altering the topography of the nanoscale surface. The attachment or adsorption of proteins onto materials can greatly affect the structure and subsequent function of those proteins, giving rise to unpredictable and potentially undesirable effects. Thus, it is essential to develop a detailed understanding of how nanostructured surface characteristics, such as atomic-scale topography, surface energy, and chemical structure may affect protein adsorption, structure, function, and stability. The presented work on gold nanoparticles (AuNP) in the forms of spheres (AuNS), rods (AuNR), cubes (AuNC) and octahedra (AuNO) will elucidate the effect of nanoparticle morphology on adsorbed model proteins lysozyme (Lyz) and α-chymotrypsin (ChT). It has been found that nanoparticle morphology does affect the structure of adsorbed proteins as well as the extent of the surface coverage; however, the final form of the nano-bio conjugate is protein specific. Lyz conjugates underwent loss of structure and rapid aggregation regardless of AuNP morphology; however, ChT conjugates exhibited no structure loss when immobilized on AuNS, and a significant, loading specific structure loss when adsorbed on AuNR. Further work will be presented on efforts to determine the role of crystal structure, surface energy, and ligand chemistry on adsorbed proteins. Wet chemical methods are used to synthesize AuNC with f100g facets and AuNO with f111g facets. Nanoparticles are characterized through electron microscopy, X-ray and electron diffraction, X-ray photoelectron spectroscopy and inductively coupled plasma mass spectroscopy. Protein conjugation and changes in protein structure are monitored through a variety of physical and spectroscopic techniques

  1. Acid properties of solid acid catalysts characterized by solid-state 31P NMR of adsorbed phosphorous probe molecules.

    PubMed

    Zheng, Anmin; Huang, Shing-Jong; Liu, Shang-Bin; Deng, Feng

    2011-09-01

    A brief review is presented on acidity characterization of solid acid catalysts by means of solid-state phosphor-31 magic-angle-spinning nuclear magnetic resonance ((31)P MAS NMR) spectroscopy using phosphor-containing molecules as probes. It is emphasized that such a simple approach using (31)P MAS NMR of adsorbed phosphorous probe molecules, namely trimethylphosphine (TMP) and trialkylphosphine oxides (R(3)PO), represents a unique technique in providing detailed qualitative and quantitative features, viz. type, strength, distribution, and concentration of acid sites in solid acid catalysts. In particular, it will be shown that when applied with a proper choice of probe molecules with varied sizes and results obtained from elemental analysis, the amounts and locations (intracrystalline vs. extracrystalline) of different types (Brønsted vs. Lewis) of acid sites may be determined. In addition, by incorporating the NMR results with that obtained from theoretical density functional theory (DFT) calculations, correlations between the (31)P chemical shifts (δ(31)P) and acidic strengths of Brønsted and Lewis acid sites may also be derived, facilitating a suitable acidity scale for solid acid catalysts.

  2. Enhanced Raman scattering by molecules adsorbed at the surface of colloidal spheroids

    NASA Astrophysics Data System (ADS)

    Wang, D.-S.; Kerker, M.

    1981-08-01

    Equations are derived and calculations are presented for the electrodynamic mechanism of enhanced Raman scattering by molecules at the surface of prolate and oblate spheroids in the small-particle limit. The molecules may be arbitrarily distributed; the particles may be arbitrarily oriented. Calculations are presented for a monolayer distributed over randomly oriented spheroids. The effects of particle shape are considered for Ag, Au, and Cu hydrosols. The peak enhancement moves to longer wavelengths, and in the case of Au and Cu the magnitude of the enhancement increases strikingly as the eccentricity increases. The relation between the dependence of the Raman enhancement upon excitation wavelength and the extinction spectra is discussed, including the precariousness of extrapolating such relations beyond the small-particle limit.

  3. Hindered and modulated rotational states and spectra of adsorbed diatomic molecules

    SciTech Connect

    Shih, Y.T.; Chuu, D.S.; Mei, W.N.

    1996-10-01

    Both vertical and horizontal adsorption configurations of a diatomic molecule were modeled as the rigid rotor with which the spatial motion was confined by a finite conical well. In addition to the polar hindering potential, a sinusoidal azimuthal modulation, which bears the local symmetry of the adsorption site, was incorporated. Eigenfunctions for different models were expressed analytically in terms of the hypergeometric functions, and eigenvalues were solved numerically. We found that the rotational energy levels exhibit oscillatory behavior when plotted as functions of the hindrance angle. This particular phenomenon was interpreted as the occurrence of resonance transmission of the rotor wave function at certain hindrance condition. We also found that the rotational levels were grouped into bands when the azimuthal modulation strength was increased. The solutions were used to calculate the rotational-state distribution of desorbed molecules, and agreement with the previous experiment was obtained. {copyright} {ital 1996 The American Physical Society.}

  4. AFM visualization at a single-molecule level of denaturated states of proteins on graphite.

    PubMed

    Barinov, Nikolay A; Prokhorov, Valery V; Dubrovin, Evgeniy V; Klinov, Dmitry V

    2016-10-01

    Different graphitic materials are either already used or believed to be advantageous in biomedical and biotechnological applications, e.g., as biomaterials or substrates for sensors. Most of these applications or associated important issues, such as biocompatibility, address the problem of adsorption of protein molecules and, in particular the conformational state of the adsorbed protein molecule on graphite. High-resolution AFM demonstrates highly oriented pyrolytic graphite (HOPG) induced denaturation of four proteins of blood plasma, such as ferritin, fibrinogen, human serum albumin (HSA) and immunoglobulin G (IgG), at a single molecule level. Protein denaturation is accompanied by the decrease of the heights of protein globules and spreading of the denatured protein fraction on the surface. In contrast, the modification of HOPG with the amphiphilic oligoglycine-hydrocarbon derivative monolayer preserves the native-like conformation and provides even more mild conditions for the protein adsorption than typically used mica. Protein unfolding on HOPG may have universal character for "soft" globular proteins.

  5. AFM visualization at a single-molecule level of denaturated states of proteins on graphite.

    PubMed

    Barinov, Nikolay A; Prokhorov, Valery V; Dubrovin, Evgeniy V; Klinov, Dmitry V

    2016-10-01

    Different graphitic materials are either already used or believed to be advantageous in biomedical and biotechnological applications, e.g., as biomaterials or substrates for sensors. Most of these applications or associated important issues, such as biocompatibility, address the problem of adsorption of protein molecules and, in particular the conformational state of the adsorbed protein molecule on graphite. High-resolution AFM demonstrates highly oriented pyrolytic graphite (HOPG) induced denaturation of four proteins of blood plasma, such as ferritin, fibrinogen, human serum albumin (HSA) and immunoglobulin G (IgG), at a single molecule level. Protein denaturation is accompanied by the decrease of the heights of protein globules and spreading of the denatured protein fraction on the surface. In contrast, the modification of HOPG with the amphiphilic oligoglycine-hydrocarbon derivative monolayer preserves the native-like conformation and provides even more mild conditions for the protein adsorption than typically used mica. Protein unfolding on HOPG may have universal character for "soft" globular proteins. PMID:27451365

  6. Switching orientation of adsorbed molecules: Reverse domino on a metal surface

    NASA Astrophysics Data System (ADS)

    Braatz, C. R.; Esat, T.; Wagner, C.; Temirov, R.; Tautz, F. S.; Jakob, P.

    2016-01-01

    A thus far unknown phase of 1,4,5,8-naphthalene-tetracarboxylic dianhydride (NTCDA) on Ag(111), characterized by an all perpendicular orientation of the planar molecules and bound to the Ag substrate through the carboxyl oxygen atoms has been identified using infrared absorption spectroscopy and scanning tunneling microscopy. Its formation process requires second layer NTCDA to squeeze into empty spaces between relaxed monolayer NTCDA molecules. Remarkably, this process causes initially parallel oriented NTCDA to likewise adopt the new, highly inclined adsorption geometry. According to our SPA-LEED and STM findings, the new phase displays a distinct long range order and shows a pronounced tendency to form 1D rows or narrow islands. We suggest that extra NTCDA preferentially transforms into the upright configuration close to existing islands and attaches to them, i.e. the transformation process proceeds in a directed and recurrent manner (reverse domino scenario). Identical processing starting with a compressed NTCDA/Ag(111) monolayer leads to a purely parallel oriented bilayer, that is, the NTCDA monolayer phase is retained and merely acts as a passive template for bilayer NTCDA. The new vertical NTCDA phase represents an unusual molecular system with π-orbitals oriented parallel to a metal surface. A substantially reduced coupling of these orbitals to Ag(111) electronic levels is conjectured, which will have a major impact on intermolecular couplings and electronically excited state lifetimes.

  7. Surface-enhanced Raman spectroscopy of organic molecules adsorbed on metallic nanoparticles.

    PubMed

    Heleg-Shabtai, Vered; Zifman, Adi; Kendler, Shai

    2012-01-01

    The improvements in Raman instrumentation have led to the development of -portable, simple to operate, Raman instruments that can be used for on-site analysis of substances relevant for homeland security purposes such as chemical and biological warfare and explosives materials.Raman spectroscopy, however, suffers from limited sensitivity which can be overcome by Surface-Enhanced Raman Spectroscopy (SERS). SERS can enhance the Raman signal of a target molecule by 6-10 orders of magnitude. The increased sensitivity, together with Raman's molecular recognition capabilities and the availability of portable Raman instruments make SERS a powerful analytical tool for on site detection.In this work we studied the effect of target molecules and SERS-active substrate properties on the obtained SERS, using a field portable Raman spectrometer. Also reported herein is the SERS detection of the chemical warfare agent sulfur mustard (HD, 2,2 dichloroethyl sulfide). This study may serve as a basis for the development of SERS platform for homeland security purposes.

  8. Tribochemical synthesis of nano-lubricant films from adsorbed molecules at sliding solid interface: Tribo-polymers from α-pinene, pinane, and n-decane

    NASA Astrophysics Data System (ADS)

    He, Xin; Barthel, Anthony J.; Kim, Seong H.

    2016-06-01

    The mechanochemical reactions of adsorbed molecules at sliding interfaces were studied for α-pinene (C10H16), pinane (C10H18), and n-decane (C10H22) on a stainless steel substrate surface. During vapor phase lubrication, molecules adsorbed at the sliding interface could be activated by mechanical shear. Under the equilibrium adsorption condition of these molecules, the friction coefficient of sliding steel surfaces was about 0.2 and a polymeric film was tribochemically produced. The synthesis yield of α-pinene tribo-polymers was about twice as much as pinane tribo-polymers. In contrast to these strained bicyclic hydrocarbons, n-decane showed much weaker activity for tribo-polymerization at the same mechanical shear condition. These results suggested that the mechanical shear at tribological interfaces could induce the opening of the strained ring structure of α-pinene and pinane, which leads to polymerization of adsorbed molecules at the sliding track. On a stainless steel surface, such polymerization reactions of adsorbed molecules do not occur under typical surface reaction conditions. The mechanical properties and boundary lubrication efficiency of the produced tribo-polymer films are discussed.

  9. Infrared spectroscopy of water clusters co-adsorbed with hydrogen molecules on a sodium chloride film

    NASA Astrophysics Data System (ADS)

    Yamakawa, Koichiro; Fukutani, Katsuyuki

    2016-06-01

    Hydrogen gas containing a trace of water vapor was dosed on a vacuum-evaporated sodium chloride film at 13 K, and water clusters formed on the substrate were investigated by infrared absorption spectroscopy. Absorption bands due to (H2O)n clusters with n = 3-6 and an induced absorption band due to hydrogen were clearly observed. With increasing gas dosage, the intensities of the cluster bands increased linearly while the intensity of the hydrogen band was constant. This suggests that the water clusters were formed in two-dimensional matrices of hydrogen. We found that the water clusters did exist on the surface upon heating even after the hydrogen molecules had desorbed. A further rise of the substrate temperature up to 27 K yielded the formation of larger clusters, (H2O)n with n > 6 . We also discuss the origins of the two bands of the trimer in terms of pseudorotation and a metastable isomer.

  10. Differential analysis of "protein corona" profile adsorbed onto different nonviral gene delivery systems.

    PubMed

    Capriotti, Anna Laura; Caracciolo, Giulio; Caruso, Giuseppe; Foglia, Patrizia; Pozzi, Daniela; Samperi, Roberto; Laganà, Aldo

    2011-12-15

    A shotgun proteomics approach was used to characterize and compare the proteins that lead to the formation of a rich "protein corona" adsorbed onto the surfaces of cationic liposomes (CLs), lipoplexes, and lipid/polycation/DNA (LPD) complexes, when they come into contact with plasma. After separation of the nanoparticle-protein complex from plasma, the protein mixture was digested, and peptides were analyzed by nanoliquid chromatography-Orbitrap LTQ-XL mass spectrometry. The number of proteins bound to lipoplexes was double that of those identified in the corona of CLs (208 vs 105), while 77 proteins were common to both coronas. The number of proteins bound to the surface of the LPD complexes (158, 133 of which are common to lipoplexes) is intermediate between those found in the protein corona of both CLs and lipoplexes. About half of them were found in the protein corona of CLs. By overlapping the three formulations, it can be seen that only 12 proteins are peculiar to LPD complexes. These results may help in designing gene delivery systems capable of binding the minimum possible quantity of proteins that influence transfection negatively, binding selectively proteins capable of helping in steering in vivo the vector toward the target, and obtaining more efficient and effective gene therapy.

  11. Can CO2 molecule adsorb effectively on Al-doped boron nitride single walled nanotube?

    NASA Astrophysics Data System (ADS)

    Shao, Peng; Kuang, Xiao-Yu; Ding, Li-Ping; Yang, Jing; Zhong, Ming-Min

    2013-11-01

    The adsorption of carbon dioxides (CO2) is very important in environmental and industrial applications. The boron nitride nanotube (BNNT) with large surface and polarity may be a good candidate as CO2 capture. Unfortunately, the pristine BNNT is almost inert to the highly stable CO2. To renew technical applications of BNNT for CO2 adsorption, we explore the possibility of CO2 adsorption on various (n, 0) (n = 6, 8, 10, 12 and 14) Al-doped BNNT by density functional theory (DFT) calculations. The results show that the Al-doped BNNT could be a potential CO2 adsorption material, and the CO2 adsorption energies are independent of BNNT diameters. Furthermore, the interactions between CO2 and exemplified (6, 0) Al-doped BNNT are investigated by density of states (DOS) and electron density. We found the interaction between CO2 and AlB-BNNT is stronger than that of CO2 and AlN-BNNT. The adsorption of CO2 can induce new density of state, as well as a local charge fluctuation due to more electron density redistribution on the atoms near CO2 molecule.

  12. Determinants of protein elution rates from preparative ion-exchange adsorbents.

    PubMed

    Angelo, James M; Lenhoff, Abraham M

    2016-04-01

    The rate processes involved in elution in preparative chromatography can affect both peak resolution and hence selectivity as well as practical factors such as facility fit. These processes depend on the physical structure of the adsorbent particles, the amount of bound solute, the solution conditions for operation or some combination of these factors. Ion-exchange adsorbents modified with covalently attached or grafted polymer layers have become widely used in preparative chromatography. Their often easily accessible microstructures offer substantial binding capacities for biomolecules, but elution has sometimes been observed to be undesirably slow. In order to determine which physicochemical phenomena control elution behavior, commercially available cellulosic, dextran-grafted and unmodified agarose materials were characterized here by their elution profiles at various conditions, including different degrees of loading. Elution data were analyzed under the assumption of purely diffusion-limited control, including the role of pore structure properties such as porosity and tortuosity. In general, effective elution rates decreased with the reduction of accessible pore volume, but differences among different proteins indicated the roles of additional factors. Additional measurements and analysis, including the use of confocal laser scanning microscopy to observe elution within single chromatographic particles, indicated the importance of protein association within the particle during elution. The use of protein stabilizing agents was explored in systems presenting atypical elution behavior, and l-arginine and disaccharide excipients were shown to alleviate the effects for one protein, lysozyme, in the presence of sodium chloride. Incorporation of these excipients into eluent buffer gave rise to faster elution and significantly lower pool volumes in elution from polymer-modified adsorbents.

  13. The leucine rich amelogenin protein (LRAP) adsorbs as monomers or dimers onto surfaces

    SciTech Connect

    Tarasevich, Barbara J.; Lea, Alan S.; Shaw, Wendy J.

    2010-03-15

    Amelogenin and amelogenin splice variants are believed to be involved in controlling the formation of the highly anisotropic and ordered hydroxyapatite crystallites that form enamel. The adsorption behavior of amelogenin proteins onto substrates is very important because protein-surface interactions are critical to it’s function. We have studied the adsorption of LRAP, a splice variant of amelogenin which may also contribute to enamel function, onto model self-assembled monolayers on gold containing of COOH, CH3, and NH2 end groups. Dynamic light scattering (DLS) experiments indicated that LRAP in phosphate buffered saline (PBS) and solutions at saturation with calcium phosphate contained aggregates of nanospheres. Null ellipsometry and atomic force microscopy (AFM) were used to study protein adsorption amounts and structures. Relatively high amounts of adsorption occurred onto the CH3 and NH2 surfaces from both calcium phosphate and PBS solutions. Adsorption was also promoted onto COOH surfaces when calcium was present in the solutions suggesting an interaction that involves calcium bridging with the negatively charged C-terminus. The ellipsometry and AFM studies suggested that the protein adsorbed onto all surfaces as LRAP monomers. We propose that the monomers adsorb onto the surfaces by disassembling or “shedding” from the nanospheres that are present in solution. This work reveals the importance of small subnanosphere-sized structures of LRAP at interfaces, structures that may be important in the biomineralization of tooth enamel.

  14. SPLINTS: small-molecule protein ligand interface stabilizers.

    PubMed

    Fischer, Eric S; Park, Eunyoung; Eck, Michael J; Thomä, Nicolas H

    2016-04-01

    Regulatory protein-protein interactions are ubiquitous in biology, and small molecule protein-protein interaction inhibitors are an important focus in drug discovery. Remarkably little attention has been given to the opposite strategy-stabilization of protein-protein interactions, despite the fact that several well-known therapeutics act through this mechanism. From a structural perspective, we consider representative examples of small molecules that induce or stabilize the association of protein domains to inhibit, or alter, signaling for nuclear hormone, GTPase, kinase, phosphatase, and ubiquitin ligase pathways. These SPLINTS (small-molecule protein ligand interface stabilizers) drive interactions that are in some cases physiologically relevant, and in others entirely adventitious. The diverse structural mechanisms employed suggest approaches for a broader and systematic search for such compounds in drug discovery. PMID:26829757

  15. Effect of side by side interactions on the thermodynamic properties of adsorbed CO molecules on the Ni(111) surface: a cluster model study

    NASA Astrophysics Data System (ADS)

    Shamkhali, Amir N.; Parsafar, Gholamabbas

    2010-05-01

    The effect of electrostatic interactions on vibrational frequencies and thermodynamic properties of CO adsorbate on the Ni(111) surface is calculated by taking the first and second nearest-neighbour interactions into account. In order to obtain reasonable results, the cluster model of various surface adsorption sites with CO adsorbate is partially optimized, using Density Functional Theory and also the MP2 method for the hcp site. Comparison between DFT and MP2 results shows that DFT results are more reliable for this system. The stretching and bending frequencies of CO adsorbate are calculated using both Partial Hessian Analysis and Cluster-Adsorbate Coupling methods. Stretching and bending frequencies are both shifted by the side by side interactions. The coupling of surface phonons and adsorbate vibrations reduces the side effects. The largest side effects on the vibrational internal energy, isochoric heat capacity, entropy and total Helmholtz free energy of adsorbed CO molecule calculated using the CAC method are found for 0.5 ML coverage. The results of the CAC method are better, but the PHA method can be used as a simple upper bound estimation. The adsorptive phase acts as an intelligent material in such a way that it changes its configuration in order to reduce the side effects.

  16. Ultra-Thin Optically Transparent Carbon Electrodes Produced from Layers of Adsorbed Proteins

    PubMed Central

    Alharthi, Sarah A.; Benavidez, Tomas E.; Garcia, Carlos D.

    2013-01-01

    This work describes a simple, versatile, and inexpensive procedure to prepare optically transparent carbon electrodes, using proteins as precursors. Upon adsorption, the protein-coated substrates were pyrolyzed under reductive conditions (5% H2) to form ultra-thin, conductive electrodes. Because proteins spontaneously adsorb to interfaces forming uniform layers, the proposed method does not require a precise control of the preparation conditions, specialized instrumentation, or expensive precursors. The resulting electrodes were characterized by a combination of electrochemical, optical, and spectroscopic means. As a proof-of-concept, the optically-transparent electrodes were also used as substrate for the development of an electrochemical glucose biosensor. The proposed films represent a convenient alternative to more sophisticated, and less available, carbon-based nanomaterials. Furthermore, these films could be formed on a variety of substrates, without classical limitations of size or shape. PMID:23421732

  17. Shotgun proteomic analytical approach for studying proteins adsorbed onto liposome surface.

    PubMed

    Capriotti, Anna Laura; Caracciolo, Giulio; Cavaliere, Chiara; Crescenzi, Carlo; Pozzi, Daniela; Laganà, Aldo

    2011-09-01

    The knowledge about the interaction between plasma proteins and nanocarriers employed for in vivo delivery is fundamental to understand their biodistribution. Protein adsorption onto nanoparticle surface (protein corona) is strongly affected by vector surface characteristics. In general, the primary interaction is thought to be electrostatic, thus surface charge of carrier is supposed to play a central role in protein adsorption. Because protein corona composition can be critical in modifying the interactive surface that is recognized by cells, characterizing its formation onto lipid particles may serve as a fundamental predictive model for the in vivo efficiency of a lipidic vector. In the present work, protein coronas adsorbed onto three differently charged cationic liposome formulations were compared by a shotgun proteomic approach based on nano-liquid chromatography-high-resolution mass spectrometry. About 130 proteins were identified in each corona, with only small differences between the different cationic liposome formulations. However, this study could be useful for the future controlled design of colloidal drug carriers and possibly in the controlled creation of biocompatible surfaces of other devices that come into contact with proteins into body fluids.

  18. Characterization of cross-linked cellulosic ion-exchange adsorbents: 2. Protein sorption and transport.

    PubMed

    Angelo, James M; Cvetkovic, Aleksandar; Gantier, Rene; Lenhoff, Abraham M

    2016-03-18

    Adsorption behavior in the HyperCel family of cellulosic ion-exchange materials (Pall Corporation) was characterized using methods to assess, quantitatively and qualitatively, the dynamics of protein uptake as well as static adsorption as a function of ionic strength and protein concentration using several model proteins. The three exchangers studied all presented relatively high adsorptive capacities under low ionic strength conditions, comparable to commercially available resins containing polymer functionalization aimed at increasing that particular characteristic. The strong cation- and anion-exchange moieties showed higher sensitivity to increasing salt concentrations, but protein affinity on the salt-tolerant STAR AX HyperCel exchanger remained strong at ionic strengths normally used in downstream processing to elute material fully during ion-exchange chromatography. Very high uptake rates were observed in both batch kinetics experiments and time-series confocal laser scanning microscopy, suggesting low intraparticle transport resistances relative to external film resistance, even at higher bulk protein concentrations where the opposite is typically observed. Electron microscopy imaging of protein adsorbed phases provided additional insight into particle structure that could not be resolved in previous work on the bare resins.

  19. Influence of fluoride-detergent combinations on the visco-elasticity of adsorbed salivary protein films.

    PubMed

    Veeregowda, Deepak H; van der Mei, Henny C; Busscher, Henk J; Sharma, Prashant K

    2011-02-01

    The visco-elasticity of salivary-protein films is related to mouthfeel, lubrication, biofilm formation, and protection against erosion and is influenced by the adsorption of toothpaste components. The thickness and the visco-elasticity of hydrated films (determined using a quartz crystal microbalance) of 2-h-old in vitro-adsorbed salivary-protein films were 43.5 nm and 9.4 MHz, respectively, whereas the dehydrated thickness, measured using X-ray photoelectron spectroscopy, was 2.4 nm. Treatment with toothpaste slurries decreased the thickness of the film, depending on the fluoride-detergent combination involved. Secondary exposure to saliva resulted in a regained thickness of the film to a level similar to its original thickness; however, no association was found between the thickness of hydrated and dehydrated films, indicating differences in film structure. Treatment with stannous fluoride/sodium lauryl sulphate (SnF(2)/SLS)-containing toothpaste slurries yielded a strong, immediate two-fold increase in characteristic film frequency (f(c)) with respect to untreated films, indicating cross-linking in adsorbed salivary-protein films by Sn(2+) that was absent when SLS was replaced with sodium hexametaphosphate (NaHMP). Secondary exposure to saliva of films treated with SnF(2) caused a strong, six-fold increase in f(c) compared with primary salivary-protein films, regardless of whether SLS or NaHMP was the detergent. This suggests that ionized stannous is not directly available for cross-linking in combination with highly negatively charged NaHMP, but becomes slowly available after initial treatment to cause cross-linking during secondary exposure to saliva.

  20. Protein mechanics: from single molecules to functional biomaterials.

    PubMed

    Li, Hongbin; Cao, Yi

    2010-10-19

    Elastomeric proteins act as the essential functional units in a wide variety of biomechanical machinery and serve as the basic building blocks for biological materials that exhibit superb mechanical properties. These proteins provide the desired elasticity, mechanical strength, resilience, and toughness within these materials. Understanding the mechanical properties of elastomeric protein-based biomaterials is a multiscale problem spanning from the atomistic/molecular level to the macroscopic level. Uncovering the design principles of individual elastomeric building blocks is critical both for the scientific understanding of multiscale mechanics of biomaterials and for the rational engineering of novel biomaterials with desirable mechanical properties. The development of single-molecule force spectroscopy techniques has provided methods for characterizing mechanical properties of elastomeric proteins one molecule at a time. Single-molecule atomic force microscopy (AFM) is uniquely suited to this purpose. Molecular dynamic simulations, protein engineering techniques, and single-molecule AFM study have collectively revealed tremendous insights into the molecular design of single elastomeric proteins, which can guide the design and engineering of elastomeric proteins with tailored mechanical properties. Researchers are focusing experimental efforts toward engineering artificial elastomeric proteins with mechanical properties that mimic or even surpass those of natural elastomeric proteins. In this Account, we summarize our recent experimental efforts to engineer novel artificial elastomeric proteins and develop general and rational methodologies to tune the nanomechanical properties of elastomeric proteins at the single-molecule level. We focus on general design principles used for enhancing the mechanical stability of proteins. These principles include the development of metal-chelation-based general methodology, strategies to control the unfolding hierarchy of

  1. Targeted Mutagenesis and Combinatorial Library Screening Enables Control of Protein Orientation on Surfaces and Increased Activity of Adsorbed Proteins.

    PubMed

    Cruz-Teran, Carlos A; Carlin, Kevin B; Efimenko, Kirill; Genzer, Jan; Rao, Balaji M

    2016-08-30

    While nonspecific adsorption is widely used for immobilizing proteins on solid surfaces, the random nature of protein adsorption may reduce the activity of immobilized proteins due to occlusion of the active site. We hypothesized that the orientation a protein assumes on a given surface can be controlled by systematically introducing mutations into a region distant from its active site, thereby retaining activity of the immobilized protein. To test this hypothesis, we generated a combinatorial protein library by randomizing six targeted residues in a binding protein derived from highly stable, nonimmunoglobulin Sso7d scaffold; mutations were targeted in a region that is distant from the binding site. This library was screened to isolate binders that retain binding to its cognate target (chicken immunoglobulin Y, cIgY) as well as exhibit adsorption on unmodified silica at pH 7.4 and high ionic strength conditions. A single mutant, Sso7d-2B5, was selected for further characterization. Sso7d-2B5 retained binding to cIgY with an apparent dissociation constant similar to that of the parent protein; both mutant and parent proteins saturated the surface of silica with similar densities. Strikingly, however, silica beads coated with Sso7d-2B5 could achieve up to 7-fold higher capture of cIgY than beads coated with the parent protein. These results strongly suggest that mutations introduced in Sso7d-2B5 alter its orientation relative to the parent protein, when adsorbed on silica surfaces. Our approach also provides a generalizable strategy for introducing mutations in proteins so as to improve their activity upon immobilization, and has direct relevance to development of protein-based biosensors and biocatalysts. PMID:27490089

  2. The Single-Molecule Approach to Membrane Protein Stoichiometry.

    PubMed

    Nichols, Michael G; Hallworth, Richard

    2016-01-01

    The advent of techniques for imaging solitary fluorescent molecules has made possible many new kinds of biological experiments. Here, we describe the application of single-molecule imaging to the problem of subunit stoichiometry in membrane proteins. A membrane protein of unknown stoichiometry, prestin, is coupled to the fluorescent enhanced green fluorescent protein (eGFP) and synthesized in the human embryonic kidney (HEK) cell line. We prepare adherent membrane fragments containing prestin-eGFP by osmotic lysis. The molecules are then exposed to continuous low-level excitation until their fluorescence reaches background levels. Their fluorescence decreases in discrete equal-amplitude steps, consistent with the photobleaching of single fluorophores. We count the number of steps required to photobleach each molecule. The molecular stoichiometry is then deduced using a binomial model.

  3. Inhibition of Lipid Oxidation in Oil-in-Water Emulsions by Interface-Adsorbed Myofibrillar Protein.

    PubMed

    Yang, Jiayi; Xiong, Youling L

    2015-10-14

    This study investigated the role of interfacial myofibrillar protein (MFP) in the oxidative stabilization of meat emulsions. Emulsions with 10% oil were prepared using either 2% (w/v) Tween 20 or 0.25, 0.5, and 1% (w/v) MFP and then subjected to hydroxyl radical oxidation at 4 °C for 0, 2, and 24 h. MFP was more readily oxidized (intrinsic fluorescence quenching, sulfur losses, and carbonyl formation) than oil [conjugated dienes and 2-thiobarbituric acid-reactive substances (TBARS)]. However, oxidized MFP in the continuous phase stimulated lipid oxidation after 24 h, sharply contrasting with interface-adsorbed MFP that inhibited TBARS formation nearly 90% (p < 0.05). Interfacial MFP from 2 h oxidized samples exhibited greater losses of fluorescence and more extensive polymerization of myosin (detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) than MFP present in the continuous phase. Results indicated that, due to the physical localization, interface-adsorbed MFP in general and myosin in particular provided accentuated protection of emulsions against oxidation. PMID:26414649

  4. Metamaterial-enhanced vibrational absorption spectroscopy for the detection of protein molecules

    NASA Astrophysics Data System (ADS)

    Bui, Tung S.; Dao, Thang D.; Dang, Luu H.; Vu, Lam D.; Ohi, Akihiko; Nabatame, Toshihide; Lee, Youngpak; Nagao, Tadaaki; Hoang, Chung V.

    2016-08-01

    From visible to mid-infrared frequencies, molecular sensing has been a major successful application of plasmonics because of the enormous enhancement of the surface electromagnetic nearfield associated with the induced collective motion of surface free carriers excited by the probe light. However, in the lower-energy terahertz (THz) region, sensing by detecting molecular vibrations is still challenging because of low sensitivity, complicated spectral features, and relatively little accumulated knowledge of molecules. Here, we report the use of a micron-scale thin-slab metamaterial (MM) architecture, which functions as an amplifier for enhancing the absorption signal of the THz vibration of an ultrathin adsorbed layer of large organic molecules. We examined bovine serum albumin (BSA) as a prototype large protein molecule and Rhodamine 6G (Rh6G) and 3,3‧-diethylthiatricarbocyanine iodide (DTTCI) as examples of small molecules. Among them, our MM significantly magnified only the signal strength of bulky BSA. On the other hand, DTTCI and Rh6G are inactive, as they lack low-frequency vibrational modes in this frequency region. The results obtained here clearly demonstrate the promise of MM-enhanced absorption spectroscopy in the THz region for detection and structural monitoring of large biomolecules such as proteins or pathogenic enzymes.

  5. Metamaterial-enhanced vibrational absorption spectroscopy for the detection of protein molecules.

    PubMed

    Bui, Tung S; Dao, Thang D; Dang, Luu H; Vu, Lam D; Ohi, Akihiko; Nabatame, Toshihide; Lee, YoungPak; Nagao, Tadaaki; Hoang, Chung V

    2016-01-01

    From visible to mid-infrared frequencies, molecular sensing has been a major successful application of plasmonics because of the enormous enhancement of the surface electromagnetic nearfield associated with the induced collective motion of surface free carriers excited by the probe light. However, in the lower-energy terahertz (THz) region, sensing by detecting molecular vibrations is still challenging because of low sensitivity, complicated spectral features, and relatively little accumulated knowledge of molecules. Here, we report the use of a micron-scale thin-slab metamaterial (MM) architecture, which functions as an amplifier for enhancing the absorption signal of the THz vibration of an ultrathin adsorbed layer of large organic molecules. We examined bovine serum albumin (BSA) as a prototype large protein molecule and Rhodamine 6G (Rh6G) and 3,3'-diethylthiatricarbocyanine iodide (DTTCI) as examples of small molecules. Among them, our MM significantly magnified only the signal strength of bulky BSA. On the other hand, DTTCI and Rh6G are inactive, as they lack low-frequency vibrational modes in this frequency region. The results obtained here clearly demonstrate the promise of MM-enhanced absorption spectroscopy in the THz region for detection and structural monitoring of large biomolecules such as proteins or pathogenic enzymes. PMID:27555217

  6. Metamaterial-enhanced vibrational absorption spectroscopy for the detection of protein molecules

    PubMed Central

    Bui, Tung S.; Dao, Thang D.; Dang, Luu H.; Vu, Lam D.; Ohi, Akihiko; Nabatame, Toshihide; Lee, YoungPak; Nagao, Tadaaki; Hoang, Chung V.

    2016-01-01

    From visible to mid-infrared frequencies, molecular sensing has been a major successful application of plasmonics because of the enormous enhancement of the surface electromagnetic nearfield associated with the induced collective motion of surface free carriers excited by the probe light. However, in the lower-energy terahertz (THz) region, sensing by detecting molecular vibrations is still challenging because of low sensitivity, complicated spectral features, and relatively little accumulated knowledge of molecules. Here, we report the use of a micron-scale thin-slab metamaterial (MM) architecture, which functions as an amplifier for enhancing the absorption signal of the THz vibration of an ultrathin adsorbed layer of large organic molecules. We examined bovine serum albumin (BSA) as a prototype large protein molecule and Rhodamine 6G (Rh6G) and 3,3′-diethylthiatricarbocyanine iodide (DTTCI) as examples of small molecules. Among them, our MM significantly magnified only the signal strength of bulky BSA. On the other hand, DTTCI and Rh6G are inactive, as they lack low-frequency vibrational modes in this frequency region. The results obtained here clearly demonstrate the promise of MM-enhanced absorption spectroscopy in the THz region for detection and structural monitoring of large biomolecules such as proteins or pathogenic enzymes. PMID:27555217

  7. Small molecule annotation for the Protein Data Bank.

    PubMed

    Sen, Sanchayita; Young, Jasmine; Berrisford, John M; Chen, Minyu; Conroy, Matthew J; Dutta, Shuchismita; Di Costanzo, Luigi; Gao, Guanghua; Ghosh, Sutapa; Hudson, Brian P; Igarashi, Reiko; Kengaku, Yumiko; Liang, Yuhe; Peisach, Ezra; Persikova, Irina; Mukhopadhyay, Abhik; Narayanan, Buvaneswari Coimbatore; Sahni, Gaurav; Sato, Junko; Sekharan, Monica; Shao, Chenghua; Tan, Lihua; Zhuravleva, Marina A

    2014-01-01

    The Protein Data Bank (PDB) is the single global repository for three-dimensional structures of biological macromolecules and their complexes, and its more than 100,000 structures contain more than 20,000 distinct ligands or small molecules bound to proteins and nucleic acids. Information about these small molecules and their interactions with proteins and nucleic acids is crucial for our understanding of biochemical processes and vital for structure-based drug design. Small molecules present in a deposited structure may be attached to a polymer or may occur as a separate, non-covalently linked ligand. During curation of a newly deposited structure by wwPDB annotation staff, each molecule is cross-referenced to the PDB Chemical Component Dictionary (CCD). If the molecule is new to the PDB, a dictionary description is created for it. The information about all small molecule components found in the PDB is distributed via the ftp archive as an external reference file. Small molecule annotation in the PDB also includes information about ligand-binding sites and about covalent and other linkages between ligands and macromolecules. During the remediation of the peptide-like antibiotics and inhibitors present in the PDB archive in 2011, it became clear that additional annotation was required for consistent representation of these molecules, which are quite often composed of several sequential subcomponents including modified amino acids and other chemical groups. The connectivity information of the modified amino acids is necessary for correct representation of these biologically interesting molecules. The combined information is made available via a new resource called the Biologically Interesting molecules Reference Dictionary, which is complementary to the CCD and is now routinely used for annotation of peptide-like antibiotics and inhibitors.

  8. Small molecule annotation for the Protein Data Bank.

    PubMed

    Sen, Sanchayita; Young, Jasmine; Berrisford, John M; Chen, Minyu; Conroy, Matthew J; Dutta, Shuchismita; Di Costanzo, Luigi; Gao, Guanghua; Ghosh, Sutapa; Hudson, Brian P; Igarashi, Reiko; Kengaku, Yumiko; Liang, Yuhe; Peisach, Ezra; Persikova, Irina; Mukhopadhyay, Abhik; Narayanan, Buvaneswari Coimbatore; Sahni, Gaurav; Sato, Junko; Sekharan, Monica; Shao, Chenghua; Tan, Lihua; Zhuravleva, Marina A

    2014-01-01

    The Protein Data Bank (PDB) is the single global repository for three-dimensional structures of biological macromolecules and their complexes, and its more than 100,000 structures contain more than 20,000 distinct ligands or small molecules bound to proteins and nucleic acids. Information about these small molecules and their interactions with proteins and nucleic acids is crucial for our understanding of biochemical processes and vital for structure-based drug design. Small molecules present in a deposited structure may be attached to a polymer or may occur as a separate, non-covalently linked ligand. During curation of a newly deposited structure by wwPDB annotation staff, each molecule is cross-referenced to the PDB Chemical Component Dictionary (CCD). If the molecule is new to the PDB, a dictionary description is created for it. The information about all small molecule components found in the PDB is distributed via the ftp archive as an external reference file. Small molecule annotation in the PDB also includes information about ligand-binding sites and about covalent and other linkages between ligands and macromolecules. During the remediation of the peptide-like antibiotics and inhibitors present in the PDB archive in 2011, it became clear that additional annotation was required for consistent representation of these molecules, which are quite often composed of several sequential subcomponents including modified amino acids and other chemical groups. The connectivity information of the modified amino acids is necessary for correct representation of these biologically interesting molecules. The combined information is made available via a new resource called the Biologically Interesting molecules Reference Dictionary, which is complementary to the CCD and is now routinely used for annotation of peptide-like antibiotics and inhibitors. PMID:25425036

  9. Watching Individual Proteins Acting on Single Molecules of DNA

    PubMed Central

    Amitani, Ichiro; Liu, Bian; Dombrowski, Christopher C.; Baskin, Ronald J.; Kowalczykowski, Stephen C.

    2011-01-01

    In traditional biochemical experiments, the behavior of individual proteins is obscured by ensemble averaging. To better understand the behavior of proteins that bind to and/or translocate on DNA, we have developed instrumentation that uses optical trapping, microfluidic solution delivery, and fluorescent microscopy to visualize either individual proteins or assemblies of proteins acting on single molecules of DNA. The general experimental design involves attaching a single DNA molecule to a polystyrene microsphere that is then used as a microscopic handle to manipulate individual DNA molecules with a laser trap. Visualization is achieved by fluorescently labeling either the DNA or the protein of interest, followed by direct imaging using high-sensitivity fluorescence microscopy. We describe the sample preparation and instrumentation used to visualize the interaction of individual proteins with single molecules of DNA. As examples, we describe the application of these methods to the study of proteins involved in recombination-mediated DNA repair, a process essential for the maintenance of genomic integrity. PMID:20580968

  10. A theoretical study of rotational and translational diffusion dynamics of molecules with a six-fold point symmetry adsorbed on a hexagonal lattice by neutron scattering.

    PubMed

    Calvo-Almazán; Miret-Artés; Fouquet

    2012-03-14

    A complete analytical model for the rotational and translational diffusion of molecules with a six-fold point symmetry on a hexagonal lattice is presented. It can be applied, in particular, to the diffusion of benzene molecules adsorbed flat on the basal plane of graphite in the case of incoherent scattering. Under the weak hindered approximation, the classical mechanics framework and making use of the van Hove formalism of correlation functions, the intermediate scattering function and its Fourier transform, the scattering law, are both obtained. They can be expressed as sums of exponential decays or Lorentzian functions, respectively, containing the contribution of each of the dynamical processes taking place. In the case of benzene lying flat on the substrate we expect translational diffusion, continuous rotations of isolated molecules and hindered rotations of molecules within clusters. Each particular diffusive mechanism can be recognized owing to its particular signature in the dependence of the quasi-elastic broadening on the momentum transfer.

  11. Evaluation of the Effectiveness of Surfactants and Denaturants to Elute and Denature Adsorbed Protein on Different Surface Chemistries.

    PubMed

    Thyparambil, Aby A; Wei, Yang; Latour, Robert A

    2015-11-01

    The elution and/or denaturation of proteins from material surfaces by chemical excipients such as surfactants and denaturants is important for numerous applications including medical implant reprocessing, bioanalyses, and biodefense. The objective of this study was to develop and apply methods to quantitatively assess how surface chemistry and adsorption conditions influence the effectiveness of three commonly used surfactants (sodium dodecyl sulfate, n-octyl-β-d-glucoside, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) and two denaturants (guanidium hydrochloride and urea) to elute protein (hen egg white lysozyme and bovine pancreatic ribonuclease A) from three different surface chemistries (silica glass, poly(methyl methacrylate), and high-density polyethylene). The structure and bioactivity of residual protein on the surface following elution were characterized using circular dichroism spectropolarimetry and enzyme assays to assess the extent of protein denaturation. Our results indicate that the denaturants were generally more effective than the surfactants in removing the adsorbed proteins from each type of surface. Also, the denaturing capacity of these excipients on the residual proteins on the surfaces was distinctly different from their influence on the proteins in solution and was unique for each of the adsorption conditions. Taken altogether, these results reveal that the effectiveness of surfactants and denaturants to elute and denature adsorbed protein is significantly influenced by surface chemistry and the conditions from which the protein was adsorbed. These results provide a basis for the selection, design, and further development of chemical agents for protein elution and surface decontamination.

  12. Evaluation of the Effectiveness of Surfactants and Denaturants to Elute and Denature Adsorbed Protein on Different Surface Chemistries.

    PubMed

    Thyparambil, Aby A; Wei, Yang; Latour, Robert A

    2015-11-01

    The elution and/or denaturation of proteins from material surfaces by chemical excipients such as surfactants and denaturants is important for numerous applications including medical implant reprocessing, bioanalyses, and biodefense. The objective of this study was to develop and apply methods to quantitatively assess how surface chemistry and adsorption conditions influence the effectiveness of three commonly used surfactants (sodium dodecyl sulfate, n-octyl-β-d-glucoside, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) and two denaturants (guanidium hydrochloride and urea) to elute protein (hen egg white lysozyme and bovine pancreatic ribonuclease A) from three different surface chemistries (silica glass, poly(methyl methacrylate), and high-density polyethylene). The structure and bioactivity of residual protein on the surface following elution were characterized using circular dichroism spectropolarimetry and enzyme assays to assess the extent of protein denaturation. Our results indicate that the denaturants were generally more effective than the surfactants in removing the adsorbed proteins from each type of surface. Also, the denaturing capacity of these excipients on the residual proteins on the surfaces was distinctly different from their influence on the proteins in solution and was unique for each of the adsorption conditions. Taken altogether, these results reveal that the effectiveness of surfactants and denaturants to elute and denature adsorbed protein is significantly influenced by surface chemistry and the conditions from which the protein was adsorbed. These results provide a basis for the selection, design, and further development of chemical agents for protein elution and surface decontamination. PMID:26449787

  13. Adsorption of peptides and small proteins with control access polymer permeation to affinity binding sites. Part I: Polymer permeation-immobilized metal ion affinity chromatography separation adsorbents with polyethylene glycol and immobilized metal ions.

    PubMed

    González-Ortega, Omar; Porath, Jerker; Guzmán, Roberto

    2012-03-01

    Despite the many efforts to develop efficient protein purification techniques, the isolation of peptides and small proteins on a larger than analytical scale remains a significant challenge. Recovery of small biomolecules from diluted complex biological mixtures, such as human serum, employing porous adsorbents is a difficult task mainly due to the presence of concentrated large biomolecules that can add undesired effects in the system such as blocking of adsorbent pores, impairing diffusion of small molecules, or competition for adsorption sites. Adsorption and size exclusion chromatography (AdSEC) controlled access media, using polyethylene glycol (PEG) as a semi-permeable barrier on a polysaccharide matrix, have been developed and explored in this work to overcome such effects and to preferentially adsorb small molecules while rejecting large ones. In the first part of this work, adsorption studies were performed with small peptides and proteins from synthetic mixtures using controlled access polymer permeation adsorption (CAPPA) media created by effectively grafting PEG on an immobilized metal affinity chromatography (IMAC) agarose resin, where chelating agents and immobilized metal ions were used as the primary affinity binding sites. Synthetic mixtures consisted of bovine serum albumin (BSA) with small proteins, peptides, amino acids (such as histidine or Val⁴-Angiotensin III), and small molecules-spiked human serum. The synthesized hybrid adsorbent consisted of agarose beads modified with iminodiacetic (IDA) groups, loaded with immobilized Cu(II) ions, and PEG. These CAPPA media with grafted PEG on the interior and exterior surfaces of the agarose matrix were effective in rejecting high molecular weight proteins. Different PEG grafting densities and PEG of different molecular weight were tested to determine their effect in rejecting and controlling adsorbent permeation properties. Low grafting density of high molecular weight PEG was found to be as

  14. Surface passivation for single-molecule protein studies.

    PubMed

    Chandradoss, Stanley D; Haagsma, Anna C; Lee, Young Kwang; Hwang, Jae-Ho; Nam, Jwa-Min; Joo, Chirlmin

    2014-04-24

    Single-molecule fluorescence spectroscopy has proven to be instrumental in understanding a wide range of biological phenomena at the nanoscale. Important examples of what this technique can yield to biological sciences are the mechanistic insights on protein-protein and protein-nucleic acid interactions. When interactions of proteins are probed at the single-molecule level, the proteins or their substrates are often immobilized on a glass surface, which allows for a long-term observation. This immobilization scheme may introduce unwanted surface artifacts. Therefore, it is essential to passivate the glass surface to make it inert. Surface coating using polyethylene glycol (PEG) stands out for its high performance in preventing proteins from non-specifically interacting with a glass surface. However, the polymer coating procedure is difficult, due to the complication arising from a series of surface treatments and the stringent requirement that a surface needs to be free of any fluorescent molecules at the end of the procedure. Here, we provide a robust protocol with step-by-step instructions. It covers surface cleaning including piranha etching, surface functionalization with amine groups, and finally PEG coating. To obtain a high density of a PEG layer, we introduce a new strategy of treating the surface with PEG molecules over two rounds, which remarkably improves the quality of passivation. We provide representative results as well as practical advice for each critical step so that anyone can achieve the high quality surface passivation.

  15. Single-molecule imaging studies of protein dynamics

    NASA Astrophysics Data System (ADS)

    Zareh, Shannon Kian G.

    2011-12-01

    Single-molecule fluorescence imaging is a powerful method for studying biological events. The work of this thesis primarily focuses on single molecule studies of the dynamics of Green Fluorescent Protein (GFP) and other fluorescent-labeled proteins by utilizing Total Internal Reflection Fluorescence (TIRF) microscopy and imaging. The single molecule experiments of this thesis covered three broad topics. First, the adsorption mechanisms of proteins onto hydrophobic and hydrophilic fused silica surfaces were imaged and reversible and irreversible adsorption mechanisms were observed. The second topic covered a new technique for measuring the diffusion coefficient of Brownian diffusing proteins, in particular GFP, in solution via a single image. The corresponding experiments showed a relationship between the intensity profile width and the diffusion coefficient of the diffusing molecules. The third topic covered an in vivo experiment involving imaging and quantifying prokaryotic cell metabolism protein dynamics inside the Bacillus subtilis bacteria, in which a helical diffusion pattern for the protein was observed. These topics are presented in the chronological order of the experiments conducted.

  16. Microsecond protein dynamics observed at the single-molecule level

    PubMed Central

    Otosu, Takuhiro; Ishii, Kunihiko; Tahara, Tahei

    2015-01-01

    How polypeptide chains acquire specific conformations to realize unique biological functions is a central problem of protein science. Single-molecule spectroscopy, combined with fluorescence resonance energy transfer, is utilized to study the conformational heterogeneity and the state-to-state transition dynamics of proteins on the submillisecond to second timescales. However, observation of the dynamics on the microsecond timescale is still very challenging. This timescale is important because the elementary processes of protein dynamics take place and direct comparison between experiment and simulation is possible. Here we report a new single-molecule technique to reveal the microsecond structural dynamics of proteins through correlation of the fluorescence lifetime. This method, two-dimensional fluorescence lifetime correlation spectroscopy, is applied to clarify the conformational dynamics of cytochrome c. Three conformational ensembles and the microsecond transitions in each ensemble are indicated from the correlation signal, demonstrating the importance of quantifying microsecond dynamics of proteins on the folding free energy landscape. PMID:26151767

  17. Microsecond protein dynamics observed at the single-molecule level

    NASA Astrophysics Data System (ADS)

    Otosu, Takuhiro; Ishii, Kunihiko; Tahara, Tahei

    2015-07-01

    How polypeptide chains acquire specific conformations to realize unique biological functions is a central problem of protein science. Single-molecule spectroscopy, combined with fluorescence resonance energy transfer, is utilized to study the conformational heterogeneity and the state-to-state transition dynamics of proteins on the submillisecond to second timescales. However, observation of the dynamics on the microsecond timescale is still very challenging. This timescale is important because the elementary processes of protein dynamics take place and direct comparison between experiment and simulation is possible. Here we report a new single-molecule technique to reveal the microsecond structural dynamics of proteins through correlation of the fluorescence lifetime. This method, two-dimensional fluorescence lifetime correlation spectroscopy, is applied to clarify the conformational dynamics of cytochrome c. Three conformational ensembles and the microsecond transitions in each ensemble are indicated from the correlation signal, demonstrating the importance of quantifying microsecond dynamics of proteins on the folding free energy landscape.

  18. Analysis of single nucleic acid molecules with protein nanopores

    PubMed Central

    Maglia, Giovanni; Heron, Andrew J.; Stoddart, David; Japrung, Deanpen; Bayley, Hagan

    2011-01-01

    We describe the methods used in our laboratory for the analysis of single nucleic acid molecules with protein nanopores. The technical section is preceded by a review of the variety of experiments that can be done with protein nanopores. The end goal of much of this work is single-molecule DNA sequencing, although sequencing is not discussed explicitly here. The technical section covers the equipment required for nucleic acid analysis, the preparation and storage of the necessary materials, and aspects of signal processing and data analysis. PMID:20627172

  19. FTMAP: extended protein mapping with user-selected probe molecules

    PubMed Central

    Ngan, Chi Ho; Bohnuud, Tanggis; Mottarella, Scott E.; Beglov, Dmitri; Villar, Elizabeth A.; Hall, David R.; Kozakov, Dima; Vajda, Sandor

    2012-01-01

    Binding hot spots, protein sites with high-binding affinity, can be identified using X-ray crystallography or NMR by screening libraries of small organic molecules that tend to cluster at such regions. FTMAP, a direct computational analog of the experimental screening approaches, globally samples the surface of a target protein using small organic molecules as probes, finds favorable positions, clusters the conformations and ranks the clusters on the basis of the average energy. The regions that bind several probe clusters predict the binding hot spots, in good agreement with experimental results. Small molecules discovered by fragment-based approaches to drug design also bind at the hot spot regions. To identify such molecules and their most likely bound positions, we extend the functionality of FTMAP (http://ftmap.bu.edu/param) to accept any small molecule as an additional probe. In its updated form, FTMAP identifies the hot spots based on a standard set of probes, and for each additional probe shows representative structures of nearby low energy clusters. This approach helps to predict bound poses of the user-selected molecules, detects if a compound is not likely to bind in the hot spot region, and provides input for the design of larger ligands. PMID:22589414

  20. Common fluorescent proteins for single-molecule localization microscopy

    NASA Astrophysics Data System (ADS)

    Klementieva, Natalia V.; Bozhanova, Nina G.; Mishina, Natalie M.; Zagaynova, Elena V.; Lukyanov, Konstantin A.; Mishin, Alexander S.

    2015-07-01

    Super-resolution techniques for breaking the diffraction barrier are spread out over multiple studies nowadays. Single-molecule localization microscopy such as PALM, STORM, GSDIM, etc allow to get super-resolved images of cell ultrastructure by precise localization of individual fluorescent molecules via their temporal isolation. However, these methods are supposed the use of fluorescent dyes and proteins with special characteristics (photoactivation/photoconversion). At the same time, there is a need for retaining high photostability of fluorophores during long-term acquisition. Here, we first showed the potential of common red fluorescent protein for single-molecule localization microscopy based on spontaneous intrinsic blinking. Also, we assessed the effect of different imaging media on photobleaching of these fluorescent proteins. Monomeric orange and red fluorescent proteins were examined for stochastic switching from a dark state to a bright fluorescent state. We studied fusions with cytoskeletal proteins in NIH/3T3 and HeLa cells. Imaging was performed on the Nikon N-STORM system equipped with EMCCD camera. To define the optimal imaging conditions we tested several types of cell culture media and buffers. As a result, high-resolution images of cytoskeleton structure were obtained. Essentially, low-intensity light was sufficient to initiate the switching of tested red fluorescent protein reducing phototoxicity and provide long-term live-cell imaging.

  1. Engineered kinesin motor proteins amenable to small-molecule inhibition.

    PubMed

    Engelke, Martin F; Winding, Michael; Yue, Yang; Shastry, Shankar; Teloni, Federico; Reddy, Sanjay; Blasius, T Lynne; Soppina, Pushpanjali; Hancock, William O; Gelfand, Vladimir I; Verhey, Kristen J

    2016-01-01

    The human genome encodes 45 kinesin motor proteins that drive cell division, cell motility, intracellular trafficking and ciliary function. Determining the cellular function of each kinesin would benefit from specific small-molecule inhibitors. However, screens have yielded only a few specific inhibitors. Here we present a novel chemical-genetic approach to engineer kinesin motors that can carry out the function of the wild-type motor yet can also be efficiently inhibited by small, cell-permeable molecules. Using kinesin-1 as a prototype, we develop two independent strategies to generate inhibitable motors, and characterize the resulting inhibition in single-molecule assays and in cells. We further apply these two strategies to create analogously inhibitable kinesin-3 motors. These inhibitable motors will be of great utility to study the functions of specific kinesins in a dynamic manner in cells and animals. Furthermore, these strategies can be used to generate inhibitable versions of any motor protein of interest. PMID:27045608

  2. Engineered kinesin motor proteins amenable to small-molecule inhibition

    PubMed Central

    Engelke, Martin F.; Winding, Michael; Yue, Yang; Shastry, Shankar; Teloni, Federico; Reddy, Sanjay; Blasius, T. Lynne; Soppina, Pushpanjali; Hancock, William O.; Gelfand, Vladimir I.; Verhey, Kristen J.

    2016-01-01

    The human genome encodes 45 kinesin motor proteins that drive cell division, cell motility, intracellular trafficking and ciliary function. Determining the cellular function of each kinesin would benefit from specific small-molecule inhibitors. However, screens have yielded only a few specific inhibitors. Here we present a novel chemical-genetic approach to engineer kinesin motors that can carry out the function of the wild-type motor yet can also be efficiently inhibited by small, cell-permeable molecules. Using kinesin-1 as a prototype, we develop two independent strategies to generate inhibitable motors, and characterize the resulting inhibition in single-molecule assays and in cells. We further apply these two strategies to create analogously inhibitable kinesin-3 motors. These inhibitable motors will be of great utility to study the functions of specific kinesins in a dynamic manner in cells and animals. Furthermore, these strategies can be used to generate inhibitable versions of any motor protein of interest. PMID:27045608

  3. Exploring one-state downhill protein folding in single molecules

    PubMed Central

    Liu, Jianwei; Campos, Luis A.; Cerminara, Michele; Wang, Xiang; Ramanathan, Ravishankar; English, Douglas S.; Muñoz, Victor

    2012-01-01

    A one-state downhill protein folding process is barrierless at all conditions, resulting in gradual melting of native structure that permits resolving folding mechanisms step-by-step at atomic resolution. Experimental studies of one-state downhill folding have typically focused on the thermal denaturation of proteins that fold near the speed limit (ca. 106 s-1) at their unfolding temperature, thus being several orders of magnitude too fast for current single-molecule methods, such as single-molecule FRET. An important open question is whether one-state downhill folding kinetics can be slowed down to make them accessible to single-molecule approaches without turning the protein into a conventional activated folder. Here we address this question on the small helical protein BBL, a paradigm of one-state downhill thermal (un)folding. We decreased 200-fold the BBL folding-unfolding rate by combining chemical denaturation and low temperature, and carried out free-diffusion single-molecule FRET experiments with 50-μs resolution and maximal photoprotection using a recently developed Trolox-cysteamine cocktail. These experiments revealed a single conformational ensemble at all denaturing conditions. The chemical unfolding of BBL was then manifested by the gradual change of this unique ensemble, which shifts from high to low FRET efficiency and becomes broader at increasing denaturant. Furthermore, using detailed quantitative analysis, we could rule out the possibility that the BBL single-molecule data are produced by partly overlapping folded and unfolded peaks. Thus, our results demonstrate the one-state downhill folding regime at the single-molecule level and highlight that this folding scenario is not necessarily associated with ultrafast kinetics. PMID:22184219

  4. Changes in solvent exposure reveal the kinetics and equilibria of adsorbed protein unfolding in hydrophobic interaction chromatography.

    PubMed

    Deitcher, R W; O'Connell, J P; Fernandez, E J

    2010-08-27

    Hydrogen exchange has been a useful technique for studying the conformational state of proteins, both in bulk solution and at interfaces, for several decades. Here, we propose a physically based model of simultaneous protein adsorption, unfolding and hydrogen exchange in HIC. An accompanying experimental protocol, utilizing mass spectrometry to quantify deuterium labeling, enables the determination of both the equilibrium partitioning between conformational states and pseudo-first order rate constants for folding and unfolding of adsorbed protein. Unlike chromatographic techniques, which rely on the interpretation of bulk phase behavior, this methodology utilizes the measurement of a molecular property (solvent exposure) and provides insight into the nature of the unfolded conformation in the adsorbed phase. Three model proteins of varying conformational stability, alpha-chymotrypsinogen A, beta-lactoglobulin B, and holo alpha-lactalbumin, are studied on Sepharose HIC resins possessing assorted ligand chemistries and densities. alpha-Chymotrypsinogen, conformationally the most stable protein in the set, exhibits no change in solvent exposure at all the conditions studied, even when isocratic pulse-response chromatography suggests nearly irreversible adsorption. Apparent unfolding energies of adsorbed beta-lactoglobulin B and holo alpha-lactalbumin range from -4 to 3 kJ/mol and are dependent on resin properties and salt concentration. Characteristic pseudo-first order rate constants for surface-induced unfolding are 0.2-0.9 min(-1). While poor protein recovery in HIC is often associated with irreversible unfolding, this study documents that non-eluting behavior can occur when surface unfolding is reversible or does not occur at all. Further, this hydrogen exchange technique can be used to assess the conformation of adsorbed protein under conditions where the protein is non-eluting and chromatographic methods are not applicable.

  5. Viscoelastic properties of adsorbed and cross-linked polypeptide and protein layers at a solid-liquid interface.

    PubMed

    Dutta, Amit K; Nayak, Arpan; Belfort, Georges

    2008-08-01

    The real-time changes in viscoelasticity of adsorbed poly(L-lysine) (PLL) and adsorbed histone (lysine rich fraction) due to cross-linking by glutaraldehyde and corresponding release of associated water were investigated using a quartz crystal microbalance with dissipation monitoring (QCM-D) and attenuated total reflection Fourier transform infrared spectroscopy (ATR/FTIR). The kinetics of PLL and histone adsorption were measured through changes in mass adsorbed onto a gold-coated quartz surface from changes in frequency and dissipation and using the Voigt viscoelastic model. Prior to cross-linking, the shear viscosity and shear modulus of the adsorbed PLL layer were approximately 3.0 x 10(-3) Pas and approximately 2.5 x 10(5) Pa, respectively, while after cross-linking, they increased to approximately 17.5 x 10(-3) Pas and approximately 2.5 x 10(6) Pa, respectively. For the adsorbed histone layer, shear viscosity and shear modulus increased modestly from approximately 1.3 x 10(-3) to approximately 2.0 x 10(-3) Pas and from approximately 1.2 x 10(4) to approximately 1.6 x 10(4) Pa, respectively. The adsorbed mass estimated from the Sauerbrey equation (perfectly elastic) and the Voigt viscoelastic model differ appreciably prior to cross-linking whereas after cross-linking they converged. This is because trapped water molecules were released during cross-linking. This was confirmed experimentally via ATR/FTIR measurements. The variation in viscoelastic properties increased substantially after cross-linking presumably due to fluctuation of the randomly cross-linked network structure. An increase in fluctuation of the viscoelastic properties and the loss of imbibed water could be used as a signature of the formation of a cross-linked network and the amount of cross-linking, respectively. PMID:18508070

  6. Zeta potential, contact angles, and AFM imaging of protein conformation adsorbed on hybrid nanocomposite surfaces.

    PubMed

    Pinho, Ana C; Piedade, Ana P

    2013-08-28

    The sputtering deposition of gold (Au) and poly(tetrafluoroethylene) (PTFE) was used to prepare a nanocomposite hybrid thin film suitable for protein adsorption while maintaining the native conformation of the biological material. The monolithic PTFE and the nanocomposite PTFE/Au thin films, with Au content up to 1 at %, were co-deposited by r.f. magnetron sputtering using argon as a discharge gas and deposited onto 316L stainless steel substrates, the most commonly used steel in biomaterials. The deposited thin films, before and after bovine serum albumin (BSA) adsorption, were thoroughly characterized with special emphasis on the surface properties/characteristics by atomic force microscopy (AFM), zeta potential, and static and dynamic contact angle measurements, in order to assess the relationship between structure and conformational changes. The influence of a pre-adsorbed peptide (RGD) was also evaluated. The nanotopographic and chemical changes induced by the presence of gold in the nanocomposite thin films enable RGD bonding, which is critical for the maintenance of the BSA native conformation after adsorption.

  7. Single-molecule protein sequencing through fingerprinting: computational assessment

    NASA Astrophysics Data System (ADS)

    Yao, Yao; Docter, Margreet; van Ginkel, Jetty; de Ridder, Dick; Joo, Chirlmin

    2015-10-01

    Proteins are vital in all biological systems as they constitute the main structural and functional components of cells. Recent advances in mass spectrometry have brought the promise of complete proteomics by helping draft the human proteome. Yet, this commonly used protein sequencing technique has fundamental limitations in sensitivity. Here we propose a method for single-molecule (SM) protein sequencing. A major challenge lies in the fact that proteins are composed of 20 different amino acids, which demands 20 molecular reporters. We computationally demonstrate that it suffices to measure only two types of amino acids to identify proteins and suggest an experimental scheme using SM fluorescence. When achieved, this highly sensitive approach will result in a paradigm shift in proteomics, with major impact in the biological and medical sciences.

  8. Single-molecule mechanics of protein-labelled DNA handles.

    PubMed

    Jadhav, Vivek S; Brüggemann, Dorothea; Wruck, Florian; Hegner, Martin

    2016-01-01

    DNA handles are often used as spacers and linkers in single-molecule experiments to isolate and tether RNAs, proteins, enzymes and ribozymes, amongst other biomolecules, between surface-modified beads for nanomechanical investigations. Custom DNA handles with varying lengths and chemical end-modifications are readily and reliably synthesized en masse, enabling force spectroscopic measurements with well-defined and long-lasting mechanical characteristics under physiological conditions over a large range of applied forces. Although these chemically tagged DNA handles are widely used, their further individual modification with protein receptors is less common and would allow for additional flexibility in grabbing biomolecules for mechanical measurements. In-depth information on reliable protocols for the synthesis of these DNA-protein hybrids and on their mechanical characteristics under varying physiological conditions are lacking in literature. Here, optical tweezers are used to investigate different protein-labelled DNA handles in a microfluidic environment under different physiological conditions. Digoxigenin (DIG)-dsDNA-biotin handles of varying sizes (1000, 3034 and 4056 bp) were conjugated with streptavidin or neutravidin proteins. The DIG-modified ends of these hybrids were bound to surface-modified polystyrene (anti-DIG) beads. Using different physiological buffers, optical force measurements showed consistent mechanical characteristics with long dissociation times. These protein-modified DNA hybrids were also interconnected in situ with other tethered biotinylated DNA molecules. Electron-multiplying CCD (EMCCD) imaging control experiments revealed that quantum dot-streptavidin conjugates at the end of DNA handles remain freely accessible. The experiments presented here demonstrate that handles produced with our protein-DNA labelling procedure are excellent candidates for grasping single molecules exposing tags suitable for molecular recognition in time

  9. Catalytic in vivo protein knockdown by small-molecule PROTACs

    PubMed Central

    Bondeson, Daniel P; Mares, Alina; Smith, Ian E D; Ko, Eunhwa; Campos, Sebastien; Miah, Afjal H; Mulholland, Katie E; Routly, Natasha; Buckley, Dennis L; Gustafson, Jeffrey L; Zinn, Nico; Grandi, Paola; Shimamura, Satoko; Bergamini, Giovanna; Faelth-Savitski, Maria; Bantscheff, Marcus; Cox, Carly; Gordon, Deborah A; Willard, Ryan R; Flanagan, John J; Casillas, Linda N; Votta, Bartholomew J; den Besten, Willem; Famm, Kristoffer; Kruidenier, Laurens; Carter, Paul S; Harling, John D; Churcher, Ian; Crews, Craig M

    2015-01-01

    The current predominant theapeutic paradigm is based on maximizing drug-receptor occupancy to achieve clinical benefit. This strategy, however, generally requires excessive drug concentrations to ensure sufficient occupancy, often leading to adverse side effects. Here, we describe major improvements to the proteolysis targeting chimeras (PROTACs) method, a chemical knockdown strategy in which a heterobifunctional molecule recruits a specific protein target to an E3 ubiquitin ligase, resulting in the target’s ubiquitination and degradation. These compounds behave catalytically in their ability to induce the ubiquitination of super-stoichiometric quantities of proteins, providing efficacy that is not limited by equilibrium occupancy. We present two PROTACs that are capable of specifically reducing protein levels by >90% at nanomolar concentrations. In addition, mouse studies indicate that they provide broad tissue distribution and knockdown of the targeted protein in tumor xenografts. Together, these data demonstrate a protein knockdown system combining many of the favorable properties of small-molecule agents with the potent protein knockdown of RNAi and CRISPR. PMID:26075522

  10. Autophagy proteins in antigen processing for presentation on MHC molecules.

    PubMed

    Münz, Christian

    2016-07-01

    Autophagy describes catabolic pathways that deliver cytoplasmic constituents for lysosomal degradation. Since major histocompatibility complex (MHC) molecules sample protein degradation products and present them to T cells for adaptive immunity, it is maybe not too surprising that autophagy contributes to this protein antigen processing for MHC presentation. However, the recently recognized breath of pathways, by which autophagy contributes to MHC antigen processing, is exciting. Macroautophagy does not only seem to deliver intracellular but facilitates also extracellular antigen processing by lysosomal hydrolysis for MHC class II presentation. Moreover, even MHC class I molecules that usually display proteasomal products are regulated by macroautophagy, probably using a pool of these molecules outside the endoplasmic reticulum, where MHC class I molecules are loaded with peptide during canonical MHC class I antigen processing. This review aims to summarize these recent developments and point out gaps of knowledge, which should be filled by further investigation, in order to harness the different antigen-processing pathways via autophagy for vaccine improvement. PMID:27319339

  11. Single-molecule mechanics of protein-labelled DNA handles

    PubMed Central

    Wruck, Florian

    2016-01-01

    Summary DNA handles are often used as spacers and linkers in single-molecule experiments to isolate and tether RNAs, proteins, enzymes and ribozymes, amongst other biomolecules, between surface-modified beads for nanomechanical investigations. Custom DNA handles with varying lengths and chemical end-modifications are readily and reliably synthesized en masse, enabling force spectroscopic measurements with well-defined and long-lasting mechanical characteristics under physiological conditions over a large range of applied forces. Although these chemically tagged DNA handles are widely used, their further individual modification with protein receptors is less common and would allow for additional flexibility in grabbing biomolecules for mechanical measurements. In-depth information on reliable protocols for the synthesis of these DNA–protein hybrids and on their mechanical characteristics under varying physiological conditions are lacking in literature. Here, optical tweezers are used to investigate different protein-labelled DNA handles in a microfluidic environment under different physiological conditions. Digoxigenin (DIG)-dsDNA-biotin handles of varying sizes (1000, 3034 and 4056 bp) were conjugated with streptavidin or neutravidin proteins. The DIG-modified ends of these hybrids were bound to surface-modified polystyrene (anti-DIG) beads. Using different physiological buffers, optical force measurements showed consistent mechanical characteristics with long dissociation times. These protein-modified DNA hybrids were also interconnected in situ with other tethered biotinylated DNA molecules. Electron-multiplying CCD (EMCCD) imaging control experiments revealed that quantum dot–streptavidin conjugates at the end of DNA handles remain freely accessible. The experiments presented here demonstrate that handles produced with our protein–DNA labelling procedure are excellent candidates for grasping single molecules exposing tags suitable for molecular recognition

  12. Real-time single-molecule coimmunoprecipitation of weak protein-protein interactions.

    PubMed

    Lee, Hong-Won; Ryu, Ji Young; Yoo, Janghyun; Choi, Byungsan; Kim, Kipom; Yoon, Tae-Young

    2013-10-01

    Coimmunoprecipitation (co-IP) analysis is a useful method for studying protein-protein interactions. It currently involves electrophoresis and western blotting, which are not optimized for detecting weak and transient interactions. In this protocol we describe an advanced version of co-IP analysis that uses real-time, single-molecule fluorescence imaging as its detection scheme. Bait proteins are pulled down onto the imaging plane of a total internal reflection (TIR) microscope. With unpurified cells or tissue extracts kept in reaction chambers, we observe single protein-protein interactions between the surface-immobilized bait and the fluorescent protein-labeled prey proteins in real time. Such direct recording provides an improvement of five orders of magnitude in the time resolution of co-IP analysis. With the single-molecule sensitivity and millisecond time resolution, which distinguish our method from other methods for measuring weak protein-protein interactions, it is possible to quantify the interaction kinetics and active fraction of native, unlabeled bait proteins. Real-time single-molecule co-IP analysis, which takes ∼4 h to complete from lysate preparation to kinetic analysis, provides a general avenue for revealing the rich kinetic picture of target protein-protein interactions, and it can be used, for example, to investigate the molecular lesions that drive individual cancers at the level of protein-protein interactions.

  13. Light-assisted small molecule screening against protein kinases

    PubMed Central

    Inglés-Prieto, Álvaro; Reichhart, Eva; Muellner, Markus K.; Nowak, Matthias; Nijman, Sebastian M.; Grusch, Michael; Janovjak, Harald

    2015-01-01

    High-throughput live-cell screens are intricate elements of systems biology studies and drug discovery pipelines. Here, we demonstrate an optogenetics-assisted method that obviates the addition of chemical activators and reporters, reduces the number of operational steps and increases information content in a cell-based small molecule screen against human protein kinases including an orphan receptor tyrosine kinase. This blueprint for all-optical screening can be adapted to many drug targets and cellular processes. PMID:26457372

  14. Can the state of platinum species be unambiguously determined by the stretching frequency of an adsorbed CO probe molecule?

    PubMed

    Aleksandrov, Hristiyan A; Neyman, Konstantin M; Hadjiivanov, Konstantin I; Vayssilov, Georgi N

    2016-08-10

    The paper addresses possible ambiguities in the determination of the state of platinum species by the stretching frequency of a CO probe, which is a common technique for characterization of platinum-containing catalytic systems. We present a comprehensive comparison of the available experimental data with our theoretical modeling (density functional) results of pertinent systems - platinum surfaces, nanoparticles and clusters as well as reduced or oxidized platinum moieties on a ceria support. Our results for CO adsorbed on-top on metallic Pt(0), with C-O vibrational frequencies in the region 2018-2077 cm(-1), suggest that a decrease of the coordination number of the platinum atom, to which CO is bound, by one lowers the CO frequency by about 7 cm(-1). This trend corroborates the Kappers-van der Maas correlation derived from the analysis of the experimental stretching frequency of CO adsorbed on platinum-containing samples on different supports. We also analyzed the effect of the charge of platinum species on the CO frequency. Based on the calculated vibrational frequencies of CO in various model systems, we concluded that the actual state of the platinum species may be mistaken based only on the measured value of the C-O vibrational frequency due to overlapping regions of frequencies corresponding to different types of species. In order to identify the actual state of platinum species one has to combine this powerful technique with other approaches. PMID:27444400

  15. Altering adsorbed proteins or cellular gene expression in bone-metastatic cancer cells affects PTHrP and Gli2 without altering cell growth.

    PubMed

    Page, Jonathan M; Merkel, Alyssa R; Ruppender, Nazanin S; Guo, Ruijing; Dadwal, Ushashi C; Cannonier, Shellese; Basu, Sandip; Guelcher, Scott A; Sterling, Julie A

    2015-09-01

    The contents of this data in brief are related to the article titled "Matrix Rigidity Regulates the Transition of Tumor Cells to a Bone-Destructive Phenotype through Integrin β3 and TGF-β Receptor Type II". In this DIB we will present our supplemental data investigating Integrin expression, attachment of cells to various adhesion molecules, and changes in gene expression in multiple cancer cell lines. Since the interactions of Integrins with adsorbed matrix proteins are thought to affect the ability of cancer cells to interact with their underlying substrates, we examined the expression of Integrin β1, β3, and β5 in response to matrix rigidity. We found that only Iβ3 increased with increasing substrate modulus. While it was shown that fibronectin greatly affects the expression of tumor-produced factors associated with bone destruction (parathyroid hormone-related protein, PTHrP, and Gli2), poly-l-lysine, vitronectin and type I collagen were also analyzed as potential matrix proteins. Each of the proteins was independently adsorbed on both rigid and compliant polyurethane films which were subsequently used to culture cancer cells. Poly-l-lysine, vitronectin and type I collagen all had negligible effects on PTHrP or Gli2 expression, but fibronectin was shown to have a dose dependent effect. Finally, altering the expression of Iβ3 demonstrated that it is required for tumor cells to respond to the rigidity of the matrix, but does not affect other cell growth or viability. Together these data support the data presented in our manuscript to show that the rigidity of bone drives Integrinβ3/TGF-β crosstalk, leading to increased expression of Gli2 and PTHrP.

  16. Urea transporter proteins as targets for small-molecule diuretics

    PubMed Central

    Esteva-Font, Cristina; Anderson, Marc O.; Verkman, Alan S.

    2016-01-01

    Conventional diuretics such as furosemide and thiazides target salt transporters in kidney tubules, but urea transporters (UTs) have emerged as alternative targets. UTs are a family of transmembrane channels expressed in a variety of mammalian tissues, in particular the kidney. UT knockout mice and humans with UT mutations exhibit reduced maximal urinary osmolality, demonstrating that UTs are necessary for the concentration of urine. Small-molecule screening has identified potent and selective inhibitors of UT-A, the UT protein expressed in renal tubule epithelial cells, and UT-B, the UT protein expressed in vasa recta endothelial cells. Data from UT knockout mice and from rodents administered UT inhibitors support the diuretic action of UT inhibition. The kidney-specific expression of UT-A1, together with high selectivity of the small-molecule inhibitors, means that off-target effects of such small-molecule drugs should be minimal. This Review summarizes the structure, expression and function of UTs, and looks at the evidence supporting the validity of UTs as targets for the development of salt-sparing diuretics with a unique mechanism of action. UT-targeted inhibitors may be useful alone or in combination with conventional diuretics for therapy of various oedemas and hyponatraemias, potentially including those refractory to treatment with current diuretics. PMID:25488859

  17. Inhibition of Protein-Protein Interactions and Signaling by Small Molecules

    NASA Astrophysics Data System (ADS)

    Freire, Ernesto

    2010-03-01

    Protein-protein interactions are at the core of cell signaling pathways as well as many bacterial and viral infection processes. As such, they define critical targets for drug development against diseases such as cancer, arthritis, obesity, AIDS and many others. Until now, the clinical inhibition of protein-protein interactions and signaling has been accomplished with the use of antibodies or soluble versions of receptor molecules. Small molecule replacements of these therapeutic agents have been extremely difficult to develop; either the necessary potency has been hard to achieve or the expected biological effect has not been obtained. In this presentation, we show that a rigorous thermodynamic approach that combines differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC) provides a unique platform for the identification and optimization of small molecular weight inhibitors of protein-protein interactions. Recent advances in the development of cell entry inhibitors of HIV-1 using this approach will be discussed.

  18. Formation of metal nanoclusters on specific surface sites of protein molecules.

    PubMed

    Braun, Nathalie; Meining, Winfried; Hars, Ulrike; Fischer, Markus; Ladenstein, Rudolf; Huber, Robert; Bacher, Adelbert; Weinkauf, Sevil; Bachmann, Luis

    2002-08-01

    During vacuum condensation of metals on frozen proteins, nanoclusters are preferentially formed at specific surface sites (decoration). Understanding the nature of metal/protein interaction is of interest for structure analysis and is also important in the fields of biocompatibility and sensor development. Studies on the interaction between metal and distinct areas on the protein which enhance or impede the probability for cluster formation require information on the structural details of the protein's surface underlying the metal clusters. On three enzyme complexes, lumazine synthase from Bacillus subtilis, proteasome from Thermoplasma acidophilum and GTP cyclohydrolase I from Escherichia coli, the decoration sites as determined by electron microscopy (EM) were correlated with their atomic surface structures as obtained by X-ray crystallography. In all three cases, decoration of the same protein results in different cluster distributions for gold and silver. Gold decorates surface areas consisting of polar but uncharged residues and with rough relief whereas silver clusters are preferentially formed on top of protein pores outlined by charged and hydrophilic residues and filled with frozen buffer under the experimental conditions. A common quality of both metals is that they strictly avoid condensation on hydrophobic sites lacking polar and charged residues. The results open ways to analyse the binding mechanism of nanoclusters to small specific sites on the surface of hydrated biomacromolecules by non-microscopic, physical-chemical methods. Understanding the mechanism may lead to advanced decoration techniques resulting in fewer background clusters. This would improve the analysis of single molecules with regard to their symmetries and their orientation in the adsorbed state and in precrystalline assemblies as well as facilitate the detection of point defects in crystals caused by misorientation or by impurities. PMID:12144790

  19. The role of Cu atoms on silver electrodes in surface enhanced Raman scattering from pyridine: Giant enhancement by a minority of adsorbed molecules

    NASA Astrophysics Data System (ADS)

    Moerl, Ludwig; Pettinger, Bruno

    1982-08-01

    Surface enhanced Raman scattering (SERS) has been studied for pyridine molecules adsorbed at Ag electrodes covered with submonolayers of Cu ( θ = 0.003 - 0.1). Depending on the amount of Cu coverage the frequencies of the breathing vibrations shift, and new breathing modes appear. Obviously two types of pyridine complexes are formed, differing in the nature of the bonding provided either by copper or silver surface atoms. The generation and quenching behaviour of SERS at rough electrodes evidence the importance of metastable atomic surface structures for SERS and indicate the cooperation of local and non-local enhancement processes. Since active sites can be stabilized with traces of Cu at the silver electrode, the enhancement factor on a molecular basis appears to be by one order of magnitude larger than earlier anticipated, and ranges from 2 × 10 6 to 1.6 × 10 7 for an exciting wavelength at 514.5 nm or 647.1 nm, respectively.

  20. David Adler Lectureship Award Talk: Friction and energy dissipation mechanisms in adsorbed molecules and molecularly thin films

    NASA Astrophysics Data System (ADS)

    Krim, Jacqueline

    2015-03-01

    Studies of the fundamental origins of friction have undergone rapid progress in recent years, with the development of new experimental and computational techniques for measuring and simulating friction at atomic length and time scales. The increased interest has sparked a variety of discussions and debates concerning the nature of the atomic-scale and quantum mechanisms that dominate the dissipative process by which mechanical energy is transformed into heat. Measurements of the sliding friction of physisorbed monolayers and bilayers can provide information on the relative contributions of these various dissipative mechanisms. Adsorbed films, whether intentionally applied or present as trace levels of physisorbed contaminants, moreover are ubiquitous at virtually all surfaces. As such, they impact a wide range of applications whose progress depends on precise control and/or knowledge of surface diffusion processes. Examples include nanoscale assembly, directed transport of Brownian particles, material flow through restricted geometries such as graphene membranes and molecular sieves, passivation and edge effects in carbon-based lubricants, and the stability of granular materials associated with frictional and frictionless contacts. Work supported by NSFDMR1310456.

  1. Probing the enhancement mechanisms of SERS with p-aminothiophenol molecules adsorbed on self-assembled gold colloidal nanoparticles

    NASA Astrophysics Data System (ADS)

    Baia, M.; Toderas, F.; Baia, L.; Popp, J.; Astilean, S.

    2006-04-01

    Gold colloidal nanoparticles were immobilized upon a glass substrate and their morphology and optical properties are analyzed with TEM and UV-Vis absorption spectroscopy. The substrate suitability for surface-enhanced Raman spectroscopy (SERS) in visible and near-infrared spectral region is demonstrated with four excitation lines using p-aminothiophenol. The SERS spectra of probing molecules exhibit a clear signature of electromagnetic and charge-transfer enhancement mechanisms, which critically depend on the laser lines. The large tunability of surface plasmon excitation combined with the advantage of highly chemical affinity to gold of probe molecules recommends this SERS-active system as a useful model for probing the mechanisms of Raman enhancement.

  2. On the widths of Stokes lines in Raman scattering from molecules adsorbed at metal surfaces and in molecular conduction junctions

    NASA Astrophysics Data System (ADS)

    Gao, Yi; Galperin, Michael; Nitzan, Abraham

    2016-06-01

    Within a generic model we analyze the Stokes linewidth in surface enhanced Raman scattering (SERS) from molecules embedded as bridges in molecular junctions. We identify four main contributions to the off-resonant Stokes signal and show that under zero voltage bias (a situation pertaining also to standard SERS experiments) and at low bias junctions only one of these contributions is pronounced. The linewidth of this component is determined by the molecular vibrational relaxation rate, which is dominated by interactions with the essentially bosonic thermal environment when the relevant molecular electronic energy is far from the metal(s) Fermi energy(ies). It increases when the molecular electronic level is close to the metal Fermi level so that an additional vibrational relaxation channel due to electron-hole (eh) exciton in the molecule opens. Other contributions to the Raman signal, of considerably broader linewidths, can become important at larger junction bias.

  3. Coacervate delivery systems for proteins and small molecule drugs

    PubMed Central

    Johnson, Noah R; Wang, Yadong

    2015-01-01

    Coacervates represent an exciting new class of drug delivery vehicles, developed in the past decade as carriers of small molecule drugs and proteins. This review summarizes several well-described coacervate systems, including Elastin-like peptides for delivery of anti-cancer therapeutics,Heparin-based coacervates with synthetic polycations for controlled growth factor delivery,Carboxymethyl chitosan aggregates for oral drug delivery,Mussel adhesive protein and hyaluronic acid coacervates. Coacervates present advantages in their simple assembly and easy incorporation into tissue engineering scaffolds or as adjuncts to cell therapies. They are also amenable to functionalization such as for targeting or for enhancing the bioactivity of their cargo. These new drug carriers are anticipated to have broad applications and noteworthy impact in the near future. PMID:25138695

  4. Coacervate delivery systems for proteins and small molecule drugs.

    PubMed

    Johnson, Noah R; Wang, Yadong

    2014-12-01

    Coacervates represent an exciting new class of drug delivery vehicles, developed in the past decade as carriers of small molecule drugs and proteins. This review summarizes several well-described coacervate systems, including: i) elastin-like peptides for delivery of anticancer therapeutics; ii) heparin-based coacervates with synthetic polycations for controlled growth factor delivery; iii) carboxymethyl chitosan aggregates for oral drug delivery; iv) Mussel adhesive protein and hyaluronic acid coacervates. Coacervates present advantages in their simple assembly and easy incorporation into tissue engineering scaffolds or as adjuncts to cell therapies. They are also amenable to functionalization such as for targeting or for enhancing the bioactivity of their cargo. These new drug carriers are anticipated to have broad applications and noteworthy impact in the near future.

  5. Semisynthetic protein nanoreactor for single-molecule chemistry

    PubMed Central

    Lee, Joongoo; Bayley, Hagan

    2015-01-01

    The covalent chemistry of individual reactants bound within a protein pore can be monitored by observing the ionic current flow through the pore, which acts as a nanoreactor responding to bond-making and bond-breaking events. In the present work, we incorporated an unnatural amino acid into the α-hemolysin (αHL) pore by using solid-phase peptide synthesis to make the central segment of the polypeptide chain, which forms the transmembrane β-barrel of the assembled heptamer. The full-length αHL monomer was obtained by native chemical ligation of the central synthetic peptide to flanking recombinant polypeptides. αHL pores with one semisynthetic subunit were then used as nanoreactors for single-molecule chemistry. By introducing an amino acid with a terminal alkyne group, we were able to visualize click chemistry at the single-molecule level, which revealed a long-lived (4.5-s) reaction intermediate. Additional side chains might be introduced in a similar fashion, thereby greatly expanding the range of single-molecule covalent chemistry that can be investigated by the nanoreactor approach. PMID:26504203

  6. Single-molecule studies of unconventional motor protein myosin VI

    NASA Astrophysics Data System (ADS)

    Kim, HyeongJun

    Myosin VI is one of the myosin superfamily members that are actin-based molecular motors. It has received special attention due to its distinct features as compared to other myosins, such as its opposite directionality and a much larger step size than expected given the length of its "leg". This dissertation presents the author.s graduate work of several single-molecule studies on myosin VI. Special attention was paid to some of myosin VI.s tail domains that consist of proximal tail (PT), medial tail (MT), distal tail (DT) domains and cargo-binding domain (CBD). The functional form of myosin VI in cells is still under debate. Although full length myosin VI proteins in cytosolic extracts of cells were monomers from earlier studies, there are several reasons why it is now believed that myosin VI could exist as a dimer. If this is true and dimerization occurs, the next logical question would be which parts of myosin VI are dimerization regions? One model claimed that the CBD is the sole dimerization region. A competing model claimed that there must be another region that could be involved in dimerization, based on their observation that a construct without the CBD could still dimerize. Our single-molecule experiment with progressively truncated myosin VI constructs showed that the MT domain is a dimerization region, supporting the latter model. Additional single-molecule experiments and molecular dynamics (MD) simulation done with our collaborators suggest that electrostatic salt bridges formed between positive and negative amino acid residues are mainly responsible for the MT domain dimerization. After resolving this, we are left with another important question which is how myosin VI can take such a large step. Recent crystal structure showed that one of the tail domains preceding the MT domain, called the PT domain, is a three-helix bundle. The most easily conceivable way might be an unfolding of the three-helix bundle upon dimerization, allowing the protein to

  7. Single-molecule studies of kinesin family motor proteins

    NASA Astrophysics Data System (ADS)

    Fordyce, Polly

    Kinesin family motor proteins drive many essential cellular processes, including cargo transport and mitotic spindle assembly and regulation. They accomplish these tasks by converting the chemical energy released from the hydrolysis of adenosine triphosphate (ATP) directly into mechanical motion along microtubules in cells. Optical traps allow us to track and apply force to individual motor proteins, and have already revealed many details of the movement of conventional kinesin, although the precise mechanism by which chemical energy is converted into mechanical motion is unclear. Other kinesin family members remain largely uncharacterized. This dissertation details the use of a novel optical-trapping assay to study Eg5, a Kinesin-5 family member involved in both spindle assembly and pole separation during mitosis. We demonstrate that individual Eg5 dimers are relatively slow and force-insensitive motors that take about 8 steps, on average, before detaching from the microtubule. Key differences in processivity and force-response between Eg5 and conventional kinesin suggest ways in which the two motors might have evolved to perform very different tasks in cells. This dissertation also details efforts to unravel how chemical energy is converted into mechanical motion by simultaneously measuring mechanical transitions (with an optical trap) and nucleotide binding and release (with single-molecule fluorescence) for individual conventional kinesin motors. We constructed a combined instrument, demonstrated its capabilities by unzipping fluorescently-labeled DNA duplexes, and used this instrument to record the motion of individual conventional kinesin motors powered by the hydrolysis of fluorescent nucleotides. Preliminary data reveal the challenges inherent in such measurements and guide proposals for future experimental approaches. Finally, this dissertation includes several chapters intended to serve as practical guides to understanding, constructing, and maintaining

  8. Performance of the TPSS Functional on Predicting Core Level Binding Energies of Main Group Elements Containing Molecules: A Good Choice for Molecules Adsorbed on Metal Surfaces.

    PubMed

    Pueyo Bellafont, Noèlia; Viñes, Francesc; Illas, Francesc

    2016-01-12

    Here we explored the performance of Hartree-Fock (HF), Perdew-Burke-Ernzerhof (PBE), and Tao-Perdew-Staroverov-Scuseria (TPSS) functionals in predicting core level 1s binding energies (BEs) and BE shifts (ΔBEs) for a large set of 68 molecules containing a wide variety of functional groups for main group elements B → F and considering up to 185 core levels. A statistical analysis comparing with X-ray photoelectron spectroscopy (XPS) experiments shows that BEs estimations are very accurate, TPSS exhibiting the best performance. Considering ΔBEs, the three methods yield very similar and excellent results, with mean absolute deviations of ∼0.25 eV. When considering relativistic effects, BEs deviations drop approaching experimental values. So, the largest mean percentage deviation is of 0.25% only. Linear trends among experimental and estimated values have been found, gaining offsets with respect to ideality. By adding relativistic effects to offsets, HF and TPSS methods underestimate experimental values by solely 0.11 and 0.05 eV, respectively, well within XPS chemical precision. TPSS is posed as an excellent choice for the characterization, by XPS, of molecules on metal solid substrates, given its suitability in describing metal substrates bonds and atomic and/or molecular orbitals.

  9. Optical and electronic properties of SO2 molecule adsorbed on Si-doped (8, 0) boron nitride nanotube

    NASA Astrophysics Data System (ADS)

    Guo, Shuang-Shuang; Wei, Xiu-Mei; Zhang, Jian-Min; Zhu, Gang-Qiang; Guo, Wan-Jin

    2016-09-01

    The study of the optical properties of pristine BNNT, Si-doped BNNTs and SO2 molecule adsorption on Si-doped BNNTs is that, to our knowledge, few relevant research have ever been found. In this paper, the adsorption behaviors of Sulfur dioxide (SO2) molecule on Si-doped Boron nitride nanotubes (BNNTs) are investigated applying the first-principles calculations. The main contribution of this paper is that the foremost investigation for the optical properties of the pristine BNNT, Si-doped BNNTs and SO2 adsorption on Si-doped BNNTs. Additionally, the electronic properties and the structural properties are also presented. In our calculations of optical properties, the dielectric constant, the refractive index and the absorption coefficient are obtained. Comparing the pristine BNNT, our results indicate that, the blue-shifts (in the main peaks of the dielectric constant of SiB -BNNT and SO2-SiB -BNNT), and the red-shifts (in the main peaks of the refractive index of SiN -BNNT and SO2-SiN -BNNT) are appeared. Under these conditions, Si-doped BNNT and Si-doped BNNT with SO2 adsorption, the gaps are reduced both for the speculated optical band gaps and the electronic structure band gaps.

  10. Synthesis of adsorbents with dendronic structures for protein hydrophobic interaction chromatography.

    PubMed

    Mata-Gómez, Marco A; Yaman, Sena; Valencia-Gallegos, Jesus A; Tari, Canan; Rito-Palomares, Marco; González-Valdez, José

    2016-04-22

    Here, we introduced a new technology based on the incorporation of dendrons-branched chemical structures-onto supports for synthesis of HIC adsorbents. In doing so we studied the synthesis and performance of these novel HIC dendron-based adsorbents. The adsorbents were synthesized in a facile two-step reaction. First, Sepharose 4FF (R) was chemically modified with polyester dendrons of different branching degrees i.e. third (G3) or fifth (G5) generations. Then, butyl-end valeric acid ligands were coupled to dendrons via ester bond formation. UV-vis spectrophotometry and FTIR analyses of the modified resins confirmed the presence of the dendrons and their ligands on them. Inclusion of dendrons allowed the increment of ligand density, 82.5 ± 11 and 175.6 ± 5.7 μmol ligand/mL resin for RG3 and RG5, respectively. Static adsorption capacity of modified resins was found to be ∼ 60 mg BSA/mL resin. Interestingly, dynamic binding capacity was higher at high flow rates, 62.5 ± 0.8 and 58.0 ± 0.5mg/mL for RG3 and RG5, respectively. RG3 was able to separate lipase, β-lactoglobulin and α-chymotrypsin selectively as well as fractionating of a whole proteome from yeast. This innovative technology will improve the existing HIC resin synthesis methods. It will also allow the reduction of the amount of adsorbent used in a chromatographic procedure and thus permit the use of smaller columns resulting in faster processes. Furthermore, this method could potentially be considered as a green technology since both, dendrons and ligands, are formed by ester bonds that are more biodegradable allowing the disposal of used resin waste in a more ecofriendly manner when compared to other exiting resins.

  11. Synthesis of adsorbents with dendronic structures for protein hydrophobic interaction chromatography.

    PubMed

    Mata-Gómez, Marco A; Yaman, Sena; Valencia-Gallegos, Jesus A; Tari, Canan; Rito-Palomares, Marco; González-Valdez, José

    2016-04-22

    Here, we introduced a new technology based on the incorporation of dendrons-branched chemical structures-onto supports for synthesis of HIC adsorbents. In doing so we studied the synthesis and performance of these novel HIC dendron-based adsorbents. The adsorbents were synthesized in a facile two-step reaction. First, Sepharose 4FF (R) was chemically modified with polyester dendrons of different branching degrees i.e. third (G3) or fifth (G5) generations. Then, butyl-end valeric acid ligands were coupled to dendrons via ester bond formation. UV-vis spectrophotometry and FTIR analyses of the modified resins confirmed the presence of the dendrons and their ligands on them. Inclusion of dendrons allowed the increment of ligand density, 82.5 ± 11 and 175.6 ± 5.7 μmol ligand/mL resin for RG3 and RG5, respectively. Static adsorption capacity of modified resins was found to be ∼ 60 mg BSA/mL resin. Interestingly, dynamic binding capacity was higher at high flow rates, 62.5 ± 0.8 and 58.0 ± 0.5mg/mL for RG3 and RG5, respectively. RG3 was able to separate lipase, β-lactoglobulin and α-chymotrypsin selectively as well as fractionating of a whole proteome from yeast. This innovative technology will improve the existing HIC resin synthesis methods. It will also allow the reduction of the amount of adsorbent used in a chromatographic procedure and thus permit the use of smaller columns resulting in faster processes. Furthermore, this method could potentially be considered as a green technology since both, dendrons and ligands, are formed by ester bonds that are more biodegradable allowing the disposal of used resin waste in a more ecofriendly manner when compared to other exiting resins. PMID:27018188

  12. THE APPLICATION OF HIGH RESOLUTION ELECTRON ENERGY LOSS SPECTROSCOPY TO THE CHARACTERIZATION OF ADSORBED MOLECULES ON RHODIUM SINGLE CRYSTAL SURFACES

    SciTech Connect

    Dubois, L.H.; Somorjai, G.A.

    1980-01-01

    The scattering of low energy electrons by metal surfaces has been studied for many years now. The electron's ease of generation and detection and high surface sensitivity (low penetration depth) make it an ideal probe for surface scientists. The impinging electron can interact with the surface in basically two ways: it can either elastically reflect (or diffract) from the surface without losing energy or lose a portion of it's incident energy and inelastically scatter. In this paper we will be concerned with only one of many possible inelastic scattering processes: the loss of the electron's energy to the vibrational modes of atoms and molecules chemisorbed on the surface. This technique is known as high resolution electron energy loss spectroscopy (or ELS, EELS, HRELS, HREELS, etc.).

  13. Analysis and Interpretation of Single Molecule Protein Unfolding Kinetics

    NASA Astrophysics Data System (ADS)

    Lannon, Herbert; Brujic, Jasna

    2012-02-01

    The kinetics of protein unfolding under a stretching force has been extensively studied by atomic force microscopy (AFM) over the past decade [1]. Experimental artifacts at the single molecule level introduce uncertainties in the data analysis that have led to several competing physical models for the unfolding process. For example, the unfolding dynamics of the protein ubiquitin under constant force has been described by probability distributions as diverse as exponential [2,3], a sum of exponentials, log-normal [4], and more recently a function describing static disorder in the Arrhenius model [5]. A new method for data analysis is presented that utilizes maximum likelihood estimation (MLE) combined with other traditional statistical tests to unambiguously rank the consistency of these and other models with the experimental data. These techniques applied to the ubiquitin unfolding data shows that the probability of unfolding is best fit with a stretched exponential distribution, with important implications on the complexity of the mechanism of protein unfolding. [4pt] [1] Carrion-Vazquez, et. al. Springer Series in Biophys. 2006 [0pt] [2] Fernandez et. al. Science 2004 [0pt] [3] Brujic et. al. Nat. Phys 2006 [0pt] [4] Garcia-Manyes et. al. Biophys. J. 2007 [0pt] [5] Kuo et. al. PNAS 2010

  14. Hybrid molecules synergistically acting against protein aggregation diseases.

    PubMed

    Korth, Carsten; Klingenstein, Ralf; Müller-Schiffmann, Andreas

    2013-01-01

    An emerging common feature of the age-associated neurodegenerative disorders like Alzheimer's disease (AD) and Creutzfeldt-Jakob disease (CJD) is the ability of many disease-associated protein aggregates to induce conversion of a normal counterpart conformer leading to an acceleration of disease progression. Curative pharmacotherapy has not been achieved so far despite successes in elucidating pathomechanisms. Here, we review the pharmaceutical strategy of generating hybrid compounds, i.e. compounds consisting of several independently acting moieties with synergistic effects, on key molecular players in AD and CJD. For prion diseases, we review hybrid compounds consisting of two different heterocyclic compounds, their synergistic effects on prion replication in a cell culture model and their ability to prolong survival of experimentally prion-infected mice in vivo. While a combination therapy of several antiprion compounds including quinacrine, clomipramine, simvastatin and tocopherol prolonged survival time to 10-25%, administration of hybrid compound quinpramine alone, a chimera of acridine and iminodibenzyl scaffolds, led to 10% survival time extension. For AD, we review a hybrid compound consisting of an Aβ recognizing D-peptide fused to a small molecule β-sheet breaker, an aminopyrazole. This molecule was able to diminish Aβ oligomers in cell culture and significantly decrease synaptotoxicity as measured by miniature excitatory postsynaptic responses in vitro. Hybrid compounds can dramatically increase potency of their single moieties and lead to novel functions when they act in a simultaneous or sequential manner thereby revealing synergistic properties. Their systematic generation combining different classes of compounds from peptides to small molecules has the potential to significantly accelerate drug discovery. PMID:24059335

  15. Maleimide photolithography for single-molecule protein-protein interaction analysis in micropatterns.

    PubMed

    Waichman, Sharon; You, Changjiang; Beutel, Oliver; Bhagawati, Maniraj; Piehler, Jacob

    2011-01-15

    Spatial organization of proteins into microscopic structures has important applications in fundamental and applied research. Preserving the function of proteins in such microstructures requires generic methods for site-specific capturing through affinity handles. Here, we present a versatile bottom-up surface micropatterning approach based on surface functionalization with maleimides, which selectively react with organic thiols. Upon UV irradiation through a photomask, the functionality of illuminated maleimide groups was efficiently destroyed. Remaining maleimides in nonilluminated regions were further reacted with different thiol-functionalized groups for site-specific protein immobilization under physiological conditions. Highly selective immobilization of His-tagged proteins into tris(nitrilotriacetic acid) functionalized microstructures with very high contrast was possible even by direct capturing of proteins from crude cell lysates. Moreover, we employed phosphopantetheinyl transfer from surface-immobilized coenzyme A to ybbR-tagged proteins in order to implement site-specific, covalent protein immobilization into microstructures. The functional integrity of the immobilized protein was confirmed by monitoring protein-protein interactions in real time. Moreover, we demonstrate quantitative single-molecule analysis of protein-protein interactions with proteins selectively captured into these high-contrast micropatterns.

  16. Probing Protein Channel Dynamics At The Single Molecule Level.

    NASA Astrophysics Data System (ADS)

    Lee, M. Ann; Dunn, Robert C.

    1997-03-01

    It would be difficult to overstate the importance played by protein ion channels in cellular function. These macromolecular pores allow the passage of ions across the cellular membrane and play indispensable roles in all aspects of neurophysiology. While the patch-clamp technique continues to provide elegant descriptions of the kinetic processes involved in ion channel gating, the associated conformational changes remain a mystery. We are using the spectroscopic capabilities and single molecule fluorescence sensitivity of near-field scanning optical microscopy (NSOM) to probe these dynamics at the single channel level. Using a newly developed cantilevered NSOM probe capable of probing soft biological samples with single molecule fluorescence sensitivity, we have begun mapping the location of single NMDA receptors in intact rat cortical neurons with <100 nm spatial resolution. We will also present recent results exploring the conformational changes accompanying activation of nuclear pore channels located in the nuclear membrane of Xenopus oocytes. Our recent NSOM and AFM measurements on single nuclear pore complexes reveal large conformational changes taking place upon activation, providing rich, new molecular level details of channel function.

  17. Small Molecule Inhibitors Targeting Activator Protein 1 (AP-1)

    PubMed Central

    2015-01-01

    Activator protein 1 (AP-1) is a pivotal transcription factor that regulates a wide range of cellular processes including proliferation, apoptosis, differentiation, survival, cell migration, and transformation. Accumulating evidence supports that AP-1 plays an important role in several severe disorders including cancer, fibrosis, and organ injury, as well as inflammatory disorders such as asthma, psoriasis, and rheumatoid arthritis. AP-1 has emerged as an actively pursued drug discovery target over the past decade. Excitingly, a selective AP-1 inhibitor T-5224 (51) has been investigated in phase II human clinical trials. Nevertheless, no effective AP-1 inhibitors have yet been approved for clinical use. Despite significant advances achieved in understanding AP-1 biology and function, as well as the identification of small molecules modulating AP-1 associated signaling pathways, medicinal chemistry efforts remain an urgent need to yield selective and efficacious AP-1 inhibitors as a viable therapeutic strategy for human diseases. PMID:24831826

  18. High ionic strength narrows the population of sites participating in protein ion-exchange adsorption: A single-molecule study

    PubMed Central

    Kisley, Lydia; Chen, Jixin; Mansur, Andrea P.; Dominguez-Medina, Sergio; Kulla, Eliona; Kang, Marci; Shuang, Bo; Kourentzi, Katerina; Poongavanam, Mohan-Vivekanandan; Dhamane, Sagar; Willson, Richard C.; Landes, Christy F.

    2014-01-01

    The retention and elution of proteins in ion-exchange chromatography is routinely controlled by adjusting the mobile phase salt concentration. It has repeatedly been observed, as judged from adsorption isotherms, that the apparent heterogeneity of adsorption is lower at more-eluting, higher ionic strength. Here, we present an investigation into the mechanism of this phenomenon using a single-molecule, super-resolution imaging technique called motion-blur Points Accumulation for Imaging in Nanoscale Topography (mbPAINT). We observed that the number of functional adsorption sites was smaller at high ionic strength and that these sites had reduced desorption kinetic heterogeneity, and thus narrower predicted elution profiles, for the anion-exchange adsorption of α-lactalbumin on an agarose-supported, clustered-charge ligand stationary phase. Explanations for the narrowing of the functional population such as inter-protein interactions and protein or support structural changes were investigated through kinetic analysis, circular dichroism spectroscopy, and microscopy of agarose microbeads, respectively. The results suggest the reduction of heterogeneity is due to both electrostatic screening between the protein and ligand and tuning the steric availability within the agarose support. Overall, we have shown that single molecule spectroscopy can aid in understanding the influence of ionic strength on the population of functional adsorbent sites participating in the ion-exchange chromatographic separation of proteins. PMID:24751557

  19. Structure and mechanics of proteins from single molecules to cells

    NASA Astrophysics Data System (ADS)

    Brown, Andre E.

    2009-07-01

    Physical factors drive evolution and play important roles in motility and attachment as well as in differentiation. As animal cells adhere to survive, they generate force and "feel" various mechanical features of their surroundings and respond to externally applied forces. This mechanosensitivity requires a substrate for cells to adhere to and a mechanism for cells to apply force, followed by a cellular response to the mechanical properties of the substrate. We have taken an outside-in approach to characterize several aspects of cellular mechanosensitivity. First, we used single molecule force spectroscopy to measure how fibrinogen, an extracellular matrix protein that forms the scaffold of blood clots, responds to applied force and found that it rapidly unfolds in 23 nm steps at forces around 100 pN. Second, we used tensile testing to measure the force-extension behavior of fibrin gels and found that they behave almost linearly to strains of over 100%, have extensibilities of 170 +/- 15%, and undergo a large volume decrease that corresponds to a large and negative peak in compressibility at low strain, which indicates a structural transition. Using electron microscopy and X-ray scattering we concluded that these properties are likely due to coiled-coil unfolding, as observed at the single molecule level in fibrinogen. Moving inside cells, we used total internal reflection fluorescence and atomic force microscopy to image self-assembled myosin filaments. These filaments of motor proteins that are responsible for cell and muscle contractility were found to be asymmetric, with an average of 32% more force generating heads on one half than the other. This could imply a force imbalance, so that rather than being simply contractile, myosin filaments may also be motile in cells.

  20. Affinity binding of antibodies to supermacroporous cryogel adsorbents with immobilized protein A for removal of anthrax toxin protective antigen.

    PubMed

    Ingavle, Ganesh C; Baillie, Les W J; Zheng, Yishan; Lis, Elzbieta K; Savina, Irina N; Howell, Carol A; Mikhalovsky, Sergey V; Sandeman, Susan R

    2015-05-01

    Polymeric cryogels are efficient carriers for the immobilization of biomolecules because of their unique macroporous structure, permeability, mechanical stability and different surface chemical functionalities. The aim of the study was to demonstrate the potential use of macroporous monolithic cryogels for biotoxin removal using anthrax toxin protective antigen (PA), the central cell-binding component of the anthrax exotoxins, and covalent immobilization of monoclonal antibodies. The affinity ligand (protein A) was chemically coupled to the reactive hydroxyl and epoxy-derivatized monolithic cryogels and the binding efficiencies of protein A, monoclonal antibodies to the cryogel column were determined. Our results show differences in the binding capacity of protein A as well as monoclonal antibodies to the cryogel adsorbents caused by ligand concentrations, physical properties and morphology of surface matrices. The cytotoxicity potential of the cryogels was determined by an in vitro viability assay using V79 lung fibroblast as a model cell and the results reveal that the cryogels are non-cytotoxic. Finally, the adsorptive capacities of PA from phosphate buffered saline (PBS) were evaluated towards a non-glycosylated, plant-derived human monoclonal antibody (PANG) and a glycosylated human monoclonal antibody (Valortim(®)), both of which were covalently attached via protein A immobilization. Optimal binding capacities of 108 and 117 mg/g of antibody to the adsorbent were observed for PANG attached poly(acrylamide-allyl glycidyl ether) [poly(AAm-AGE)] and Valortim(®) attached poly(AAm-AGE) cryogels, respectively, This indicated that glycosylation status of Valortim(®) antibody could significantly increase (8%) its binding capacity relative to the PANG antibody on poly(AAm-AGE)-protien-A column (p < 0.05). The amounts of PA which remained in the solution after passing PA spiked PBS through PANG or Valortim bound poly(AAm-AGE) cryogel were significantly (p < 0

  1. Affinity binding of antibodies to supermacroporous cryogel adsorbents with immobilized protein A for removal of anthrax toxin protective antigen.

    PubMed

    Ingavle, Ganesh C; Baillie, Les W J; Zheng, Yishan; Lis, Elzbieta K; Savina, Irina N; Howell, Carol A; Mikhalovsky, Sergey V; Sandeman, Susan R

    2015-05-01

    Polymeric cryogels are efficient carriers for the immobilization of biomolecules because of their unique macroporous structure, permeability, mechanical stability and different surface chemical functionalities. The aim of the study was to demonstrate the potential use of macroporous monolithic cryogels for biotoxin removal using anthrax toxin protective antigen (PA), the central cell-binding component of the anthrax exotoxins, and covalent immobilization of monoclonal antibodies. The affinity ligand (protein A) was chemically coupled to the reactive hydroxyl and epoxy-derivatized monolithic cryogels and the binding efficiencies of protein A, monoclonal antibodies to the cryogel column were determined. Our results show differences in the binding capacity of protein A as well as monoclonal antibodies to the cryogel adsorbents caused by ligand concentrations, physical properties and morphology of surface matrices. The cytotoxicity potential of the cryogels was determined by an in vitro viability assay using V79 lung fibroblast as a model cell and the results reveal that the cryogels are non-cytotoxic. Finally, the adsorptive capacities of PA from phosphate buffered saline (PBS) were evaluated towards a non-glycosylated, plant-derived human monoclonal antibody (PANG) and a glycosylated human monoclonal antibody (Valortim(®)), both of which were covalently attached via protein A immobilization. Optimal binding capacities of 108 and 117 mg/g of antibody to the adsorbent were observed for PANG attached poly(acrylamide-allyl glycidyl ether) [poly(AAm-AGE)] and Valortim(®) attached poly(AAm-AGE) cryogels, respectively, This indicated that glycosylation status of Valortim(®) antibody could significantly increase (8%) its binding capacity relative to the PANG antibody on poly(AAm-AGE)-protien-A column (p < 0.05). The amounts of PA which remained in the solution after passing PA spiked PBS through PANG or Valortim bound poly(AAm-AGE) cryogel were significantly (p < 0

  2. Modeling adsorption: Investigating adsorbate and adsorbent properties

    NASA Astrophysics Data System (ADS)

    Webster, Charles Edwin

    1999-12-01

    Surface catalyzed reactions play a major role in current chemical production technology. Currently, 90% of all chemicals are produced by heterogeneously catalyzed reactions. Most of these catalyzed reactions involve adsorption, concentrating the substrate(s) (the adsorbate) on the surface of the solid (the adsorbent). Pore volumes, accessible surface areas, and the thermodynamics of adsorption are essential in the understanding of solid surface characteristics fundamental to catalyst and adsorbent screening and selection. Molecular properties such as molecular volumes and projected molecular areas are needed in order to convert moles adsorbed to surface volumes and areas. Generally, these molecular properties have been estimated from bulk properties, but many assumptions are required. As a result, different literature values are employed for these essential molecular properties. Calculated molar volumes and excluded molecular areas are determined and tabulated for a variety of molecules. Molecular dimensions of molecules are important in the understanding of molecular exclusion as well as size and shape selectivity, diffusion, and adsorbent selection. Molecular dimensions can also be used in the determination of the effective catalytic pore size of a catalyst. Adsorption isotherms, on zeolites, (crystalline mineral oxides) and amorphous solids, can be analyzed with the Multiple Equilibrium Analysis (MEA) description of adsorption. The MEA produces equilibrium constants (Ki), capacities (ni), and thermodynamic parameters (enthalpies, ΔHi, and entropies, ΔSi) of adsorption for each process. Pore volumes and accessible surface areas are calculated from the process capacities. Adsorption isotherms can also be predicted for existing and new adsorbate-adsorbent systems with the MEA. The results show that MEA has the potential of becoming a standard characterization method for microporous solids that will lead to an increased understanding of their behavior in gas

  3. Adsorption of proteins at physiological concentrations on pegylated surfaces and the compatibilizing role of adsorbed albumin with respect to other proteins according to optical waveguide lightmode spectroscopy (OWLS).

    PubMed

    Leclercq, Laurent; Modena, Enrico; Vert, Michel

    2013-01-01

    In literature, contacts between pegylated compounds and blood proteins are generally discussed in terms of excluded volume-related repulsions although adsorption and compatibility have been reported for some of these proteins occasionally. The major problem to investigate the behavior of blood in contact with pegylated surfaces is the complexity of the medium and especially the presence of albumin in large excess. In a model approach, optical waveguide lightmode spectroscopy (OWLS) was used to monitor the fate of albumin, fibrinogen, and γ-globulins at physiological concentrations in pH = 7.4 isotonic HEPES buffer after contact with SiTiO2 chips coated with diblock poly(DL-lactic acid)-block-poly(ethylene oxide)s and triblock poly(DL-lactic acid)-block-poly(ethylene oxide)-block-poly(DL-lactic acid) copolymers. Corresponding homopolymers were used as controls. The three protein systems were investigated separately, as a mixture and when added successively according to different orders of addition. OWLS gave access to the mass and the thickness of adhering protein layers that resist washing with HEPES buffer. Protein depositions were detected regardless of the presence of poly(ethylene glycol) segments on surfaces. Adsorption depended on the protein, on the surface and also on the presence of the other proteins. Unexpectedly any surface coated with a layer of adsorbed albumin prevented deposition of other proteins, including albumin itself. This outstanding finding suggests that it was the presence of albumin adsorbed on a surface, pegylated or not, that made that surface compatible with other proteins. As a consequence, dipping a device to be in contact with the blood of a patient in a solution of albumin could be a very simple means to avoid further protein deposition and maybe platelets adhesion after in vivo implantation.

  4. Protein frustratometer: a tool to localize energetic frustration in protein molecules

    PubMed Central

    Jenik, Michael; Parra, R. Gonzalo; Radusky, Leandro G.; Turjanski, Adrian; Wolynes, Peter G.; Ferreiro, Diego U.

    2012-01-01

    The frustratometer is an energy landscape theory-inspired algorithm that aims at quantifying the location of frustration manifested in protein molecules. Frustration is a useful concept for gaining insight to the proteins biological behavior by analyzing how the energy is distributed in protein structures and how mutations or conformational changes shift the energetics. Sites of high local frustration often indicate biologically important regions involved in binding or allostery. In contrast, minimally frustrated linkages comprise a stable folding core of the molecule that is conserved in conformational changes. Here, we describe the implementation of these ideas in a webserver freely available at the National EMBNet node—Argentina, at URL: http://lfp.qb.fcen.uba.ar/embnet/. PMID:22645321

  5. Protein frustratometer: a tool to localize energetic frustration in protein molecules.

    PubMed

    Jenik, Michael; Parra, R Gonzalo; Radusky, Leandro G; Turjanski, Adrian; Wolynes, Peter G; Ferreiro, Diego U

    2012-07-01

    The frustratometer is an energy landscape theory-inspired algorithm that aims at quantifying the location of frustration manifested in protein molecules. Frustration is a useful concept for gaining insight to the proteins biological behavior by analyzing how the energy is distributed in protein structures and how mutations or conformational changes shift the energetics. Sites of high local frustration often indicate biologically important regions involved in binding or allostery. In contrast, minimally frustrated linkages comprise a stable folding core of the molecule that is conserved in conformational changes. Here, we describe the implementation of these ideas in a webserver freely available at the National EMBNet node-Argentina, at URL: http://lfp.qb.fcen.uba.ar/embnet/.

  6. Expansion Hamiltonian model for a diatomic molecule adsorbed on a surface: Vibrational states of the CO/Cu(100) system including surface vibrations

    SciTech Connect

    Meng, Qingyong; Meyer, Hans-Dieter

    2015-10-28

    Molecular-surface studies are often done by assuming a corrugated, static (i.e., rigid) surface. To be able to investigate the effects that vibrations of surface atoms may have on spectra and cross sections, an expansion Hamiltonian model is proposed on the basis of the recently reported [R. Marquardt et al., J. Chem. Phys. 132, 074108 (2010)] SAP potential energy surface (PES), which was built for the CO/Cu(100) system with a rigid surface. In contrast to other molecule-surface coupling models, such as the modified surface oscillator model, the coupling between the adsorbed molecule and the surface atoms is already included in the present expansion SAP-PES model, in which a Taylor expansion around the equilibrium positions of the surface atoms is performed. To test the quality of the Taylor expansion, a direct model, that is avoiding the expansion, is also studied. The latter, however, requests that there is only one movable surface atom included. On the basis of the present expansion and direct models, the effects of a moving top copper atom (the one to which CO is bound) on the energy levels of a bound CO/Cu(100) system are studied. For this purpose, the multiconfiguration time-dependent Hartree calculations are carried out to obtain the vibrational fundamentals and overtones of the CO/Cu(100) system including a movable top copper atom. In order to interpret the results, a simple model consisting of two coupled harmonic oscillators is introduced. From these calculations, the vibrational levels of the CO/Cu(100) system as function of the frequency of the top copper atom are discussed.

  7. Sum Frequency Generation Vibrational Spectroscopy of Adsorbed Amino Acids, Peptides and Proteins of Hydrophilic and Hydrophobic Solid-Water Interfaces

    SciTech Connect

    Holinga IV, George Joseph

    2010-09-01

    Sum frequency generation (SFG) vibrational spectroscopy was used to investigate the interfacial properties of several amino acids, peptides, and proteins adsorbed at the hydrophilic polystyrene solid-liquid and the hydrophobic silica solid-liquid interfaces. The influence of experimental geometry on the sensitivity and resolution of the SFG vibrational spectroscopy technique was investigated both theoretically and experimentally. SFG was implemented to investigate the adsorption and organization of eight individual amino acids at model hydrophilic and hydrophobic surfaces under physiological conditions. Biointerface studies were conducted using a combination of SFG and quartz crystal microbalance (QCM) comparing the interfacial structure and concentration of two amino acids and their corresponding homopeptides at two model liquid-solid interfaces as a function of their concentration in aqueous solutions. The influence of temperature, concentration, equilibration time, and electrical bias on the extent of adsorption and interfacial structure of biomolecules were explored at the liquid-solid interface via QCM and SFG. QCM was utilized to quantify the biological activity of heparin functionalized surfaces. A novel optical parametric amplifier was developed and utilized in SFG experiments to investigate the secondary structure of an adsorbed model peptide at the solid-liquid interface.

  8. Protein Frustratometer 2: a tool to localize energetic frustration in protein molecules, now with electrostatics

    PubMed Central

    Parra, R. Gonzalo; Schafer, Nicholas P.; Radusky, Leandro G.; Tsai, Min-Yeh; Guzovsky, A. Brenda; Wolynes, Peter G.; Ferreiro, Diego U.

    2016-01-01

    The protein frustratometer is an energy landscape theory-inspired algorithm that aims at localizing and quantifying the energetic frustration present in protein molecules. Frustration is a useful concept for analyzing proteins’ biological behavior. It compares the energy distributions of the native state with respect to structural decoys. The network of minimally frustrated interactions encompasses the folding core of the molecule. Sites of high local frustration often correlate with functional regions such as binding sites and regions involved in allosteric transitions. We present here an upgraded version of a webserver that measures local frustration. The new implementation that allows the inclusion of electrostatic energy terms, important to the interactions with nucleic acids, is significantly faster than the previous version enabling the analysis of large macromolecular complexes within a user-friendly interface. The webserver is freely available at URL: http://frustratometer.qb.fcen.uba.ar. PMID:27131359

  9. Inhibition of α-helix-mediated protein-protein interactions using designed molecules

    NASA Astrophysics Data System (ADS)

    Azzarito, Valeria; Long, Kérya; Murphy, Natasha S.; Wilson, Andrew J.

    2013-03-01

    Inhibition of protein-protein interactions (PPIs) represents a significant challenge because it is unclear how they can be effectively and selectively targeted using small molecules. Achieving this goal is critical given the defining role of these interactions in biological processes. A rational approach to inhibitor design based on the secondary structure at the interface is the focus of much research, and different classes of designed ligands have emerged, some of which effectively and selectively disrupt targeted PPIs. This Review discusses the relevance of PPIs and, in particular, the importance of α-helix-mediated PPIs to chemical biology and drug discovery with a focus on designing inhibitors, including constrained peptides, foldamers and proteomimetic-derived ligands. In doing so, key challenges and major advances in developing generic approaches for the elaboration of PPI inhibitors are highlighted. The challenges faced in developing such ligands as drug leads -- and how criteria applied to these may differ from conventional small-molecule drugs -- are summarized.

  10. [Surface-enhanced Raman spectroscopy of biopolymers: membrane proteins, bacteriorhodopsin and rhodopsin adsorbed on silver electrodes and silver hydrosols].

    PubMed

    Nabiev, I R; Efremov, R G; Chumanov, G D

    1986-01-01

    Surface-enhanced Raman (SER) spectra of purple membranes of Halobacterium halobium and photoreceptor disks of the rod outer segments adsorbed on silver hydrosols were analysed. It has been shown that the intensity of SER spectra of bacterial and visual rhodopsins increases 5 X 10(4) times at adsorption. Concentration relationship of the signal intensity of SER spectra has the maximum at bacteriorhodopsin concentration about 2 X 10(-7) M. It has been shown that adsorption on silver hydrosol leads to fixation of light-induced photochemical transformations in bacterial and visual rhodopsins. Adsorption on the "smooth" electrodes at the potential of the zero charge of silver does not affect the photocycle of bacteriorhodopsin. An increase or decrease of the electrode potential relative to the zero charge point of silver leads to the accumulation of kinetic intermediate K610 and a decrease of the concentration of the form BRh570. It has been shown that on the "smooth" electrode primarily the long-range component of the SER mechanism is realized. Bands corresponding to the vibrations of the atom groups directly contacting with the metal are mainly intensified after redox cycle which increases the concentration of chemosorption centres. A conclusion is drawn that the method of SER spectroscopy of biomolecules adsorbed on "smooth" electrodes, permits obtaining information similar to that obtained from the analysis of Raman spectra of unadsorbed molecules, but at concentrations by two orders less. Adsorption on the electrodes treated with the help of redox cycle permits to obtain highly oriented preparations and to study topography of biopolymers in water solutions and suspensions.

  11. Plant nuclear hormone receptors: a role for small molecules in protein-protein interactions.

    PubMed

    Lumba, Shelley; Cutler, Sean; McCourt, Peter

    2010-01-01

    Plant hormones are a group of chemically diverse small molecules that direct processes ranging from growth and development to biotic and abiotic stress responses. Surprisingly, genome analyses suggest that classic animal nuclear hormone receptor homologs do not exist in plants. It now appears that plants have co-opted several protein families to perceive hormones within the nucleus. In one solution to the problem, the hormones auxin and jasmonate (JA) act as “molecular glue” that promotes protein-protein interactions between receptor F-boxes and downstream corepressor targets. In another solution, gibberellins (GAs) bind and elicit a conformational change in a novel soluble receptor family related to hormone-sensitive lipases. Abscisic acid (ABA), like GA, also acts through an allosteric mechanism involving a START-domain protein. The molecular identification of plant nuclear hormone receptors will allow comparisons with animal nuclear receptors and testing of fundamental questions about hormone function in plant development and evolution.

  12. Elastic response of a protein monolayer adsorbed at decorated water surface

    NASA Astrophysics Data System (ADS)

    Singh, Amarjeet; Konovalov, Oleg

    2015-05-01

    Under the in-plane isothermal compression the self-assembled protein monolayer expand in the direction perpendicular to the applied force as a function of applied compression. The structure finally buckle beyond a critical compression, which finally returns to the initial structure when the compression force was removed, behaving like an elastic body. We modelled the layer as homogeneous elastic medium and calculated elastic constants. Young's modulus of the protein layer is 2 orders of magnitude smaller than the bulk lysozyme crystals. It is of fundamental significance to be able to predict the elastic properties of the proteins at air-water interface since protein remains in their natural environment unlike protein crystals.

  13. Porous ceramic/agarose composite adsorbents for fast protein liquid chromatography.

    PubMed

    Xia, Haifeng; Jin, Xionghua; Wu, Puqiang; Zheng, Zhiyong

    2012-02-01

    Porous ceramic/agarose composite adsorbents were designed and prepared with silica ceramic beads and 4% agarose gel, and then functionalized with a special ligand carboxymethyl. A novel method was introduced to fabricating of the porous silica ceramic beads. The morphology of SEM shows a spherical shape and a porous structure of the ceramic beads. Nitrogen adsorption-desorption analysis gives an average pore size of 287.5 Å, a BET surface area of 29.33 m²/g and a porosity of 41.8%, respectively. Additionally, X-ray diffraction pattern indicates that the amorphous silica has been transformed into two crystal phases of quartz and cristobalite, leading to a porous and rigid skeleton and ensuring the application of the composite beads at high flow velocities. Lysozyme of hen egg-white with the activity of 12,700 U/mg was purified by the composite ion-exchanger in one step and the recovery and purification factor reaches 95.2% and 7.9, respectively.

  14. Identification of protein/target molecule interactions using yeast surface-displayed cDNA libraries

    PubMed Central

    Bidlingmaier, Scott; Liu, Bin

    2011-01-01

    We describe a novel expression cloning method based on screening yeast surface-displayed human cDNA libraries by direct affinity interaction to identify cellular proteins binding to a broad spectrum of target molecules. Being a eukaryote, yeast protein expression pathways are similar to those found in mammalian cells, and therefore mammalian protein fragments displayed on the yeast cell wall are more likely to be properly folded and functional than proteins displayed using prokaryotic systems. Yeast surface displayed human cDNA libraries have been successfully used to screen for proteins that bind to post-translationally modified phosphorylated peptides, small signaling molecule phosphatidylinositides, and monoclonal antibodies. In this article we describe protocols for using yeast surface-displayed cDNA libraries, coupled with fluorescence-activated cell sorting (FACS), to select protein fragments with affinity for various target molecules including post-translationally modified peptides, small signaling molecules, monoclonal phage antibodies, and monoclonal IgG molecules. PMID:21365493

  15. Sensitized anti-Stokes luminescence centers in microcrystals of Zn0.6Cd0.4S solid solutions with adsorbed dye molecules and few-atomic silver clusters

    NASA Astrophysics Data System (ADS)

    Ovchinnikov, O. V.; Kosyakova, E. A.; Leonova, L. Yu.; Smirnov, M. S.; Evlev, A. B.; Latyshev, A. N.; Utekhin, A. N.

    2008-03-01

    The sensitized anti-Stokes luminescence excited by radiation with wavelengths from 610 to 750 nm and flux densities of 1014 1015 quanta/(cm2·s) is detected for microcrystals of Zn 0.6 Cd 0.4 S solid solutions with adsorbed organic malachite green and methylene blue dye molecules. The position of its excitation spectra coincides with that of the absorption spectra of adsorbed dye molecules, which suggests the cooperative mechanism of its occurrence. The possibility of amplification of the anti-Stokes luminescence by means of adsorption of silver atoms and few-atomic silver clusters, in addition to the dye molecules, on the Zn 0.6 Cd 0.4 S surface is investigated. It is assumed that in the latter case, the anti-Stokes luminescence is excited as a result of two-quantum optical transitions with electron or electron excitation energy transfer from the dye molecules adsorbed on the Zn 0.6 Cd 0.4 S surface to silver atoms and few-atomic silver clusters creating deep local states with photoionization energies of 1.8 2.0 eV in the gap.

  16. Effects of positively charged redox molecules on disulfide-coupled protein folding.

    PubMed

    Okumura, Masaki; Shimamoto, Shigeru; Nakanishi, Takeyoshi; Yoshida, Yu-ichiro; Konogami, Tadafumi; Maeda, Shogo; Hidaka, Yuji

    2012-11-01

    In vitro folding of disulfide-containing proteins is generally regulated by redox molecules, such as glutathione. However, the role of the cross-disulfide-linked species formed between the redox molecule and the protein as a folding intermediate in the folding mechanism is poorly understood. In the present study, we investigated the effect of the charge on a redox molecule on disulfide-coupled protein folding. Several types of aliphatic thiol compounds including glutathione were examined for the folding of disulfide-containing-proteins, such as lysozyme and prouroguanylin. The results indicate that the positive charge and its dispersion play a critical role in accelerating disulfide-coupled protein folding.

  17. Quantitative proteomics analysis of adsorbed plasma proteins classifies nanoparticles with different surface properties and size

    SciTech Connect

    Zhang, Haizhen; Burnum, Kristin E.; Luna, Maria L.; Petritis, Brianne O.; Kim, Jong Seo; Qian, Weijun; Moore, Ronald J.; Heredia-Langner, Alejandro; Webb-Robertson, Bobbie-Jo M.; Thrall, Brian D.; Camp, David G.; Smith, Richard D.; Pounds, Joel G.; Liu, Tao

    2011-12-01

    In biofluids (e.g., blood plasma) nanoparticles are readily embedded in layers of proteins that can affect their biological activity and biocompatibility. Herein, we report a study on the interactions between human plasma proteins and nanoparticles with a controlled systematic variation of properties using stable isotope labeling and liquid chromatography-mass spectrometry (LC-MS) based quantitative proteomics. Novel protocol has been developed to simplify the isolation of nanoparticle bound proteins and improve the reproducibility. Plasma proteins associated with polystyrene nanoparticles with three different surface chemistries and two sizes as well as for four different exposure times (for a total of 24 different samples) were identified and quantified by LC-MS analysis. Quantitative comparison of relative protein abundances were achieved by spiking an 18 O-labeled 'universal reference' into each individually processed unlabeled sample as an internal standard, enabling simultaneous application of both label-free and isotopic labeling quantitation across the sample set. Clustering analysis of the quantitative proteomics data resulted in distinctive pattern that classifies the nanoparticles based on their surface properties and size. In addition, data on the temporal study indicated that the stable protein 'corona' that was isolated for the quantitative analysis appeared to be formed in less than 5 minutes. The comprehensive results obtained herein using quantitative proteomics have potential implications towards predicting nanoparticle biocompatibility.

  18. Antidiabetic effects of glucokinase regulatory protein small-molecule disruptors

    NASA Astrophysics Data System (ADS)

    Lloyd, David J.; St Jean, David J.; Kurzeja, Robert J. M.; Wahl, Robert C.; Michelsen, Klaus; Cupples, Rod; Chen, Michelle; Wu, John; Sivits, Glenn; Helmering, Joan; Komorowski, Renée; Ashton, Kate S.; Pennington, Lewis D.; Fotsch, Christopher; Vazir, Mukta; Chen, Kui; Chmait, Samer; Zhang, Jiandong; Liu, Longbin; Norman, Mark H.; Andrews, Kristin L.; Bartberger, Michael D.; van, Gwyneth; Galbreath, Elizabeth J.; Vonderfecht, Steven L.; Wang, Minghan; Jordan, Steven R.; Véniant, Murielle M.; Hale, Clarence

    2013-12-01

    Glucose homeostasis is a vital and complex process, and its disruption can cause hyperglycaemia and type II diabetes mellitus. Glucokinase (GK), a key enzyme that regulates glucose homeostasis, converts glucose to glucose-6-phosphate in pancreatic β-cells, liver hepatocytes, specific hypothalamic neurons, and gut enterocytes. In hepatocytes, GK regulates glucose uptake and glycogen synthesis, suppresses glucose production, and is subject to the endogenous inhibitor GK regulatory protein (GKRP). During fasting, GKRP binds, inactivates and sequesters GK in the nucleus, which removes GK from the gluconeogenic process and prevents a futile cycle of glucose phosphorylation. Compounds that directly hyperactivate GK (GK activators) lower blood glucose levels and are being evaluated clinically as potential therapeutics for the treatment of type II diabetes mellitus. However, initial reports indicate that an increased risk of hypoglycaemia is associated with some GK activators. To mitigate the risk of hypoglycaemia, we sought to increase GK activity by blocking GKRP. Here we describe the identification of two potent small-molecule GK-GKRP disruptors (AMG-1694 and AMG-3969) that normalized blood glucose levels in several rodent models of diabetes. These compounds potently reversed the inhibitory effect of GKRP on GK activity and promoted GK translocation both in vitro (isolated hepatocytes) and in vivo (liver). A co-crystal structure of full-length human GKRP in complex with AMG-1694 revealed a previously unknown binding pocket in GKRP distinct from that of the phosphofructose-binding site. Furthermore, with AMG-1694 and AMG-3969 (but not GK activators), blood glucose lowering was restricted to diabetic and not normoglycaemic animals. These findings exploit a new cellular mechanism for lowering blood glucose levels with reduced potential for hypoglycaemic risk in patients with type II diabetes mellitus.

  19. Mechanical design of proteins studied by single-molecule force spectroscopy and protein engineering.

    PubMed

    Carrion-Vazquez, M; Oberhauser, A F; Fisher, T E; Marszalek, P E; Li, H; Fernandez, J M

    2000-01-01

    Mechanical unfolding and refolding may regulate the molecular elasticity of modular proteins with mechanical functions. The development of the atomic force microscopy (AFM) has recently enabled the dynamic measurement of these processes at the single-molecule level. Protein engineering techniques allow the construction of homomeric polyproteins for the precise analysis of the mechanical unfolding of single domains. alpha-Helical domains are mechanically compliant, whereas beta-sandwich domains, particularly those that resist unfolding with backbone hydrogen bonds between strands perpendicular to the applied force, are more stable and appear frequently in proteins subject to mechanical forces. The mechanical stability of a domain seems to be determined by its hydrogen bonding pattern and is correlated with its kinetic stability rather than its thermodynamic stability. Force spectroscopy using AFM promises to elucidate the dynamic mechanical properties of a wide variety of proteins at the single molecule level and provide an important complement to other structural and dynamic techniques (e.g., X-ray crystallography, NMR spectroscopy, patch-clamp). PMID:11106807

  20. X-ray Reflectivity Studies of Adsorbed Proteins on Langmuir Layers

    NASA Astrophysics Data System (ADS)

    Málková, Šárka; Pingali, Sai V.; Long, Fei; Cho, Wonhwa; Schlossman, Mark L.

    2002-03-01

    Synchrotron X-ray reflectivity is used to study the interaction of the C2 domain of cytosolic phospholipase A2 (cPLA_2-C2) with a phospholipid membrane. SOPC (1-steraoyl-2-oleoyl-sn-glycero-3-phosphocholine) monolayer has been chosen as our model membrane. SOPC monolayer is supported on a buffered, Ca^2+ containing aqueous solution. The original phospholipid layer, which can be described by a two layer model roughened by capillary wave theory, is modified and a three layer model is necessary to fit the data after the protein is injected. The data analysis indicates that a third layer attached to the phospholipid headgroup region is formed. This additional layer corresponds to proteins bound to the phospholipid. The mechanism of the protein binding to the lipid depends on the initial lipid pressure with adsorption being less pronounced at higher pressures. Two control experiments are performed. Buffer containing no Ca^2+ is used in the first and F35/L39A mutant of cPLA_2-C2 (the mutation is in the region containing ligands for multiple Ca^2+ ions) is used in the second. Reflectivity curves do not show any evidence of protein adsorption to the phospholipid monolayer in any of our two control experiments. Our results thus support the idea that the initial pressure of the lipid, Ca^2+ ions and Ca^2+ binding sites of cPLA_2-C2 are important factors for the membrane binding of this protein.

  1. Characterisation of the Ru/MgF2 catalyst with adsorbed O2, NO, CO probe molecules by EPR and IR spectroscopy

    NASA Astrophysics Data System (ADS)

    Goslar, J.; Wojciechowska, M.; Zieliński, M.

    2006-07-01

    Electron paramagnetic resonance (EPR) and infrared (IR) spectroscopy were used to study the formation of ruthenium and adsorbed species appearing on the catalyst during O2, NO, and CO adsorption at room temperature on 1 wt% Ru/MgF2 catalysts prepared from Ru3(CO)12 . Both EPR and IR results provided clear evidence for the interaction between surface ruthenium and probe molecules. No EPR signals due to ruthenium (Ru) species were recorded at 300 and 77 K after H2-reduction of the catalyst at 673 K. However, at 4.2 K a very weak EPR spectrum due to low-spin (4d5) Ru3+ complexes was detected. A weak anisotropic O2 radicals signal with g∣∣=2.017 and g⊥=2.003 superimposed on a broad (ΔBpp=120 mT), slightly asymmetric line at g=2.45(1) was identified after O2 admission to the reduced sample. Adsorption of NO gives only a broad, Gaussian-shaped EPR line at g=2.43(1) indicating that the admission of NO, similarly to O2 adsorption, brings about an oxidation of Ru species in the course of the NO decomposition reaction. Introduction of NO over the CO preadsorbed catalyst leads to EPR spectrum with parameters g⊥=1.996, g∣∣=1.895, and A⊥N=2.9 mT assigned to surface NO species associated with Ru ions. The IR spectra recorded after adsorption of NO or CO probe molecules showed the bands in the range of frequency characteristic of ruthenium nitrosyl, nitro, and nitrate/nitrite species and the bands characteristic of ruthenium mono-and multicarbonyls, respectively. Addition of CO after NO admission to the catalyst leads to appearance in the IR spectrum, beside the ones characteristic of NO adsorption, the bands which can be attributed to Ru CO2 and Ru NCO species, indicating that the reaction between NO and CO occurs. These species were also detected after CO adsorption followed by NO adsorption, additionally to the band at 1850 cm-1 being due to cis type ▪ species.

  2. Small Molecule Control of Intracellular Protein Levels Through Modulation of the Ubiquitin Proteasome System

    PubMed Central

    Buckley, Dennis L.

    2015-01-01

    Traditionally, biological probes and drugs have targeted the activities of proteins (such as enzymes and receptors) that can be easily controlled by small molecules. The remaining majority of the proteome has been deemed “undruggable”. By using small molecule modulators of the ubiquitin proteasome, protein levels, rather than protein activities can be targeted instead, increasing the number of druggable targets. While targeting the proteasome itself can lead to a global increase in protein levels, targeting other components of the UPS (e.g., the hundreds of E3 ubiquitin ligases) can lead to an increase in protein levels in a more targeted fashion. Alternatively, multiple strategies for inducing protein degradation with small molecule probes are emerging. With the ability to induce and inhibit the degradation of targeted proteins, small molecule modulators of the UPS have the potential to significantly expand the druggable portion of the proteome beyond traditional targets such as enzymes and receptors. PMID:24459094

  3. Single-molecule pull-down for investigating protein-nucleic acid interactions.

    PubMed

    Fareh, Mohamed; Loeff, Luuk; Szczepaniak, Malwina; Haagsma, Anna C; Yeom, Kyu-Hyeon; Joo, Chirlmin

    2016-08-01

    The genome and transcriptome are constantly modified by proteins in the cell. Recent advances in single-molecule techniques allow for high spatial and temporal observations of these interactions between proteins and nucleic acids. However, due to the difficulty of obtaining functional protein complexes, it remains challenging to study the interactions between macromolecular protein complexes and nucleic acids. Here, we combined single-molecule fluorescence with various protein complex pull-down techniques to determine the function and stoichiometry of ribonucleoprotein complexes. Through the use of three examples of protein complexes from eukaryotic cells (Drosha, Dicer, and TUT4 protein complexes), we provide step-by-step guidance for using novel single-molecule techniques. Our single-molecule methods provide sub-second and nanometer resolution and can be applied to other nucleoprotein complexes that are essential for cellular processes. PMID:27017911

  4. A threading approach to protein structure prediction: Studies on TNF-like molecules, Rev proteins, and protein kinases

    NASA Astrophysics Data System (ADS)

    Ihm, Yungok

    The main focus of this dissertation is the application of the threading approach to specific biological problems. The threading scheme developed in our group targets incorporating important structural features necessary for detecting structural similarity between the target sequence and the template structure. This enables us to use our threading method to solve problems for which sequence-based methods are not very much useful. We applied our threading method to predict the three-dimensional structures of lentivirus (EIAV, HIV-1, FIV, SIV) Rev proteins. Predicted structures of Rev proteins suggest that they share a structural similarity among themselves (four-helix bundle). Also, the threading approach has been utilized for screening for potential TNF-like molecules in Arabidopsis. The threading approach identified 35 potential TNF-like proteins in Arabidopsis, six of which are particularly interesting to be tested for the receptor kinase ligand activity. Threading method has also been used to identify potentially new protein kinases, which are not included in the protein kinase data base of C. elegans and Arabidopis. We identified eleven potentially new protein kinases and an additional protein worth investigating for protein kinase activity in C. elegans. Further, we identified ten potentially new protein kinases and additional four proteins worth investigating for the protein kinase activity in Arabidopsis.

  5. Colloid-probe AFM studies of the interaction forces of proteins adsorbed on colloidal crystals.

    PubMed

    Singh, Gurvinder; Bremmell, Kristen E; Griesser, Hans J; Kingshott, Peter

    2015-04-28

    In recent years, colloid-probe AFM has been used to measure the direct interaction forces between colloidal particles of different size or surface functionality in aqueous media, as one can study different forces in symmerical systems (i.e., sphere-sphere geometry). The present study investigates the interaction between protein coatings on colloid probes and hydrophilic surfaces decorated with hexagonally close packed single particle layers that are either uncoated or coated with proteins. Controlled solvent evaporation from aqueous suspensions of colloidal particles (coated with or without lysozyme and albumin) produces single layers of close-packed colloidal crystals over large areas on a solid support. The measurements have been carried out in an aqueous medium at different salt concentrations and pH values. The results show changes in the interaction forces as the surface charge of the unmodified or modified particles, and ionic strength or pH of the solution is altered. At high ionic strength or pH, electrostatic interactions are screened, and a strong repulsive force at short separation below 5 nm dominates, suggesting structural changes in the absorbed protein layer on the particles. We also study the force of adhesion, which decreases with an increment in the salt concentration, and the interaction between two different proteins indicating a repulsive interaction on approach and adhesion on retraction. PMID:25758979

  6. Small-molecule inhibitors of protein-protein interactions: progressing towards the reality

    PubMed Central

    Arkin, Michelle R.; Tang, Yinyan; Wells, James A.

    2014-01-01

    Summary The past twenty years have seen many advances in our understanding of protein-protein interactions (PPI) and how to target them with small-molecule therapeutics. In 2004, we reviewed some early successes; since then, potent inhibitors have been developed for diverse protein complexes, and compounds are now in clinical trials for six targets. Surprisingly, many of these PPI clinical candidates have efficiency metrics typical of ‘lead-like’ or ‘drug-like’ molecules and are orally available. Successful discovery efforts have integrated multiple disciplines and make use of all the modern tools of target-based discovery - structure, computation, screening, and biomarkers. PPI become progressively more challenging as the interfaces become more complex, i.e., as binding epitopes are displayed on primary, secondary, or tertiary structures. Here, we review the last ten years of progress, focusing on the properties of PPI inhibitors that have advanced to clinical trials and prospects for the future of PPI drug discovery. PMID:25237857

  7. Expanded bed adsorption/desorption of proteins with Streamline Direct CST I adsorbent.

    PubMed

    Li, Ping; Xiu, Guohua; Mata, Vera G; Grande, Carlos A; Rodrigues, Alirio E

    2006-08-20

    Streamline Direct CST I is a new type of ion exchanger with multi-modal functional groups, specially designed for an expanded bed adsorption (EBA) process, which can capture directly the proteins from the high ionic strength feedstocks with a high binding capacity. In this study, an experimental study is carried out for two-component proteins (BSA and myoglobin) competitive adsorption and desorption in an expanded bed packed with Streamline Direct CST I. Based on the measurements of the single- and two-component bovine serum albumin (BSA)/myoglobin adsorption isotherm on Streamline Direct CST I, the binding and elution conditions for the whole EBA process are selected; and then frontal analysis for a longer timescale and column displacement experiments in a fixed bed (XK16/20 column) are carried out to evaluate the two-component proteins (BSA and myoglobin) competitive adsorption and displacement on Streamline Direct CST I. Finally, the feasibility of capturing both BSA and myoglobin by an expanded bed packed with Streamline Direct CST I is addressed in a Streamline 50 column packed with 300 mL Streamline Direct CST I. PMID:16572450

  8. Restored physiology in protein-deficient yeast by a small molecule channel

    PubMed Central

    Cioffi, Alexander G.; Hou, Jennifer; Grillo, Anthony S.; Diaz, Katrina A.; Burke, Martin D.

    2015-01-01

    Deficiencies of protein ion channels underlie many currently incurable human diseases. Robust networks of pumps and channels are usually responsible for the directional movement of specific ions in organisms ranging from microbes to humans. We thus questioned whether minimally selective small molecule mimics of missing protein channels might be capable of collaborating with the corresponding protein ion pumps to restore physiology. Here we report vigorous and sustainable restoration of yeast cell growth by replacing missing protein ion channels with imperfect small molecule mimics. We further provide evidence that this tolerance for imperfect mimicry is attributable to collaboration between the channel-forming small molecule and protein ion pumps. These results illuminate a mechanistic framework for pursuing small molecule replacements for deficient protein ion channels that underlie a range of challenging human diseases. PMID:26230309

  9. Quantifying the Entropy of Binding for Water Molecules in Protein Cavities by Computing Correlations

    PubMed Central

    Huggins, David J.

    2015-01-01

    Protein structural analysis demonstrates that water molecules are commonly found in the internal cavities of proteins. Analysis of experimental data on the entropies of inorganic crystals suggests that the entropic cost of transferring such a water molecule to a protein cavity will not typically be greater than 7.0 cal/mol/K per water molecule, corresponding to a contribution of approximately +2.0 kcal/mol to the free energy. In this study, we employ the statistical mechanical method of inhomogeneous fluid solvation theory to quantify the enthalpic and entropic contributions of individual water molecules in 19 protein cavities across five different proteins. We utilize information theory to develop a rigorous estimate of the total two-particle entropy, yielding a complete framework to calculate hydration free energies. We show that predictions from inhomogeneous fluid solvation theory are in excellent agreement with predictions from free energy perturbation (FEP) and that these predictions are consistent with experimental estimates. However, the results suggest that water molecules in protein cavities containing charged residues may be subject to entropy changes that contribute more than +2.0 kcal/mol to the free energy. In all cases, these unfavorable entropy changes are predicted to be dominated by highly favorable enthalpy changes. These findings are relevant to the study of bridging water molecules at protein-protein interfaces as well as in complexes with cognate ligands and small-molecule inhibitors. PMID:25692597

  10. Structure-based design of small-molecule protein-protein interaction modulators: the story so far.

    PubMed

    Falchi, Federico; Caporuscio, Fabiana; Recanatini, Maurizio

    2014-03-01

    As the pivotal role of protein-protein interactions in cell growth, transcriptional activity, intracellular trafficking, signal transduction and pathological conditions has been assessed, experimental and in silico strategies have been developed to design protein-protein interaction modulators. State-of-the-art structure-based design methods, mainly pharmacophore modeling and docking, which have succeeded in the identification of enzyme inhibitors, receptor agonists and antagonists, and new tools specifically conceived to target protein-protein interfaces (e.g., hot-spot and druggable pocket prediction methods) have been applied in the search for small-molecule protein-protein interaction modulators. Many successful applications of structure-based design approaches that, despite the challenge of targeting protein-protein interfaces with small molecules, have led to the identification of micromolar and submicromolar hits are reviewed here.

  11. Atomic force microscopy compatible device for stretching cells and adsorbed proteins

    NASA Astrophysics Data System (ADS)

    De Jong, K. L.; MacLeod, H. C.; Norton, P. R.; Petersen, N. O.; Jasnin, M. F.

    2006-02-01

    A device that we term a "microrack" was designed to provide a means to stretch cells and proteins and to permit the measurement of any changes in adhesion forces that might occur as a result of the strain, with an atomic force microscope. The device requires an elastic material that allows adsorption of proteins and attachment of cells. The elastomer polydimethylsiloxane (PDMS) was chosen, and its suitability for short-term cell studies was tested by comparing cell morphology and fiber distribution on PDMS with cells grown on glass, a conventional substrate for cell study. Atomic force microscopy (AFM) images and section analysis of beads and scrape marks on the PDMS surface before and after stretching indicate that the microrack can provide up to 21%-29% deformation of PDMS. AFM images of cells grown on PDMS show that material attached to the surface is also affected by stretching of the microrack. The rupture of the cell after stretching and rippling of the cell under compression can be attributed to the fixation treatment, but indicates that the cell morphology is significantly affected by the movement of the substrate on the microrack.

  12. Adsorbent phosphates

    NASA Technical Reports Server (NTRS)

    Watanabe, S.

    1983-01-01

    An adsorbent which uses as its primary ingredient phosphoric acid salts of zirconium or titanium is presented. Production methods are discussed and several examples are detailed. Measurements of separating characteristics of some gases using the salts are given.

  13. Hide and seek: Identification and confirmation of small molecule protein targets.

    PubMed

    Ursu, Andrei; Waldmann, Herbert

    2015-08-15

    Target identification and confirmation for small molecules is often the rate limiting step in drug discovery. A robust method to identify proteins addressed by small molecules is affinity chromatography using chemical probes. These usually consist of the compound of interest equipped with a linker molecule and a proper tag. Recently, methods emerged that allow the identification of protein targets without prior functionalization of the small molecule of interest. The digest offers an update on the newest developments in the area of target identification with special focus on confirmation techniques. PMID:26115575

  14. Single Molecule and Collective Dynamics of Motor Protein Coupled with Mechano-Sensitive Chemical Reaction

    NASA Astrophysics Data System (ADS)

    Iwaki, Mitsuhiro; Marcucci, Lorenzo; Togashi, Yuichi; Yanagida, Toshio

    2013-12-01

    Motor proteins such as myosin and kinesin hydrolyze ATP into ADP and Pi to convert chemical energy into mechanical work. This resultsin various motile processes like muscle contraction, vesicle transport and cell division. Recent single molecule experiments have revealed that external load applied to these motor proteins perturb not only the mechanical motion, but the ATP hydrolysis cycle as well, making these molecules mechano-enzymes. Here, we describe our single molecule detection techniques to reveal the mechano-enzymatic properties of myosin and introduce recent progress from both experimental and theoretical approaches at the single- and multiple-molecule level.

  15. Extending the range of low energy electron diffraction (LEED) surface structure determination: Co-adsorbed molecules, incommensurate overlayers and alloy surface order studied by new video and electron counting LEED techniques

    SciTech Connect

    Ogletree, D.F.

    1986-11-01

    LEED multiple scattering theory is briefly summarized, and aspects of electron scattering with particular significance to experimental measurements such as electron beam coherence, instrument response and phonon scattering are analyzed. Diffuse LEED experiments are discussed. New techniques that enhance the power of LEED are described, including a real-time video image digitizer applied to LEED intensity measurements, along with computer programs to generate I-V curves. The first electron counting LEED detector using a ''wedge and strip'' position sensitive anode and digital electronics is described. This instrument uses picoampere incident beam currents, and its sensitivity is limited only by statistics and counting times. Structural results on new classes of surface systems are presented. The structure of the c(4 x 2) phase of carbon monoxide adsorbed on Pt(111) has been determined, showing that carbon monoxide molecules adsorb in both top and bridge sites, 1.85 +- 0.10 A and 1.55 +- 0.10 A above the metal surface, respectively. The structure of an incommensurate graphite overlayer on Pt(111) is analyzed. The graphite layer is 3.70 +- 0.05 A above the metal surface, with intercalated carbon atoms located 1.25 +- 0.10 A above hollow sites supporting it. The (2..sqrt..3 x 4)-rectangular phase of benzene and carbon monoxide coadsorbed on Pt(111) is analyzed. Benzene molecules adsorb in bridge sites parallel to and 2.10 +- 0.10 A above the surface. The carbon ring is expanded, with an average C-C bond length of 1.72 +- 0.15 A. The carbon monoxide molecules also adsorb in bridge sites. The structure of the (..sqrt..3 x ..sqrt..3) reconstruction on the (111) face of the ..cap alpha..-CuAl alloy has been determined.

  16. Single-Molecule Study of Proteins by Biological Nanopore Sensors

    PubMed Central

    Wu, Dongmei; Bi, Sheng; Zhang, Liyu; Yang, Jun

    2014-01-01

    Nanopore technology has been developed for detecting properties of proteins through monitoring of ionic current modulations as protein passes via a nanosize pore. As a real-time, sensitive, selective and stable technology, biological nanopores are of widespread concern. Here, we introduce the background of nanopore researches in the area of α-hemolysin (α-HL) nanopores in protein conformation detections and protein–ligand interactions. Moreover, several original biological nanopores are also introduced with various features and functions. PMID:25268917

  17. Green algae (Chlorella pyrenoidosa) adsorbs Bacillus thurigiensis (Bt) toxin, Cry1Ca insecticidal protein, without an effect on growth.

    PubMed

    Wang, Jiamei; Chen, Xiuping; Li, Yunhe; Su, Changqing; Ding, Jiatong; Peng, Yufa

    2014-08-01

    The effect of purified Cry1Ca insecticidal protein on the growth of Chlorella pyrenoidosa was studied in a three-generation toxicity test. The C. pyrenoidosa medium with a density of 5.4 × 10(5) cells/mL was subcultured for three generations with added Cry1Ca at 0, 10, 100, and 1000 µg/L, and cell numbers were determined daily. To explore the distribution of Cry1Ca in C. pyrenoidosa and the culture medium, Cry1Ca was added at 1000 µg/L to algae with a high density of 4.8 × 10(6) cells/mL, and Cry1Ca content was determined daily in C. pyrenoidosa and the culture medium by enzyme-linked immunosorbent assays. Our results showed that the growth curves of C. pyrenoidosa exposed to 10, 100, and 1000 µg/L of Cry1Ca almost overlapped with that of the blank control, and there were no statistically significant differences among the four treatments from day 0 to day 7, regardless of generation. Moreover, the Cry1Ca content in the culture medium and in C. pyrenoidosa sharply decreased under exposure of 1000 µg/L Cry1Ca with high initial C. pyrenoidosa cell density. The above results demonstrate that Cry1Ca in water can be rapidly adsorbed and degraded by C. pyrenoidosa, but it has no suppressive or stimulative effect on algae growth.

  18. Proton transfer via a transient linear water-molecule chain in a membrane protein

    PubMed Central

    Freier, Erik; Wolf, Steffen; Gerwert, Klaus

    2011-01-01

    High-resolution protein ground-state structures of proton pumps and channels have revealed internal protein-bound water molecules. Their possible active involvement in protein function has recently come into focus. An illustration of the formation of a protonated protein-bound water cluster that is actively involved in proton transfer was described for the membrane protein bacteriorhodopsin (bR) [Garczarek F, Gerwert K (2006) Nature 439:109–112]. Here we show through a combination of time-resolved FTIR spectroscopy and molecular dynamics simulations that three protein-bound water molecules are rearranged by a protein conformational change that resulted in a transient Grotthuss-type proton-transfer chain extending through a hydrophobic protein region of bR. This transient linear water chain facilitates proton transfer at an intermediate conformation only, thereby directing proton transfer within the protein. The rearrangement of protein-bound water molecules that we describe, from inactive positions in the ground state to an active chain in an intermediate state, appears to be energetically favored relative to transient incorporation of water molecules from the bulk. Our discovery provides insight into proton-transfer mechanisms through hydrophobic core regions of ubiquitous membrane spanning proteins such as G-protein coupled receptors or cytochrome C oxidases. PMID:21709261

  19. Accumulation of small protein molecules in a macroscopic complex coacervate.

    PubMed

    Lindhoud, Saskia; Claessens, Mireille M A E

    2016-01-14

    To obtain insight into the accumulation of proteins into macroscopic complex coacervate phases, the lysozyme concentration in complex coacervates containing the cationic polyelectrolyte poly-(N,N dimethylaminoethyl methacrylate) and the anionic polyelectrolyte polyacrylic acid was investigated as a function of the mixing ratio, protein concentration and ionic strength. Maximal protein enrichment of the complex coacervate phase was observed to require the presence of all three macromolecules. Under optimized conditions the protein concentrations in the complex coacervate were as high as 200 g L(-1). Such high concentrations are comparable to the protein concentration in the cytosol, suggesting that these interesting liquid phases may serve a suitable model system for the phase behavior of the cytosol and genesis and function of membrane-less organelles. The high stability of the complexes and the salt dependent uptake of protein suggest that complex coacervates may provide a way to store hydrated proteins at high concentrations and might therefore be of interest in the formulation of high protein foods.

  20. Kinetics of single DNA molecule denaturation by T4 Gene 32 protein

    NASA Astrophysics Data System (ADS)

    Pant, Kiran; Karpel, Richard L.; Williams, Mark C.

    2003-03-01

    Bacteriophage T4 gene 32 protein (32 protein) specifically binds single-stranded DNA, a property essential for its role in DNA replication, recombination, and repair. Although on a thermodynamic basis, single-stranded DNA binding proteins should lower the thermal melting temperature of double-stranded DNA (dsDNA), 32 protein does not. Using single molecule force spectroscopy, we show for the first time that 32 protein is capable of slowly destabilizing natural dsDNA. Direct measurements of single DNA molecule denaturation and renaturation kinetics in the presence of 32 protein and its proteolytic fragments reveal three types of kinetic behavior, attributable to specific protein structural domains, which regulate 32 protein's helix-destabilizing capabilities. This regulation is potentially biologically significant because uncontrolled helix-destabilization would be lethal to the cell. We also obtain equilibrium measurements of the DNA helix-coil transition free energy in the presence of these proteins for the first time.

  1. Small Molecule-Photoactive Yellow Protein Labeling Technology in Live Cell Imaging.

    PubMed

    Gao, Feng; Gao, Tang; Zhou, Kechao; Zeng, Wenbin

    2016-01-01

    Characterization of the chemical environment, movement, trafficking and interactions of proteins in live cells is essential to understanding their functions. Labeling protein with functional molecules is a widely used approach in protein research to elucidate the protein location and functions both in vitro and in live cells or in vivo. A peptide or a protein tag fused to the protein of interest and provides the opportunities for an attachment of small molecule probes or other fluorophore to image the dynamics of protein localization. Here we reviewed the recent development of no-wash small molecular probes for photoactive yellow protein (PYP-tag), by the means of utilizing a quenching mechanism based on the intramolecular interactions, or an environmental-sensitive fluorophore. Several fluorogenic probes have been developed, with fast labeling kinetics and cell permeability. This technology allows quick live-cell imaging of cell-surface and intracellular proteins without a wash-out procedure. PMID:27589715

  2. High-Throughput Single-Molecule Studies of Protein-DNA Interactions

    PubMed Central

    Robison, Aaron D.; Finkelstein, Ilya J.

    2014-01-01

    Fluorescence and force-based single-molecule studies of protein-nucleic acid interactions continue to shed critical insights into many aspects of DNA and RNA processing. As single-molecule assays are inherently low-throughput, obtaining statistically relevant datasets remains a major challenge. Additionally, most fluorescence-based single-molecule particle-tracking assays are limited to observing fluorescent proteins that are in the low-nanomolar range, as spurious background signals predominate at higher fluorophore concentrations. These technical limitations have traditionally limited the types of questions that could be addressed via single-molecule methods. In this review, we describe new approaches for high-throughput and high-concentration single-molecule biochemical studies. We conclude with a discussion of outstanding challenges for the single-molecule biologist and how these challenges can be tackled to further approach the biochemical complexity of the cell. PMID:24859086

  3. Single-molecule measurements of proteins using carbon nanotube field-effect transistors

    NASA Astrophysics Data System (ADS)

    Sims, Patrick Craig

    Single-walled carbon nanotube (SWCNT) field-effect transistors (FETs) provide a promising platform for investigating proteins at the single-molecule level. Recently, we have demonstrated that SWCNT FETs have sufficient sensitivity and bandwidth to monitor the conformational motions and processivity of an individual T4 lysozyme molecule. This is accomplished by functionalizing a SWCNT FET device with a single protein and measuring the conductance versus time through the device as it is submerged in an electrolyte solution. To generalize this approach for the study of a wide variety of proteins at the single-molecule level, this dissertation investigates the conjugation process to determine and isolate the key parameters involved in functionalizing a SWCNT with a single protein, the physical basis for transducing conformational motion of a protein into an electrical signal, and finally, the general application of the technique to monitor the binary and ternary complex formation of cAMP-dependent protein kinase (PKA).

  4. What are preferred water-aromatic interactions in proteins and crystal structures of small molecules?

    PubMed

    Janjić, Goran V; Malkov, Saša N; Zivković, Miodrag V; Zarić, Snežana D

    2014-11-21

    The distribution of water molecules around aromatic rings in the protein structures and crystal structures of small molecules shows quite a small number of the strongest OH-π interactions, a larger number of parallel interactions, and the largest number of the weakest CH-O interactions.

  5. Getting across the cell membrane: an overview for small molecules, peptides, and proteins.

    PubMed

    Yang, Nicole J; Hinner, Marlon J

    2015-01-01

    The ability to efficiently access cytosolic proteins is desired in both biological research and medicine. However, targeting intracellular proteins is often challenging, because to reach the cytosol, exogenous molecules must first traverse the cell membrane. This review provides a broad overview of how certain molecules are thought to cross this barrier, and what kinds of approaches are being made to enhance the intracellular delivery of those that are impermeable. We first discuss rules that govern the passive permeability of small molecules across the lipid membrane, and mechanisms of membrane transport that have evolved in nature for certain metabolites, peptides, and proteins. Then, we introduce design strategies that have emerged in the development of small molecules and peptides with improved permeability. Finally, intracellular delivery systems that have been engineered for protein payloads are surveyed. Viewpoints from varying disciplines have been brought together to provide a cohesive overview of how the membrane barrier is being overcome.

  6. Getting Across the Cell Membrane: An Overview for Small Molecules, Peptides, and Proteins

    PubMed Central

    Yang, Nicole J.; Hinner, Marlon J.

    2016-01-01

    The ability to efficiently access cytosolic proteins is desired in both biological research and medicine. However, targeting intracellular proteins is often challenging, because to reach the cytosol, exogenous molecules must first traverse the cell membrane. This review provides a broad overview of how certain molecules are thought to cross this barrier, and what kinds of approaches are being made to enhance the intracellular delivery of those that are impermeable. We first discuss rules that govern the passive permeability of small molecules across the lipid membrane, and mechanisms of membrane transport that have evolved in nature for certain metabolites, peptides, and proteins. Then, we introduce design strategies that have emerged in the development of small molecules and peptides with improved permeability. Finally, intracellular delivery systems that have been engineered for protein payloads are surveyed. Viewpoints from varying disciplines have been brought together to provide a cohesive overview of how the membrane barrier is being overcome. PMID:25560066

  7. Utilizing Yeast Surface Human Proteome Display Libraries to Identify Small Molecule-Protein Interactions.

    PubMed

    Bidlingmaier, Scott; Liu, Bin

    2015-01-01

    The identification of proteins that interact with small bioactive molecules is a critical but often difficult and time-consuming step in understanding cellular signaling pathways or molecular mechanisms of drug action. Numerous methods for identifying small molecule-interacting proteins have been developed and utilized, including affinity-based purification followed by mass spectrometry analysis, protein microarrays, phage display, and three-hybrid approaches. Although all these methods have been used successfully, there remains a need for additional techniques for analyzing small molecule-protein interactions. A promising method for identifying small molecule-protein interactions is affinity-based selection of yeast surface-displayed human proteome libraries. Large and diverse libraries displaying human protein fragments on the surface of yeast cells have been constructed and subjected to FACS-based enrichment followed by comprehensive exon microarray-based output analysis to identify protein fragments with affinity for small molecule ligands. In a recent example, a proteome-wide search has been successfully carried out to identify cellular proteins binding to the signaling lipids PtdIns(4,5)P2 and PtdIns(3,4,5)P3. Known phosphatidylinositide-binding proteins such as pleckstrin homology domains were identified, as well as many novel interactions. Intriguingly, many novel nuclear phosphatidylinositide-binding proteins were discovered. Although the existence of an independent pool of nuclear phosphatidylinositides has been known about for some time, their functions and mechanism of action remain obscure. Thus, the identification and subsequent study of nuclear phosphatidylinositide-binding proteins is expected to bring new insights to this important biological question. Based on the success with phosphatidylinositides, it is expected that the screening of yeast surface-displayed human proteome libraries will be of general use for the discovery of novel small

  8. [Unfolding chaperone as a prion protein relating molecule].

    PubMed

    Hachiya, Naomi S; Sakasegawa, Yuji; Kaneko, Kiyotoshi

    2003-11-01

    Prion protein exists in two different isoforms, a normal cellular isoform (PrPc) and an abnormal infectious isoform (PrPSc), the latter is a causative agent of prion disease such as mad cow disease and Creutzfeldt-Jakob disease. Amino acid sequences of PrPc and PrPSc are identical, but their conformations are rather different; PrPc rich in non beta-sheet vs. PrPSc rich in beta-sheet isoform. Since the two isoforms have quite different conformation, this host factor might be a molecular chaperone, which enables to override an energy barrier between PrPc and PrPSc. To examine the protein unfolding activities against collectively folded structure exist or not, we constructed an assay system and purified a novel molecular chaperone. Unfolding, from S. cerevisiae. Unfolding consists of oligomeric ring-like structure with the central cavity and has an ATP-dependent protein Unfoldingg activity with broad specificity in vitro, of which targets included PrP in beta-sheet form, alpha-synuclein, and A beta protein. We have also found that mouse neuroblastoma N2a cells contained the activity. Treatment of this factor with an ATP-hydrolyzing enzyme, apyrase, caused the decrease in its protein Unfoldingg activity. It was suggested that the purified protein probably formed homo-oligomer consisting of 4-5 subunits and its activity was ATP-dependent. PMID:15152473

  9. Mapping lipid and detergent molecules at the surface of membrane proteins.

    PubMed

    Cogdell, Richard J; Gardiner, Alastair T; Roszak, Aleksander W; Stončius, Sigitas; Kočovský, Pavel; Isaacs, Neil W

    2011-06-01

    Electron-density maps for the crystal structures of membrane proteins often show features suggesting binding of lipids and/or detergent molecules on the hydrophobic surface, but usually it is difficult to identify the bound molecules. In our studies, heavy-atom-labelled phospholipids and detergents have been used to unequivocally identify these binding sites at the surfaces of test membrane proteins, the reaction centres from Rhodobacter sphaeroides and Blastochloris viridis. The generality of this method is discussed in the present article.

  10. Atomic force microscopy study of the adsorption of protein molecules on transferred Langmuir monolayer

    SciTech Connect

    Gainutdinov, R. V. Tolstikhina, A. L.; Stepina, N. D.; Novikova, N. N.; Yur'eva, E. A.; Khripunov, A. K.

    2010-09-15

    Ordered protein films have been obtained by the adsorption of protein molecules on a Langmuir monolayer, which had previously formed on a silicon substrate, using the Langmuir-Blodgett and molecular self-organization methods. A mixture of cholesterol with dipalmitoylphosphatidylcholine (DPPC) and a polymer-cellulose acetopivalinate-were used as immobilization materials. Protein molecules (catalase and alkaline phosphatase) immobilized on solid substrates have been investigated by atomic force micros-copy. It was shown that the developed combined technique provides a deposition of homogeneous ultrathin protein films with a high degree of filling.

  11. Chemical methods for degradation of target proteins using designed light-activatable organic molecules.

    PubMed

    Tanimoto, Shuho; Takahashi, Daisuke; Toshima, Kazunobu

    2012-08-11

    Molecular design, chemical synthesis, and biological evaluation of several designed organic molecules, which target-selectively degrade proteins upon photo-irradiation, are introduced. The designed molecules for protein photo-degradation include 2-phenylquinoline-steroid hormone hybrids and porphyrin derivatives, both of which selectively photo-degrade estrogen receptor-α, and fullerene-sugar and -sulfonic acid hybrids, which selectively photo-degrade HIV-1 protease and amyloid β, respectively. The information will provide a novel and effective way to control specific functions of proteins, and contribute to the molecular design of novel protein photo-degrading agents, which should find wide application in chemistry, biology, and medicine. PMID:22739361

  12. Protein Adsorption in Three Dimensions

    PubMed Central

    Vogler, Erwin A.

    2011-01-01

    Recent experimental and theoretical work clarifying the physical chemistry of blood-protein adsorption from aqueous-buffer solution to various kinds of surfaces is reviewed and interpreted within the context of biomaterial applications, especially toward development of cardiovascular biomaterials. The importance of this subject in biomaterials surface science is emphasized by reducing the “protein-adsorption problem” to three core questions that require quantitative answer. An overview of the protein-adsorption literature identifies some of the sources of inconsistency among many investigators participating in more than five decades of focused research. A tutorial on the fundamental biophysical chemistry of protein adsorption sets the stage for a detailed discussion of the kinetics and thermodynamics of protein adsorption, including adsorption competition between two proteins for the same adsorbent immersed in a binary-protein mixture. Both kinetics and steady-state adsorption can be rationalized using a single interpretive paradigm asserting that protein molecules partition from solution into a three-dimensional (3D) interphase separating bulk solution from the physical-adsorbent surface. Adsorbed protein collects in one-or-more adsorbed layers, depending on protein size, solution concentration, and adsorbent surface energy (water wettability). The adsorption process begins with the hydration of an adsorbent surface brought into contact with an aqueous-protein solution. Surface hydration reactions instantaneously form a thin, pseudo-2D interface between the adsorbent and protein solution. Protein molecules rapidly diffuse into this newly-formed interface, creating a truly 3D interphase that inflates with arriving proteins and fills to capacity within milliseconds at mg/mL bulk-solution concentrations CB. This inflated interphase subsequently undergoes time-dependent (minutes-to-hours) decrease in volume VI by expulsion of either-or-both interphase water and

  13. Distinct effect of xenobiotics on the metal-binding properties of protein molecules.

    PubMed

    Novikova, Natalia; Kovalchuk, Mikhail; Stepina, Nina; Gaynutdinov, Radmir; Chukhrai, Elena; Yurieva, Eleonora

    2015-07-01

    The X-ray standing-wave method was applied to study the elemental composition and molecular organization of ordered protein films of alkaline phosphatase exposed to different xenobiotics (drug compounds, lead). Binding of metal ions from triply distilled water to protein molecules has been experimentally observed. Definite differences in the arrangement of impurity metal ions in the films have been established. The considerable enhancement of protein-metal interactions is attributed to partial rearrangement of the protein native structure, induced by xenobiotics.

  14. Sperm specific proteins-potential candidate molecules for fertility control.

    PubMed

    Suri, Anil

    2004-03-10

    The increase in population growth rate warrants the development of additional contraceptive methods that are widely acceptable, free from side effects and less expensive. Immunocontraception, and in particular the targeting of antibodies to gamete-specific antigens implicated in sperm egg binding and fertilization, offers an attractive approach to control fertility. The development of a contraceptive vaccine based on sperm antigen represents a promising approach to contraception. In mammals, fertilization is completed by the direct interaction of sperm and egg, a process mediated primarily by sperm surface proteins. Sperm have proteins that are unique, cell specific, immunogenic and accessible to antibodies. A few of the sperm specific proteins have been isolated and characterized. The antibodies raised against the sperm specific antigens have proved to be extremely effective at reducing sperm-egg interaction in vitro; fertility trials in sub-human primates would eventually prove the effectiveness of the sperm antigens in terms of contraceptive efficacy.

  15. Sperm specific proteins-potential candidate molecules for fertility control

    PubMed Central

    Suri, Anil

    2004-01-01

    The increase in population growth rate warrants the development of additional contraceptive methods that are widely acceptable, free from side effects and less expensive. Immunocontraception, and in particular the targeting of antibodies to gamete-specific antigens implicated in sperm egg binding and fertilization, offers an attractive approach to control fertility. The development of a contraceptive vaccine based on sperm antigen represents a promising approach to contraception. In mammals, fertilization is completed by the direct interaction of sperm and egg, a process mediated primarily by sperm surface proteins. Sperm have proteins that are unique, cell specific, immunogenic and accessible to antibodies. A few of the sperm specific proteins have been isolated and characterized. The antibodies raised against the sperm specific antigens have proved to be extremely effective at reducing sperm-egg interaction in vitro; fertility trials in sub-human primates would eventually prove the effectiveness of the sperm antigens in terms of contraceptive efficacy. PMID:15012833

  16. Single-Molecule Manipulation Studies of a Mechanically Activated Protein

    NASA Astrophysics Data System (ADS)

    Botello, Eric; Harris, Nolan; Choi, Huiwan; Bergeron, Angela; Dong, Jing-Fei; Kiang, Ching-Hwa

    2009-10-01

    Plasma von Willebrand factor (pVWF) is the largest multimeric adhesion ligand found in human blood and must be adhesively activated by exposure to shear stress, like at sites of vascular injury, to initiate blood clotting. Sheared pVWF (sVWF) will undergo a conformational change from a loose tangled coil to elongated strings forming adhesive fibers by binding with other sVWF. VWF's adhesion activity is also related to its length, with the ultra-large form of VWF (ULVWF) being hyper-actively adhesive without exposure to shear stress; it has also been shown to spontaneously form fibers. We used single molecule manipulation techniques with the AFM to stretch pVWF, sVWF and ULVWF and monitor the forces as a function of molecular extension. We showed a similar increase in resistance to unfolding for sVWF and ULVWF when compared to pVWF. This mechanical resistance to forced unfolding is reduced when other molecules known to disrupt their fibril formation are present. Our results show that sVWF and ULVWF domains unfold at higher forces than pVWF, which is consistent with the hypothesis that shear stress induces lateral association that alters adhesion activity of pVWF.

  17. Quasiparticle excitations of adsorbates on doped graphene

    NASA Astrophysics Data System (ADS)

    Lischner, Johannes; Wickenburg, Sebastian; Wong, Dillon; Karrasch, Christoph; Wang, Yang; Lu, Jiong; Omrani, Arash A.; Brar, Victor; Tsai, Hsin-Zon; Wu, Qiong; Corsetti, Fabiano; Mostofi, Arash; Kawakami, Roland K.; Moore, Joel; Zettl, Alex; Louie, Steven G.; Crommie, Mike

    Adsorbed atoms and molecules can modify the electronic structure of graphene, but in turn it is also possible to control the properties of adsorbates via the graphene substrate. In my talk, I will discuss the electronic structure of F4-TCNQ molecules on doped graphene and present a first-principles based theory of quasiparticle excitations that captures the interplay of doping-dependent image charge interactions between substrate and adsorbate and electron-electron interaction effects on the molecule. The resulting doping-dependent quasiparticle energies will be compared to experimental scanning tunnelling spectra. Finally, I will also discuss the effects of charged adsorbates on the electronic structure of doped graphene.

  18. Rheumatoid Rescue of Misfolded Cellular Proteins by MHC Class II Molecules: A New Hypothesis for Autoimmune Diseases.

    PubMed

    Arase, Hisashi

    2016-01-01

    Misfolded proteins localized in the endoplasmic reticulum are degraded promptly and thus are not transported outside cells. However, misfolded proteins in the endoplasmic reticulum are rescued from protein degradation upon association with major histocompatibility complex (MHC) class II molecules and are transported to the cell surface by MHC class II molecules without being processed to peptides. Studies on the misfolded proteins rescued by MHC class II molecules have revealed that misfolded proteins associated with MHC class II molecules are specific targets for autoantibodies produced in autoimmune diseases. Furthermore, a strong correlation has been observed between autoantibody binding to misfolded proteins associated with MHC class II molecules and the autoimmune disease susceptibility conferred by each MHC class II allele. These new insights into MHC class II molecules suggest that misfolded proteins rescued from protein degradation by MHC class II molecules are recognized as "neo-self" antigens by immune system and are involved in autoimmune diseases as autoantibody targets.

  19. Probing Protein Fluctuations, Folding and Misfolding at Single-molecule Resolution

    NASA Astrophysics Data System (ADS)

    Deniz, Ashok

    2010-03-01

    The conformational fluctuations and folding of proteins are key for their function in cells and organisms. Conversely, misfolding and aggregation can cause disease, although amyloids with functional significance are also being identified. To better understand these aspects of protein biophysics, we utilize single-molecule fluorescence and complementary methods to directly study complex protein dynamics, structural distributions, and conformational transitions. In one example, we used these methods to investigate disorder and disorder-to-order transitions in intrinsically disordered proteins (IDPs). IDPs are an interesting class of proteins which are relatively unstructured in isolation, but can often fold by interacting with binding partners. These complex systems are increasingly found to play major roles in biology and disease. In one case, we used a combination of single-molecule FRET (smFRET), coincidence and correlation analyses to probe the native structural features of a yeast protein Sup35, whose amyloid state is believed to be used in a beneficial context in yeast. We find that the monomeric protein populates a compact and rapidly fluctuating ensemble of conformations. In another case, we studied the binding-coupled folding of the IDP alpha-synuclein, whose misfolding and aggregation have been linked to Parkinson's disease. Single-molecule measurements directly revealed a complex multi-state folding landscape for this protein. Observations of a transient folding intermediate using microfluidic mixing, and links to misfolding and aggregation will also be discussed. Our results highlight single-molecule methodology that is broadly applicable to map protein folding and misfolding landscapes.

  20. Influence of surface chemistry on the structural organization of monomolecular protein layers adsorbed to functionalized aqueous interfaces.

    PubMed Central

    Lösche, M; Piepenstock, M; Diederich, A; Grünewald, T; Kjaer, K; Vaknin, D

    1993-01-01

    The molecular organization of streptavidin (SA) bound to aqueous surface monolayers of biotin-functionalized lipids and binary lipid mixtures has been investigated with neutron reflectivity and electron and fluorescence microscopy. The substitution of deuterons (2H) for protons (1H), both in subphase water molecules and in the alkyl chains of the lipid surface monolayer, was utilized to determine the interface structure on the molecular length scale. In all cases studied, the protein forms monomolecular layers underneath the interface with thickness values of approximately 40 A. A systematic dependence of the structural properties of such self-assembled SA monolayers on the surface chemistry was observed: the lateral protein density depends on the length of the spacer connecting the biotin moiety and its hydrophobic anchor. The hydration of the lipid head groups in the protein-bound state depends on the dipole moment density at the interface. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 5 FIGURE 11 FIGURE 12 FIGURE A1 PMID:8298041

  1. Single molecule surface enhanced resonance Raman scattering (SERRS) of the enhanced green fluorescent protein (EGFP)

    NASA Astrophysics Data System (ADS)

    Hofkens, Johan; De Schryver, Frans C.; Cotlet, Mircea; Habuchi, Satoshi

    2004-06-01

    One of the most intriguing findings in single molecule spectroscopy (SMS) is the observation of Raman spectra of individual molecules, despite the small cross section of the transitions involved. The observation of the spectra can be explained by the surface enhanced Raman scattering (SERRS) effect. At the single-molecule level, the SERRS-spectra recorded as a function of time reveal inhomogeneous behaviour such as on/off blinking, spectral diffusion, intensity fluctuations of vibrational line, and even splitting of some lines within the spectrum of one molecule. Single-molecule SERRS (SM-SERRS) spectroscopy opens up exciting opportunities in the field of biophysics and biomedical spectroscopy. The first example of single protein SERRS was performed on hemoglobin. However, the possibility of extracting the heme group by silver sols can not be excluded. Here we report on SM-SERRS spectra of enhanced green fluorescent protein (EGFP) in which the chromophore is kept in the protein. The time series of SM-SERRS spectra suggest the conversion of the EGFP chromophore between the deprotonated and the protonated form. Autocorrelation analysis of SM-SERRS trajectory reveals the presence of fast dynamics taking place in the protein. Our findings show the potential of the technique to study structural dynamics of protein molecules.

  2. Interaction between bound water molecules and local protein structures: A statistical analysis of the hydrogen bond structures around bound water molecules.

    PubMed

    Hong, Seungpyo; Kim, Dongsup

    2016-01-01

    Water molecules play an important role in protein folding and protein interactions through their structural association with proteins. Examples of such structural association can be found in protein crystal structures, and can often explain protein functionality in the context of structure. We herein report the systematic analysis of the local structures of proteins interacting with water molecules, and the characterization of their geometric features. We first examined the interaction of water molecules with a large local interaction environment by comparing the preference of water molecules in three regions, namely, the protein-protein interaction (PPI) interfaces, the crystal contact (CC) interfaces, and the non-interfacial regions. High preference of water molecules to the PPI and CC interfaces was found. In addition, the bound water on the PPI interface was more favorably associated with the complex interaction structure, implying that such water-mediated structures may participate in the shaping of the PPI interface. The pairwise water-mediated interaction was then investigated, and the water-mediated residue-residue interaction potential was derived. Subsequently, the types of polar atoms surrounding the water molecules were analyzed, and the preference of the hydrogen bond acceptor was observed. Furthermore, the geometries of the structures interacting with water were analyzed, and it was found that the major structure on the protein surface exhibited planar geometry rather than tetrahedral geometry. Several previously undiscovered characteristics of water-protein interactions were unfolded in this study, and are expected to lead to a better understanding of protein structure and function.

  3. Terminal supraparticle assemblies from similarly charged protein molecules and nanoparticles

    NASA Astrophysics Data System (ADS)

    Park, Jai Il; Nguyen, Trung Dac; de Queirós Silveira, Gleiciani; Bahng, Joong Hwan; Srivastava, Sudhanshu; Zhao, Gongpu; Sun, Kai; Zhang, Peijun; Glotzer, Sharon C.; Kotov, Nicholas A.

    2014-05-01

    Self-assembly of proteins and inorganic nanoparticles into terminal assemblies makes possible a large family of uniformly sized hybrid colloids. These particles can be compared in terms of utility, versatility and multifunctionality to other known types of terminal assemblies. They are simple to make and offer theoretical tools for designing their structure and function. To demonstrate such assemblies, we combine cadmium telluride nanoparticles with cytochrome C protein and observe spontaneous formation of spherical supraparticles with a narrow size distribution. Such self-limiting behaviour originates from the competition between electrostatic repulsion and non-covalent attractive interactions. Experimental variation of supraparticle diameters for several assembly conditions matches predictions obtained in simulations. Similar to micelles, supraparticles can incorporate other biological components as exemplified by incorporation of nitrate reductase. Tight packing of nanoscale components enables effective charge and exciton transport in supraparticles and bionic combination of properties as demonstrated by enzymatic nitrate reduction initiated by light absorption in the nanoparticle.

  4. Small Molecule-facilitated Degradation of ANO1 Protein

    PubMed Central

    Bill, Anke; Hall, Michelle Lynn; Borawski, Jason; Hodgson, Catherine; Jenkins, Jeremy; Piechon, Philippe; Popa, Oana; Rothwell, Christopher; Tranter, Pamela; Tria, Scott; Wagner, Trixie; Whitehead, Lewis; Gaither, L. Alex

    2014-01-01

    ANO1, a calcium-activated chloride channel, is highly expressed and amplified in human cancers and is a critical survival factor in these cancers. The ANO1 inhibitor CaCCinh-A01 decreases proliferation of ANO1-amplified cell lines; however, the mechanism of action remains elusive. We explored the mechanism behind the inhibitory effect of CaCCinh-A01 on cell proliferation using a combined experimental and in silico approach. We show that inhibition of ANO1 function is not sufficient to diminish proliferation of ANO1-dependent cancer cells. We report that CaCCinh-A01 reduces ANO1 protein levels by facilitating endoplasmic reticulum-associated, proteasomal turnover of ANO1. Washout of CaCCinh-A01 rescued ANO1 protein levels and resumed cell proliferation. Proliferation of newly derived CaCCinh-A01-resistant cell pools was not affected by CaCCinh-A01 as compared with the parental cells. Consistently, CaCCinh-A01 failed to reduce ANO1 protein levels in these cells, whereas ANO1 currents were still inhibited by CaCCinh-A01, indicating that CaCCinh-A01 inhibits cell proliferation by reducing ANO1 protein levels. Furthermore, we employed in silico methods to elucidate novel biological functions of ANO1 inhibitors. Specifically, we derived a pharmacophore model to describe inhibitors capable of promoting ANO1 degradation and report new inhibitors of ANO1-dependent cell proliferation. In summary, our data demonstrate that inhibition of the channel activity of ANO1 is not sufficient to inhibit ANO1-dependent cell proliferation, indicating that the role of ANO1 in cancer only partially depends on its function as a channel. Our results provide an impetus for gaining a deeper understanding of ANO1 modulation in cells and introduce a new targeting approach for antitumor therapy in ANO1-amplified cancers. PMID:24599954

  5. Allantoin as a solid phase adsorbent for removing endotoxins.

    PubMed

    Vagenende, Vincent; Ching, Tim-Jang; Chua, Rui-Jing; Gagnon, Pete

    2013-10-01

    In this study we present a simple and robust method for removing endotoxins from protein solutions by using crystals of the small-molecule compound 2,5-dioxo-4-imidazolidinyl urea (allantoin) as a solid phase adsorbent. Allantoin crystalline powder is added to a protein solution at supersaturated concentrations, endotoxins bind and undissolved allantoin crystals with bound endotoxins are removed by filtration or centrifugation. This method removes an average of 99.98% endotoxin for 20 test proteins. The average protein recovery is ∼80%. Endotoxin binding is largely independent of pH, conductivity, reducing agent and various organic solvents. This is consistent with a hydrogen-bond based binding mechanism. Allantoin does not affect protein activity and stability, and the use of allantoin as a solid phase adsorbent provides better endotoxin removal than anion exchange, polymixin affinity and biological affinity methods for endotoxin clearance.

  6. A Multi-technique Characterization of Adsorbed Protein Films: Orientation and Structure by ToF-SIMS, NEXAFS, SFG, and XPS

    NASA Astrophysics Data System (ADS)

    Baio, Joseph E.

    There are many techniques that allow surface scientists to study interfaces. However, few are routinely applied to probe biological surfaces. The work presented here demonstrates how detailed information about the conformation, orientation, chemical state, and molecular structure of biological molecules immobilized onto a surface can be assessed by electron spectroscopy, mass spectrometry, and nonlinear vibrational spectroscopy techniques. This investigation began with the development of simple model systems (small proteins, and peptides) and evolved into a study of more complex --- real world systems. Initially, two model systems based on the chemical and electrostatic immobilization of a small rigid protein (Protein G B1 domain, 6kDa) were built to develop the capabilities of time-of-flight secondary ion mass spectrometry (ToFSIMS), near edge X-ray absorption fine structure spectroscopy (NEXAFS) and sum frequency generation (SFG) spectroscopy as tools to probe the structure of surface immobilized proteins. X-ray photoelectron spectroscopy (XPS) was used to measure the amount of immobilized protein and ToF-SIMS sampled the amino acid composition of the exposed surface of the protein film. Within the ToF-SIMS spectra, an enrichment of secondary ions from amino acids located at opposite ends of the proteins were used to describe protein orientation. SFG spectral peaks characteristic of ordered alpha-helix and beta-sheet elements were observed for both systems and the phase of the peaks indicated a predominantly upright orientation for both the covalent and electrostatic configurations. Polarization dependence of the NEXAFS signal from the N 1s to pi* transition of the peptide bonds that make up the beta-sheets also indicated protein ordering at the surface. Building upon the Protein G B1 studies, the orientation and structure of a surface immobilized antibody (HuLys Fv: variant of humanized anti-lysozyme variable fragment, 26kDa) was characterized across two

  7. Screening of the Binding of Small Molecules to Proteins by Desorption Electrospray Ionization Mass Spectrometry Combined with Protein Microarray

    NASA Astrophysics Data System (ADS)

    Yao, Chenxi; Wang, Tao; Zhang, Buqing; He, Dacheng; Na, Na; Ouyang, Jin

    2015-11-01

    The interaction between bioactive small molecule ligands and proteins is one of the important research areas in proteomics. Herein, a simple and rapid method is established to screen small ligands that bind to proteins. We designed an agarose slide to immobilize different proteins. The protein microarrays were allowed to interact with different small ligands, and after washing, the microarrays were screened by desorption electrospray ionization mass spectrometry (DESI MS). This method can be applied to screen specific protein binding ligands and was shown for seven proteins and 34 known ligands for these proteins. In addition, a high-throughput screening was achieved, with the analysis requiring approximately 4 s for one sample spot. We then applied this method to determine the binding between the important protein matrix metalloproteinase-9 (MMP-9) and 88 small compounds. The molecular docking results confirmed the MS results, demonstrating that this method is suitable for the rapid and accurate screening of ligands binding to proteins.

  8. Lotus-leaf-like topography predominates over adsorbed ECM proteins in poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) surface/cell interactions.

    PubMed

    Zheng, Jun; Li, Dan; Yuan, Lin; Liu, Xiaoli; Chen, Hong

    2013-06-26

    It is well-known that extracellular matrix (ECM) proteins mediate cell/surface interactions. However, introduction of a specific surface topography may disturb the correlation between ECM proteins adsorption and cells adhesion on a given surface. In present study, lotus-leaf-like topography was introduced on the surface of a biodegradable material, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx). Protein adsorption and cell interactions with this lotus-leaf-like surface (designated PHBHHx-L) were investigated. Water contact angle data indicated that the hydrophobicity of PHBHHx was enhanced by the introduction of lotus-leaf-like topography. The adsorption of extracellular matrix proteins (fibronectin and vitronectin) on PHBHHx-L was measured by enzyme linked immunosorbent assay (ELISA). Compared with flat PHBHHx, adsorption on the PHBHHx-L surface increased by ~260% for fibronectin and ~40% for vitronectin. In contrast, fibroblast and endothelial cell adhesion and proliferation were reduced on the PHBHHx-L compared to the flat polymer surface. These results suggest that the inhibition of cell adhesion and proliferation caused by the lotus-leaf-like topography dominates over the effect of the adsorbed adhesive proteins in promoting adhesion and proliferation. It can be concluded that the lotus-leaf-like topography plays a dominant role in cell/PHBHHx-L interactions. The present findings indicate the complexity of the interplay among surface topography, adsorbed proteins, and cell-surface interactions.

  9. Single-molecule spectroscopy of protein conformational dynamics in live eukaryotic cells.

    PubMed

    König, Iwo; Zarrine-Afsar, Arash; Aznauryan, Mikayel; Soranno, Andrea; Wunderlich, Bengt; Dingfelder, Fabian; Stüber, Jakob C; Plückthun, Andreas; Nettels, Daniel; Schuler, Benjamin

    2015-08-01

    Single-molecule methods have become widely used for quantifying the conformational heterogeneity and structural dynamics of biomolecules in vitro. Their application in vivo, however, has remained challenging owing to shortcomings in the design and reproducible delivery of labeled molecules, the range of applicable analysis methods, and suboptimal cell culture conditions. By addressing these limitations in an integrated approach, we demonstrate the feasibility of probing protein dynamics from milliseconds down to the nanosecond regime in live eukaryotic cells with confocal single-molecule Förster resonance energy transfer (FRET) spectroscopy. We illustrate the versatility of the approach by determining the dimensions and submicrosecond chain dynamics of an intrinsically disordered protein; by detecting even subtle changes in the temperature dependence of protein stability, including in-cell cold denaturation; and by quantifying the folding dynamics of a small protein. The methodology opens possibilities for assessing the effect of the cellular environment on biomolecular conformation, dynamics and function.

  10. One, two or three? Probing the stoichiometry of membrane proteins by single-molecule localization microscopy

    PubMed Central

    Fricke, Franziska; Beaudouin, Joel; Eils, Roland; Heilemann, Mike

    2015-01-01

    Probing the oligomeric state of abundant molecules, such as membrane proteins in intact cells, is essential, but has not been straightforward. We address this challenge with a simple counting strategy that is capable of reporting the oligomeric state of dense, membrane-bound protein complexes. It is based on single-molecule localization microscopy to super-resolve protein structures in intact cells and basic quantitative evaluation. We validate our method with membrane-bound monomeric CD86 and dimeric cytotoxic T-lymphocyte-associated protein as model proteins and confirm their oligomeric states. We further detect oligomerization of CD80 and vesicular stomatitis virus glycoprotein and propose coexistence of monomers and dimers for CD80 and trimeric assembly of the viral protein at the cell membrane. This approach should prove valuable for researchers striving for reliable molecular counting in cells. PMID:26358640

  11. Analyte induced water adsorbability in gas phase biosensors: the influence of ethinylestradiol on the water binding protein capacity.

    PubMed

    Snopok, Borys; Kruglenko, Ivanna

    2015-05-01

    An ultra-sensitive gas phase biosensor/tracer/bio-sniffer is an emerging technology platform designed to provide real-time information on air-borne analytes, or those in liquids, through classical headspace analysis. The desired bio-sniffer measures gaseous 17α- ethinylestradiol (ETED) as frequency changes on a quartz crystal microbalance (QCM), which is a result of the interactions of liquid sample components in the headspace (ETED and water) with a biorecognition layer. The latter was constructed by immobilization of polyclonal antiserum against a phenolic A-ring of estrogenic receptors through protein A. The QCM response exhibited stretched exponential kinetics of negative frequency shifts with reversible and "irreversible" components of mass uptake onto the sensor surface in static headspace conditions when exposed to water solutions of ETED over the sensor working range, from 10(-10) to 10(-17) g L(-1). It was shown that the variations in the QCM response characteristics are due to the change of the water-binding capacity of the sensing layer induced by protein transformations initiated by the binding of ETED molecules. This result is well correlated with the natural physiological function of estrogens in controlling the homeostasis of body fluids in living beings. PMID:25763411

  12. Alternative modulation of protein–protein interactions by small molecules

    PubMed Central

    Fischer, Gerhard; Rossmann, Maxim; Hyvönen, Marko

    2015-01-01

    Protein–protein interactions (PPI) have become increasingly popular drug targets, with a number of promising compounds currently in clinical trials. Recent research shows, that PPIs can be modulated in more ways than direct inhibition, where novel non-competitive modes of action promise a solution for the difficult nature of PPI drug discovery. Here, we review recently discovered PPI modulators in light of their mode of action and categorise them as disrupting versus stabilising, orthosteric versus allosteric and by their ability to affect the proteins’ dynamics. We also give recent examples of compounds successful in the clinic, analyse their physicochemical properties and discuss how to overcome the hurdles in discovering alternative modes of modulation. PMID:25935873

  13. Discovery of Small Molecules that Inhibit the Disordered Protein, p27Kip1

    SciTech Connect

    Iconaru, Luigi I.; Ban, David; Bharatham, Kavitha; Ramanathan, Arvind; Zhang, Weixing; Shelat, Anang A.; Zuo, Jian; Kriwacki, Richard W.

    2015-10-28

    In disordered proteins we see that they are highly prevalent in biological systems. They control myriad signaling and regulatory processes, and their levels and/or cellular localization are often altered in human disease. In contrast to folded proteins, disordered proteins, due to conformational heterogeneity and dynamics, are not considered viable drug targets. We challenged this paradigm by identifying through NMR-based screening small molecules that bound specifically, albeit weakly, to the disordered cell cycle regulator, p27Kip1 (p27). Moreover, two groups of molecules bound to sites created by transient clusters of aromatic residues within p27. Conserved chemical features within these two groups of small molecules exhibited complementarity to their binding sites within p27, establishing structure-activity relationships for small molecule: disordered protein interactions. Finally, one compound counteracted the Cdk2/cyclin A inhibitory function of p27 in vitro, providing proof-of- principle that small molecules can inhibit the function of a disordered protein (p27) through sequestration in a conformation incapable of folding and binding to a natural regulatory target (Cdk2/cyclin A).

  14. DNA binding fluorescent proteins for the direct visualization of large DNA molecules

    PubMed Central

    Lee, Seonghyun; Oh, Yeeun; Lee, Jungyoon; Choe, Sojeong; Lim, Sangyong; Lee, Hyun Soo; Jo, Kyubong; Schwartz, David C.

    2016-01-01

    Fluorescent proteins that also bind DNA molecules are useful reagents for a broad range of biological applications because they can be optically localized and tracked within cells, or provide versatile labels for in vitro experiments. We report a novel design for a fluorescent, DNA-binding protein (FP-DBP) that completely ‘paints’ entire DNA molecules, whereby sequence-independent DNA binding is accomplished by linking a fluorescent protein to two small peptides (KWKWKKA) using lysine for binding to the DNA phosphates, and tryptophan for intercalating between DNA bases. Importantly, this ubiquitous binding motif enables fluorescent proteins (Kd = 14.7 μM) to confluently stain DNA molecules and such binding is reversible via pH shifts. These proteins offer useful robust advantages for single DNA molecule studies: lack of fluorophore mediated photocleavage and staining that does not perturb polymer contour lengths. Accordingly, we demonstrate confluent staining of naked DNA molecules presented within microfluidic devices, or localized within live bacterial cells. PMID:26264666

  15. Discovery of Small Molecules that Inhibit the Disordered Protein, p27Kip1

    DOE PAGES

    Iconaru, Luigi I.; Ban, David; Bharatham, Kavitha; Ramanathan, Arvind; Zhang, Weixing; Shelat, Anang A.; Zuo, Jian; Kriwacki, Richard W.

    2015-10-28

    In disordered proteins we see that they are highly prevalent in biological systems. They control myriad signaling and regulatory processes, and their levels and/or cellular localization are often altered in human disease. In contrast to folded proteins, disordered proteins, due to conformational heterogeneity and dynamics, are not considered viable drug targets. We challenged this paradigm by identifying through NMR-based screening small molecules that bound specifically, albeit weakly, to the disordered cell cycle regulator, p27Kip1 (p27). Moreover, two groups of molecules bound to sites created by transient clusters of aromatic residues within p27. Conserved chemical features within these two groups ofmore » small molecules exhibited complementarity to their binding sites within p27, establishing structure-activity relationships for small molecule: disordered protein interactions. Finally, one compound counteracted the Cdk2/cyclin A inhibitory function of p27 in vitro, providing proof-of- principle that small molecules can inhibit the function of a disordered protein (p27) through sequestration in a conformation incapable of folding and binding to a natural regulatory target (Cdk2/cyclin A).« less

  16. Multiplex single-molecule interaction profiling of DNA-barcoded proteins.

    PubMed

    Gu, Liangcai; Li, Chao; Aach, John; Hill, David E; Vidal, Marc; Church, George M

    2014-11-27

    In contrast with advances in massively parallel DNA sequencing, high-throughput protein analyses are often limited by ensemble measurements, individual analyte purification and hence compromised quality and cost-effectiveness. Single-molecule protein detection using optical methods is limited by the number of spectrally non-overlapping chromophores. Here we introduce a single-molecular-interaction sequencing (SMI-seq) technology for parallel protein interaction profiling leveraging single-molecule advantages. DNA barcodes are attached to proteins collectively via ribosome display or individually via enzymatic conjugation. Barcoded proteins are assayed en masse in aqueous solution and subsequently immobilized in a polyacrylamide thin film to construct a random single-molecule array, where barcoding DNAs are amplified into in situ polymerase colonies (polonies) and analysed by DNA sequencing. This method allows precise quantification of various proteins with a theoretical maximum array density of over one million polonies per square millimetre. Furthermore, protein interactions can be measured on the basis of the statistics of colocalized polonies arising from barcoding DNAs of interacting proteins. Two demanding applications, G-protein coupled receptor and antibody-binding profiling, are demonstrated. SMI-seq enables 'library versus library' screening in a one-pot assay, simultaneously interrogating molecular binding affinity and specificity.

  17. Revealing Two-State Protein-Protein Interaction of Calmodulin by Single-Molecule Spectroscopy

    SciTech Connect

    Liu, Ruchuan; Hu, Dehong; Tan, Xin; Lu, H PETER.

    2006-08-09

    We report a single-molecule fluorescence resonance energy transfer (FRET) and polarization study of conformational dynamics of calmodulin (CaM) interacting with a target peptide, C28W of 28 amino-acid oligomer. The C28W peptide represents the essential binding sequence domain of the Ca-ATPase protein interacting with CaM, which is important in cellular signaling for the regulation of energy in metabolism. However, the mechanism of the CaM-C28W recognition complex formation is still unclear. The amino-terminal (N-terminal) domain of the CaM was labeled with a fluorescein-based arsenical hairpin binder (F1AsH) that enables our unambiguously probing the CaM N-terminal target-binding domain motions at a millisecond timescale without convolution of the probe-dye random motions. By analyzing the distribution of FRET efficiency between F1AsH labeled CaM and Texas Red labeled C28W and the polarization fluctuation dynamics and distributions of the CaM N-terminal domain, we reveal slow (at sub-second time scale) binding-unbinding motions of the N-terminal domain of the CaM in CaM-C28W complexes, which is a strong evidence of a two-state binding interaction of CaM-mediated cell signaling.

  18. Revealing Two-State Protein-Protein Interactions of Calmodulin by Single-Molecule Spectroscopy

    SciTech Connect

    Liu, Ruchuan; Hu, Dehong; Tan, Xin; Lu, H. Peter

    2006-08-01

    We report a single-molecule fluorescence resonance energy transfer (FRET) and polarization study of conformational dynamics of calmodulin (CaM) interacting with a target peptide, C28W of a 28 amino acid oligomer. The C28W peptide represents the essential binding sequence domain of the Ca-ATPase protein interacting with CaM, which is important in cellular signaling for the regulation of energy in metabolism. However, the mechanism of the CaM/C28W recognition complex formation is still unclear. The amino-terminal (N-terminal) domain of the CaM was labeled with a fluorescein-based arsenical hairpin binder (FlAsH) that enables our unambiguous probing of the CaM N-terminal target-binding domain motions on a millisecond time scale without convolution of the probe-dye random motions. Finally, by analyzing the distribution of FRET efficiency between FlAsH labeled CaM and Texas Red labeled C28W and the polarization fluctuation dynamics and distributions of the CaM N-terminal domain, we reveal binding-unbinding motions of the N-terminal domain of the CaM in CaM/C28W complexes, which is strong evidence of a two-state binding interaction of CaM-mediated cell signaling.

  19. Different combinations of atomic interactions predict protein-small molecule and protein-DNA/RNA affinities with similar accuracy.

    PubMed

    Dias, Raquel; Kolazckowski, Bryan

    2015-11-01

    Interactions between proteins and other molecules play essential roles in all biological processes. Although it is widely held that a protein's ligand specificity is determined primarily by its three-dimensional structure, the general principles by which structure determines ligand binding remain poorly understood. Here we use statistical analyses of a large number of protein-ligand complexes with associated binding-affinity measurements to quantitatively characterize how combinations of atomic interactions contribute to ligand affinity. We find that there are significant differences in how atomic interactions determine ligand affinity for proteins that bind small chemical ligands, those that bind DNA/RNA and those that interact with other proteins. Although protein-small molecule and protein-DNA/RNA binding affinities can be accurately predicted from structural data, models predicting one type of interaction perform poorly on the others. Additionally, the particular combinations of atomic interactions required to predict binding affinity differed between small-molecule and DNA/RNA data sets, consistent with the conclusion that the structural bases determining ligand affinity differ among interaction types. In contrast to what we observed for small-molecule and DNA/RNA interactions, no statistical models were capable of predicting protein-protein affinity with >60% correlation. We demonstrate the potential usefulness of protein-DNA/RNA binding prediction as a possible tool for high-throughput virtual screening to guide laboratory investigations, suggesting that quantitative characterization of diverse molecular interactions may have practical applications as well as fundamentally advancing our understanding of how molecular structure translates into function.

  20. Binding of Solvent Molecules to a Protein Surface in Binary Mixtures Follows a Competitive Langmuir Model.

    PubMed

    Kulschewski, Tobias; Pleiss, Jürgen

    2016-09-01

    The binding of solvent molecules to a protein surface was modeled by molecular dynamics simulations of of Candida antarctica (C. antarctica) lipase B in binary mixtures of water, methanol, and toluene. Two models were analyzed: a competitive Langmuir model which assumes identical solvent binding sites with a different affinity toward water (KWat), methanol (KMet), and toluene (KTol) and a competitive Langmuir model with an additional interaction between free water and already bound water (KWatWat). The numbers of protein-bound molecules of both components of a binary mixture were determined for different compositions as a function of their thermodynamic activities in the bulk phase, and the binding constants were simultaneously fitted to the six binding curves (two components of three different mixtures). For both Langmuir models, the values of KWat, KMet, and KTol were highly correlated. The highest binding affinity was found for methanol, which was almost 4-fold higher than the binding affinities of water and toluene (KMet ≫ KWat ≈ KTol). Binding of water was dominated by the water-water interaction (KWatWat). Even for the three protein surface patches of highest water affinity, the binding affinity of methanol was 2-fold higher than water and 8-fold higher than toluene (KMet > KWat > KTol). The Langmuir model provides insights into the protein destabilizing mechanism of methanol which has a high binding affinity toward the protein surface. Thus, destabilizing solvents compete with intraprotein interactions and disrupt the tertiary structure. In contrast, benign solvents such as water or toluene have a low affinity toward the protein surface. Water is a special solvent: only few water molecules bind directly to the protein; most water molecules bind to already bound water molecules thus forming water patches. A quantitative mechanistic model of protein-solvent interactions that includes competition and miscibility of the components contributes a robust basis

  1. Quantum theory of IR spectroscopy of dipole-forbidden vibrational modes of adsorbed molecules on the surface of a metal in the frequency range of the anomalous skin effect

    SciTech Connect

    Volokitin, A.I.; Persson, B.N.J.

    1995-09-01

    A completely quantum-mechanical calculation of the IR spectrum for the dipole-forbidden vibrational modes of adsorbed molecules on a metal surface is performed. IT is shown for broad-band metals with a simple band structure that the asymmetry of the line shape is determined by nonlocal effects, while the nonadiabaticity makes a small contribution. In the region of the limiting anomalous skin effect ({omega}/{omega}{sub 1}{much_lt} 1, where {omega} is the frequency of the IR radiation, {omega}{sub 1}={upsilon}{sub F}/{delta}, {upsilon}{sub F} is the Fermi velocity, {delta}=c/{omega}{sub p} is the depth of the skin layer, and {omega}{sub p} is the plasma frequency) the broad-band absorption spectrum caused by the adsorbed molecules has an asymptotic limit {omega}{sup O}. The theory is compared with new absolute measurements of the IR spectrum of the CO/Cu(100) system. 20 refs., 2 figs.

  2. Measurement of Globular Protein Molecules by Electron Microscopy

    PubMed Central

    Hall, Cecil E.

    1960-01-01

    A series of molecular species with approximately spherical shape and with molecular weights between 35,000 and 250,000 were shadowed with platinum while resting on a cleaved mica surface. They were backed, stripped from the surface, and examined by electron microscopy. Materials examined were: pepsin, liver alcohol dehydrogenase, yeast alcohol dehydrogenase, glutamic dehydrogenase, polyhedral virus protein (insect), fibrinogen substructure, alkaline phosphatase, and microsomal particles from Escherichia coli. Measurements were made of widths perpendicular to the shadowing direction and heights were deduced from shadow lengths. For those molecular species with well established molecular weights the average heights correlate very well with the diameter of the theoretical sphere but the average widths are too great by 50 to 80 A due to the lateral growth of the deposited metal. Although the distortion in shape of shadowed particles is relatively large, with standardized conditions for shadowing, it is possible to make allowance for the distortion and to obtain reasonably reliable estimates of the dimensions of spherical organic particles down to a molecular weight of about 35,000. PMID:14399016

  3. Monitoring dynamic binding of chromatin proteins in vivo by single-molecule tracking.

    PubMed

    Mazza, Davide; Ganguly, Sourav; McNally, James G

    2013-01-01

    Single-molecule fluorescence microscopy has been used for decades to quantify macromolecular dynamics occurring in specimens that are in direct contact with a coverslip. This has permitted in vitro analysis of single-molecule motion in various biochemically reconstituted systems as well as in vivo studies of single-molecule motion on cell membranes. More recently, thanks to improvements in fluorescent tags and microscopes, it has been possible to follow individual molecules inside thicker specimens such as the nucleus of living cells. This has enabled a detailed description of the live-cell binding of nuclear proteins to DNA. In this protocol we describe a method to quantify intranuclear binding using single-molecule tracking (SMT). PMID:23980004

  4. TNF Superfamily Protein–Protein Interactions: Feasibility of Small-Molecule Modulation

    PubMed Central

    Song, Yun; Buchwald, Peter

    2015-01-01

    The tumor necrosis factor (TNF) superfamily (TNFSF) contains about thirty structurally related receptors (TNFSFRs) and about twenty protein ligands that bind to one or more of these receptors. Almost all of these cell surface protein-protein interactions (PPIs) represent high-value therapeutic targets for inflammatory or immune modulation in autoimmune diseases, transplant recipients, or cancers, and there are several biologics including antibodies and fusion proteins targeting them that are in various phases of clinical development. Small-molecule inhibitors or activators could represent possible alternatives if the difficulties related to the targeting of protein-protein interactions by small molecules can be addressed. Compounds proving the feasibility of such approaches have been identified through different drug discovery approaches for a number of these TNFSFR-TNFSF type PPIs including CD40-CD40L, BAFFR-BAFF, TRAIL-DR5, and OX40-OX40L. Corresponding structural, signaling, and medicinal chemistry aspects are briefly reviewed here. While none of these small-molecule modulators identified so far seems promising enough to be pursued for clinical development, they provide proof-of-principle evidence that these interactions are susceptible to small-molecule modulation and can serve as starting points toward the identification of more potent and selective candidates. PMID:25706111

  5. Quantitative Fluorescent Labeling of Aldehyde-Tagged Proteins for Single-Molecule Imaging

    PubMed Central

    Shi, Xinghua; Jung, Yonil; Lin, Li-Jung; Liu, Cheng; Wu, Cong; Cann, Isaac K. O.; Ha, Taekjip

    2012-01-01

    A major hurdle for molecular mechanistic studies of many proteins is the lack of a general method for fluorescent labeling with high efficiency, specificity, and speed. By incorporating an aldehyde motif genetically into a protein and improving the labeling kinetics substantially under mild conditions, we achieved fast, site-specific labeling of a protein with ~100% efficiency while maintaining the biological function. We demonstrate that an aldehyde-tagged protein can be specifically labeled in cell extracts without protein purification and then can be used in single-molecule pull-down analysis. We further show the unique power of our method in a series of single-molecule studies on the transient interactions and switching between two quantitatively labeled DNA polymerases on their processivity factor. PMID:22466795

  6. Stochastic protein expression in individual cells at the single molecule level

    NASA Astrophysics Data System (ADS)

    Cai, Long; Friedman, Nir; Xie, X. Sunney

    2006-03-01

    In a living cell, gene expression-the transcription of DNA to messenger RNA followed by translation to protein-occurs stochastically, as a consequence of the low copy number of DNA and mRNA molecules involved. These stochastic events of protein production are difficult to observe directly with measurements on large ensembles of cells owing to lack of synchronization among cells. Measurements so far on single cells lack the sensitivity to resolve individual events of protein production. Here we demonstrate a microfluidic-based assay that allows real-time observation of the expression of β-galactosidase in living Escherichia coli cells with single molecule sensitivity. We observe that protein production occurs in bursts, with the number of molecules per burst following an exponential distribution. We show that the two key parameters of protein expression-the burst size and frequency-can be either determined directly from real-time monitoring of protein production or extracted from a measurement of the steady-state copy number distribution in a population of cells. Application of this assay to probe gene expression in individual budding yeast and mouse embryonic stem cells demonstrates its generality. Many important proteins are expressed at low levels, and are thus inaccessible by current genomic and proteomic techniques. This microfluidic single cell assay opens up possibilities for system-wide characterization of the expression of these low copy number proteins.

  7. Quality control in single-molecule studies of kinesins and microtubule-associated proteins.

    PubMed

    Brouhard, Gary J

    2010-01-01

    Commercial microscopes capable of single-molecule experiments have made it simple for researchers to adopt these powerful techniques. This chapter is meant to help newcomers assess whether their data is of sufficient quality to warrant time-intensive analysis. Two problems can hamper single-molecule experiments: (1) non-specific aggregation of the proteins of interest and (2) detection thresholds from a poor microscope setup. I outline four steps that researchers can take to overcome these problems and convince themselves that they are observing bona fide single molecules.

  8. Can radiation damage to protein crystals be reduced using small-molecule compounds?

    SciTech Connect

    Kmetko, Jan; Warkentin, Matthew; Englich, Ulrich; Thorne, Robert E.

    2011-10-01

    Free-radical scavengers that are known to be effective protectors of proteins in solution are found to increase global radiation damage to protein crystals. Protective mechanisms may become deleterious in the protein-dense environment of a crystal. Recent studies have defined a data-collection protocol and a metric that provide a robust measure of global radiation damage to protein crystals. Using this protocol and metric, 19 small-molecule compounds (introduced either by cocrystallization or soaking) were evaluated for their ability to protect lysozyme crystals from radiation damage. The compounds were selected based upon their ability to interact with radiolytic products (e.g. hydrated electrons, hydrogen, hydroxyl and perhydroxyl radicals) and/or their efficacy in protecting biological molecules from radiation damage in dilute aqueous solutions. At room temperature, 12 compounds had no effect and six had a sensitizing effect on global damage. Only one compound, sodium nitrate, appeared to extend crystal lifetimes, but not in all proteins and only by a factor of two or less. No compound provided protection at T = 100 K. Scavengers are ineffective in protecting protein crystals from global damage because a large fraction of primary X-ray-induced excitations are generated in and/or directly attack the protein and because the ratio of scavenger molecules to protein molecules is too small to provide appreciable competitive protection. The same reactivity that makes some scavengers effective radioprotectors in protein solutions may explain their sensitizing effect in the protein-dense environment of a crystal. A more productive focus for future efforts may be to identify and eliminate sensitizing compounds from crystallization solutions.

  9. Water molecules inside protein structure affect binding of monosaccharides with HIV-1 antibody 2G12.

    PubMed

    Ueno-Noto, Kaori; Takano, Keiko

    2016-10-01

    Water molecules inside biomolecules constitute integral parts of their structure and participate in the functions of the proteins. Some of the X-ray crystallographic data are insufficient for analyzing a series of ligand-protein complexes in the same condition. We theoretically investigated antibody binding abilities of saccharide ligands and the effects of the inner water molecules of ligand-antibody complexes. Classical molecular dynamics and quantum chemical simulations using a model with possible water molecules inside the protein were performed with saccharide ligands and Human Immunodeficiency Virus 1 neutralizing antibody 2G12 complexes to estimate how inner water molecules of the protein affect the dynamics of the complexes as well as the ligand-antibody interaction. Our results indicate the fact that d-fructose's strong affinity to the antibody was partly due to the good retentiveness of solvent water molecules of the ligand and its stability of the ligand's conformation and relative position in the active site. © 2016 Wiley Periodicals, Inc.

  10. Water molecules inside protein structure affect binding of monosaccharides with HIV-1 antibody 2G12.

    PubMed

    Ueno-Noto, Kaori; Takano, Keiko

    2016-10-01

    Water molecules inside biomolecules constitute integral parts of their structure and participate in the functions of the proteins. Some of the X-ray crystallographic data are insufficient for analyzing a series of ligand-protein complexes in the same condition. We theoretically investigated antibody binding abilities of saccharide ligands and the effects of the inner water molecules of ligand-antibody complexes. Classical molecular dynamics and quantum chemical simulations using a model with possible water molecules inside the protein were performed with saccharide ligands and Human Immunodeficiency Virus 1 neutralizing antibody 2G12 complexes to estimate how inner water molecules of the protein affect the dynamics of the complexes as well as the ligand-antibody interaction. Our results indicate the fact that d-fructose's strong affinity to the antibody was partly due to the good retentiveness of solvent water molecules of the ligand and its stability of the ligand's conformation and relative position in the active site. © 2016 Wiley Periodicals, Inc. PMID:27388036

  11. Targeted adsorption of molecules in the colon with the novel adsorbent-based medicinal product, DAV132: A proof of concept study in healthy subjects.

    PubMed

    de Gunzburg, Jean; Ducher, Annie; Modess, Christiane; Wegner, Danilo; Oswald, Stefan; Dressman, Jennifer; Augustin, Violaine; Feger, Céline; Andremont, Antoine; Weitschies, Werner; Siegmund, Werner

    2015-01-01

    During antibiotic treatments, active residuals reaching the colon profoundly affect the bacterial flora resulting in the emergence of resistance. To prevent these effects, we developed an enteric-coated formulated activated-charcoal based product, DAV132, meant to deliver its adsorbent to the ileum and neutralize antibiotic residues in the proximal colon. In a randomized, control, crossover study, the plasma pharmacokinetics of the probe drugs amoxicillin (500 mg) absorbed in the proximal intestine, and sulfapyridine (25 mg) metabolized from sulfasalazine in the cecum and rapidly absorbed, were compared after a single administration in 18 healthy subjects who had received DAV132, uncoated formulated activated charcoal (FAC) or water 16 and 8 hours before, concomitantly with the probe drugs, and 8 hours thereafter. The AUC0-96 h of amoxicillin was reduced by more than 70% when it was taken with FAC, but bioequivalent when it was taken with water or DAV132. By contrast, the AUC0-96 h of sulfapyridine was reduced by more than 90% when administered with either FAC or DAV132 in comparison with water. The results show that DAV132 can selectively adsorb drug compounds in the proximal colon, without interfering with drug absorption in the proximal small intestine, thereby constituting a proof of concept that DAV132 actually functions in humans.

  12. Integrated system for temperature-controlled fast protein liquid chromatography comprising improved copolymer modified beaded agarose adsorbents and a travelling cooling zone reactor arrangement.

    PubMed

    Müller, Tobias K H; Cao, Ping; Ewert, Stephanie; Wohlgemuth, Jonas; Liu, Haiyang; Willett, Thomas C; Theodosiou, Eirini; Thomas, Owen R T; Franzreb, Matthias

    2013-04-12

    An integrated approach to temperature-controlled chromatography, involving copolymer modified agarose adsorbents and a novel travelling cooling zone reactor (TCZR) arrangement, is described. Sepharose CL6B was transformed into a thermoresponsive cation exchange adsorbent (thermoCEX) in four synthetic steps: (i) epichlorohydrin activation; (ii) amine capping; (iii) 4,4'-azobis(4-cyanovaleric acid) immobilization; and 'graft from' polymerization of poly(N-isopropylacrylamide-co-N-tert-butylacrylamide-co-acrylic acid-co-N,N'-methylenebisacrylamide). FT-IR, (1)H NMR, gravimetry and chemical assays allowed precise determination of the adsorbent's copolymer composition and loading, and identified the initial epoxy activation step as a critical determinant of 'on-support' copolymer loading, and in turn, protein binding performance. In batch binding studies with lactoferrin, thermoCEX's binding affinity and maximum adsorption capacity rose smoothly with temperature increase from 20 to 50 °C. In temperature shifting chromatography experiments employing thermoCEX in thermally jacketed columns, 44-51% of the lactoferrin adsorbed at 42 °C could be desorbed under binding conditions by cooling the column to 22 °C, but the elution peaks exhibited strong tailing. To more fully exploit the potential of thermoresponsive chromatography adsorbents, a new column arrangement, the TCZR, was developed. In TCZR chromatography, a narrow discrete cooling zone (special assembly of copper blocks and Peltier elements) is moved along a bespoke fixed-bed separation columnfilled with stationary phase. In tests with thermoCEX, it was possible to recover 65% of the lactoferrin bound at 35 °C using 8 successive movements of the cooling zone at a velocity of 0.1mm/s; over half of the recovered protein was eluted in the first peak in more concentrated form than in the feed. Intra-particle diffusion of desorbed protein out of the support pores, and the ratio between the velocities of the cooling

  13. Development of high-productivity, strong cation-exchange adsorbers for protein capture by graft polymerization from membranes with different pore sizes

    PubMed Central

    Chenette, Heather C.S.; Robinson, Julie R.; Hobley, Eboni; Husson, Scott M.

    2012-01-01

    This paper describes the surface modification of macroporous membranes using ATRP (atom transfer radical polymerization) to create cation-exchange adsorbers with high protein binding capacity at high product throughput. The work is motivated by the need for a more economical and rapid capture step in downstream processing of protein therapeutics. Membranes with three reported nominal pore sizes (0.2, 0.45, 1.0 μm) were modified with poly(3-sulfopropyl methacrylate, potassium salt) tentacles, to create a high density of protein binding sites. A special formulation was used in which the monomer was protected by a crown ether to enable surface-initiated ATRP of this cationic polyelectrolyte. Success with modification was supported by chemical analysis using Fourier-transform infrared spectroscopy and indirectly by measurement of pure water flux as a function of polymerization time. Uniformity of modification within the membranes was visualized with confocal laser scanning microscopy. Static and dynamic binding capacities were measured using lysozyme protein to allow comparisons with reported performance data for commercial cation-exchange materials. Dynamic binding capacities were measured for flow rates ranging from 13 to 109 column volumes (CV)/min. Results show that this unique ATRP formulation can be used to fabricate cation-exchange membrane adsorbers with dynamic binding capacities as high as 70 mg/mL at a throughput of 100 CV/min and unprecedented productivity of 300 mg/mL/min. PMID:23175597

  14. A general method for the covalent labeling of fusion proteins with small molecules in vivo.

    PubMed

    Keppler, Antje; Gendreizig, Susanne; Gronemeyer, Thomas; Pick, Horst; Vogel, Horst; Johnsson, Kai

    2003-01-01

    Characterizing the movement, interactions, and chemical microenvironment of a protein inside the living cell is crucial to a detailed understanding of its function. Most strategies aimed at realizing this objective are based on genetically fusing the protein of interest to a reporter protein that monitors changes in the environment of the coupled protein. Examples include fusions with fluorescent proteins, the yeast two-hybrid system, and split ubiquitin. However, these techniques have various limitations, and considerable effort is being devoted to specific labeling of proteins in vivo with small synthetic molecules capable of probing and modulating their function. These approaches are currently based on the noncovalent binding of a small molecule to a protein, the formation of stable complexes between biarsenical compounds and peptides containing cysteines, or the use of biotin acceptor domains. Here we describe a general method for the covalent labeling of fusion proteins in vivo that complements existing methods for noncovalent labeling of proteins and that may open up new ways of studying proteins in living cells.

  15. Highly Precise Quantification of Protein Molecules per Cell During Stress and Starvation Responses in Bacillus subtilis *

    PubMed Central

    Maaβ, Sandra; Wachlin, Gerhild; Bernhardt, Jörg; Eymann, Christine; Fromion, Vincent; Riedel, Katharina; Becher, Dörte; Hecker, Michael

    2014-01-01

    Systems biology based on high quality absolute quantification data, which are mandatory for the simulation of biological processes, successively becomes important for life sciences. We provide protein concentrations on the level of molecules per cell for more than 700 cytosolic proteins of the Gram-positive model bacterium Bacillus subtilis during adaptation to changing growth conditions. As glucose starvation and heat stress are typical challenges in B. subtilis' natural environment and induce both, specific and general stress and starvation proteins, these conditions were selected as models for starvation and stress responses. Analyzing samples from numerous time points along the bacterial growth curve yielded reliable and physiologically relevant data suitable for modeling of cellular regulation under altered growth conditions. The analysis of the adaptational processes based on protein molecules per cell revealed stress-specific modulation of general adaptive responses in terms of protein amount and proteome composition. Furthermore, analysis of protein repartition during glucose starvation showed that biomass seems to be redistributed from proteins involved in amino acid biosynthesis to enzymes of the central carbon metabolism. In contrast, during heat stress most resources of the cell, namely those from amino acid synthetic pathways, are used to increase the amount of chaperones and proteases. Analysis of dynamical aspects of protein synthesis during heat stress adaptation revealed, that these proteins make up almost 30% of the protein mass accumulated during early phases of this stress. PMID:24878497

  16. Wheat proteins enhance stability and function of adhesion molecules in cryopreserved hepatocytes.

    PubMed

    Grondin, Mélanie; Hamel, Francine; Averill-Bates, Diana A; Sarhan, Fathey

    2009-01-01

    Cryopreserved hepatocytes with good hepatospecific functions upon thawing are important for clinical transplantation and for in vitro drug toxicity testing. However, cryopreservation reduces viability and certain hepatospecific functions, but the most pronounced change is diminished attachment efficiency of hepatocytes. Adhesion of cells to the extracellular matrix and cell-cell contacts are crucial for many aspects of cellular function. These processes are partly mediated and controlled by cellular adhesion molecules. The mechanisms responsible for reduced attachment efficiency of cryopreserved hepatocytes are not well understood. To address this question, we investigated the effect of a new cryopreservation procedure, using wheat proteins (WPs) or mixtures of recombinant forms of wheat freezing tolerance-associated proteins, on the stability of three important adhesion molecules (beta1-integrin, E-cadherin, and beta-catenin). Immunoblot analyses revealed that the levels of beta1-integrin, E-cadherin, and beta-catenin were much lower in cryopreserved rat hepatocytes, when compared to fresh cells. Protein expression of the adhesion molecules was generally lower in cells cryopreserved with DMSO, compared to WPs. Moreover, the stability of the adhesion molecules was not affected by cryopreservation to the same degree, with more pronounced decreases occurring for beta1-integrin (62-74%) > beta-catenin (51-58%) > E-cadherin (21-37%). However, when hepatocytes were cryopreserved with partially purified WPs (SulWPE, AcWPE) or with mixtures of recombinant wheat proteins, there was a clear protective effect against the loss of protein expression of beta1-integrin, E-cadherin, and beta-catenin. Protein expression was only 10-20% lower than that observed in fresh hepatocytes. These findings clearly demonstrate that WPs, and more particularly, partially purified WPs and recombinant wheat proteins, were more efficient for cryopreservation of rat hepatocytes by maintaining good

  17. OPLS3: A Force Field Providing Broad Coverage of Drug-like Small Molecules and Proteins.

    PubMed

    Harder, Edward; Damm, Wolfgang; Maple, Jon; Wu, Chuanjie; Reboul, Mark; Xiang, Jin Yu; Wang, Lingle; Lupyan, Dmitry; Dahlgren, Markus K; Knight, Jennifer L; Kaus, Joseph W; Cerutti, David S; Krilov, Goran; Jorgensen, William L; Abel, Robert; Friesner, Richard A

    2016-01-12

    The parametrization and validation of the OPLS3 force field for small molecules and proteins are reported. Enhancements with respect to the previous version (OPLS2.1) include the addition of off-atom charge sites to represent halogen bonding and aryl nitrogen lone pairs as well as a complete refit of peptide dihedral parameters to better model the native structure of proteins. To adequately cover medicinal chemical space, OPLS3 employs over an order of magnitude more reference data and associated parameter types relative to other commonly used small molecule force fields (e.g., MMFF and OPLS_2005). As a consequence, OPLS3 achieves a high level of accuracy across performance benchmarks that assess small molecule conformational propensities and solvation. The newly fitted peptide dihedrals lead to significant improvements in the representation of secondary structure elements in simulated peptides and native structure stability over a number of proteins. Together, the improvements made to both the small molecule and protein force field lead to a high level of accuracy in predicting protein-ligand binding measured over a wide range of targets and ligands (less than 1 kcal/mol RMS error) representing a 30% improvement over earlier variants of the OPLS force field. PMID:26584231

  18. OPLS3: A Force Field Providing Broad Coverage of Drug-like Small Molecules and Proteins.

    PubMed

    Harder, Edward; Damm, Wolfgang; Maple, Jon; Wu, Chuanjie; Reboul, Mark; Xiang, Jin Yu; Wang, Lingle; Lupyan, Dmitry; Dahlgren, Markus K; Knight, Jennifer L; Kaus, Joseph W; Cerutti, David S; Krilov, Goran; Jorgensen, William L; Abel, Robert; Friesner, Richard A

    2016-01-12

    The parametrization and validation of the OPLS3 force field for small molecules and proteins are reported. Enhancements with respect to the previous version (OPLS2.1) include the addition of off-atom charge sites to represent halogen bonding and aryl nitrogen lone pairs as well as a complete refit of peptide dihedral parameters to better model the native structure of proteins. To adequately cover medicinal chemical space, OPLS3 employs over an order of magnitude more reference data and associated parameter types relative to other commonly used small molecule force fields (e.g., MMFF and OPLS_2005). As a consequence, OPLS3 achieves a high level of accuracy across performance benchmarks that assess small molecule conformational propensities and solvation. The newly fitted peptide dihedrals lead to significant improvements in the representation of secondary structure elements in simulated peptides and native structure stability over a number of proteins. Together, the improvements made to both the small molecule and protein force field lead to a high level of accuracy in predicting protein-ligand binding measured over a wide range of targets and ligands (less than 1 kcal/mol RMS error) representing a 30% improvement over earlier variants of the OPLS force field.

  19. Small molecule proteostasis regulators that reprogram the ER to reduce extracellular protein aggregation

    PubMed Central

    Plate, Lars; Cooley, Christina B; Chen, John J; Paxman, Ryan J; Gallagher, Ciara M; Madoux, Franck; Genereux, Joseph C; Dobbs, Wesley; Garza, Dan; Spicer, Timothy P; Scampavia, Louis; Brown, Steven J; Rosen, Hugh; Powers, Evan T; Walter, Peter; Hodder, Peter; Wiseman, R Luke; Kelly, Jeffery W

    2016-01-01

    Imbalances in endoplasmic reticulum (ER) proteostasis are associated with etiologically-diverse degenerative diseases linked to excessive extracellular protein misfolding and aggregation. Reprogramming of the ER proteostasis environment through genetic activation of the Unfolded Protein Response (UPR)-associated transcription factor ATF6 attenuates secretion and extracellular aggregation of amyloidogenic proteins. Here, we employed a screening approach that included complementary arm-specific UPR reporters and medium-throughput transcriptional profiling to identify non-toxic small molecules that phenocopy the ATF6-mediated reprogramming of the ER proteostasis environment. The ER reprogramming afforded by our molecules requires activation of endogenous ATF6 and occurs independent of global ER stress. Furthermore, our molecules phenocopy the ability of genetic ATF6 activation to selectively reduce secretion and extracellular aggregation of amyloidogenic proteins. These results show that small molecule-dependent ER reprogramming, achieved through preferential activation of the ATF6 transcriptional program, is a promising strategy to ameliorate imbalances in ER function associated with degenerative protein aggregation diseases. DOI: http://dx.doi.org/10.7554/eLife.15550.001 PMID:27435961

  20. Small molecule proteostasis regulators that reprogram the ER to reduce extracellular protein aggregation.

    PubMed

    Plate, Lars; Cooley, Christina B; Chen, John J; Paxman, Ryan J; Gallagher, Ciara M; Madoux, Franck; Genereux, Joseph C; Dobbs, Wesley; Garza, Dan; Spicer, Timothy P; Scampavia, Louis; Brown, Steven J; Rosen, Hugh; Powers, Evan T; Walter, Peter; Hodder, Peter; Wiseman, R Luke; Kelly, Jeffery W

    2016-01-01

    Imbalances in endoplasmic reticulum (ER) proteostasis are associated with etiologically-diverse degenerative diseases linked to excessive extracellular protein misfolding and aggregation. Reprogramming of the ER proteostasis environment through genetic activation of the Unfolded Protein Response (UPR)-associated transcription factor ATF6 attenuates secretion and extracellular aggregation of amyloidogenic proteins. Here, we employed a screening approach that included complementary arm-specific UPR reporters and medium-throughput transcriptional profiling to identify non-toxic small molecules that phenocopy the ATF6-mediated reprogramming of the ER proteostasis environment. The ER reprogramming afforded by our molecules requires activation of endogenous ATF6 and occurs independent of global ER stress. Furthermore, our molecules phenocopy the ability of genetic ATF6 activation to selectively reduce secretion and extracellular aggregation of amyloidogenic proteins. These results show that small molecule-dependent ER reprogramming, achieved through preferential activation of the ATF6 transcriptional program, is a promising strategy to ameliorate imbalances in ER function associated with degenerative protein aggregation diseases. PMID:27435961

  1. Visualizing repetitive diffusion activity of double-strand RNA binding proteins by single molecule fluorescence assays.

    PubMed

    Koh, Hye Ran; Wang, Xinlei; Myong, Sua

    2016-08-01

    TRBP, one of double strand RNA binding proteins (dsRBPs), is an essential cofactor of Dicer in the RNA interference pathway. Previously we reported that TRBP exhibits repetitive diffusion activity on double strand (ds)RNA in an ATP independent manner. In the TRBP-Dicer complex, the diffusion mobility of TRBP facilitates Dicer-mediated RNA cleavage. Such repetitive diffusion of dsRBPs on a nucleic acid at the nanometer scale can be appropriately captured by several single molecule detection techniques. Here, we provide a step-by-step guide to four different single molecule fluorescence assays by which the diffusion activity of dsRBPs on dsRNA can be detected. One color assay, termed protein induced fluorescence enhancement enables detection of unlabeled protein binding and diffusion on a singly labeled RNA. Two-color Fluorescence Resonance Energy Transfer (FRET) in which labeled dsRBPs is applied to labeled RNA, allows for probing the motion of protein along the RNA axis. Three color FRET reports on the diffusion movement of dsRBPs from one to the other end of RNA. The single molecule pull down assay provides an opportunity to collect dsRBPs from mammalian cells and examine the protein-RNA interaction at single molecule platform. PMID:27012177

  2. Efficient Isothermal Titration Calorimetry Technique Identifies Direct Interaction of Small Molecule Inhibitors with the Target Protein.

    PubMed

    Gal, Maayan; Bloch, Itai; Shechter, Nelia; Romanenko, Olga; Shir, Ofer M

    2016-01-01

    Protein-protein interactions (PPI) play a critical role in regulating many cellular processes. Finding novel PPI inhibitors that interfere with specific binding of two proteins is considered a great challenge, mainly due to the complexity involved in characterizing multi-molecular systems and limited understanding of the physical principles governing PPIs. Here we show that the combination of virtual screening techniques, which are capable of filtering a large library of potential small molecule inhibitors, and a unique secondary screening by isothermal titration calorimetry, a label-free method capable of observing direct interactions, is an efficient tool for finding such an inhibitor. In this study we applied this strategy in a search for a small molecule capable of interfering with the interaction of the tumor-suppressor p53 and the E3-ligase MDM2. We virtually screened a library of 15 million small molecules that were filtered to a final set of 80 virtual hits. Our in vitro experimental assay, designed to validate the activity of mixtures of compounds by isothermal titration calorimetry, was used to identify an active molecule against MDM2. At the end of the process the small molecule (4S,7R)-4-(4-chlorophenyl)-5-hydroxy-2,7-dimethyl-N-(6-methylpyridin-2-yl)-4,6,7,8 tetrahydrIoquinoline-3-carboxamide was found to bind MDM2 with a dissociation constant of ~2 µM. Following the identification of this single bioactive compound, spectroscopic measurements were used to further characterize the interaction of the small molecule with the target protein. 2D NMR spectroscopy was used to map the binding region of the small molecule, and fluorescence polarization measurement confirmed that it indeed competes with p53.

  3. Protein Adsorption in Three Dimensions

    PubMed Central

    Vogler, Erwin A.

    2011-01-01

    Recent experimental and theoretical work clarifying the physical chemistry of blood-protein adsorption from aqueous-buffer solution to various kinds of surfaces is reviewed and interpreted within the context of biomaterial applications, especially toward development of cardiovascular biomaterials. The importance of this subject in biomaterials surface science is emphasized by reducing the “protein-adsorption problem” to three core questions that require quantitative answer. An overview of the protein-adsorption literature identifies some of the sources of inconsistency among many investigators participating in more than five decades of focused research. A tutorial on the fundamental biophysical chemistry of protein adsorption sets the stage for a detailed discussion of the kinetics and thermodynamics of protein adsorption, including adsorption competition between two proteins for the same adsorbent immersed in a binary-protein mixture. Both kinetics and steady-state adsorption can be rationalized using a single interpretive paradigm asserting that protein molecules partition from solution into a three-dimensional (3D) interphase separating bulk solution from the physical-adsorbent surface. Adsorbed protein collects in one-or-more adsorbed layers, depending on protein size, solution concentration, and adsorbent surface energy (water wettability). The adsorption process begins with the hydration of an adsorbent surface brought into contact with an aqueous-protein solution. Surface hydration reactions instantaneously form a thin, pseudo-2D interface between the adsorbent and protein solution. Protein molecules rapidly diffuse into this newly-formed interface, creating a truly 3D interphase that inflates with arriving proteins and fills to capacity within milliseconds at mg/mL bulk-solution concentrations CB. This inflated interphase subsequently undergoes time-dependent (minutes-to-hours) decrease in volume VI by expulsion of either-or-both interphase water and

  4. Multivariate Analysis of Side Effects of Drug Molecules Based on Knowledge of Protein Bindings and ProteinProtein Interactions.

    PubMed

    Hasegawa, Kiyoshi; Funatsu, Kimito

    2014-12-01

    Here, we examined the relationships between 969 side effects associated with 658 drugs and their 1368 human protein targets using our hybrid approaches. Firstly, L-shaped PLS (LPLS) was used to construct a multivariate model of side effects and protein bindings of drug molecules. LPLS is an extension of standard PLS regression, where, in addition to the response matrix Y and the regressor matrix X, an extra data matrix Z is constructed that summarizes the background information of X. X and Y are matrices comprising drugs-target proteins, and drugs-side effects, respectively. The Z matrix is the proteinprotein interaction data. From the loading plot of Y, we could identify two remarkable side effects (urinary incontinence and increased salivation) From the corresponding loading plot of X, the responsible protein targets causing each side effect could be estimated (sodium channels and gamma-aminobutyric acid (GABA) receptors). The loading plot of the Z matrix indicated that the GABA receptors interact with each other and they heavily influence the side effect of increased salivation. Secondly, Bayesian classifier methods were separately applied to the cases of the two side effects. That is, the Bayesian classifier method was used to classify drug molecules as binding or not binding to the responsible protein targets associated with each side effect. Using atom-coloring techniques, it was possible to estimate which fragments on the drug molecule might cause the specific side effects. This information is valuable for drug design to avoid specific side effects.

  5. Graft copolymer composed of cationic backbone and bottle brush-like side chains as a physically adsorbed coating for protein separation by capillary electrophoresis.

    PubMed

    Zhou, Dan; Xiang, Lina; Zeng, Rongju; Cao, Fuhu; Zhu, Xiaoxi; Wang, Yanmei

    2011-12-01

    To stabilize electroosmotic flow (EOF) and suppress protein adsorption onto the silica capillary inner wall, a cationic hydroxyethylcellulose-graft-poly (poly(ethylene glycol) methyl ether methacrylate) (cat-HEC-g-PPEGMA) graft copolymer composed of cationic backbone and bottle brush-like side chains was synthesized for the first time and used as a novel physically adsorbed coating for protein separation by capillary electrophoresis. Reversed (anodal) and very stable EOF was obtained in cat-HEC-g-PPEGMA-coated capillary at pH 2.2-7.8. The effects of degree of cationization, PEGMA grafting ratio, PEGMA molecular mass, and buffer pH on the separation of basic proteins were investigated. A systematic comparative study of protein separation in bare and HEC-coated capillaries and in cat-HEC-g-PPEGMA-coated capillary was also performed. The basic proteins can be well separated in cat-HEC-g-PPEGMA-coated capillary over the pH range of 2.8-6.8 with good repeatability and high separation efficiency, because the coating combines good protein-resistant property of bottle brush-like PPEGMA side chains with excellent coating ability of cat-HEC backbone. Besides its success in separation of basic proteins, the cat-HEC-g-PPEGMA coating was also superior in the fast separation of other protein samples, such as protein mixture, egg white, and saliva, which indicates that it is a promising coating for further proteomics analysis. PMID:22038787

  6. Kinetics of small molecule interactions with membrane proteins in single cells measured with mechanical amplification

    PubMed Central

    Guan, Yan; Shan, Xiaonan; Zhang, Fenni; Wang, Shaopeng; Chen, Hong-Yuan; Tao, Nongjian

    2015-01-01

    Measuring small molecule interactions with membrane proteins in single cells is critical for understanding many cellular processes and for screening drugs. However, developing such a capability has been a difficult challenge. We show that molecular interactions with membrane proteins induce a mechanical deformation in the cellular membrane, and real-time monitoring of the deformation with subnanometer resolution allows quantitative analysis of small molecule–membrane protein interaction kinetics in single cells. This new strategy provides mechanical amplification of small binding signals, making it possible to detect small molecule interactions with membrane proteins. This capability, together with spatial resolution, also allows the study of the heterogeneous nature of cells by analyzing the interaction kinetics variability between different cells and between different regions of a single cell. PMID:26601298

  7. Proteomic analysis of egg white heparin-binding proteins: towards the identification of natural antibacterial molecules

    PubMed Central

    Guyot, Nicolas; Labas, Valérie; Harichaux, Grégoire; Chessé, Magali; Poirier, Jean-Claude; Nys, Yves; Réhault-Godbert, Sophie

    2016-01-01

    The chicken egg resists most environmental microbes suggesting that it potentially contains efficient antimicrobial molecules. Considering that some heparin-binding proteins in mammals are antibacterial, we investigated the presence and the antimicrobial activity of heparin-binding proteins from chicken egg white. Mass spectrometry analysis of the proteins recovered after heparin-affinity chromatography, revealed 20 proteins, including known antimicrobial proteins (avidin, lysozyme, TENP, ovalbumin-related protein X and avian bêta-defensin 11). The antibacterial activity of three new egg candidates (vitelline membrane outer layer protein 1, beta-microseminoprotein-like (LOC101750704) and pleiotrophin) was demonstrated against Listeria monocytogenes and/or Salmonella enterica Enteritidis. We showed that all these molecules share the property to inhibit bacterial growth through their heparin-binding domains. However, vitelline membrane outer layer 1 has additional specific structural features that can contribute to its antimicrobial potential. Moreover, we identified potential supplementary effectors of innate immunity including mucin 5B, E-selectin ligand 1, whey acidic protein 3, peptidyl prolyl isomerase B and retinoic acid receptor responder protein 2. These data support the concept of using heparin affinity combined to mass spectrometry to obtain an overview of the various effectors of innate immunity composing biological milieus, and to identify novel antimicrobial candidates of interest in the race for alternatives to antibiotics. PMID:27294500

  8. Proteomic analysis of egg white heparin-binding proteins: towards the identification of natural antibacterial molecules.

    PubMed

    Guyot, Nicolas; Labas, Valérie; Harichaux, Grégoire; Chessé, Magali; Poirier, Jean-Claude; Nys, Yves; Réhault-Godbert, Sophie

    2016-01-01

    The chicken egg resists most environmental microbes suggesting that it potentially contains efficient antimicrobial molecules. Considering that some heparin-binding proteins in mammals are antibacterial, we investigated the presence and the antimicrobial activity of heparin-binding proteins from chicken egg white. Mass spectrometry analysis of the proteins recovered after heparin-affinity chromatography, revealed 20 proteins, including known antimicrobial proteins (avidin, lysozyme, TENP, ovalbumin-related protein X and avian bêta-defensin 11). The antibacterial activity of three new egg candidates (vitelline membrane outer layer protein 1, beta-microseminoprotein-like (LOC101750704) and pleiotrophin) was demonstrated against Listeria monocytogenes and/or Salmonella enterica Enteritidis. We showed that all these molecules share the property to inhibit bacterial growth through their heparin-binding domains. However, vitelline membrane outer layer 1 has additional specific structural features that can contribute to its antimicrobial potential. Moreover, we identified potential supplementary effectors of innate immunity including mucin 5B, E-selectin ligand 1, whey acidic protein 3, peptidyl prolyl isomerase B and retinoic acid receptor responder protein 2. These data support the concept of using heparin affinity combined to mass spectrometry to obtain an overview of the various effectors of innate immunity composing biological milieus, and to identify novel antimicrobial candidates of interest in the race for alternatives to antibiotics. PMID:27294500

  9. X-ray crystallography: Assessment and validation of protein-small molecule complexes for drug discovery

    PubMed Central

    Cooper, David R.; Porebski, Przemyslaw J.; Chruszcz, Maksymilian; Minor, Wladek

    2011-01-01

    Introduction Crystallography is the key initial component for structure-based and fragment-based drug design and can often generate leads that can be developed into high potency drugs. Therefore, huge sums of money are committed based on the outcome of crystallography experiments and their interpretation. Areas covered This review discusses how to evaluate the correctness of an X-ray structure, focusing on the validation of small molecule-protein complexes. Various types of inaccuracies found within the PDB are identified and the ramifications of these errors are discussed. The reader will gain an understanding of the key parameters that need to be inspected before a structure can be used in drug discovery efforts, as well as an appreciation of the difficulties of correctly interpreting electron density for small molecules. The reader will also be introduced to methods for validating small molecules within the context of a macromolecular structure. Expert opinion One of the reasons that ligand identification and positioning, within a macromolecular crystal structure, is so difficult is that the quality of small molecules widely varies in the PDB. For this reason, the PDB can not always be considered a reliable repository of structural information pertaining to small molecules, and this makes the derivation of general principles that govern small molecule-protein interactions more difficult. PMID:21779303

  10. Predicting metabolic pathways of small molecules and enzymes based on interaction information of chemicals and proteins.

    PubMed

    Gao, Yu-Fei; Chen, Lei; Cai, Yu-Dong; Feng, Kai-Yan; Huang, Tao; Jiang, Yang

    2012-01-01

    Metabolic pathway analysis, one of the most important fields in biochemistry, is pivotal to understanding the maintenance and modulation of the functions of an organism. Good comprehension of metabolic pathways is critical to understanding the mechanisms of some fundamental biological processes. Given a small molecule or an enzyme, how may one identify the metabolic pathways in which it may participate? Answering such a question is a first important step in understanding a metabolic pathway system. By utilizing the information provided by chemical-chemical interactions, chemical-protein interactions, and protein-protein interactions, a novel method was proposed by which to allocate small molecules and enzymes to 11 major classes of metabolic pathways. A benchmark dataset consisting of 3,348 small molecules and 654 enzymes of yeast was constructed to test the method. It was observed that the first order prediction accuracy evaluated by the jackknife test was 79.56% in identifying the small molecules and enzymes in a benchmark dataset. Our method may become a useful vehicle in predicting the metabolic pathways of small molecules and enzymes, providing a basis for some further analysis of the pathway systems.

  11. Monitoring Single-Molecule Protein Dynamics with a Carbon Nanotube Transistor

    NASA Astrophysics Data System (ADS)

    Collins, Philip G.

    2014-03-01

    Nanoscale electronic devices like field-effect transistors have long promised to provide sensitive, label-free detection of biomolecules. Single-walled carbon nanotubes press this concept further by not just detecting molecules but also monitoring their dynamics in real time. Recent measurements have demonstrated this premise by monitoring the single-molecule processivity of three different enzymes: lysozyme, protein Kinase A, and the Klenow fragment of DNA polymerase I. With all three enzymes, single molecules tethered to nanotube transistors were electronically monitored for 10 or more minutes, allowing us to directly observe a range of activity including rare transitions to chemically inactive and hyperactive conformations. The high bandwidth of the nanotube transistors further allow every individual chemical event to be clearly resolved, providing excellent statistics from tens of thousands of turnovers by a single enzyme. Initial success with three different enzymes indicates the generality and attractiveness of the nanotube devices as a new tool to complement other single-molecule techniques. Research on transduction mechanisms provides the design rules necessary to further generalize this architecture and apply it to other proteins. The purposeful incorporation of just one amino acid is sufficient to fabricate effective, single molecule sensors from a wide range of enzymes or proteins.

  12. Conformational dynamics of single protein molecules studied by direct mechanical manipulation.

    PubMed

    Heidarsson, Pétur O; Naqvi, Mohsin M; Sonar, Punam; Valpapuram, Immanuel; Cecconi, Ciro

    2013-01-01

    Advances in single-molecule manipulation techniques have recently enabled researchers to study a growing array of biological processes in unprecedented detail. Individual molecules can now be manipulated with subnanometer precision along a simple and well-defined reaction coordinate, the molecular end-to-end distance, and their conformational changes can be monitored in real time with ever-improving time resolution. The behavior of biomolecules under tension continues to unravel at an accelerated pace and often in combination with computational studies that reveal the atomistic details of the process under investigation. In this chapter, we explain the basic principles of force spectroscopy techniques, with a focus on optical tweezers, and describe some of the theoretical models used to analyze and interpret single-molecule manipulation data. We then highlight some recent and exciting results that have emerged from this research field on protein folding and protein-ligand interactions.

  13. Shedding light on protein folding, structural and functional dynamics by single molecule studies.

    PubMed

    Bavishi, Krutika; Hatzakis, Nikos S

    2014-01-01

    The advent of advanced single molecule measurements unveiled a great wealth of dynamic information revolutionizing our understanding of protein dynamics and behavior in ways unattainable by conventional bulk assays. Equipped with the ability to record distribution of behaviors rather than the mean property of a population, single molecule measurements offer observation and quantification of the abundance, lifetime and function of multiple protein states. They also permit the direct observation of the transient and rarely populated intermediates in the energy landscape that are typically averaged out in non-synchronized ensemble measurements. Single molecule studies have thus provided novel insights about how the dynamic sampling of the free energy landscape dictates all aspects of protein behavior; from its folding to function. Here we will survey some of the state of the art contributions in deciphering mechanisms that underlie protein folding, structural and functional dynamics by single molecule fluorescence microscopy techniques. We will discuss a few selected examples highlighting the power of the emerging techniques and finally discuss the future improvements and directions. PMID:25429564

  14. Impact of temperature and electrical potentials on the stability and structure of collagen adsorbed on the gold electrode

    NASA Astrophysics Data System (ADS)

    Meiners, Frank; Ahlers, Michael; Brand, Izabella; Wittstock, Gunther

    2015-01-01

    The morphology and structure of collagen type I adsorbed on gold electrodes were studied as a function of electrode potential and temperature by means of capacitance measurements, polarization modulation infrared reflection-absorption spectroscopy and scanning force microscopy at temperatures of 37 °C, 43 °C and 50 °C. The selected temperatures corresponded to the normal body temperature, temperature of denaturation of collagen molecules and denaturation of collagen fibrils, respectively. Independently of the solution temperature, collagen was adsorbed on gold electrodes in the potential range - 0.7 V < E < 0.4 V vs. Ag/AgCl, where the protein film was very stable. Fragments of collagen molecules made a direct contact to the gold surface and water was present in the film. Protein molecules were oriented preferentially with their long axis towards the gold surface. Collagen molecules in the adsorbed state preserved their native triple helical structure even at temperatures corresponding to collagen denaturation in aqueous solutions. Application of E < - 0.75 V vs. Ag/AgCl leads to the swelling of the protein film by water and desorption from the electrode surface. IR spectra provided no evidence of the thermal denaturation of adsorbed collagen molecules. A temperature increase to 50 °C leads to a distortion of the collagen film. The processes of aggregation and fibrilization were preferred over thermal denaturation for collagen adsorbed on the electrode surface and exposed to changing potentials.

  15. RNA binding protein and binding site useful for expression of recombinant molecules

    DOEpatents

    Mayfield, Stephen P.

    2006-10-17

    The present invention relates to a gene expression system in eukaryotic and prokaryotic cells, preferably plant cells and intact plants. In particular, the invention relates to an expression system having a RB47 binding site upstream of a translation initiation site for regulation of translation mediated by binding of RB47 protein, a member of the poly(A) binding protein family. Regulation is further effected by RB60, a protein disulfide isomerase. The expression system is capable of functioning in the nuclear/cytoplasm of cells and in the chloroplast of plants. Translation regulation of a desired molecule is enhanced approximately 100 fold over that obtained without RB47 binding site activation.

  16. RNA binding protein and binding site useful for expression of recombinant molecules

    DOEpatents

    Mayfield, Stephen

    2000-01-01

    The present invention relates to a gene expression system in eukaryotic and prokaryotic cells, preferably plant cells and intact plants. In particular, the invention relates to an expression system having a RB47 binding site upstream of a translation initiation site for regulation of translation mediated by binding of RB47 protein, a member of the poly(A) binding protein family. Regulation is further effected by RB60, a protein disulfide isomerase. The expression system is capable of functioning in the nuclear/cytoplasm of cells and in the chloroplast of plants. Translation regulation of a desired molecule is enhanced approximately 100 fold over that obtained without RB47 binding site activation.

  17. Exploiting Transient Protein States for the Design of Small-Molecule Stabilizers of Mutant p53

    PubMed Central

    Joerger, Andreas C.; Bauer, Matthias R.; Wilcken, Rainer; Baud, Matthias G.J.; Harbrecht, Hannes; Exner, Thomas E.; Boeckler, Frank M.; Spencer, John; Fersht, Alan R.

    2015-01-01

    Summary The destabilizing p53 cancer mutation Y220C creates an extended crevice on the surface of the protein that can be targeted by small-molecule stabilizers. Here, we identify different classes of small molecules that bind to this crevice and determine their binding modes by X-ray crystallography. These structures reveal two major conformational states of the pocket and a cryptic, transiently open hydrophobic subpocket that is modulated by Cys220. In one instance, specifically targeting this transient protein state by a pyrrole moiety resulted in a 40-fold increase in binding affinity. Molecular dynamics simulations showed that both open and closed states of this subsite were populated at comparable frequencies along the trajectories. Our data extend the framework for the design of high-affinity Y220C mutant binders for use in personalized anticancer therapy and, more generally, highlight the importance of implementing protein dynamics and hydration patterns in the drug-discovery process. PMID:26636255

  18. Visualization of a protein-protein interaction at a single-molecule level by atomic force microscopy.

    PubMed

    Bonazza, Klaus; Rottensteiner, Hanspeter; Seyfried, Birgit K; Schrenk, Gerald; Allmaier, Günter; Turecek, Peter L; Friedbacher, Gernot

    2014-02-01

    Atomic force microscopy is unmatched in terms of high-resolution imaging under ambient conditions. Over the years, substantial progress has been made using this technique to improve our understanding of biological systems on the nanometer scale, such as visualization of single biomolecules. For monitoring also the interaction between biomolecules, in situ high-speed imaging is making enormous progress. Here, we describe an alternative ex situ imaging method where identical molecules are recorded before and after reaction with a binding partner. Relocation of the identical molecules on the mica surface was thereby achieved by using a nanoscale scratch as marker. The method was successfully applied to study the complex formation between von Willebrand factor (VWF) and factor VIII (FVIII), two essential haemostatic components of human blood. FVIII binding was discernible by an appearance of globular domains appended to the N-terminal large globular domains of VWF. The specificity of the approach could be demonstrated by incubating VWF with FVIII in the presence of a high salt buffer which inhibits the interaction between these two proteins. The results obtained indicate that proteins can maintain their reactivity for subsequent interactions with other molecules when gently immobilized on a solid substrate and subjected to intermittent drying steps. The technique described opens up a new analytical perspective for studying protein-protein interactions as it circumvents some of the obstacles encountered by in situ imaging and other ex situ techniques. PMID:24363113

  19. Alpha-helical regions of the protein molecule as organic nanotubes

    NASA Astrophysics Data System (ADS)

    Suprun, Anatol D.; Shmeleva, Liudmyla V.

    2014-05-01

    An α-helical region of protein molecule was considered in a model of nanotube. The molecule is in conditions of quantum excitations. Such model corresponds to a one-dimensional molecular nanocrystal with three molecules in an elementary cell at the presence of excitation. For the analysis of different types of conformational response of the α-helical area of the protein molecule on excitation, the nonlinear response of this area to the intramolecular quantum excitation caused by hydrolysis of adenosine triphosphate (ATP) is taken into account. It has been established that in the simplest case, three types of excitation are realized. As estimates show, each of them `serves' different kinds of protein. The symmetrical type of excitation, most likely, is realized in the reduction of traversal-striped skeletal muscles. It has the highest excitation energy. This well protects from casual actions. Antisymmetric excitations have intermediate energy (between symmetrical and asymmetrical). They, most likely, are realized in membranous and nucleic proteins. It is shown that the conformational response of the α-helical region of the protein is (in angstroms) a quantity of order N c /5, where N c is the number of spiral turns. For the number of turns typical in this case: N c ~ 10, displacement compounds are a quantity of order 2 Å. It qualitatively corresponds to observable values. Asymmetrical excitations have the lowest energy. Therefore, most likely, they are realized in enzymatic proteins. It was shown that at this type of excitation, the bending of the α-helix is formally directed to the opposite side with respect to the antisymmetric excitations. Also, it has a greater value than the antisymmetric case for N c ≤ 14 and smaller for N c > 14.

  20. Single-molecule spectroscopy of the temperature-induced collapse of unfolded proteins

    PubMed Central

    Nettels, Daniel; Müller-Späth, Sonja; Küster, Frank; Hofmann, Hagen; Haenni, Dominik; Rüegger, Stefan; Reymond, Luc; Hoffmann, Armin; Kubelka, Jan; Heinz, Benjamin; Gast, Klaus; Best, Robert B.; Schuler, Benjamin

    2009-01-01

    We used single-molecule FRET in combination with other biophysical methods and molecular simulations to investigate the effect of temperature on the dimensions of unfolded proteins. With single-molecule FRET, this question can be addressed even under near-native conditions, where most molecules are folded, allowing us to probe a wide range of denaturant concentrations and temperatures. We find a compaction of the unfolded state of a small cold shock protein with increasing temperature in both the presence and the absence of denaturant, with good agreement between the results from single-molecule FRET and dynamic light scattering. Although dissociation of denaturant from the polypeptide chain with increasing temperature accounts for part of the compaction, the results indicate an important role for additional temperature-dependent interactions within the unfolded chain. The observation of a collapse of a similar extent in the extremely hydrophilic, intrinsically disordered protein prothymosin α suggests that the hydrophobic effect is not the sole source of the underlying interactions. Circular dichroism spectroscopy and replica exchange molecular dynamics simulations in explicit water show changes in secondary structure content with increasing temperature and suggest a contribution of intramolecular hydrogen bonding to unfolded state collapse. PMID:19933333

  1. Single-molecule spectroscopy of the temperature-induced collapse of unfolded proteins.

    PubMed

    Nettels, Daniel; Müller-Späth, Sonja; Küster, Frank; Hofmann, Hagen; Haenni, Dominik; Rüegger, Stefan; Reymond, Luc; Hoffmann, Armin; Kubelka, Jan; Heinz, Benjamin; Gast, Klaus; Best, Robert B; Schuler, Benjamin

    2009-12-01

    We used single-molecule FRET in combination with other biophysical methods and molecular simulations to investigate the effect of temperature on the dimensions of unfolded proteins. With single-molecule FRET, this question can be addressed even under near-native conditions, where most molecules are folded, allowing us to probe a wide range of denaturant concentrations and temperatures. We find a compaction of the unfolded state of a small cold shock protein with increasing temperature in both the presence and the absence of denaturant, with good agreement between the results from single-molecule FRET and dynamic light scattering. Although dissociation of denaturant from the polypeptide chain with increasing temperature accounts for part of the compaction, the results indicate an important role for additional temperature-dependent interactions within the unfolded chain. The observation of a collapse of a similar extent in the extremely hydrophilic, intrinsically disordered protein prothymosin alpha suggests that the hydrophobic effect is not the sole source of the underlying interactions. Circular dichroism spectroscopy and replica exchange molecular dynamics simulations in explicit water show changes in secondary structure content with increasing temperature and suggest a contribution of intramolecular hydrogen bonding to unfolded state collapse.

  2. Direct-reversible binding of small molecules to G protein βγ subunits

    PubMed Central

    Seneviratne, AMPB; Burroughs, Michael; Giralt, Ernest; Smrcka, Alan V.

    2011-01-01

    Heterotrimeric guanine nucleotide-binding proteins (G proteins) composed of three subunits α, β, γ mediate activation of multiple intracellular signaling cascades initiated by G protein-coupled receptors (GPCRs). Previously our laboratory identified small molecules that bind to Gβγ and interfere with or enhance binding of select effectors with Gβγ. To understand the molecular mechanisms of selectivity and assess binding of compounds to Gβγ, we used biophysical and biochemical approaches to directly monitor small molecule binding to Gβγ. Surface plasmon resonance (SPR) analysis indicated that multiple compounds bound directly to Gβγ with affinities in the high nanomolar to low micromolar range but with surprisingly slow on and off rate kinetics. While the koff was slow for most of the compounds in physiological buffers, they could be removed from Gβγ with mild chaotropic salts or mildly dissociating collision energy in a mass-spectrometer indicating that compound-Gβγ interactions were non-covalent. Finally, at concentrations used to observe maximal biological effects the stoichiometry of binding was 1:1. The results from this study show that small molecule modulation of Gβγ-effector interactions is by specific direct non-covalent and reversible binding of small molecules to Gβγ. This is highly relevant to development of Gβγ targeting as a therapeutic approach since reversible, direct binding is a prerequisite for drug development and important for specificity. PMID:21621014

  3. Protein-bound water molecules in primate red- and green-sensitive visual pigments.

    PubMed

    Katayama, Kota; Furutani, Yuji; Imai, Hiroo; Kandori, Hideki

    2012-02-14

    Protein-bound water molecules play crucial roles in the structure and function of proteins. The functional role of water molecules has been discussed for rhodopsin, the light sensor for twilight vision, on the basis of X-ray crystallography, Fourier transform infrared (FTIR) spectroscopy, and a radiolytic labeling method, but nothing is known about the protein-bound waters in our color visual pigments. Here we apply low-temperature FTIR spectroscopy to monkey red (MR)- and green (MG)-sensitive color pigments at 77 K and successfully identify water vibrations using D(2)O and D(2)(18)O in the whole midinfrared region. The observed water vibrations are 6-8 for MR and MG, indicating that several water molecules are present near the retinal chromophore and change their hydrogen bonds upon retinal photoisomerization. In this sense, color visual pigments possess protein-bound water molecules essentially similar to those of rhodopsin. The absence of strongly hydrogen-bonded water molecules (O-D stretch at <2400 cm(-1)) is common between rhodopsin and color pigments, which greatly contrasts with the case of proton-pumping microbial rhodopsins. On the other hand, two important differences are observed in water signal between rhodopsin and color pigments. First, the water vibrations are identical between the 11-cis and 9-cis forms of rhodopsin, but different vibrational bands are observed at >2550 cm(-1) for both MR and MG. Second, strongly hydrogen-bonded water molecules (2303 cm(-1) for MR and 2308 cm(-1) for MG) are observed for the all-trans form after retinal photoisomerization, which is not the case for rhodopsin. These specific features of MR and MG can be explained by the presence of water molecules in the Cl(-)-biding site, which are located near positions C11 and C9 of the retinal chromophore. The averaged frequencies of the observed water O-D stretching vibrations for MR and MG are lower as the λ(max) is red-shifted, suggesting that water molecules are involved in

  4. Screening of the binding of small molecules to proteins by desorption electrospray ionization mass spectrometry combined with protein microarray.

    PubMed

    Yao, Chenxi; Wang, Tao; Zhang, Buqing; He, Dacheng; Na, Na; Ouyang, Jin

    2015-11-01

    The interaction between bioactive small molecule ligands and proteins is one of the important research areas in proteomics. Herein, a simple and rapid method is established to screen small ligands that bind to proteins. We designed an agarose slide to immobilize different proteins. The protein microarrays were allowed to interact with different small ligands, and after washing, the microarrays were screened by desorption electrospray ionization mass spectrometry (DESI MS). This method can be applied to screen specific protein binding ligands and was shown for seven proteins and 34 known ligands for these proteins. In addition, a high-throughput screening was achieved, with the analysis requiring approximately 4 s for one sample spot. We then applied this method to determine the binding between the important protein matrix metalloproteinase-9 (MMP-9) and 88 small compounds. The molecular docking results confirmed the MS results, demonstrating that this method is suitable for the rapid and accurate screening of ligands binding to proteins. Graphical Abstract ᅟ. PMID:26174365

  5. Screening of the binding of small molecules to proteins by desorption electrospray ionization mass spectrometry combined with protein microarray.

    PubMed

    Yao, Chenxi; Wang, Tao; Zhang, Buqing; He, Dacheng; Na, Na; Ouyang, Jin

    2015-11-01

    The interaction between bioactive small molecule ligands and proteins is one of the important research areas in proteomics. Herein, a simple and rapid method is established to screen small ligands that bind to proteins. We designed an agarose slide to immobilize different proteins. The protein microarrays were allowed to interact with different small ligands, and after washing, the microarrays were screened by desorption electrospray ionization mass spectrometry (DESI MS). This method can be applied to screen specific protein binding ligands and was shown for seven proteins and 34 known ligands for these proteins. In addition, a high-throughput screening was achieved, with the analysis requiring approximately 4 s for one sample spot. We then applied this method to determine the binding between the important protein matrix metalloproteinase-9 (MMP-9) and 88 small compounds. The molecular docking results confirmed the MS results, demonstrating that this method is suitable for the rapid and accurate screening of ligands binding to proteins. Graphical Abstract ᅟ.

  6. The L1 family of cell adhesion molecules: a sickening number of mutations and protein functions.

    PubMed

    Hortsch, Michael; Nagaraj, Kakanahalli; Mualla, Rula

    2014-01-01

    L1-type proteins are transmembrane cell adhesion molecules with an evolutionary well-conserved protein domain structure of usually six immunoglobulin and five fibronectin type III domains. By engaging in many different protein-protein interactions they are involved in a multitude of molecular functions and are important players during the formation and maintenance of metazoan nervous systems. As a result, mutations in L1-type genes cause a great variety of phenotypes, most of which are neurological in nature. In humans, mutations in the L1CAM gene are responsible for L1 syndrome and other L1-type genes have been implicated in conditions as varied as mental retardation, autism, schizophrenia, multiple sclerosis, and other disorders. Equally, the overexpression of L1-type proteins appears to have deleterious effects in various types of human tumor cells, where they generally contribute to an increase in cell mobility and metastatic potential. PMID:25300138

  7. Local composition in the vicinity of a protein molecule in an aqueous mixed solvent.

    PubMed

    Shulgin, Ivan L; Ruckenstein, Eli

    2007-04-19

    This paper is focused on the composition of a cosolvent in the vicinity of a protein surface (local composition) and its dependence on various factors. First, the Kirkwood-Buff theory of solution is used to obtain analytical expressions that connect the excess or deficit number of cosolvent and water molecules in the vicinity of a protein surface with experimentally measurable quantities such as the bulk concentration of the mixed solvent, the preferential binding parameter, and the molar volumes of water and cosolvent. Using these expressions, relations between the preferential binding parameter (at a molal concentration scale) and the above excesses (or deficits) are established. In addition, the obtained expressions are used to examine the effect of the nonideality of the water + cosolvent mixtures and of the molar volume of the cosolvent on the excess (or deficit) number of cosolvent molecules in the vicinity of the protein surface. It is shown that at least for the mixed solvents considered (water + urea and water + glucose) the nonideality of the mixed solvent is not an important factor in the local compositions around a protein molecule and that the main contribution is provided by the nonidealities of the protein-water and protein-cosolvent mixtures. Special attention is paid to urea as cosolvent, because urea is one of only a few compounds with a concentration at the protein surface larger than its concentration in the bulk. The composition dependence of the excess of urea around a protein molecule is calculated for the water + lysozyme + urea mixture at pH = 7.0 and 2.0. At pH = 7.0, the excess of urea becomes almost composition independent at high urea concentrations. Such independence could be explained by assuming that urea totally replaces water in some areas of the protein surface, whereas on the remaining areas of the protein surface both water and urea are present with concentration comparable to those in the bulk. The Schellman exchange model was

  8. Single-molecule kinetics under force: probing protein folding and enzymatic activity with optical tweezers

    NASA Astrophysics Data System (ADS)

    Wong, Wesley

    2010-03-01

    Weak non-covalent bonds between and within single molecules govern many aspects of biological structure and function (e.g. DNA base-paring, receptor-ligand binding, protein folding, etc.) In living systems, these interactions are often subject to mechanical forces, which can greatly alter their kinetics and activity. My group develops and applies novel single-molecule manipulation techniques to explore and quantify these force-dependent kinetics. Using optical tweezers, we have quantified the force-dependent unfolding and refolding kinetics of different proteins, including the cytoskeletal protein spectrin in collaboration with E. Evans's group [1], and the A2 domain of the von Willebrand factor blood clotting protein in collaboration with T. Springer's group [2]. Furthermore, we have studied the kinetics of the ADAMTS13 enzyme acting on a single A2 domain, and have shown that physiolgical forces in the circulation can act as a cofactor for enzymatic cleavage, regulating hemostatic activity [2]. References: 1. E. Evans, K. Halvorsen, K. Kinoshita, and W.P. Wong, Handbook of Single Molecule Biophysics, P. Hinterdorfer, ed., Springer (2009). 2. X. Zhang, K. Halvorsen, C.-Z. Zhang, W.P. Wong, and T.A. Springer, Science 324 (5932), 1330-1334 (2009).

  9. Knowledge-based annotation of small molecule binding sites in proteins

    PubMed Central

    2010-01-01

    Background The study of protein-small molecule interactions is vital for understanding protein function and for practical applications in drug discovery. To benefit from the rapidly increasing structural data, it is essential to improve the tools that enable large scale binding site prediction with greater emphasis on their biological validity. Results We have developed a new method for the annotation of protein-small molecule binding sites, using inference by homology, which allows us to extend annotation onto protein sequences without experimental data available. To ensure biological relevance of binding sites, our method clusters similar binding sites found in homologous protein structures based on their sequence and structure conservation. Binding sites which appear evolutionarily conserved among non-redundant sets of homologous proteins are given higher priority. After binding sites are clustered, position specific score matrices (PSSMs) are constructed from the corresponding binding site alignments. Together with other measures, the PSSMs are subsequently used to rank binding sites to assess how well they match the query and to better gauge their biological relevance. The method also facilitates a succinct and informative representation of observed and inferred binding sites from homologs with known three-dimensional structures, thereby providing the means to analyze conservation and diversity of binding modes. Furthermore, the chemical properties of small molecules bound to the inferred binding sites can be used as a starting point in small molecule virtual screening. The method was validated by comparison to other binding site prediction methods and to a collection of manually curated binding site annotations. We show that our method achieves a sensitivity of 72% at predicting biologically relevant binding sites and can accurately discriminate those sites that bind biological small molecules from non-biological ones. Conclusions A new algorithm has been

  10. Molecular Mechanics of Single Protein Molecules Measured with the Atomic Force Microscope

    NASA Astrophysics Data System (ADS)

    Hansma, Paul K.

    2000-03-01

    After a short history of AFM development in our lab, including recent developments with small cantilevers, this talk will focus on 1) pulling single protein molecules to explore the forces involved in unfolding and 2) watching single protein molecules in action to learn how they function mechanically. 1) Pulling experiments on proteins used as marine adhesives in abalone shells and other biological composite materials reveal modules bound together by sacrificial bonds that are weaker than the backbone bonds in the polypeptide chain.1 These self-healing modules provide effective energy absorption and appear to be a real key to understanding the impressive fracture resistance of biological composite materials. For example, the abalone shell is 3000 times more fracture resistant than a single crystal of calcium carbonate, despite the fact that 97% of the mass of the shell is crystalline calcium carbonate. 2) It is becoming possible, again with AFMs, to learn how some enzymes, nature's nanomachines, do their exquisite materials synthesis and processing. The talk will focus on the chaperonin system of GroEL and GroES that processes incorrectly folded proteins and assists them in refolding correctly. It is becoming possible not only to see single molecule events such as the association and disassociation of the GroEL-Gro-ES complex, but also to measure potential energy functions for the molecules in various conformational states. These new measurements, together with detailed structural measurements from other techniques, give new clues about how these proteins use the energy of ATP to do their work. Since the AFMs of today are very far from fundamental limits, this is only the beginning. 1. B. L. Smith, T. E. Schaffer, M. Viani, J. B. Thompson, N. A. Frederick, J. Kindt, A. Belcher, G. D. Stucky, D. E. Morse and P. K. Hansma, Nature 399, 761 (1999)

  11. Single molecule DNA interaction kinetics of retroviral nucleic acid chaperone proteins

    NASA Astrophysics Data System (ADS)

    Williams, Mark

    2010-03-01

    Retroviral nucleocapsid (NC) proteins are essential for several viral replication processes including specific genomic RNA packaging and reverse transcription. The nucleic acid chaperone activity of NC facilitates the latter process. In this study, we use single molecule biophysical methods to quantify the DNA interactions of wild type and mutant human immunodeficiency virus type 1 (HIV-1) NC and Gag and human T-cell leukemia virus type 1 (HTLV-1) NC. We find that the nucleic acid interaction properties of these proteins differ significantly, with HIV-1 NC showing rapid protein binding kinetics, significant duplex destabilization, and strong DNA aggregation, all properties that are critical components of nucleic acid chaperone activity. In contrast, HTLV-1 NC exhibits significant destabilization activity but extremely slow DNA interaction kinetics and poor aggregating capability, which explains why HTLV-1 NC is a poor nucleic acid chaperone. To understand these results, we developed a new single molecule method for quantifying protein dissociation kinetics, and applied this method to probe the DNA interactions of wild type and mutant HIV-1 and HTLV-1 NC. We find that mutations to aromatic and charged residues strongly alter the proteins' nucleic acid interaction kinetics. Finally, in contrast to HIV-1 NC, HIV-1 Gag, the nucleic acid packaging protein that contains NC as a domain, exhibits relatively slow binding kinetics, which may negatively impact its ability to act as a nucleic acid chaperone.

  12. Inhibition of Heterotrimeric G Protein Signaling by a Small Molecule Acting on Gα Subunit

    PubMed Central

    Ayoub, Mohammed Akli; Damian, Marjorie; Gespach, Christian; Ferrandis, Eric; Lavergne, Olivier; De Wever, Olivier; Banères, Jean-Louis; Pin, Jean-Philippe; Prévost, Grégoire Pierre

    2009-01-01

    The simultaneous activation of many distinct G protein-coupled receptors (GPCRs) and heterotrimeric G proteins play a major role in various pathological conditions. Pan-inhibition of GPCR signaling by small molecules thus represents a novel strategy to treat various diseases. To better understand such therapeutic approach, we have characterized the biomolecular target of BIM-46187, a small molecule pan-inhibitor of GPCR signaling. Combining bioluminescence and fluorescence resonance energy transfer techniques in living cells as well as in reconstituted receptor-G protein complexes, we observed that, by direct binding to the Gα subunit, BIM-46187 prevents the conformational changes of the receptor-G protein complex associated with GPCR activation. Such a binding prevents the proper interaction of receptors with the G protein heterotrimer and inhibits the agonist-promoted GDP/GTP exchange. These observations bring further evidence that inhibiting G protein activation through direct binding to the Gα subunit is feasible and should constitute a new strategy for therapeutic intervention. PMID:19648112

  13. Visualizing Protein Movement on DNA at the Single-molecule Level using DNA Curtains

    PubMed Central

    Silverstein, Timothy D.; Gibb, Bryan; Greene, Eric C.

    2014-01-01

    A fundamental feature of many nucleic-acid binding proteins is their ability to move along DNA either by diffusion-based mechanisms or by ATP-hydrolysis driven translocation. For example, most site-specific DNA-binding proteins must diffuse to some extent along DNA to either find their target sites, or to otherwise fulfill their biological roles. Similarly, nucleic-acid translocases such as helicases and polymerases must move along DNA to fulfill their functions. In both instances, the proteins must also be capable of moving in crowded environments while navigating through DNA-bound obstacles. These types of behaviors can be challenging to analyze by bulk biochemical methods because of the transient nature of the interactions, and/or heterogeneity of the reaction intermediates. The advent of single-molecule methodologies has overcome some of these problems, and has led to many new insights into the mechanisms that contribute to protein motion along DNA. We have developed DNA curtains as a tool to facilitate single molecule observations of protein-nucleic acid interactions, and we have applied these new research tools to systems involving both diffusive-based motion as well as ATP directed translocation. Here we highlight these studies by first discussing how diffusion contributes to target searches by proteins involved in post-replicative mismatch repair. We then discuss DNA curtain assays of two different DNA translocases, RecBCD and FtsK, which participate in homologous DNA recombination and site-specific DNA recombination, respectively. PMID:24598576

  14. MARCKS-like protein is an initiating molecule in axolotl appendage regeneration.

    PubMed

    Sugiura, Takuji; Wang, Heng; Barsacchi, Rico; Simon, Andras; Tanaka, Elly M

    2016-03-10

    Identifying key molecules that launch regeneration has been a long-sought goal. Multiple regenerative animals show an initial wound-associated proliferative response that transits into sustained proliferation if a considerable portion of the body part has been removed. In the axolotl, appendage amputation initiates a round of wound-associated cell cycle induction followed by continued proliferation that is dependent on nerve-derived signals. A wound-associated molecule that triggers the initial proliferative response to launch regeneration has remained obscure. Here, using an expression cloning strategy followed by in vivo gain- and loss-of-function assays, we identified axolotl MARCKS-like protein (MLP) as an extracellularly released factor that induces the initial cell cycle response during axolotl appendage regeneration. The identification of a regeneration-initiating molecule opens the possibility of understanding how to elicit regeneration in other animals.

  15. MARCKS-like protein is an initiating molecule in axolotl appendage regeneration.

    PubMed

    Sugiura, Takuji; Wang, Heng; Barsacchi, Rico; Simon, Andras; Tanaka, Elly M

    2016-03-10

    Identifying key molecules that launch regeneration has been a long-sought goal. Multiple regenerative animals show an initial wound-associated proliferative response that transits into sustained proliferation if a considerable portion of the body part has been removed. In the axolotl, appendage amputation initiates a round of wound-associated cell cycle induction followed by continued proliferation that is dependent on nerve-derived signals. A wound-associated molecule that triggers the initial proliferative response to launch regeneration has remained obscure. Here, using an expression cloning strategy followed by in vivo gain- and loss-of-function assays, we identified axolotl MARCKS-like protein (MLP) as an extracellularly released factor that induces the initial cell cycle response during axolotl appendage regeneration. The identification of a regeneration-initiating molecule opens the possibility of understanding how to elicit regeneration in other animals. PMID:26934225

  16. Direct Observation of the Three-State Folding of a Single Protein Molecule

    NASA Astrophysics Data System (ADS)

    Cecconi, Ciro; Shank, Elizabeth A.; Bustamante, Carlos; Marqusee, Susan

    2005-09-01

    We used force-measuring optical tweezers to induce complete mechanical unfolding and refolding of individual Escherichia coli ribonuclease H (RNase H) molecules. The protein unfolds in a two-state manner and refolds through an intermediate that correlates with the transient molten globule-like intermediate observed in bulk studies. This intermediate displays unusual mechanical compliance and unfolds at substantially lower forces than the native state. In a narrow range of forces, the molecule hops between the unfolded and intermediate states in real time. Occasionally, hopping was observed to stop as the molecule crossed the folding barrier directly from the intermediate, demonstrating that the intermediate is on-pathway. These studies allow us to map the energy landscape of RNase H.

  17. Control of Pre-Tilt Angles of Liquid Crystal Molecules Using a Chemically Adsorbed Monomolecular Layer as an Alignment Film in Liquid Crystal Cells

    NASA Astrophysics Data System (ADS)

    Ogawa, Kazufumi; Ohtake, Tadashi; Nomura, Takaiki

    2002-11-01

    Photoaligned monomolecular layers containing two materials were formed to control pre-tilt angles (θp) of liquid crystal molecules for twisted nematic (TN) type liquid crystal displays (LCDs) by a chemical adsorption (CA) technique and a photoalignment technique. One was a new chlorosilane type surfactant, 4‧-(6-trichlorosilyloxyhexyloxy) chalcone (CO), having photopolymerizablity, and the other was a surfactant having a straight carbon chain (SC). Although we tried screening six different surfectants as an additive to CO, a surfactant having a long straight hydrocarbon chain (octadecyl-trichlorosilane: C18) was the most suitable for the TN type LCDs. By changing the molecular ratio of CO and C18, pre-tilt angles of liquid crystal molecules in a test liquid crystal (LC) cell could be controlled from 0 to 8° with perfect mono-domain alignment. When surfactants having short hydrocarbon chains and those having fluorocarbon chains were used, the quality of the TN type LC cells obtained was not good.

  18. Three-dimensional Nanowire Structures for Ultra-Fast Separation of DNA, Protein and RNA Molecules

    PubMed Central

    Rahong, Sakon; Yasui, Takao; Yanagida, Takeshi; Nagashima, Kazuki; Kanai, Masaki; Meng, Gang; He, Yong; Zhuge, Fuwei; Kaji, Noritada; Kawai, Tomoji; Baba, Yoshinobu

    2015-01-01

    Separation and analysis of biomolecules represent crucial processes for biological and biomedical engineering development; however, separation resolution and speed for biomolecules analysis still require improvements. To achieve separation and analysis of biomolecules in a short time, the use of highly-ordered nanostructures fabricated by top-down or bottom-up approaches have been proposed. Here, we reported on the use of three-dimensional (3D) nanowire structures embedded in microchannels fabricated by a bottom-up approach for ultrafast separation of small biomolecules, such as DNA, protein, and RNA molecules. The 3D nanowire structures could analyze a mixture of DNA molecules (50–1000 bp) within 50 s, a mixture of protein molecules (20–340 kDa) within 5 s, and a mixture of RNA molecules (100–1000 bases) within 25 s. And, we could observe the electrophoretic mobility difference of biomolecules as a function of molecular size in the 3D nanowire structures. Since the present methodology allows users to control the pore size of sieving materials by varying the number of cycles for nanowire growth, the 3D nanowire structures have a good potential for use as alternatives for other sieving materials. PMID:26073192

  19. Simple horizontal magnetic tweezers for micromanipulation of single DNA molecules and DNA-protein complexes.

    PubMed

    McAndrew, Christopher P; Tyson, Christopher; Zischkau, Joseph; Mehl, Patrick; Tuma, Pamela L; Pegg, Ian L; Sarkar, Abhijit

    2016-01-01

    We report the development of a simple-to-implement magnetic force transducer that can apply a wide range of piconewton (pN) scale forces on single DNA molecules and DNA-protein complexes in the horizontal plane. The resulting low-noise force-extension data enable very high-resolution detection of changes in the DNA tether's extension: ~0.05 pN in force and <10 nm change in extension. We have also verified that we can manipulate DNA in near equilibrium conditions through the wide range of forces by ramping the force from low to high and back again, and observing minimal hysteresis in the molecule's force response. Using a calibration technique based on Stokes' drag law, we have confirmed our force measurements from DNA force-extension experiments obtained using the fluctuation-dissipation theorem applied to transverse fluctuations of the magnetic microsphere. We present data on the force-distance characteristics of a DNA molecule complexed with histones. The results illustrate how the tweezers can be used to study DNA binding proteins at the single molecule level.

  20. Chemokines, costimulatory molecules and fusion proteins for the immunotherapy of solid tumors.

    PubMed

    Lechner, Melissa G; Russell, Sarah M; Bass, Rikki S; Epstein, Alan L

    2011-11-01

    In this article, the role of chemokines and costimulatory molecules in the immunotherapy of experimental murine solid tumors and immunotherapy used in ongoing clinical trials are presented. Chemokine networks regulate physiologic cell migration that may be disrupted to inhibit antitumor immune responses or co-opted to promote tumor growth and metastasis in cancer. Recent studies highlight the potential use of chemokines in cancer immunotherapy to improve innate and adaptive cell interactions and to recruit immune effector cells into the tumor microenvironment. Another critical component of antitumor immune responses is antigen priming and activation of effector cells. Reciprocal expression and binding of costimulatory molecules and their ligands by antigen-presenting cells and naive lymphocytes ensures robust expansion, activity and survival of tumor-specific effector cells in vivo. Immunotherapy approaches using agonist antibodies or fusion proteins of immunomodulatory molecules significantly inhibit tumor growth and boost cell-mediated immunity. To localize immune stimulation to the tumor site, a series of fusion proteins consisting of a tumor-targeting monoclonal antibody directed against tumor necrosis and chemokines or costimulatory molecules were generated and tested in tumor-bearing mice. While several of these reagents were initially shown to have therapeutic value, combination therapies with methods to delete suppressor cells had the greatest effect on tumor growth. In conclusion, a key conclusion that has emerged from these studies is that successful immunotherapy will require both advanced methods of immunostimulation and the removal of immunosuppression in the host.

  1. A novel multitarget tracking algorithm for Myosin VI protein molecules on actin filaments in TIRFM sequences.

    PubMed

    Li, G; Sanchez, V; Nagaraj, P C S B; Khan, S; Rajpoot, N

    2015-12-01

    We propose a novel multitarget tracking framework for Myosin VI protein molecules in total internal reflection fluorescence microscopy sequences which integrates an extended Hungarian algorithm with an interacting multiple model filter. The extended Hungarian algorithm, which is a linear assignment problem based method, helps to solve measurement assignment and spot association problems commonly encountered when dealing with multiple targets, although a two-motion model interacting multiple model filter increases the tracking accuracy by modelling the nonlinear dynamics of Myosin VI protein molecules on actin filaments. The evaluation of our tracking framework is conducted on both real and synthetic total internal reflection fluorescence microscopy sequences. The results show that the framework achieves higher tracking accuracies compared to the state-of-the-art tracking methods, especially for sequences with high spot density. PMID:26259144

  2. Ab Initio Calculations of the Electronic Structures and Biological Functions of Protein Molecules

    NASA Astrophysics Data System (ADS)

    Zheng, Haoping

    2003-04-01

    The self-consistent cluster-embedding (SCCE) calculation method reduces the computational effort from M3 to about M1 (M is the number of atoms in the system) with unchanged calculation precision. So the ab initio, all-electron calculation of the electronic structure and biological function of protein molecule becomes a reality, which will promote new proteomics considerably. The calculated results of two real protein molecules, the trypsin inhibitor from the seeds of squash Cucurbita maxima (CMTI-I, 436 atoms) and the Ascaris trypsin inhibitor (912 atoms, two three-dimensional structures), are presented. The reactive sites of the inhibitors are determined and explained. The precision of structure determination of inhibitors are tested theoretically.

  3. Ab Initio Calculations of the Electronic Structures and Biological Functions of Protein Molecules

    NASA Astrophysics Data System (ADS)

    Zheng, Haoping

    The self-consistent cluster-embedding (SCCE) calculation method reduces the computational effort from M3 to about M1 (M is the number of atoms in the system) with precise calculations. Thus the ab initio, all-electron calculation of the electronic structure and biological function of protein molecule has become a reality, which will promote new proteomics considerably. The calculated results of two real protein molecules, the trypsin inhibitor from the seeds of squash Cucurbita maxima (CMTI-I, 436 atoms) and the ascaris trypsin inhibitor (912 atoms, two three-dimensional structures), will be presented in this paper. The reactive sites of the inhibitors are determined and explained. The accuracy of structure determination of the inhibitors are tested theoretically.

  4. Engineered Protein Polymer-Gold Nanoparticle Hybrid Materials for Small Molecule Delivery

    PubMed Central

    Dai, Min; Frezzo, JA; Sharma, E; Chen, R; Singh, N; Yuvienco, C; Caglar, E; Xiao, S; Saxena, A; Montclare, JK

    2016-01-01

    We have fabricated protein polymer-gold nanoparticle (P-GNP) nanocomposites that exhibit enhanced binding and delivery properties of the small hydrophobic molecule drug, curcumin, to the model breast cancer cell line, MCF-7. These hybrid biomaterials are constructed via in situ GNP templated-synthesis with genetically engineered histidine tags. The P-GNP nanocomposites exhibit enhanced small molecule loading, sustained release and increased uptake by MCF-7 cells. When compared to the proteins polymers alone, the P-GNPs demonstrate a greater than 7-fold increase in curcumin binding, a nearly 50% slower release profile and more than 2-fold increase in cellular uptake of curcumin. These results suggest that P-GNP nanocomposites serve as promising candidates for drug delivery vehicles. PMID:27081576

  5. Identification of small molecule binding sites within proteins using phage display technology.

    SciTech Connect

    Rodi, D. J.; Agoston, G. E.; Manon, R.; Lapcevich, R.; Green, S. J.; Makowski, L.; Biosciences Division; EntreMed Inc.; Florida State Univ.

    2001-11-01

    Affinity selection of peptides displayed on phage particles was used as the basis for mapping molecular contacts between small molecule ligands and their protein targets. Analysis of the crystal structures of complexes between proteins and small molecule ligands revealed that virtually all ligands of molecular weight 300 Da or greater have a continuous binding epitope of 5 residues or more. This observation led to the development of a technique for binding site identification which involves statistical analysis of an affinity-selected set of peptides obtained by screening of libraries of random, phage-displayed peptides against small molecules attached to solid surfaces. A random sample of the selected peptides is sequenced and used as input for a similarity scanning program which calculates cumulative similarity scores along the length of the putative receptor. Regions of the protein sequence exhibiting the highest similarity with the selected peptides proved to have a high probability of being involved in ligand binding. This technique has been employed successfully to map the contact residues in multiple known targets of the anticancer drugs paclitaxel (Taxol), docetaxel (Taxotere) and 2-methoxyestradiol and the glycosaminoglycan hyaluronan, and to identify a novel paclitaxel receptor [1]. These data corroborate the observation that the binding properties of peptides displayed on the surface of phage particles can mimic the binding properties of peptides in naturally occurring proteins. It follows directly that structural context is relatively unimportant for determining the binding properties of these disordered peptides. This technique represents a novel, rapid, high resolution method for identifying potential ligand binding sites in the absence of three-dimensional information and has the potential to greatly enhance the speed of development of novel small molecule pharmaceuticals.

  6. Single-molecule spectroscopy of protein folding in a chaperonin cage

    PubMed Central

    Hofmann, Hagen; Hillger, Frank; Pfeil, Shawn H.; Hoffmann, Armin; Streich, Daniel; Haenni, Dominik; Nettels, Daniel; Lipman, Everett A.; Schuler, Benjamin

    2010-01-01

    Molecular chaperones are known to be essential for avoiding protein aggregation in vivo, but it is still unclear how they affect protein folding mechanisms. We use single-molecule Förster resonance energy transfer to follow the folding of a protein inside the GroEL/GroES chaperonin cavity over a time range from milliseconds to hours. Our results show that confinement in the chaperonin decelerates the folding of the C-terminal domain in the substrate protein rhodanese, but leaves the folding rate of the N-terminal domain unaffected. Microfluidic mixing experiments indicate that strong interactions of the substrate with the cavity walls impede the folding process, but the folding hierarchy is preserved. Our results imply that no universal chaperonin mechanism exists. Rather, a competition between intra- and intermolecular interactions determines the folding rates and mechanisms of a substrate inside the GroEL/GroES cage. PMID:20547872

  7. See me, feel me: methods to concurrently visualize and manipulate single DNA molecules and associated proteins

    PubMed Central

    van Mameren, Joost; Peterman, Erwin J. G.; Wuite, Gijs J. L.

    2008-01-01

    Direct visualization of DNA and proteins allows researchers to investigate DNA–protein interactions with great detail. Much progress has been made in this area as a result of increasingly sensitive single-molecule fluorescence techniques. At the same time, methods that control the conformation of DNA molecules have been improving constantly. The combination of both techniques has appealed to researchers ever since single-molecule measurements have become possible and indeed first implementations of such combined approaches have proven useful in the study of several DNA-binding proteins in real time. Here, we describe the technical state-of-the-art of various integrated manipulation-and-visualization methods. We first discuss methods that allow only little control over the DNA conformation, such as DNA combing. We then describe DNA flow-stretching approaches that allow more control, and end with the full control on position and extension obtained by manipulating DNA with optical tweezers. The advantages and limitations of the various techniques are discussed, as well as several examples of applications to biophysical or biochemical questions. We conclude with an outlook describing potential future technical developments in combining fluorescence microscopy with DNA micromanipulation technology. PMID:18586820

  8. The theory of bio-energy transport in the protein molecules and its properties

    NASA Astrophysics Data System (ADS)

    Pang, Xiao-feng

    2011-10-01

    The bio-energy transport is a basic problem in life science and related to many biological processes. Therefore to establish the mechanism of bio-energy transport and its theory have an important significance. Based on different properties of structure of α-helical protein molecules some theories of bio-energy transport along the molecular chains have been proposed and established, where the energy is released by hydrolysis of adenosine triphosphate (ATP). A brief survey of past researches on different models and theories of bio-energy, including Davydov's, Takeno's, Yomosa's, Brown et al.'s, Schweitzer's, Cruzeiro-Hansson's, Forner's and Pang's models were first stated in this paper. Subsequently we studied and reviewed mainly and systematically the properties, thermal stability and lifetimes of the carriers (solitons) transporting the bio-energy at physiological temperature 300 K in Pang's and Davydov's theories. From these investigations we know that the carrier (soliton) of bio-energy transport in the α-helical protein molecules in Pang's model has a higher binding energy, higher thermal stability and larger lifetime at 300 K relative to those of Davydov's model, in which the lifetime of the new soliton at 300 K is enough large and belongs to the order of 10 -10 s or τ/τ⩾700. Thus we can conclude that the soliton in Pang's model is exactly the carrier of the bio-energy transport, Pang's theory is appropriate to α-helical protein molecules.

  9. The cellular membrane as a mediator for small molecule interaction with membrane proteins.

    PubMed

    Mayne, Christopher G; Arcario, Mark J; Mahinthichaichan, Paween; Baylon, Javier L; Vermaas, Josh V; Navidpour, Latifeh; Wen, Po-Chao; Thangapandian, Sundarapandian; Tajkhorshid, Emad

    2016-10-01

    The cellular membrane constitutes the first element that encounters a wide variety of molecular species to which a cell might be exposed. Hosting a large number of structurally and functionally diverse proteins associated with this key metabolic compartment, the membrane not only directly controls the traffic of various molecules in and out of the cell, it also participates in such diverse and important processes as signal transduction and chemical processing of incoming molecular species. In this article, we present a number of cases where details of interaction of small molecular species such as drugs with the membrane, which are often experimentally inaccessible, have been studied using advanced molecular simulation techniques. We have selected systems in which partitioning of the small molecule with the membrane constitutes a key step for its final biological function, often binding to and interacting with a protein associated with the membrane. These examples demonstrate that membrane partitioning is not only important for the overall distribution of drugs and other small molecules into different compartments of the body, it may also play a key role in determining the efficiency and the mode of interaction of the drug with its target protein. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg. PMID:27163493

  10. Single molecule photobleaching (SMPB) technology for counting of RNA, DNA, protein and other molecules in nanoparticles and biological complexes by TIRF instrumentation.

    PubMed

    Zhang, Hui; Guo, Peixuan

    2014-05-15

    Direct counting of biomolecules within biological complexes or nanomachines is demanding. Single molecule counting using optical microscopy is challenging due to the diffraction limit. The single molecule photobleaching (SMPB) technology for direct counting developed by our team (Shu et al., 2007 [18]; Zhang et al., 2007 [19]) offers a simple and straightforward method to determine the stoichiometry of molecules or subunits within biocomplexes or nanomachines at nanometer scales. Stoichiometry is determined by real-time observation of the number of descending steps resulted from the photobleaching of individual fluorophore. This technology has now been used extensively for single molecule counting of protein, RNA, and other macromolecules in a variety of complexes or nanostructures. Here, we elucidate the SMPB technology, using the counting of RNA molecules within a bacteriophage phi29 DNA-packaging biomotor as an example. The method described here can be applied to the single molecule counting of other molecules in other systems. The construction of a concise, simple and economical single molecule total internal reflection fluorescence (TIRF) microscope combining prism-type and objective-type TIRF is described. The imaging system contains a deep-cooled sensitive EMCCD camera with single fluorophore detection sensitivity, a laser combiner for simultaneous dual-color excitation, and a Dual-View™ imager to split the multiple outcome signals to different detector channels based on their wavelengths. Methodology of the single molecule photobleaching assay used to elucidate the stoichiometry of RNA on phi29 DNA packaging motor and the mechanism of protein/RNA interaction are described. Different methods for single fluorophore labeling of RNA molecules are reviewed. The process of statistical modeling to reveal the true copy number of the biomolecules based on binomial distribution is also described.

  11. The Protein Architecture of Human Secretory Vesicles Reveals Differential Regulation of Signaling Molecule Secretion by Protein Kinases

    PubMed Central

    Taupenot, Laurent; Ziegler, Michael; O'Connor, Daniel T.; Ma, Qi; Smoot, Michael; Ideker, Trey; Hook, Vivian

    2012-01-01

    Secretory vesicles are required for release of chemical messengers to mediate intercellular signaling among human biological systems. It is necessary to define the organization of the protein architecture of the ‘human’ dense core secretory vesicles (DCSV) to understand mechanisms for secretion of signaling molecules essential for cellular regulatory processes. This study, therefore, conducted extensive quantitative proteomics and systems biology analyses of human DCSV purified from human pheochromocytoma. Over 600 human DCSV proteins were identified with quantitative evaluation of over 300 proteins, revealing that most proteins participate in producing peptide hormones and neurotransmitters, enzymes, and the secretory machinery. Systems biology analyses provided a model of interacting DCSV proteins, generating hypotheses for differential intracellular protein kinases A and C signaling pathways. Activation of cellular PKA and PKC pathways resulted in differential secretion of neuropeptides, catecholamines, and β-amyloid of Alzheimer's disease for mediating cell-cell communication. This is the first study to define a model of the protein architecture of human DCSV for human disease and health. PMID:22916103

  12. Influence of the water molecules near surface of viral protein on virus activation process

    NASA Astrophysics Data System (ADS)

    Shepelenko, S. O.; Salnikov, A. S.; Rak, S. V.; Goncharova, E. P.; Ryzhikov, A. B.

    2009-06-01

    The infection of a cell with influenza virus comprises the stages of receptor binding to the cell membrane, endocytosis of virus particle, and fusion of the virus envelope and cell endosome membrane, which is determined by the conformational changes in hemagglutinin, a virus envelope protein, caused by pH decrease within the endosome. The pH value that induces conformation rearrangements of hemagglutinin molecule considerably varies for different influenza virus strains, first and foremost, due to the differences in amino acid structure of the corresponding proteins. The main goal of this study was to construct a model making it possible to assess the critical pH value characterizing the fusogenic activity of influenza virus hemagglutinin from the data on hemagglutinin structure and experimental verification of this model. Under this model, we assume that when the electrostatic force between interacting hemagglutinin molecules in the virus envelop exceeds a certain value, the hemagglutinin HA1 subunits are arranged so that they form a cavity sufficient for penetration of water molecules. This event leads to an irreversible hydration of the inner fragments of hemagglutinin molecule in a trimer and to the completion of conformational changes. The geometry of electrostatic field in hemagglutinin trimer was calculated taking into account the polarization effects near the interface of two dielectrics, aqueous medium and protein macromolecule. The critical pH values for the conformational changes in hemagglutinin were measured by the erythrocyte hemolysis induced by influenza virus particles when decreasing pH. The critical pH value conditionally separating the pH range into the regions with and without the conformational changes was calculated for several influenza virus H1N1 and H3N2 strains based on the data on the amino acid structure of the corresponding hemagglutinin molecules. Comparison of the theoretical and experimental values of critical pH values for

  13. Fluorescence dynamics of microsphere-adsorbed sunscreens

    NASA Astrophysics Data System (ADS)

    Krishnan, R.

    2005-03-01

    Sunscreens are generally oily substances which are prepared in organic solvents, emulsions or dispersions with micro- or nanoparticles. These molecules adsorb to and integrate into skin cells. In order to understand the photophysical properties of the sunscreen, we compare steady-state and time-resolved fluorescence in organic solvent of varying dielectric constant ɛ and adsorbed to polystyrene microspheres and dispersed in water. Steady-state fluorescence is highest and average fluorescence lifetime longest in toluene, the solvent of lowest ɛ. However, there is no uniform dependence on ɛ. Sunscreens PABA and padimate-O show complex emission spectra. Microsphere-adsorbed sunscreens exhibit highly non-exponential decay, illustrative of multiple environments of the adsorbed molecule. The heterogeneous fluorescence dynamics likely characterizes sunscreen adsorbed to cells.

  14. Screening of Small-Molecule Inhibitors of Protein-Protein Interaction with Capillary Electrophoresis Frontal Analysis.

    PubMed

    Xu, Mei; Liu, Chao; Zhou, Mi; Li, Qing; Wang, Renxiao; Kang, Jingwu

    2016-08-16

    A simple and effective method for identifying inhibitors of protein-protein interactions (PPIs) was developed by using capillary electrophoresis frontal analysis (CE-FA). Antiapoptotic B-cell-2 (Bcl-2) family member Bcl-XL protein, a 5-carboxyfluorescein labeled peptide truncated from the BH3 domain of Bid (F-Bid) as the ligand, and a known Bcl-XL-Bid interaction inhibitor ABT-263 were employed as an experimental model for the proof of concept. In CE-FA, the free ligand is separated from the protein and protein-ligand complex to permit the measurement of the equilibrium concentration of the ligand, hence the dissociation constant of the protein-ligand complex. In the presence of inhibitors, formation of the protein-ligand complex is hindered, thereby the inhibition can be easily identified by the raised plateau height of the ligand and the decayed plateau of the complex. Further, we proposed an equation used to convert the IC50 value into the inhibition constant Ki value, which is more useful than the former for comparison. In addition, the sample pooling strategy was employed to improve the screening throughput more than 10 times. A small chemical library composed of synthetic compounds and natural extracts were screened with the method, two natural products, namely, demethylzeylasteral and celastrol, were identified as new inhibitors to block the Bcl-XL-Bid interaction. Cell-based assay was performed to validate the activity of the identified compounds. The result demonstrated that CE-FA represents a straightforward and robust technique for screening of PPI inhibitors. PMID:27425825

  15. Rational design of selective small-molecule inhibitors for β-catenin/B-cell lymphoma 9 protein-protein interactions.

    PubMed

    Hoggard, Logan R; Zhang, Yongqiang; Zhang, Min; Panic, Vanja; Wisniewski, John A; Ji, Haitao

    2015-09-30

    Selective inhibition of α-helix-mediated protein-protein interactions (PPIs) with small organic molecules provides great potential for the discovery of chemical probes and therapeutic agents. Protein Data Bank data mining using the HippDB database indicated that (1) the side chains of hydrophobic projecting hot spots at positions i, i + 3, and i + 7 of an α-helix had few orientations when interacting with the second protein and (2) the hot spot pockets of PPI complexes had different sizes, shapes, and chemical groups when interacting with the same hydrophobic projecting hot spots of α-helix. On the basis of these observations, a small organic molecule, 4'-fluoro-N-phenyl-[1,1'-biphenyl]-3-carboxamide, was designed as a generic scaffold that itself directly mimics the binding mode of the side chains of hydrophobic projecting hot spots at positions i, i + 3, and i + 7 of an α-helix. Convenient decoration of this generic scaffold led to the selective disruption of α-helix-mediated PPIs. A series of small-molecule inhibitors selective for β-catenin/B-cell lymphoma 9 (BCL9) over β-catenin/cadherin PPIs was designed and synthesized. The binding mode of new inhibitors was characterized by site-directed mutagenesis and structure-activity relationship studies. This new class of inhibitors can selectively disrupt β-catenin/BCL9 over β-catenin/cadherin PPIs, suppress the transactivation of canonical Wnt signaling, downregulate the expression of Wnt target genes, and inhibit the growth of Wnt/β-catenin-dependent cancer cells. PMID:26352795

  16. Targeted studies on the interaction of nicotine and morin molecules with amyloid β-protein.

    PubMed

    Boopathi, Subramaniam; Kolandaivel, Ponmalai

    2014-03-01

    Alzheimer's disease (AD) is a neurodegenerative disorder that occurs due to progressive deposition of amyloid β-protein (Aβ) in the brain. Stable conformations of solvated Aβ₁₋₄₂ protein were predicted by molecular dynamics (MD) simulation using the OPLSAA force field. The seven residue peptide (Lys-Leu-Val-Phe-Phe-Ala-Glu) Aβ₁₆₋₂₂ associated with AD was studied and reported in this paper. Since effective therapeutic agents have not yet been studied in detail, attention has focused on the use of natural products as effective anti-aggregation compounds, targeting the Aβ₁₋₄₂ protein directly. Experimental and theoretical investigation suggests that some compounds extracted from natural products might be useful, but detailed insights into the mechanism by which they might act remains elusive. The molecules nicotine and morin are found in cigarettes and beverages. Here, we report the results of interaction studies of these compounds at each hydrophobic residue of Aβ₁₆₋₂₂ peptide using the hybrid ONIOM (B3LYP/6-31G**:UFF) method. It was found that interaction with nicotine produced higher deformation in the Aβ₁₆₋₂₂ peptide than interaction with morin. MD simulation studies revealed that interaction of the nicotine molecule with the β-sheet of Aβ₁₆₋₂₂ peptide transforms the β-sheet to an α-helical structure, which helps prohibit the aggregation of Aβ-protein.

  17. Overview of Recent Progress in Protein-Expression Technologies for Small-Molecule Screening.

    PubMed

    Cuozzo, John W; Soutter, Holly H

    2014-08-01

    Production of novel soluble and membrane-localized protein targets for functional and affinity-based screening has often been limited by the inability of traditional protein-expression systems to generate recombinant proteins that have properties similar to those of their endogenous counterparts. Such targets have often been labeled as challenging. Although biological validation of these challenging targets for specific disease areas may be strong, discovery of small-molecule modulators can be greatly delayed or completely halted due to target-expression issues. In this article, the limitations of traditional protein-expression systems will be discussed along with new systems designed to overcome these challenges. Recent work in this field has focused on two major areas for both soluble and membrane targets: construct-design strategies to improve expression levels and new hosts that can carry out the posttranslational modifications necessary for proper target folding and function. Another area of active research has been on the reconstitution of solubilized membrane targets for both structural analysis and screening. Finally, the potential impact of these new systems on the output of small-molecule screening campaigns will be discussed. PMID:24525871

  18. SwissDock, a protein-small molecule docking web service based on EADock DSS

    PubMed Central

    Grosdidier, Aurélien; Zoete, Vincent; Michielin, Olivier

    2011-01-01

    Most life science processes involve, at the atomic scale, recognition between two molecules. The prediction of such interactions at the molecular level, by so-called docking software, is a non-trivial task. Docking programs have a wide range of applications ranging from protein engineering to drug design. This article presents SwissDock, a web server dedicated to the docking of small molecules on target proteins. It is based on the EADock DSS engine, combined with setup scripts for curating common problems and for preparing both the target protein and the ligand input files. An efficient Ajax/HTML interface was designed and implemented so that scientists can easily submit dockings and retrieve the predicted complexes. For automated docking tasks, a programmatic SOAP interface has been set up and template programs can be downloaded in Perl, Python and PHP. The web site also provides an access to a database of manually curated complexes, based on the Ligand Protein Database. A wiki and a forum are available to the community to promote interactions between users. The SwissDock web site is available online at http://www.swissdock.ch. We believe it constitutes a step toward generalizing the use of docking tools beyond the traditional molecular modeling community. PMID:21624888

  19. Max Delbruck Prize in Biological Physics Lecture: Single-molecule protein folding and transition paths

    NASA Astrophysics Data System (ADS)

    Eaton, William

    2012-02-01

    The transition path is the tiny fraction of an equilibrium molecular trajectory when a transition occurs by crossing the free energy barrier between two states. It is a uniquely single-molecule property, and has not yet been observed experimentally for any system in the condensed phase. The importance of the transition path in protein folding is that it contains all of the mechanistic information on how a protein folds. As a major step toward observing transition paths, we have determined the average transition-path time for a fast and a slow-folding protein from a photon-by-photon analysis of fluorescence trajectories in single-molecule FRET experiments. While the folding rate coefficients differ by 10,000-fold, surprisingly, the transition-path times differ by less than 5-fold, showing that a successful barrier crossing event takes almost the same time for a fast- and a slow-folding protein, i.e. almost the same time to fold when it actually happens.

  20. Specificity of Amyloid Precursor-like Protein 2 Interactions with MHC Class I Molecules

    PubMed Central

    Tuli, Amit; Sharma, Mahak; Naslavsky, Naava; Caplan, Steve; Solheim, Joyce C.

    2008-01-01

    The ubiquitously expressed amyloid precursor-like protein 2 (APLP2) has been previously found to regulate cell surface expression of the MHC class I molecule Kd and bind strongly to Kd. In the study reported here, we demonstrated that APLP2 binds, in varied degrees, to several other mouse MHC class I allotypes, and that the ability of APLP2 to affect cell surface expression of an MHC class I molecule is not limited to Kd. Ld, like Kd, was found associated with APLP2 in the Golgi, but Kd was also associated with APLP2 within intracellular vesicular structures. We also investigated the effect of β2m on APLP2/MHC interaction, and found that human β2m transfection increased the association of APLP2 with mouse MHC class I molecules, likely by affecting H2 class I heavy chain conformation. APLP2 was demonstrated to bind specifically to the conformation of Ld having folded outer domains, consistent with our previous results with Kd and indicating APLP2 interacts with the α1α2 region on each of these H2 class I molecules. Furthermore, we observed that binding to APLP2 involved the MHC α3/transmembrane/cytoplasmic region, suggesting that conserved as well as polymorphic regions of the H2 class I molecule may participate in interaction with APLP2. In summary, we demonstrated that APLP2′s binding, co-localization pattern, and functional impact vary among H2 class I molecules, and that APLP2/MHC association is influenced by multiple domains of the MHC class I heavy chain and by β2m’s effects on the conformation of the heavy chain. PMID:18452037

  1. Polymer-drug conjugates for intracellar molecule-targeted photoinduced inactivation of protein and growth inhibition of cancer cells

    NASA Astrophysics Data System (ADS)

    Wang, Bing; Yuan, Huanxiang; Zhu, Chunlei; Yang, Qiong; Lv, Fengting; Liu, Libing; Wang, Shu

    2012-10-01

    For most molecule-targeted anticancer systems, intracellular protein targets are very difficult to be accessed by antibodies, and also most efforts are made to inhibit protein activity temporarily rather than inactivate them permanently. In this work we firstly designed and synthesized multifunctional polymer-drug conjugates (polythiophene-tamoxifen) for intracellular molecule-targeted binding and inactivation of protein (estrogen receptor α, ERα) for growth inhibition of MCF-7 cancer cells. Small molecule drug was conjugated to polymer side chain for intracellular signal protein targeting, and simultaneously the fluorescent characteristic of polymer for tracing the cellular uptake and localization of polythiophene-drug conjugates by cell imaging. Under light irradiation, the conjugated polymer can sensitize oxygen to produce reactive oxygen species (ROS) that specifically inactivate the targeted protein, and thus inhibit the growth of tumor cells. The conjugates showed selective growth inhibition of ERα positive cancer cells, which exhibits low side effect for our intracellular molecule-targeted therapy system.

  2. Quantification of DNA-associated proteins inside eukaryotic cells using single-molecule localization microscopy.

    PubMed

    Etheridge, Thomas J; Boulineau, Rémi L; Herbert, Alex; Watson, Adam T; Daigaku, Yasukazu; Tucker, Jem; George, Sophie; Jönsson, Peter; Palayret, Matthieu; Lando, David; Laue, Ernest; Osborne, Mark A; Klenerman, David; Lee, Steven F; Carr, Antony M

    2014-10-29

    Development of single-molecule localization microscopy techniques has allowed nanometre scale localization accuracy inside cells, permitting the resolution of ultra-fine cell structure and the elucidation of crucial molecular mechanisms. Application of these methodologies to understanding processes underlying DNA replication and repair has been limited to defined in vitro biochemical analysis and prokaryotic cells. In order to expand these techniques to eukaryotic systems, we have further developed a photo-activated localization microscopy-based method to directly visualize DNA-associated proteins in unfixed eukaryotic cells. We demonstrate that motion blurring of fluorescence due to protein diffusivity can be used to selectively image the DNA-bound population of proteins. We designed and tested a simple methodology and show that it can be used to detect changes in DNA binding of a replicative helicase subunit, Mcm4, and the replication sliding clamp, PCNA, between different stages of the cell cycle and between distinct genetic backgrounds.

  3. Dual Functional Small Molecule Probes as Fluorophore and Ligand for Misfolding Proteins

    PubMed Central

    Zhang, Xueli; Ran, Chongzhao

    2013-01-01

    Misfolding of a protein is a destructive process for variety of diseases that include neurodegenerative diseases such as Alzheimer’s disease, Parkinson disease, Huntington disease, mad cow disease, amyotrophic lateral sclerosis (ALS), and frontal temporal dementia (FTD), and other non-CNS diseases such as diabetes, cystic fibrosis, and lysosomal storage diseases. Formation of various misfunctional large assembles of the misfolded protein is the primary consequence. To detect the formation of the aggregated species is very important for not only basic mechanism research but also very crucial for diagnosis of the diseases. In this review, we updated references related to the new development of the dual functional fluorescent small molecule probes for detecting the aggregated proteins in vitro and in vivo. PMID:24363605

  4. Dual Functional Small Molecule Probes as Fluorophore and Ligand for Misfolding Proteins.

    PubMed

    Zhang, Xueli; Ran, Chongzhao

    2013-03-01

    Misfolding of a protein is a destructive process for variety of diseases that include neurodegenerative diseases such as Alzheimer's disease, Parkinson disease, Huntington disease, mad cow disease, amyotrophic lateral sclerosis (ALS), and frontal temporal dementia (FTD), and other non-CNS diseases such as diabetes, cystic fibrosis, and lysosomal storage diseases. Formation of various misfunctional large assembles of the misfolded protein is the primary consequence. To detect the formation of the aggregated species is very important for not only basic mechanism research but also very crucial for diagnosis of the diseases. In this review, we updated references related to the new development of the dual functional fluorescent small molecule probes for detecting the aggregated proteins in vitro and in vivo. PMID:24363605

  5. Chemical reactivation of quenched fluorescent protein molecules enables resin-embedded fluorescence microimaging

    NASA Astrophysics Data System (ADS)

    Xiong, Hanqing; Zhou, Zhenqiao; Zhu, Mingqiang; Lv, Xiaohua; Li, Anan; Li, Shiwei; Li, Longhui; Yang, Tao; Wang, Siming; Yang, Zhongqin; Xu, Tonghui; Luo, Qingming; Gong, Hui; Zeng, Shaoqun

    2014-06-01

    Resin embedding is a well-established technique to prepare biological specimens for microscopic imaging. However, it is not compatible with modern green-fluorescent protein (GFP) fluorescent-labelling technique because it significantly quenches the fluorescence of GFP and its variants. Previous empirical optimization efforts are good for thin tissue but not successful on macroscopic tissue blocks as the quenching mechanism remains uncertain. Here we show most of the quenched GFP molecules are structurally preserved and not denatured after routine embedding in resin, and can be chemically reactivated to a fluorescent state by alkaline buffer during imaging. We observe up to 98% preservation in yellow-fluorescent protein case, and improve the fluorescence intensity 11.8-fold compared with unprocessed samples. We demonstrate fluorescence microimaging of resin-embedded EGFP/EYFP-labelled tissue block without noticeable loss of labelled structures. This work provides a turning point for the imaging of fluorescent protein-labelled specimens after resin embedding.

  6. Kinetic Ductility and Force-Spike Resistance of Proteins from Single-Molecule Force Spectroscopy.

    PubMed

    Cossio, Pilar; Hummer, Gerhard; Szabo, Attila

    2016-08-23

    Ductile materials can absorb spikes in mechanical force, whereas brittle ones fail catastrophically. Here we develop a theory to quantify the kinetic ductility of single molecules from force spectroscopy experiments, relating force-spike resistance to the differential responses of the intact protein and the unfolding transition state to an applied mechanical force. We introduce a class of unistable one-dimensional potential surfaces that encompass previous models as special cases and continuously cover the entire range from ductile to brittle. Compact analytic expressions for force-dependent rates and rupture-force distributions allow us to analyze force-clamp and force-ramp pulling experiments. We find that the force-transmitting protein domains of filamin and titin are kinetically ductile when pulled from their two termini, making them resistant to force spikes. For the mechanostable muscle protein titin, a highly ductile model reconciles data over 10 orders of magnitude in force loading rate from experiment and simulation.

  7. Chemical reactivation of quenched fluorescent protein molecules enables resin-embedded fluorescence microimaging

    PubMed Central

    Xiong, Hanqing; Zhou, Zhenqiao; Zhu, Mingqiang; Lv, Xiaohua; Li, Anan; Li, Shiwei; Li, Longhui; Yang, Tao; Wang, Siming; Yang, Zhongqin; Xu, Tonghui; Luo, Qingming; Gong, Hui; Zeng, Shaoqun

    2014-01-01

    Resin embedding is a well-established technique to prepare biological specimens for microscopic imaging. However, it is not compatible with modern green-fluorescent protein (GFP) fluorescent-labelling technique because it significantly quenches the fluorescence of GFP and its variants. Previous empirical optimization efforts are good for thin tissue but not successful on macroscopic tissue blocks as the quenching mechanism remains uncertain. Here we show most of the quenched GFP molecules are structurally preserved and not denatured after routine embedding in resin, and can be chemically reactivated to a fluorescent state by alkaline buffer during imaging. We observe up to 98% preservation in yellow-fluorescent protein case, and improve the fluorescence intensity 11.8-fold compared with unprocessed samples. We demonstrate fluorescence microimaging of resin-embedded EGFP/EYFP-labelled tissue block without noticeable loss of labelled structures. This work provides a turning point for the imaging of fluorescent protein-labelled specimens after resin embedding. PMID:24886825

  8. Quantification of DNA-associated proteins inside eukaryotic cells using single-molecule localization microscopy

    PubMed Central

    Etheridge, Thomas J.; Boulineau, Rémi L.; Herbert, Alex; Watson, Adam T.; Daigaku, Yasukazu; Tucker, Jem; George, Sophie; Jönsson, Peter; Palayret, Matthieu; Lando, David; Laue, Ernest; Osborne, Mark A.; Klenerman, David; Lee, Steven F.; Carr, Antony M.

    2014-01-01

    Development of single-molecule localization microscopy techniques has allowed nanometre scale localization accuracy inside cells, permitting the resolution of ultra-fine cell structure and the elucidation of crucial molecular mechanisms. Application of these methodologies to understanding processes underlying DNA replication and repair has been limited to defined in vitro biochemical analysis and prokaryotic cells. In order to expand these techniques to eukaryotic systems, we have further developed a photo-activated localization microscopy-based method to directly visualize DNA-associated proteins in unfixed eukaryotic cells. We demonstrate that motion blurring of fluorescence due to protein diffusivity can be used to selectively image the DNA-bound population of proteins. We designed and tested a simple methodology and show that it can be used to detect changes in DNA binding of a replicative helicase subunit, Mcm4, and the replication sliding clamp, PCNA, between different stages of the cell cycle and between distinct genetic backgrounds. PMID:25106872

  9. Kinetic Ductility and Force-Spike Resistance of Proteins from Single-Molecule Force Spectroscopy.

    PubMed

    Cossio, Pilar; Hummer, Gerhard; Szabo, Attila

    2016-08-23

    Ductile materials can absorb spikes in mechanical force, whereas brittle ones fail catastrophically. Here we develop a theory to quantify the kinetic ductility of single molecules from force spectroscopy experiments, relating force-spike resistance to the differential responses of the intact protein and the unfolding transition state to an applied mechanical force. We introduce a class of unistable one-dimensional potential surfaces that encompass previous models as special cases and continuously cover the entire range from ductile to brittle. Compact analytic expressions for force-dependent rates and rupture-force distributions allow us to analyze force-clamp and force-ramp pulling experiments. We find that the force-transmitting protein domains of filamin and titin are kinetically ductile when pulled from their two termini, making them resistant to force spikes. For the mechanostable muscle protein titin, a highly ductile model reconciles data over 10 orders of magnitude in force loading rate from experiment and simulation. PMID:27558726

  10. Probing protein multidimensional conformational fluctuations by single-molecule multiparameter photon stamping spectroscopy.

    PubMed

    Lu, Maolin; Lu, H Peter

    2014-10-16

    Conformational motions of proteins are highly dynamic and intrinsically complex. To capture the temporal and spatial complexity of conformational motions and further to understand their roles in protein functions, an attempt is made to probe multidimensional conformational dynamics of proteins besides the typical one-dimensional FRET coordinate or the projected conformational motions on the one-dimensional FRET coordinate. T4 lysozyme hinge-bending motions between two domains along α-helix have been probed by single-molecule FRET. Nevertheless, the domain motions of T4 lysozyme are rather complex involving multiple coupled nuclear coordinates and most likely contain motions besides hinge-bending. It is highly likely that the multiple dimensional protein conformational motions beyond the typical enzymatic hinged-bending motions have profound impact on overall enzymatic functions. In this report, we have developed a single-molecule multiparameter photon stamping spectroscopy integrating fluorescence anisotropy, FRET, and fluorescence lifetime. This spectroscopic approach enables simultaneous observations of both FRET-related site-to-site conformational dynamics and molecular rotational (or orientational) motions of individual Cy3-Cy5 labeled T4 lysozyme molecules. We have further observed wide-distributed rotational flexibility along orientation coordinates by recording fluorescence anisotropy and simultaneously identified multiple intermediate conformational states along FRET coordinate by monitoring time-dependent donor lifetime, presenting a whole picture of multidimensional conformational dynamics in the process of T4 lysozyme open-close hinge-bending enzymatic turnover motions under enzymatic reaction conditions. By analyzing the autocorrelation functions of both lifetime and anisotropy trajectories, we have also observed the dynamic and static inhomogeneity of T4 lysozyme multidimensional conformational fluctuation dynamics, providing a fundamental

  11. Simple horizontal magnetic tweezers for micromanipulation of single DNA molecules and DNA–protein complexes

    PubMed Central

    McAndrew, Christopher P.; Tyson, Christopher; Zischkau, Joseph; Mehl, Patrick; Tuma, Pamela L.; Pegg, Ian L.; Sarkar, Abhijit

    2016-01-01

    We report the development of a simple-to-implement magnetic force transducer that can apply a wide range of piconewton (pN) scale forces on single DNA molecules and DNA–protein complexes in the horizontal plane. The resulting low-noise force-extension data enable very high-resolution detection of changes in the DNA tether’s extension: ~0.05 pN in force and <10 nm change in extension. We have also verified that we can manipulate DNA in near equilibrium conditions through the wide range of forces by ramping the force from low to high and back again, and observing minimal hysteresis in the molecule’s force response. Using a calibration technique based on Stokes’ drag law, we have confirmed our force measurements from DNA force-extension experiments obtained using the fluctuation-dissipation theorem applied to transverse fluctuations of the magnetic microsphere. We present data on the force-distance characteristics of a DNA molecule complexed with histones. The results illustrate how the tweezers can be used to study DNA binding proteins at the single molecule level. PMID:26757808

  12. Highly sensitive immunoassay of protein molecules based on single nanoparticle fluorescence detection in a nanowell

    NASA Astrophysics Data System (ADS)

    Han, Jin-Hee; Kim, Hee-Joo; Lakshmana, Sudheendra; Gee, Shirley J.; Hammock, Bruce D.; Kennedy, Ian M.

    2011-03-01

    A nanoarray based-single molecule detection system was developed for detecting proteins with extremely high sensitivity. The nanoarray was able to effectively trap nanoparticles conjugated with biological sample into nanowells by integrating with an electrophoretic particle entrapment system (EPES). The nanoarray/EPES is superior to other biosensor using immunoassays in terms of saving the amounts of biological solution and enhancing kinetics of antibody binding due to reduced steric hindrance from the neighboring biological molecules. The nanoarray patterned onto a layer of PMMA and LOL on conductive and transparent indium tin oxide (ITO)-glass slide by using e-beam lithography. The suspension of 500 nm-fluorescent (green emission)-carboxylated polystyrene (PS) particles coated with protein-A followed by BDE 47 polyclonal antibody was added to the chip that was connected to the positive voltage. The droplet was covered by another ITO-coated-glass slide and connected to a ground terminal. After trapping the particles into the nanowells, the solution of different concentrations of anti-rabbit- IgG labeled with Alexa 532 was added for an immunoassay. A single molecule detection system could quantify the anti-rabbit IgG down to atto-mole level by counting photons emitted from the fluorescent dye bound to a single nanoparticle in a nanowell.

  13. Trafficking of MHC molecules to the cell surface creates dynamic protein patches.

    PubMed

    Blumenthal, Daniel; Edidin, Michael; Gheber, Levi A

    2016-09-01

    Major histocompatibility complex class I (MHC-I) molecules signal infection or transformation by engaging receptors on T lymphocytes. The spatial organization of MHC-I on the plasma membranes is important for this engagement. We and others have shown that MHC-I molecules, like other membrane proteins, are not uniformly distributed, but occur in patches in the plasma membrane. Here, we describe the temporal details of MHC-I patch formation and combine them with the spatial details, which we have described earlier, to yield a comprehensive quantitative description of patch formation. MHC-I is delivered to the plasma membrane in clathrin-coated vesicles, arriving at a rate of ∼2.5×10(-3) μm(-1) min(-1) (or about two arrivals per minute over the whole cell). The vesicles dock and fuse at non-random, apparently targeted, locations on the membrane and the newly delivered MHC-I molecules form patches that are a few hundred nanometers in diameter. The patches are maintained at steady state by a dynamic equilibrium between the rate of delivery and the rate of hindered diffusion of MHC-I molecules out of the patches (caused by components of the actin cytoskeleton). PMID:27466380

  14. Specific Interactions of Neutral Side Chains of an Adsorbed Protein with the Surface of α-Quartz and Silica Gel.

    PubMed

    Odinokov, Alexey V; Bagaturyants, Alexander A

    2015-07-16

    Many key features of the protein adsorption on the silica surfaces still remain unraveled. One of the open questions is the interaction of nonpolar side chains with siloxane cavities. Here, we use nonequilibrium molecular dynamics simulations for the detailed investigation of the binding of several hydrophobic and amphiphilic protein side chains with silica surface. These interactions were found to be a possible driving force for protein adsorption. The free energy gain was larger for the disordered surface of amorphous silica gel as compared to α-quartz, but the impact depended on the type of amino acid. The dependence was analyzed from the structural point of view. For every amino acid an enthalpy-entropy compensation behavior was observed. These results confirm a hypothesis of an essential role of hydrophobic interactions in protein unfolding and irreversible adsorption on the silica surface.

  15. Small-Molecule Protein-Protein Interaction Inhibitor of Oncogenic Rho Signaling.

    PubMed

    Diviani, Dario; Raimondi, Francesco; Del Vescovo, Cosmo D; Dreyer, Elisa; Reggi, Erica; Osman, Halima; Ruggieri, Lucia; Gonano, Cynthia; Cavin, Sabrina; Box, Clare L; Lenoir, Marc; Overduin, Michael; Bellucci, Luca; Seeber, Michele; Fanelli, Francesca

    2016-09-22

    Uncontrolled activation of Rho signaling by RhoGEFs, in particular AKAP13 (Lbc) and its close homologs, is implicated in a number of human tumors with poor prognosis and resistance to therapy. Structure predictions and alanine scanning mutagenesis of Lbc identified a circumscribed hot region for RhoA recognition and activation. Virtual screening targeting that region led to the discovery of an inhibitor of Lbc-RhoA interaction inside cells. By interacting with the DH domain, the compound inhibits the catalytic activity of Lbc, halts cellular responses to activation of oncogenic Lbc pathways, and reverses a number of prostate cancer cell phenotypes such as proliferation, migration, and invasiveness. This study provides insights into the structural determinants of Lbc-RhoA recognition. This is a successful example of structure-based discovery of a small protein-protein interaction inhibitor able to halt oncogenic Rho signaling in cancer cells with therapeutic implications.

  16. Interaction of HIV-1 Gag protein components with single DNA molecules

    NASA Astrophysics Data System (ADS)

    Cruceanu, Margareta; Gorelick, Robert J.; Williams, Mark C.

    2003-03-01

    The Gag protein of the HIV-1 retrovirus is cleaved into three major proteins as part of viral maturation: nucleocapsid (NC), capsid, and matrix. NC is the first of these proteins to be cleaved, and it is cleaved in three stages into NCp15, followed by NCp9, and finally NCp7. In this study, we use optical tweezers to investigate the capability of these NC proteins to alter the helix-coil transition of single DNA molecules. We have previously shown that the capability to alter the DNA helix-coil transition is an excellent probe of the nucleic acid chaperone activity of NC proteins, in which the secondary structure of nucleic acids is rearranged to facilitate reverse transcription. By examining the capability of NCp15, NCp9, and NCp7 to alter DNA stretching, the current studies will test the role of proteolytic cleavage of Gag in regulating the nucleic acid chaperone activity of NC. Whereas binding studies suggest that NCp9 and NCp15 bind more strongly to DNA than NCp7, our DNA stretching results indicate that these proteins all have similar effects on DNA stretching.

  17. MARCKS-Like Protein is an Initiating Molecule in Axolotl Appendage Regeneration

    PubMed Central

    Sugiura, Takuji; Wang, Heng; Barsacchi, Rico; Simon, Andras; Tanaka, Elly M.

    2016-01-01

    Identifying key molecules that launch regeneration has been a long sought goal. Multiple regenerative animals show an initial wound-associated proliferative response that transits into sustained proliferation if a significant portion of the body part has been removed 1-3. In the axolotl, appendage amputation initiates a round of wound-associated cell cycle induction followed by continued proliferation that is dependent on nerve-derived signals 4,5. A wound-associated molecule that triggers the initial proliferative response to launch regeneration has remained obscure. Using an expression cloning strategy followed by in vivo gain- and loss-of-function assays, we identified axolotl MARCKS like Protein (MLP) as an extracellularly released factor that induces the initial cell cycle response during axolotl appendage regeneration. PMID:26934225

  18. Chemical annotation of small and peptide-like molecules at the Protein Data Bank.

    PubMed

    Young, Jasmine Y; Feng, Zukang; Dimitropoulos, Dimitris; Sala, Raul; Westbrook, John; Zhuravleva, Marina; Shao, Chenghua; Quesada, Martha; Peisach, Ezra; Berman, Helen M

    2013-01-01

    Over the past decade, the number of polymers and their complexes with small molecules in the Protein Data Bank archive (PDB) has continued to increase significantly. To support scientific advancements and ensure the best quality and completeness of the data files over the next 10 years and beyond, the Worldwide PDB partnership that manages the PDB archive is developing a new deposition and annotation system. This system focuses on efficient data capture across all supported experimental methods. The new deposition and annotation system is composed of four major modules that together support all of the processing requirements for a PDB entry. In this article, we describe one such module called the Chemical Component Annotation Tool. This tool uses information from both the Chemical Component Dictionary and Biologically Interesting molecule Reference Dictionary to aid in annotation. Benchmark studies have shown that the Chemical Component Annotation Tool provides significant improvements in processing efficiency and data quality. Database URL: http://wwpdb.org. PMID:24291661

  19. Chemical annotation of small and peptide-like molecules at the Protein Data Bank.

    PubMed

    Young, Jasmine Y; Feng, Zukang; Dimitropoulos, Dimitris; Sala, Raul; Westbrook, John; Zhuravleva, Marina; Shao, Chenghua; Quesada, Martha; Peisach, Ezra; Berman, Helen M

    2013-01-01

    Over the past decade, the number of polymers and their complexes with small molecules in the Protein Data Bank archive (PDB) has continued to increase significantly. To support scientific advancements and ensure the best quality and completeness of the data files over the next 10 years and beyond, the Worldwide PDB partnership that manages the PDB archive is developing a new deposition and annotation system. This system focuses on efficient data capture across all supported experimental methods. The new deposition and annotation system is composed of four major modules that together support all of the processing requirements for a PDB entry. In this article, we describe one such module called the Chemical Component Annotation Tool. This tool uses information from both the Chemical Component Dictionary and Biologically Interesting molecule Reference Dictionary to aid in annotation. Benchmark studies have shown that the Chemical Component Annotation Tool provides significant improvements in processing efficiency and data quality. Database URL: http://wwpdb.org.

  20. Model for Stretching and Unfolding the Giant Multidomain Muscle Protein Using Single-Molecule Force Spectroscopy

    NASA Astrophysics Data System (ADS)

    Staple, Douglas B.; Payne, Stephen H.; Reddin, Andrew L. C.; Kreuzer, Hans Jürgen

    2008-12-01

    Single-molecule manipulation has allowed the forced unfolding of multidomain proteins. Here we outline a theory that not only explains these experiments but also points out a number of difficulties in their interpretation and makes suggestions for further experiments. For titin we reproduce force-extension curves, the dependence of break force on pulling speed, and break-force distributions and also validate two common experimental views: Unfolding titin Ig domains can be explained as stepwise increases in contour length, and increasing force peaks in native Ig sequences represent a hierarchy of bond strengths. Our theory is valid for essentially any molecule that can be unfolded in atomic force microscopy; as a further example, we present force-extension curves for the unfolding of RNA hairpins.

  1. The physics of pulling polyproteins: a review of single molecule force spectroscopy using the AFM to study protein unfolding

    NASA Astrophysics Data System (ADS)

    Hughes, Megan L.; Dougan, Lorna

    2016-07-01

    One of the most exciting developments in the field of biological physics in recent years is the ability to manipulate single molecules and probe their properties and function. Since its emergence over two decades ago, single molecule force spectroscopy has become a powerful tool to explore the response of biological molecules, including proteins, DNA, RNA and their complexes, to the application of an applied force. The force versus extension response of molecules can provide valuable insight into its mechanical stability, as well as details of the underlying energy landscape. In this review we will introduce the technique of single molecule force spectroscopy using the atomic force microscope (AFM), with particular focus on its application to study proteins. We will review the models which have been developed and employed to extract information from single molecule force spectroscopy experiments. Finally, we will end with a discussion of future directions in this field.

  2. The physics of pulling polyproteins: a review of single molecule force spectroscopy using the AFM to study protein unfolding.

    PubMed

    Hughes, Megan L; Dougan, Lorna

    2016-07-01

    One of the most exciting developments in the field of biological physics in recent years is the ability to manipulate single molecules and probe their properties and function. Since its emergence over two decades ago, single molecule force spectroscopy has become a powerful tool to explore the response of biological molecules, including proteins, DNA, RNA and their complexes, to the application of an applied force. The force versus extension response of molecules can provide valuable insight into its mechanical stability, as well as details of the underlying energy landscape. In this review we will introduce the technique of single molecule force spectroscopy using the atomic force microscope (AFM), with particular focus on its application to study proteins. We will review the models which have been developed and employed to extract information from single molecule force spectroscopy experiments. Finally, we will end with a discussion of future directions in this field.

  3. Crystal structures of bovine odorant-binding protein in complex with odorant molecules.

    PubMed

    Vincent, Florence; Ramoni, Roberto; Spinelli, Silvia; Grolli, Stefano; Tegoni, Mariella; Cambillau, Christian

    2004-10-01

    The structure of bovine odorant-binding protein (bOBP) revealed a striking feature of a dimer formed by domain swapping [Tegoni, M., Ramoni, R., Bignetti, E., Spinelli, S. & Cambillau, C. (1996) Nat. Struct. Biol.3, 863-867; Bianchet, M.A., Bains, G., Pelosi, P., Pevsner, J., Snyder, S.H., Monaco, H.L. & Amzel, L.M. (1996) Nat. Struct. Biol.3, 934-939] and the presence of a naturally occuring ligand [Ramoni, R., Vincent, F., Grolli, S., Conti, V., Malosse, C., Boyer, F.D., Nagnan-Le Meillour, P., Spinelli, S., Cambillau, C. & Tegoni, M. (2001) J. Biol. Chem.276, 7150-7155]. These features led us to investigate the binding of odorant molecules with bOBP in solution and in the crystal. The behavior of odorant molecules in bOBP resembles that observed with porcine OBP (pOBP), although the latter is monomeric and devoid of ligand when purified. The odorant molecules presented K(d) values with bOBP in the micromolar range. Most of the X-ray structures revealed that odorant molecules interact with a common set of residues forming the cavity wall and do not exhibit specific interactions. Depending on the ligand and on the monomer (A or B), a single residue--Phe89--presents alternate conformations and might control cross-talking between the subunits. Crystal data on both pOBP and bOBP, in contrast with binding and spectroscopic studies on rat OBP in solution, reveal an absence of significant conformational changes involving protein loops or backbone. Thus, the role of OBP in signal triggering remains unresolved.

  4. A small molecule inhibitor of redox-regulated protein translocation into mitochondria.

    PubMed

    Dabir, Deepa V; Hasson, Samuel A; Setoguchi, Kiyoko; Johnson, Meghan E; Wongkongkathep, Piriya; Douglas, Colin J; Zimmerman, Johannes; Damoiseaux, Robert; Teitell, Michael A; Koehler, Carla M

    2013-04-15

    The mitochondrial disulfide relay system of Mia40 and Erv1/ALR facilitates import of the small translocase of the inner membrane (Tim) proteins and cysteine-rich proteins. A chemical screen identified small molecules that inhibit Erv1 oxidase activity, thereby facilitating dissection of the disulfide relay system in yeast and vertebrate mitochondria. One molecule, mitochondrial protein import blockers from the Carla Koehler laboratory (MitoBloCK-6), attenuated the import of Erv1 substrates into yeast mitochondria and inhibited oxidation of Tim13 and Cmc1 in in vitro reconstitution assays. In addition, MitoBloCK-6 revealed an unexpected role for Erv1 in the carrier import pathway, namely transferring substrates from the translocase of the outer membrane complex onto the small Tim complexes. Cardiac development was impaired in MitoBloCK-6-exposed zebrafish embryos. Finally, MitoBloCK-6 induced apoptosis via cytochrome c release in human embryonic stem cells (hESCs) but not in differentiated cells, suggesting an important role for ALR in hESC homeostasis.

  5. Small‐Molecule and Peptide Inhibitors of the Pro‐Survival Protein Mcl‐1

    PubMed Central

    Beekman, Andrew M.

    2015-01-01

    Abstract The ability of protein–protein interactions to regulate cellular processes in both beneficial and detrimental ways has made them obvious drug targets. The Bcl‐2 family of proteins undergo a series of protein–protein interactions which regulate the intrinsic cell‐death pathway. The pro‐survival members of the Bcl‐2 family, including Bcl‐2, Bcl‐xL, and Mcl‐1, are commonly overexpressed in a number of human cancers. Effective modulators of members of the Bcl‐2 family have been developed and are undergoing clinical trials, but the efficient modulation of Mcl‐1 is still not represented in the clinic. In addition, Mcl‐1 is a major cause of resistance to radio‐ and chemotherapies, including inhibitors that target other Bcl‐2 family members. Subsequently, the inhibition of Mcl‐1 has become of significant interest to the scientific community. This review covers the progress made to date in modulating the activity of Mcl‐1, by both stapled peptides and small molecules. The development of peptides as drug candidates, and the advancement of experimental and computational techniques used to discover small molecules are also highlighted. PMID:26696548

  6. Single-Molecule Imaging Reveals the Activation Dynamics of Intracellular Protein Smad3 on Cell Membrane

    NASA Astrophysics Data System (ADS)

    Li, Nan; Yang, Yong; He, Kangmin; Zhang, Fayun; Zhao, Libo; Zhou, Wei; Yuan, Jinghe; Liang, Wei; Fang, Xiaohong

    2016-09-01

    Smad3 is an intracellular protein that plays a key role in propagating transforming growth factor β (TGF-β) signals from cell membrane to nucleus. However whether the transient process of Smad3 activation occurs on cell membrane and how it is regulated remains elusive. Using advanced live-cell single-molecule fluorescence microscopy to image and track fluorescent protein-labeled Smad3, we observed and quantified, for the first time, the dynamics of individual Smad3 molecules docking to and activation on the cell membrane. It was found that Smad3 docked to cell membrane in both unstimulated and stimulated cells, but with different diffusion rates and dissociation kinetics. The change in its membrane docking dynamics can be used to study the activation of Smad3. Our results reveal that Smad3 binds with type I TGF-β receptor (TRI) even in unstimulated cells. Its activation is regulated by TRI phosphorylation but independent of receptor endocytosis. This study offers new information on TGF-β/Smad signaling, as well as a new approach to investigate the activation of intracellular signaling proteins for a better understanding of their functions in signal transduction.

  7. Quantitative mass spectrometry-based assay development and validation: from small molecules to proteins.

    PubMed

    Božović, Andrea; Kulasingam, Vathany

    2013-04-01

    Mass spectrometry (MS) has emerged as a powerful analytical tool for the identification, characterization and quantification of various biomolecules (small molecules, drug metabolites and proteins) in biological specimens. The use of mass spectrometers in the clinical diagnostic laboratories have gained popularity due to its ease of development of new assays, ability to measure multiple analytes in a single analytical run, low volume requirements and low reagent costs. Novel technological advancements in ionization sources, instrumentation and software have increased the popularity of these platforms. Consequently, a number of home-brew assays, utilizing the power of MS, are being developed and validated for clinical diagnostic use. In this review, we will discuss the two phases that precede method implementation: method development and validation for both small molecule analysis and protein quantification using liquid chromatography tandem mass spectrometry (LC-MS/MS). Some of the challenges facing protein quantification will be highlighted and an outlook for the future of laboratory medicine and MS will be provided. PMID:23041077

  8. Single-Molecule FRET Reveals Hidden Complexity in a Protein Energy Landscape

    PubMed Central

    Tsytlonok, Maksym; Ibrahim, Shehu M.; Rowling, Pamela J.E.; Xu, Wenshu; Ruedas-Rama, Maria J.; Orte, Angel; Klenerman, David; Itzhaki, Laura S.

    2015-01-01

    Summary Here, using single-molecule FRET, we reveal previously hidden conformations of the ankyrin-repeat domain of AnkyrinR, a giant adaptor molecule that anchors integral membrane proteins to the spectrin-actin cytoskeleton through simultaneous binding of multiple partner proteins. We show that the ankyrin repeats switch between high-FRET and low-FRET states, controlled by an unstructured “safety pin” or “staple” from the adjacent domain of AnkyrinR. Opening of the safety pin leads to unravelling of the ankyrin repeat stack, a process that will dramatically affect the relative orientations of AnkyrinR binding partners and, hence, the anchoring of the spectrin-actin cytoskeleton to the membrane. Ankyrin repeats are one of the most ubiquitous molecular recognition platforms in nature, and it is therefore important to understand how their structures are adapted for function. Our results point to a striking mechanism by which the order-disorder transition and, thereby, the activity of repeat proteins can be regulated. PMID:25565106

  9. Single-Molecule Imaging Reveals the Activation Dynamics of Intracellular Protein Smad3 on Cell Membrane.

    PubMed

    Li, Nan; Yang, Yong; He, Kangmin; Zhang, Fayun; Zhao, Libo; Zhou, Wei; Yuan, Jinghe; Liang, Wei; Fang, Xiaohong

    2016-01-01

    Smad3 is an intracellular protein that plays a key role in propagating transforming growth factor β (TGF-β) signals from cell membrane to nucleus. However whether the transient process of Smad3 activation occurs on cell membrane and how it is regulated remains elusive. Using advanced live-cell single-molecule fluorescence microscopy to image and track fluorescent protein-labeled Smad3, we observed and quantified, for the first time, the dynamics of individual Smad3 molecules docking to and activation on the cell membrane. It was found that Smad3 docked to cell membrane in both unstimulated and stimulated cells, but with different diffusion rates and dissociation kinetics. The change in its membrane docking dynamics can be used to study the activation of Smad3. Our results reveal that Smad3 binds with type I TGF-β receptor (TRI) even in unstimulated cells. Its activation is regulated by TRI phosphorylation but independent of receptor endocytosis. This study offers new information on TGF-β/Smad signaling, as well as a new approach to investigate the activation of intracellular signaling proteins for a better understanding of their functions in signal transduction. PMID:27641076

  10. Single-Molecule Imaging Reveals the Activation Dynamics of Intracellular Protein Smad3 on Cell Membrane

    PubMed Central

    Li, Nan; Yang, Yong; He, Kangmin; Zhang, Fayun; Zhao, Libo; Zhou, Wei; Yuan, Jinghe; Liang, Wei; Fang, Xiaohong

    2016-01-01

    Smad3 is an intracellular protein that plays a key role in propagating transforming growth factor β (TGF-β) signals from cell membrane to nucleus. However whether the transient process of Smad3 activation occurs on cell membrane and how it is regulated remains elusive. Using advanced live-cell single-molecule fluorescence microscopy to image and track fluorescent protein-labeled Smad3, we observed and quantified, for the first time, the dynamics of individual Smad3 molecules docking to and activation on the cell membrane. It was found that Smad3 docked to cell membrane in both unstimulated and stimulated cells, but with different diffusion rates and dissociation kinetics. The change in its membrane docking dynamics can be used to study the activation of Smad3. Our results reveal that Smad3 binds with type I TGF-β receptor (TRI) even in unstimulated cells. Its activation is regulated by TRI phosphorylation but independent of receptor endocytosis. This study offers new information on TGF-β/Smad signaling, as well as a new approach to investigate the activation of intracellular signaling proteins for a better understanding of their functions in signal transduction. PMID:27641076

  11. Serology of Neisseria gonorrhoeae: coagglutination serogroups WI and WII/III correspond to different outer membrane protein I molecules.

    PubMed Central

    Sandstrom, E G; Chen, K C; Buchanan, T M

    1982-01-01

    The 125I-labeled tryptic peptides of the outer membrane protein I of 33 previously characterized serological reference strains of Neisseria gonorrhoeae were investigated by peptide maps in relation to their coagglutination W serogroup. Serogroup WI strains tended to have lower-molecular-weight protein I molecules than did WII strains, and WIII strains had the highest-molecular-weight protein I molecules, although the serogroup could not be predicted from the molecular weights of the protein I molecules for a given strain. All 13 strains belonging to serogroup WI were found to have 11 peptides in common, as judged by their migration with respect to one another and to the internal marker valine in the peptide maps. Common peptides isolated from a given strain were found to comigrate with the corresponding common peptides from other strains in the same serogroup under various electrophoretic conditions. The 20 strains belonging to serogroups WII and WIII were all found to have 10 common peptides by the same criteria. When common peptides from serogroup WI were compared with the common peptides of serogroups WII and WIII, only three of these peptides appeared to be similar. Thus, two different outer membrane protein I molecules seem to exist which are mutually exclusive. Protein IA molecules contain the antigens recognized as serogroup WI, and protein IB molecules contain the antigens that characterize serogroups WII and WIII. Images PMID:6183215

  12. A protein molecule in an aqueous mixed solvent: fluctuation theory outlook.

    PubMed

    Shulgin, Ivan L; Ruckenstein, Eli

    2005-08-01

    In the present paper a procedure to calculate the properties of proteins in aqueous mixed solvents, particularly the excesses of the constituents of the mixed solvent near the protein molecule and the preferential binding parameters, is suggested. Expressions for the Kirkwood-Buff integrals in ternary mixtures and for the preferential binding parameter were derived and used to calculate various properties of infinitely dilute proteins in aqueous mixed solvents. The derived expressions and experimental information regarding the partial molar volumes and the preferential binding parameters were used to calculate the excesses (deficits) of water and cosolvent (in comparison with the bulk concentrations of protein-free mixed solvent) in the vicinity of ribonuclease A, ribonuclease T1, and lysozyme molecules. The calculations showed that water was in excess in the vicinity of ribonuclease A for water/glycerol and water/trehalose mixtures, and the cosolvent urea was in excess in the vicinity of ribonuclease T1 and lysozyme. The derivative of the activity coefficient of the protein with respect to the mole fraction of water was also calculated. This derivative was negative for the water/glycerol and water/trehalose mixed solvents and positive for the water/urea mixture. The mixture of lysozyme in the water/urea solvent is of particular interest, because the lysozyme at pH 7.0 is in its native state up to 9.3M urea, while at pH 2.0 it is denaturated between 2.5 and 5M and higher concentrations of urea. Our results demonstrated a striking similarity in the hydration of lysozyme at both pHs. It is worthwhile to note that the excesses of urea were only weakly composition dependent on both cases.

  13. HaloPROTACS: Use of Small Molecule PROTACs to Induce Degradation of HaloTag Fusion Proteins

    PubMed Central

    Buckley, Dennis L; Raina, Kanak; Darricarrere, Nicole; Hines, John; Gustafson, Jeffrey L; Smith, Ian E.; Miah, Afjal H; Harling, John D; Crews, Craig M

    2015-01-01

    Small molecule-induced protein degradation is an attractive strategy for the development of chemical probes. One method for inducing targeted protein degradation involves the use of PROTACs, heterobifunctional molecules that can recruit specific E3 ligases to a desired protein of interest. PROTACs have been successfully used to degrade numerous proteins in cells, but the peptidic E3 ligase ligands used in previous PROTACs have hindered their development into more mature chemical probes or therapeutics. We report the design of a novel class of PROTACs that incorporate small molecule VHL ligands to successfully degrade HaloTag7 fusion proteins. These HaloPROTACs will inspire the development of future PROTACs with more drug-like properties. Additionally, these HaloPROTACs are useful chemical genetic tools, due to their ability to chemically knockdown widely used HaloTag7 fusion proteins in a general fashion. PMID:26070106

  14. Single-molecule Study of Nucleocapsid Protein Chaperoned DNA Hairpin Structural Dynamics

    NASA Astrophysics Data System (ADS)

    Zeng, Yining; Cosa, Gonzalo; Liu, Hsiao-Wei; Landes, Christy; Makarov, Dmitrii; Barbara, Paul; Musier-Forsyth, Karin

    2006-03-01

    In HIV-1 reverse transcription, the nucleocapsid protein, NC, induces secondary structure fluctuations in the transactivation response (TAR) region of DNA and RNA hairpins. Time resolved single-molecule fluorescence resonance energy transfer was used to study NC chaperoned secondary fluctuations of DNA hairpins. The experiments reveal that the NC induced secondary fluctuations are limited to the terminal stems, and the mechanism for the fluctuations is complex. The dynamic processes occur over a wide time range, i.e. ˜5 to >250 milliseconds and involve long-lived intermediates. The dynamic role of DNA loop regions and NC binding/dissociation events are discussed.

  15. Single-point single-molecule FRAP distinguishes inner and outer nuclear membrane protein distribution

    PubMed Central

    Mudumbi, Krishna C; Schirmer, Eric C; Yang, Weidong

    2016-01-01

    The normal distribution of nuclear envelope transmembrane proteins (NETs) is disrupted in several human diseases. NETs are synthesized on the endoplasmic reticulum and then transported from the outer nuclear membrane (ONM) to the inner nuclear membrane (INM). Quantitative determination of the distribution of NETs on the ONM and INM is limited in available approaches, which moreover provide no information about translocation rates in the two membranes. Here we demonstrate a single-point single-molecule FRAP microscopy technique that enables determination of distribution and translocation rates for NETs in vivo. PMID:27558844

  16. Single-point single-molecule FRAP distinguishes inner and outer nuclear membrane protein distribution.

    PubMed

    Mudumbi, Krishna C; Schirmer, Eric C; Yang, Weidong

    2016-01-01

    The normal distribution of nuclear envelope transmembrane proteins (NETs) is disrupted in several human diseases. NETs are synthesized on the endoplasmic reticulum and then transported from the outer nuclear membrane (ONM) to the inner nuclear membrane (INM). Quantitative determination of the distribution of NETs on the ONM and INM is limited in available approaches, which moreover provide no information about translocation rates in the two membranes. Here we demonstrate a single-point single-molecule FRAP microscopy technique that enables determination of distribution and translocation rates for NETs in vivo. PMID:27558844

  17. Sizing of colloidal particle and protein molecules in a hanging fluid drop

    NASA Technical Reports Server (NTRS)

    Ansari, Rafat R.; Suh, Kwang I.

    1995-01-01

    We report non-invasive particle size measurements of polystyrene latex colloidal particles and bovine serum albumin (BSA) protein molecules suspended in tiny hanging fluid drops of 30 micro-Liter volume using a newly designed fiber optic probe. The probe is based upon the principles of the technique of dynamic light scattering (DLS). The motivation for this work comes from growing protein crystals in outer space. Protein crystals have been grown previously in hanging drops in microgravity experiments on-board the space shuttle orbiter. However, obtaining quantitative information on nucleation and growth of the protein crystals in real time has always been a desired goal, but hitherto not achieved. Several protein researchers have shown interest in using DLS to monitor crystal growth process in a droplet, but elaborate instrumentation and optical alignment problems have made in-situ applications difficult. We demonstrate that such an experiment is now possible. Our system offers fast (5 seconds) determination of particle size, utilize safe levels of very low laser power (less than or equal to 0.2 mW), a small scattering volume (approximately 2 x 10(exp -5) cu mm) and high spatial coherence (Beta) values. This is a major step forward when compared to currently available DLS systems.

  18. Production of a bifunctional hybrid molecule B72.3/metallothionein-1 by protein engineering.

    PubMed Central

    Xiang, J; Koropatnick, J; Qi, Y; Luo, X; Moyana, T; Li, K; Chen, Y

    1993-01-01

    A hybrid anti-tumour B72.3 antibody/metallothionein protein B72.3MT-1 was produced by the construction of the expression vector mpSV2neo-EP1-B72.3MT-1. This vector contained the neo gene as a selection marker, the murine immunoglobulin promoter and enhancer, and the hybrid B72.3 heavy chain gene fragment with mouse metallothionein-1 cDNA gene ligated into its CH2 domain. The expression vector was transfected to the heavy chain loss mutant B72.3Mut(K) cell line. The hybrid protein B72.3MT-1 was purified from transfectant supernates using a Protein G column. We showed that the hybrid protein retained the binding reactivity for the TAG72 antigen as the original B72.3 antibody, and the metal-binding capacity of the native metallothionein molecule. Therefore, the bifunctional hybrid protein B72.3MT-1 may be very useful in cancer imaging when labelled with radionuclides such as 99mTc. Images Figure 3 Figure 6 Figure 7 PMID:8495976

  19. Single-Molecule Analysis of Protein Large-Amplitude Conformational Transitions

    NASA Astrophysics Data System (ADS)

    Yang, Haw

    2011-03-01

    Proteins have evolved to harness thermal fluctuations, rather than frustrated by them, to carry out chemical transformations and mechanical work. What are, then, the operation and design principles of protein machines? To frame the problem in a tractable way, several basic questions have been formulated to guide the experimental design: (a) How many conformational states can a protein sample on the functionally important timescale? (b) What are the inter-conversion rates between states? (c) How do ligand binding or interactions with other proteins modulate the motions? (d) What are the structural basis of flexibility and its underlying molecular mechanics? Guided by this framework, we have studied protein tyrosine phosphatase B, PtpB, from M. tuberculosis (a virulence factor of tuberculosis and a potential drug target) and adenylate kinase, AK, from E. coli (a ubiquitous energy-balancing enzyme in cells). These domain movements have been followed in real time on their respective catalytic timescales using high-resolution single-molecule Förster resonance energy transfer (FRET) spectroscopy. It is shown quantitatively that both PtpB and AK are capable of dynamically sampling two distinct states that correlate well with those observed by x-ray crystallography. Integrating these microscopic dynamics into macroscopic kinetics allows us to place the experimentally measured free-energy landscape in the context of enzymatic turnovers.

  20. High-Affinity, Small-Molecule Peptidomimetic Inhibitors of MLL1/WDR5 Protein-Protein Interaction

    SciTech Connect

    Karatas, Hacer; Townsend, Elizabeth C; Cao, Fang; Chen, Yong; Bernard, Denzil; Liu, Liu; Lei, Ming; Dou, Yali; Wang, Shaomeng

    2013-02-12

    Mixed lineage leukemia 1 (MLL1) is a histone H3 lysine 4 (H3K4) methyltransferase, and targeting the MLL1 enzymatic activity has been proposed as a novel therapeutic strategy for the treatment of acute leukemia harboring MLL1 fusion proteins. The MLL1/WDR5 protein–protein interaction is essential for MLL1 enzymatic activity. In the present study, we designed a large number of peptidomimetics to target the MLL1/WDR5 interaction based upon -CO-ARA-NH–, the minimum binding motif derived from MLL1. Our study led to the design of high-affinity peptidomimetics, which bind to WDR5 with Ki < 1 nM and function as potent antagonists of MLL1 activity in a fully reconstituted in vitro H3K4 methyltransferase assay. Determination of co-crystal structures of two potent peptidomimetics in complex with WDR5 establishes their structural basis for high-affinity binding to WDR5. Evaluation of one such peptidomimetic, MM-102, in bone marrow cells transduced with MLL1-AF9 fusion construct shows that the compound effectively decreases the expression of HoxA9 and Meis-1, two critical MLL1 target genes in MLL1 fusion protein mediated leukemogenesis. MM-102 also specifically inhibits cell growth and induces apoptosis in leukemia cells harboring MLL1 fusion proteins. Our study provides the first proof-of-concept for the design of small-molecule inhibitors of the WDR5/MLL1 protein–protein interaction as a novel therapeutic approach for acute leukemia harboring MLL1 fusion proteins.

  1. Single Molecule Science for Personalized Nanomedicine: Atomic Force Microscopy of Biopolymer-Protein Interactions

    NASA Astrophysics Data System (ADS)

    Hsueh, Carlin

    Nanotechnology has a unique and relatively untapped utility in the fields of medicine and dentistry at the level of single-biopolymer and -molecule diagnostics. In recent years atomic force microscopy (AFM) has garnered much interest due to its ability to obtain atomic-resolution of molecular structures and probe biophysical behaviors of biopolymers and proteins in a variety of biologically significant environments. The work presented in this thesis focuses on the nanoscale manipulation and observation of biopolymers to develop an innovative technology for personalized medicine while understanding complex biological systems. These studies described here primarily use AFM to observe biopolymer interactions with proteins and its surroundings with unprecedented resolution, providing a better understanding of these systems and interactions at the nanoscale. Transcriptional profiling, the measure of messenger RNA (mRNA) abundance in a single cell, is a powerful technique that detects "behavior" or "symptoms" at the tissue and cellular level. We have sought to develop an alternative approach, using our expertise in AFM and single molecule nanotechnology, to achieve a cost-effective high throughput method for sensitive detection and profiling of subtle changes in transcript abundance. The technique does not require amplification of the mRNA sample because the AFM provides three-dimensional views of molecules with unprecedented resolution, requires minimal sample preparation, and utilizes a simple tagging chemistry on cDNA molecules. AFM images showed collagen polymers in teeth and of Drebrin-A remodeling of filamentous actin structure and mechanics. AFM was used to image collagen on exposed dentine tubules and confirmed tubule occlusion with a desensitizing prophylaxis paste by Colgate-Palmolive. The AFM also superseded other microscopy tools in resolving F-actin helix remodeling and possible cooperative binding by a neuronal actin binding protein---Drebrin-A, an

  2. Observation of HIV-1 Nucleocapsid Protein induced TAR DNA melting at the single molecule level

    NASA Astrophysics Data System (ADS)

    Cosa, Gonzalo; Harbron, Elizabeth; O'Connor, Donald; Musier-Forsyth, Karin; Barbara, Paul

    2003-03-01

    Reverse transcription of the HIV-1 RNA genome involves several nucleic acid rearrangement steps, and the HIV-1 nucleocapsid protein (NC) plays a key role in this process. NC is a nucleic acid chaperone protein, which facilitates the formation of the most stable nucleic acid structures. Single molecule fluorescence resonance energy transfer (SM-FRET) measurements enable us to observe the NC-induced conformational fluctuations of a transactivation response region (TAR) DNA hairpin, which is part of the initial product of reverse transcription known as minus-strand strong-stop DNA. SM-FRET studies show that the majority of conformational fluctuations of the fluorescently-labeled TAR DNA hairpin in the presence of NC occur in <100 ms. A single molecule explores a wide range of confomations unpon NC binding, with fluctuations encompassing as many as 40 bases in both arms of the hairpin. No conformational fluctuations are observed with the dye-labeled TAR DNA hairpin in the absence of NC or when a labeled TAR DNA hairpin variant lacking bulges and internal loops is analyzed in the presence of NC. This study represents the first real-time observation of NC-mediated nucleic acid conformational fluctuations, revealing new insights into NC's nucleic acid chaperone activity.

  3. Controlling Brownian motion of single protein molecules and single fluorophores in aqueous buffer.

    PubMed

    Cohen, Adam E; Moerner, W E

    2008-05-12

    We present an Anti-Brownian Electrokinetic trap (ABEL trap) capable of trapping individual fluorescently labeled protein molecules in aqueous buffer. The ABEL trap operates by tracking the Brownian motion of a single fluorescent particle in solution, and applying a time-dependent electric field designed to induce an electrokinetic drift that cancels the Brownian motion. The trapping strength of the ABEL trap is limited by the latency of the feedback loop. In previous versions of the trap, this latency was set by the finite frame rate of the camera used for video-tracking. In the present system, the motion of the particle is tracked entirely in hardware (without a camera or image-processing software) using a rapidly rotating laser focus and lock-in detection. The feedback latency is set by the finite rate of arrival of photons. We demonstrate trapping of individual molecules of the protein GroEL in buffer, and we show confinement of single fluorophores of the dye Cy3 in water.

  4. Applications of Engineered DNA-Binding Molecules Such as TAL Proteins and the CRISPR/Cas System in Biology Research.

    PubMed

    Fujita, Toshitsugu; Fujii, Hodaka

    2015-09-24

    Engineered DNA-binding molecules such as transcription activator-like effector (TAL or TALE) proteins and the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) (CRISPR/Cas) system have been used extensively for genome editing in cells of various types and species. The sequence-specific DNA-binding activities of these engineered DNA-binding molecules can also be utilized for other purposes, such as transcriptional activation, transcriptional repression, chromatin modification, visualization of genomic regions, and isolation of chromatin in a locus-specific manner. In this review, we describe applications of these engineered DNA-binding molecules for biological purposes other than genome editing.

  5. Imaging Green Fluorescent Protein-tagged Receptor Molecules in Cellular Membranes with Near-field Scanning Optical Microscopy

    NASA Astrophysics Data System (ADS)

    Iwabuchi, Shinichiro; Hwang, Jeeseong; Goldner, Lori S.; Heinz, William; Edidin, Michael

    2001-03-01

    We report on near-field imaging of the major histocompatibility complex Class I (MHC-1) molecules in membranes of mammalian cells. The MHC-1 molecules are genetically modified to contain a green fluorescent protein moiety. Using near-field fluorescence imaging, we explore the distribution of MHC-1 molecules with resolution significantly better than what can be achieved with an ordinary microscope. We find submicron size features that are associated with the redistribution of MHC-1 proteins on the plasma membrane. Our NSOM for cell biology uses an aperture probe and permits the simultaneous optical and topographic imaging of samples both in air and in aqueous solution.

  6. Bacteroidales Secreted Antimicrobial Proteins Target Surface Molecules Necessary for Gut Colonization and Mediate Competition In Vivo

    PubMed Central

    Roelofs, Kevin G.; Coyne, Michael J.; Gentyala, Rahul R.; Chatzidaki-Livanis, Maria

    2016-01-01

    ABSTRACT We recently showed that human gut Bacteroidales species secrete antimicrobial proteins (BSAPs), and we characterized in vitro the first such BSAP produced by Bacteroides fragilis. In this study, we identified a second potent BSAP produced by the ubiquitous and abundant human gut species Bacteroides uniformis. The two BSAPs contain a membrane attack complex/perforin (MACPF) domain but share very little sequence similarity. We identified the target molecules of BSAP-sensitive cells and showed that each BSAP targets a different class of surface molecule: BSAP-1 targets an outer membrane protein of sensitive B. fragilis strains, and BSAP-2 targets the O-antigen glycan of lipopolysaccharide (LPS) of sensitive B. uniformis strains. Species-wide genomic and phenotypic analyses of B. fragilis and B. uniformis showed that BSAP-producing strains circumvent killing by synthesizing an orthologous nontargeted surface molecule. The BSAP genes are adjacent to the gene(s) encoding their target replacements, suggesting coacquisition. Using a gnotobiotic mouse competitive-colonization model, we found that the BSAP surface targets are important for colonization of the mammalian gut, thereby explaining why they are maintained in sensitive strains and why they were replaced rather than deleted in BSAP-producing strains. Using isogenic BSAP-producing, -sensitive, and -resistant strains, we show that a BSAP-producing strain outcompetes a sensitive strain but not a resistant strain in the mammalian gut. Human gut metagenomic datasets reveal that BSAP-1-sensitive strains do not cooccur with BSAP-1-producing strains in human gut microbiotas, further supporting the idea that BSAPs are important competitive factors with relevance to the strain-level composition of the human gut microbiota. PMID:27555309

  7. Solvent friction effects propagate over the entire protein molecule through low-frequency collective modes.

    PubMed

    Moritsugu, Kei; Kidera, Akinori; Smith, Jeremy C

    2014-07-24

    Protein solvation dynamics has been investigated using atom-dependent Langevin friction coefficients derived directly from molecular dynamics (MD) simulations. To determine the effect of solvation on the atomic friction coefficients, solution and vacuum MD simulations were performed for lysozyme and staphylococcal nuclease and analyzed by Langevin mode analysis. The coefficients thus derived are roughly correlated with the atomic solvent-accessible surface area (ASA), as expected from the fact that friction occurs as the result of collisions with solvent molecules. However, a considerable number of atoms with higher friction coefficients are found inside the core region. Hence, the influence of solvent friction propagates into the protein core. The internal coefficients have large contributions from the low-frequency modes, yielding a simple picture of the surface-to-core long-range damping via solvation governed by collective low-frequency modes. To make use of these findings in implicit-solvent modeling, we compare the all-atom friction results with those obtained using Langevin dynamics (LD) with two empirical representations: the constant-friction and the ASA-dependent (Pastor-Karplus) friction models. The constant-friction model overestimates the core and underestimates the surface damping whereas the ASA-dependent friction model, which damps protein atoms only on the solvent-accessible surface, reproduces well the friction coefficients for both the surface and core regions observed in the explicit-solvent MD simulations. Therefore, in LD simulation, the solvent friction coefficients should be imposed only on the protein surface. PMID:24999844

  8. Small-molecule tools for dissecting the roles of SSB/protein interactions in genome maintenance

    SciTech Connect

    Lu, Duo; Bernstein, Douglas A.; Satyshur, Kenneth A.; Keck, James L.

    2010-09-03

    Bacterial single-stranded DNA-binding proteins (SSBs) help to recruit a diverse array of genome maintenance enzymes to their sites of action through direct protein interactions. For all cases examined to date, these interactions are mediated by the evolutionarily conserved C terminus of SSB (SSB-Ct). The essential nature of SSB protein interactions makes inhibitors that block SSB complex formation valuable biochemical tools and attractive potential antibacterial agents. Here, we identify four small molecules that disrupt complexes formed between Escherichia coli SSB and Exonuclease I (ExoI), a well-studied SSB-interacting enzyme. Each compound disrupts ExoI/SSB-Ct peptide complexes and abrogates SSB stimulation of ExoI nuclease activity. Structural and biochemical studies support a model for three of the compounds in which they compete with SSB for binding to ExoI. The fourth appears to rely on an allosteric mechanism to disrupt ExoI/SSB complexes. Subsets of the inhibitors block SSB-Ct complex formation with two other SSB-interaction partners as well, which highlights their utility as reagents for investigating the roles of SSB/protein interactions in diverse DNA replication, recombination, and repair reactions.

  9. Single-molecule observation of protein folding in symmetric GroEL-(GroES)2 complexes.

    PubMed

    Takei, Yodai; Iizuka, Ryo; Ueno, Taro; Funatsu, Takashi

    2012-11-30

    The chaperonin, GroEL, is an essential molecular chaperone that mediates protein folding together with its cofactor, GroES, in Escherichia coli. It is widely believed that the two rings of GroEL alternate between the folding active state coupled to GroES binding during the reaction cycle. In other words, an asymmetric GroEL-GroES complex (the bullet-shaped complex) is formed throughout the cycle, whereas a symmetric GroEL-(GroES)(2) complex (the football-shaped complex) is not formed. We have recently shown that the football-shaped complex coexists with the bullet-shaped complex during the reaction cycle. However, how protein folding proceeds in the football-shaped complex remains poorly understood. Here, we used GFP as a substrate to visualize protein folding in the football-shaped complex by single-molecule fluorescence techniques. We directly showed that GFP folding occurs in both rings of the football-shaped complex. Remarkably, the folding was a sequential two-step reaction, and the kinetics were in excellent agreement with those in the bullet-shaped complex. These results demonstrate that the same reactions take place independently in both rings of the football-shaped complex to facilitate protein folding. PMID:23048033

  10. Solvent friction effects propagate over the entire protein molecule through low-frequency collective modes.

    PubMed

    Moritsugu, Kei; Kidera, Akinori; Smith, Jeremy C

    2014-07-24

    Protein solvation dynamics has been investigated using atom-dependent Langevin friction coefficients derived directly from molecular dynamics (MD) simulations. To determine the effect of solvation on the atomic friction coefficients, solution and vacuum MD simulations were performed for lysozyme and staphylococcal nuclease and analyzed by Langevin mode analysis. The coefficients thus derived are roughly correlated with the atomic solvent-accessible surface area (ASA), as expected from the fact that friction occurs as the result of collisions with solvent molecules. However, a considerable number of atoms with higher friction coefficients are found inside the core region. Hence, the influence of solvent friction propagates into the protein core. The internal coefficients have large contributions from the low-frequency modes, yielding a simple picture of the surface-to-core long-range damping via solvation governed by collective low-frequency modes. To make use of these findings in implicit-solvent modeling, we compare the all-atom friction results with those obtained using Langevin dynamics (LD) with two empirical representations: the constant-friction and the ASA-dependent (Pastor-Karplus) friction models. The constant-friction model overestimates the core and underestimates the surface damping whereas the ASA-dependent friction model, which damps protein atoms only on the solvent-accessible surface, reproduces well the friction coefficients for both the surface and core regions observed in the explicit-solvent MD simulations. Therefore, in LD simulation, the solvent friction coefficients should be imposed only on the protein surface.

  11. Heme protein dynamics studied by phosphorescence of an external phosphorescent probe molecule.

    PubMed

    Beckham, S; Cook, M P; Karki, L; Luchsinger, M M; Whitlock, V R; Wu, Y; Zhang, Q; Schuh, M D

    1994-05-01

    The rate constant for quenching, kq, of the phosphorescence of 6-bromo-2-naphthyl sulfate (BNS) by cytochromes c, cytochrome c peroxidase, catalase, and myoglobin has been measured as a function of temperature and solvent viscosity. In aqueous solution at pH 7.0 for cytochromes c and myoglobin the value of kq is nearly equal to the rate constant for diffusional intermolecular contact, which is estimated from the value of kq for microperoxidase-11. For cytochrome c peroxidase and catalase kq is at least 350 times smaller than the rate of diffusional quenching, which shows that quenching of BNS phosphorescence occurs predominantly over the short distance between donor and acceptor. The mechanism for cytochrome c and myoglobin is found not to involve static quenching, deep penetration of BNS into the globin, or unfolding of the protein to allow contact between heme and BNS. It is concluded that quenching occurs by interaction of BNS with the exposed heme edge and by surface insertion of BNS into the protein to a depth sufficient for quenching by the unexposed heme. The effect of rapid-diffusional enhancement on kq is small. From a comparison of the results for the heme proteins, a model emerges that describes cytochrome c and myoglobin as having dynamic surfaces. Sufficient fluctuations persist to allow penetration of polyatomic probe molecules into the protein matrix, but the dynamics and/or interior microenvironment acts to increase resistance with increasing depth of penetration. PMID:8179330

  12. Molecular insights into the stabilization of protein-protein interactions with small molecule: The FKBP12-rapamycin-FRB case study

    NASA Astrophysics Data System (ADS)

    Chaurasia, Shilpi; Pieraccini, Stefano; De Gonda, Riccardo; Conti, Simone; Sironi, Maurizio

    2013-11-01

    Targetting protein-protein interactions is a challenging task in drug discovery process. Despite the challenges, several studies provided evidences for the development of small molecules modulating protein-protein interactions. Here we consider a typical case of protein-protein interaction stabilization: the complex between FKBP12 and FRB with rapamycin. We have analyzed the stability of the complex and characterized its interactions at the atomic level by performing free energy calculations and computational alanine scanning. It is shown that rapamycin stabilizes the complex by acting as a bridge between the two proteins; and the complex is stable only in the presence of rapamycin.

  13. Antiviral compounds discovered by virtual screening of small-molecule libraries against dengue virus E protein.

    PubMed

    Zhou, Zhigang; Khaliq, Mansoora; Suk, Jae-Eun; Patkar, Chinmay; Li, Long; Kuhn, Richard J; Post, Carol Beth

    2008-12-19

    Infection by the mosquito-borne dengue virus causes dengue fever and the sometimes fatal dengue hemorrhagic fever. The increasing number of dengue infections per year suggests that the virus is becoming more virulent and its transmission is expanding. Nevertheless, no effective treatment for dengue infection currently exists. In a search for antiviral agents effective against dengue virus, we investigated the potential of targeting a structural protein site rather than an enzymatic one. Using this approach, we now report the discovery of a small molecule ligand that inhibits viral growth. Our results also provide the first evidence that the binding site, a pocket located at the hinge between domains 1 and 2 of the envelope protein (E protein) on the virus surface, is a valid target for antiviral therapy. Ligand candidates were identified from libraries of approximately 142,000 compounds using a computational high-throughput screening protocol targeting this pocket of the E protein. Cell-based assays were conducted on 23 top-ranked compounds. Among four with good antiviral activity profiles, the compound P02 was found to inhibit viral reproduction at micromolar concentrations. Using saturation transfer difference NMR spectroscopy, we also show that the compound binds virus and competes for binding E protein with the known ligand N-octyl-beta-D-glucoside. Together, the results are consistent with an inhibition mechanism against maturation or host-cell entry mediated by ligand binding to the E-protein pocket. P02 is a promising lead compound for future development of an effective treatment against dengue virus and related flaviviruses.

  14. Discovery and structural characterization of a small molecule 14-3-3 protein-protein interaction inhibitor

    PubMed Central

    Zhao, Jing; Du, Yuhong; Horton, John R.; Upadhyay, Anup K.; Lou, Bin; Bai, Yan; Zhang, Xing; Du, Lupei; Li, Minyong; Wang, Binghe; Zhang, Lixin; Barbieri, Joseph T.; Khuri, Fadlo R.; Cheng, Xiaodong; Fu, Haian

    2011-01-01

    The 14-3-3 family of phosphoserine/threonine-recognition proteins engage multiple nodes in signaling networks that control diverse physiological and pathophysiological functions and have emerged as promising therapeutic targets for such diseases as cancer and neurodegenerative disorders. Thus, small molecule modulators of 14-3-3 are much needed agents for chemical biology investigations and therapeutic development. To analyze 14-3-3 function and modulate its activity, we conducted a chemical screen and identified 4-[(2Z)-2-[4-formyl-6-methyl-5-oxo-3-(phosphonatooxymethyl)pyridin-2-ylidene]hydrazinyl]benzoate as a 14-3-3 inhibitor, which we termed FOBISIN (FOurteen-three-three BInding Small molecule INhibitor) 101. FOBISIN101 effectively blocked the binding of 14-3-3 with Raf-1 and proline-rich AKT substrate, 40 kDa and neutralized the ability of 14-3-3 to activate exoenzyme S ADP-ribosyltransferase. To provide a mechanistic basis for 14-3-3 inhibition, the crystal structure of 14-3-3ζ in complex with FOBISIN101 was solved. Unexpectedly, the double bond linking the pyridoxal-phosphate and benzoate moieties was reduced by X-rays to create a covalent linkage of the pyridoxal-phosphate moiety to lysine 120 in the binding groove of 14-3-3, leading to persistent 14-3-3 inactivation. We suggest that FOBISIN101-like molecules could be developed as an entirely unique class of 14-3-3 inhibitors, which may serve as radiation-triggered therapeutic agents for the treatment of 14-3-3-mediated diseases, such as cancer. PMID:21908710

  15. Evaluation of fluorophores to label SNAP-tag fused proteins for multicolor single-molecule tracking microscopy in live cells.

    PubMed

    Bosch, Peter J; Corrêa, Ivan R; Sonntag, Michael H; Ibach, Jenny; Brunsveld, Luc; Kanger, Johannes S; Subramaniam, Vinod

    2014-08-19

    Single-molecule tracking has become a widely used technique for studying protein dynamics and their organization in the complex environment of the cell. In particular, the spatiotemporal distribution of membrane receptors is an active field of study due to its putative role in the regulation of signal transduction. The SNAP-tag is an intrinsically monovalent and highly specific genetic tag for attaching a fluorescent label to a protein of interest. Little information is currently available on the choice of optimal fluorescent dyes for single-molecule microscopy utilizing the SNAP-tag labeling system. We surveyed 6 green and 16 red excitable dyes for their suitability in single-molecule microscopy of SNAP-tag fusion proteins in live cells. We determined the nonspecific binding levels and photostability of these dye conjugates when bound to a SNAP-tag fused membrane protein in live cells. We found that only a limited subset of the dyes tested is suitable for single-molecule tracking microscopy. The results show that a careful choice of the dye to conjugate to the SNAP-substrate to label SNAP-tag fusion proteins is very important, as many dyes suffer from either rapid photobleaching or high nonspecific staining. These characteristics appear to be unpredictable, which motivated the need to perform the systematic survey presented here. We have developed a protocol for evaluating the best dyes, and for the conditions that we evaluated, we find that Dy 549 and CF 640 are the best choices tested for single-molecule tracking. Using an optimal dye pair, we also demonstrate the possibility of dual-color single-molecule imaging of SNAP-tag fusion proteins. This survey provides an overview of the photophysical and imaging properties of a range of SNAP-tag fluorescent substrates, enabling the selection of optimal dyes and conditions for single-molecule imaging of SNAP-tagged fusion proteins in eukaryotic cell lines.

  16. Evaluation of Fluorophores to Label SNAP-Tag Fused Proteins for Multicolor Single-Molecule Tracking Microscopy in Live Cells

    PubMed Central

    Bosch, Peter J.; Corrêa, Ivan R.; Sonntag, Michael H.; Ibach, Jenny; Brunsveld, Luc; Kanger, Johannes S.; Subramaniam, Vinod

    2014-01-01

    Single-molecule tracking has become a widely used technique for studying protein dynamics and their organization in the complex environment of the cell. In particular, the spatiotemporal distribution of membrane receptors is an active field of study due to its putative role in the regulation of signal transduction. The SNAP-tag is an intrinsically monovalent and highly specific genetic tag for attaching a fluorescent label to a protein of interest. Little information is currently available on the choice of optimal fluorescent dyes for single-molecule microscopy utilizing the SNAP-tag labeling system. We surveyed 6 green and 16 red excitable dyes for their suitability in single-molecule microscopy of SNAP-tag fusion proteins in live cells. We determined the nonspecific binding levels and photostability of these dye conjugates when bound to a SNAP-tag fused membrane protein in live cells. We found that only a limited subset of the dyes tested is suitable for single-molecule tracking microscopy. The results show that a careful choice of the dye to conjugate to the SNAP-substrate to label SNAP-tag fusion proteins is very important, as many dyes suffer from either rapid photobleaching or high nonspecific staining. These characteristics appear to be unpredictable, which motivated the need to perform the systematic survey presented here. We have developed a protocol for evaluating the best dyes, and for the conditions that we evaluated, we find that Dy 549 and CF 640 are the best choices tested for single-molecule tracking. Using an optimal dye pair, we also demonstrate the possibility of dual-color single-molecule imaging of SNAP-tag fusion proteins. This survey provides an overview of the photophysical and imaging properties of a range of SNAP-tag fluorescent substrates, enabling the selection of optimal dyes and conditions for single-molecule imaging of SNAP-tagged fusion proteins in eukaryotic cell lines. PMID:25140415

  17. Dissecting the Influence of Protein Flexibility on the Location and Thermodynamic Profile of Explicit Water Molecules in Protein-Ligand Binding.

    PubMed

    Yang, Ying; Lill, Markus A

    2016-09-13

    Explicit water molecules in the binding site of proteins play a crucial role for protein-ligand association. Recent advances in computer-aided drug discovery methodology allow for an accurate prediction of the localized position and thermodynamic profile of water molecules (i.e., hydration sites) in the binding site. The underlying calculations are based on MD simulations of explicit water molecules in a restrained protein structure. However, the ligand-binding process is typically associated with protein conformational change that influences the position and thermodynamic properties of the hydration site. In this manuscript, we present the developments of two methods to incorporate the influence of protein conformational change on hydration sites either by following the conformational transition step-by-step (method I) or to match the hydration sites of the two transition end states using local coordinate systems (method II). Using these methods, we highlight the difference in the estimated protein desolvation free energy with and without inclusion of protein flexibility. To the best of our knowledge, this is the first study that explicitly studies the influence of protein conformational change on the position and thermodynamic profiles of water molecules and provides methodology to incorporate protein flexibility into the estimation of the desolvation free energy. PMID:27494046

  18. Dissecting the Influence of Protein Flexibility on the Location and Thermodynamic Profile of Explicit Water Molecules in Protein-Ligand Binding.

    PubMed

    Yang, Ying; Lill, Markus A

    2016-09-13

    Explicit water molecules in the binding site of proteins play a crucial role for protein-ligand association. Recent advances in computer-aided drug discovery methodology allow for an accurate prediction of the localized position and thermodynamic profile of water molecules (i.e., hydration sites) in the binding site. The underlying calculations are based on MD simulations of explicit water molecules in a restrained protein structure. However, the ligand-binding process is typically associated with protein conformational change that influences the position and thermodynamic properties of the hydration site. In this manuscript, we present the developments of two methods to incorporate the influence of protein conformational change on hydration sites either by following the conformational transition step-by-step (method I) or to match the hydration sites of the two transition end states using local coordinate systems (method II). Using these methods, we highlight the difference in the estimated protein desolvation free energy with and without inclusion of protein flexibility. To the best of our knowledge, this is the first study that explicitly studies the influence of protein conformational change on the position and thermodynamic profiles of water molecules and provides methodology to incorporate protein flexibility into the estimation of the desolvation free energy.

  19. Mechanical Folding and Unfolding of Protein Barnase at the Single-Molecule Level.

    PubMed

    Alemany, Anna; Rey-Serra, Blanca; Frutos, Silvia; Cecconi, Ciro; Ritort, Felix

    2016-01-01

    The unfolding and folding of protein barnase has been extensively investigated in bulk conditions under the effect of denaturant and temperature. These experiments provided information about structural and kinetic features of both the native and the unfolded states of the protein, and debates about the possible existence of an intermediate state in the folding pathway have arisen. Here, we investigate the folding/unfolding reaction of protein barnase under the action of mechanical force at the single-molecule level using optical tweezers. We measure unfolding and folding force-dependent kinetic rates from pulling and passive experiments, respectively, and using Kramers-based theories (e.g., Bell-Evans and Dudko-Hummer-Szabo models), we extract the position of the transition state and the height of the kinetic barrier mediating unfolding and folding transitions, finding good agreement with previous bulk measurements. Measurements of the force-dependent kinetic barrier using the continuous effective barrier analysis show that protein barnase verifies the Leffler-Hammond postulate under applied force and allow us to extract its free energy of folding, ΔG0. The estimated value of ΔG0 is in agreement with our predictions obtained using fluctuation relations and previous bulk studies. To address the possible existence of an intermediate state on the folding pathway, we measure the power spectrum of force fluctuations at high temporal resolution (50 kHz) when the protein is either folded or unfolded and, additionally, we study the folding transition-path time at different forces. The finite bandwidth of our experimental setup sets the lifetime of potential intermediate states upon barnase folding/unfolding in the submillisecond timescale. PMID:26745410

  20. Dihydromunduletone Is a Small-Molecule Selective Adhesion G Protein-Coupled Receptor Antagonist.

    PubMed

    Stoveken, Hannah M; Bahr, Laura L; Anders, M W; Wojtovich, Andrew P; Smrcka, Alan V; Tall, Gregory G

    2016-09-01

    Adhesion G protein-coupled receptors (aGPCRs) have emerging roles in development and tissue maintenance and is the most prevalent GPCR subclass mutated in human cancers, but to date, no drugs have been developed to target them in any disease. aGPCR extracellular domains contain a conserved subdomain that mediates self-cleavage proximal to the start of the 7-transmembrane domain (7TM). The two receptor protomers, extracellular domain and amino terminal fragment (NTF), and the 7TM or C-terminal fragment remain noncovalently bound at the plasma membrane in a low-activity state. We recently demonstrated that NTF dissociation liberates the 7TM N-terminal stalk, which acts as a tethered-peptide agonist permitting receptor-dependent heterotrimeric G protein activation. In many cases, natural aGPCR ligands are extracellular matrix proteins that dissociate the NTF to reveal the tethered agonist. Given the perceived difficulty in modifying extracellular matrix proteins to create aGPCR probes, we developed a serum response element (SRE)-luciferase-based screening approach to identify GPR56/ADGRG1 small-molecule inhibitors. A 2000-compound library comprising known drugs and natural products was screened for GPR56-dependent SRE activation inhibitors that did not inhibit constitutively active Gα13-dependent SRE activation. Dihydromunduletone (DHM), a rotenoid derivative, was validated using cell-free aGPCR/heterotrimeric G protein guanosine 5'-3-O-(thio)triphosphate binding reconstitution assays. DHM inhibited GPR56 and GPR114/ADGRG5, which have similar tethered agonists, but not the aGPCR GPR110/ADGRF1, M3 muscarinic acetylcholine, or β2 adrenergic GPCRs. DHM inhibited tethered peptide agonist-stimulated and synthetic peptide agonist-stimulated GPR56 but did not inhibit basal activity, demonstrating that it antagonizes the peptide agonist. DHM is a novel aGPCR antagonist and potentially useful chemical probe that may be developed as a future aGPCR therapeutic. PMID:27338081

  1. Identification of polymer surface adsorbed proteins implicated in pluripotent human embryonic stem cell expansion† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6bm00214e Click here for additional data file.

    PubMed Central

    Hammad, Moamen; Rao, Wei; Smith, James G. W.; Anderson, Daniel G.; Langer, Robert; Young, Lorraine E.; Barrett, David A.; Davies, Martyn C.; Denning, Chris

    2016-01-01

    Improved biomaterials are required for application in regenerative medicine, biosensing, and as medical devices. The response of cells to the chemistry of polymers cultured in media is generally regarded as being dominated by proteins adsorbed to the surface. Here we use mass spectrometry to identify proteins adsorbed from a complex mouse embryonic fibroblast (MEF) conditioned medium found to support pluripotent human embryonic stem cell (hESC) expansion on a plasma etched tissue culture polystyrene surface. A total of 71 proteins were identified, of which 14 uniquely correlated with the surface on which pluripotent stem cell expansion was achieved. We have developed a microarray combinatorial protein spotting approach to test the potential of these 14 proteins to support expansion of a hESC cell line (HUES-7) and a human induced pluripotent stem cell line (ReBl-PAT) on a novel polymer (N-(4-Hydroxyphenyl) methacrylamide). These proteins were spotted to form a primary array yielding several protein mixture ‘hits’ that enhanced cell attachment to the polymer. A second array was generated to test the function of a refined set of protein mixtures. We found that a combination of heat shock protein 90 and heat shock protein-1 encourage elevated adherence of pluripotent stem cells at a level comparable to fibronectin pre-treatment. PMID:27466628

  2. Discovery of Small-Molecule Nonfluorescent Inhibitors of Fluorogen-Fluorogen Activating Protein Binding Pair.

    PubMed

    Wu, Yang; Stauffer, Shaun R; Stanfield, Robyn L; Tapia, Phillip H; Ursu, Oleg; Fisher, Gregory W; Szent-Gyorgyi, Christopher; Evangelisti, Annette; Waller, Anna; Strouse, J Jacob; Carter, Mark B; Bologa, Cristian; Gouveia, Kristine; Poslusney, Mike; Waggoner, Alan S; Lindsley, Craig W; Jarvik, Jonathan W; Sklar, Larry A

    2016-01-01

    A new class of biosensors, fluorogen activating proteins (FAPs), has been successfully used to track receptor trafficking in live cells. Unlike the traditional fluorescent proteins (FPs), FAPs do not fluoresce unless bound to their specific small-molecule fluorogens, and thus FAP-based assays are highly sensitive. Application of the FAP-based assay for protein trafficking in high-throughput flow cytometry resulted in the discovery of a new class of compounds that interferes with the binding between fluorogens and FAP, thus blocking the fluorescence signal. These compounds are high-affinity, nonfluorescent analogs of fluorogens with little or no toxicity to the tested cells and no apparent interference with the normal function of FAP-tagged receptors. The most potent compound among these, N,4-dimethyl-N-(2-oxo-2-(4-(pyridin-2-yl)piperazin-1-yl)ethyl)benzenesulfonamide (ML342), has been investigated in detail. X-ray crystallographic analysis revealed that ML342 competes with the fluorogen, sulfonated thiazole orange coupled to diethylene glycol diamine (TO1-2p), for the same binding site on a FAP, AM2.2. Kinetic analysis shows that the FAP-fluorogen interaction is more complex than a homogeneous one-site binding process, with multiple conformational states of the fluorogen and/or the FAP, and possible dimerization of the FAP moiety involved in the process.

  3. Continuous Reduction of Protein-Bound Uraemic Toxins with Improved Oxidative Stress by Using the Oral Charcoal Adsorbent AST-120 in Haemodialysis Patients.

    PubMed

    Yamamoto, Suguru; Kazama, Junichiro J; Omori, Kentaro; Matsuo, Koji; Takahashi, Yoshimitsu; Kawamura, Kazuko; Matsuto, Takayuki; Watanabe, Hiroshi; Maruyama, Toru; Narita, Ichiei

    2015-09-23

    Accumulation of protein-bound uraemic toxins (PBUTs) is one of the reasons for the development of uraemia-related complications including cardiovascular disease; however, conventional haemodialysis is limited in its ability to remove PBUTs. We aimed to examine whether the oral charcoal adsorbent AST-120 has an additive effect on PBUT removal in haemodialysis patients. During the 4-week study, anuric patients undergoing haemodialysis received AST-120 (6 g/day) in the last 2 weeks (n = 10) or the first 2 weeks (n = 10). Serum levels of total and free PBUTs such as indoxyl sulfate, p-cresyl sulfate, and phenyl sulfate at the pre- and postdialysis sessions were measured before and after AST-120 use and after discontinuation. Levels of the oxidative stress markers oxidized albumin and 8-isoprostane were also measured. AST-120 use induced dramatic reduction of indoxyl sulfate (total, 45.7% [33.2-50.5%]; free, 70.4% [44.8-79.8%]), p-cresyl sulfate (total, 31.1% [25.0-48.0%]; free, 63.5% [49.3-70.9%]), and phenyl sulfate (free, 50.6% [32.3-71.2%]) levels; however, this effect disappeared after the discontinuation of AST-120. AST-120 use also induced substantial reduction of the oxidized albumin and 8-isoprostane levels. In conclusion, oral administration of AST-120 had additive effects on the continuous reduction of some PBUTs in anuric patients undergoing haemodialysis.

  4. Single-molecule resolution of G protein-coupled receptor (GPCR) complexes.

    PubMed

    Jonas, Kim C; Huhtaniemi, Ilpo; Hanyaloglu, Aylin C

    2016-01-01

    The organization of G protein-coupled receptors (GPCRs) into dimers and higher-order oligomers has provided a potential mechanistic system in defining complex GPCR responses. Despite being studied for nearly 20 years it has, and still is, been an area of controversy. Although technology has developed to quantitatively measure these associations in real time, identify the structural interfaces and even systems to understand the physiological significance of di/oligomerization, key questions remain outstanding including the role of each individual complex from the monomer to the higher-order oligomer, in their native system. Recently, single-molecule microscopy approaches have provided the tools to directly visualize individual GPCRs in dimers and oligomers, though as with any technological development each have their advantages and limitations. This chapter will describe these recent developments in single-molecule fluorescent microscopy, focusing on our recent application of super-resolution imaging of the GPCR for the luteinizing hormone/chorionic gonadotropin to quantify GPCR monomers and formation of protomers in to dimers and distinct oligomeric forms. We present our approach, considerations, strategy, and challenges to visualize this receptor beyond the light diffraction limit via photoactivated localization microscopy with photoactivatable dyes. The addition of super-resolution approaches to the GPCR "nano-tool kit" will pave the way for novel avenues to answer outstanding questions regarding the existence and significance of these complexes to GPCR signaling. PMID:26928539

  5. Directly measuring single molecule heterogeneity in proteins and RNA using force spectroscopy

    NASA Astrophysics Data System (ADS)

    Hinczewski, Michael; Hyeon, Changbong; Thirumalai, Devarajan

    One of the most intriguing results of single molecule experiments on proteins and nucleic acids is the discovery of functional heterogeneity: the observation that complex cellular machines exhibit multiple, biologically active conformations. The structural differences between these conformations may be subtle, but each distinct state can be remarkably long-lived, with stochastic interconversions occurring only at macroscopic timescales, fractions of a second or longer. Though we now have proof of functional heterogeneity in a handful of systems--enzymes, motors, adhesion complexes--identifying and measuring it remains a formidable challenge. We show that evidence of this phenomenon is more widespread than previously known, encoded in data collected from some of the most well-established single molecule techniques: AFM or optical tweezer pulling experiments. We present a theoretical procedure for analyzing distributions of rupture/unfolding forces recorded at different pulling speeds. This results in a single parameter, quantifying the degree of heterogeneity, and also leads to bounds on the equilibration and conformational interconversion timescales. Our work suggests experimental approaches for estimating the timescales of these fluctuations with unprecedented accuracy.

  6. Force microscopy imaging of individual protein molecules with sub-pico Newton force sensitivity.

    PubMed

    Patil, Shivprasad; Martinez, Nicolas F; Lozano, Jose R; Garcia, Ricardo

    2007-01-01

    The capability of atomic force microscopes (AFM) to generate atomic or nanoscale resolution images of surfaces has deeply transformed the study of materials. However, high resolution imaging of biological systems has proved more difficult than obtaining atomic resolution images of crystalline surfaces. In many cases, the forces exerted by the tip on the molecules (1-10 nN) either displace them laterally or break the noncovalent bonds that hold the biomolecules together. Here, we apply a force microscope concept based on the simultaneous excitation of the first two flexural modes of the cantilever. The coupling of the modes generated by the tip-molecule forces enables imaging under the application of forces ( approximately 35 pN) which are smaller than those needed to break noncovalent bonds. With this instrument we have resolved the intramolecular structure of antibodies in monomer and pentameric forms. Furthermore, the instrument has a force sensitivity of 0.2 pN which enables the identification of compositional changes along the protein fragments. PMID:17918769

  7. Force microscopy imaging of individual protein molecules with sub-pico Newton force sensitivity.

    PubMed

    Patil, Shivprasad; Martinez, Nicolas F; Lozano, Jose R; Garcia, Ricardo

    2007-01-01

    The capability of atomic force microscopes (AFM) to generate atomic or nanoscale resolution images of surfaces has deeply transformed the study of materials. However, high resolution imaging of biological systems has proved more difficult than obtaining atomic resolution images of crystalline surfaces. In many cases, the forces exerted by the tip on the molecules (1-10 nN) either displace them laterally or break the noncovalent bonds that hold the biomolecules together. Here, we apply a force microscope concept based on the simultaneous excitation of the first two flexural modes of the cantilever. The coupling of the modes generated by the tip-molecule forces enables imaging under the application of forces ( approximately 35 pN) which are smaller than those needed to break noncovalent bonds. With this instrument we have resolved the intramolecular structure of antibodies in monomer and pentameric forms. Furthermore, the instrument has a force sensitivity of 0.2 pN which enables the identification of compositional changes along the protein fragments.

  8. Slowing down single-molecule trafficking through a protein nanopore reveals intermediates for peptide translocation

    NASA Astrophysics Data System (ADS)

    Mereuta, Loredana; Roy, Mahua; Asandei, Alina; Lee, Jong Kook; Park, Yoonkyung; Andricioaei, Ioan; Luchian, Tudor

    2014-01-01

    The microscopic details of how peptides translocate one at a time through nanopores are crucial determinants for transport through membrane pores and important in developing nano-technologies. To date, the translocation process has been too fast relative to the resolution of the single molecule techniques that sought to detect its milestones. Using pH-tuned single-molecule electrophysiology and molecular dynamics simulations, we demonstrate how peptide passage through the α-hemolysin protein can be sufficiently slowed down to observe intermediate single-peptide sub-states associated to distinct structural milestones along the pore, and how to control residence time, direction and the sequence of spatio-temporal state-to-state dynamics of a single peptide. Molecular dynamics simulations of peptide translocation reveal the time- dependent ordering of intermediate structures of the translocating peptide inside the pore at atomic resolution. Calculations of the expected current ratios of the different pore-blocking microstates and their time sequencing are in accord with the recorded current traces.

  9. Single-molecule resolution of G protein-coupled receptor (GPCR) complexes.

    PubMed

    Jonas, Kim C; Huhtaniemi, Ilpo; Hanyaloglu, Aylin C

    2016-01-01

    The organization of G protein-coupled receptors (GPCRs) into dimers and higher-order oligomers has provided a potential mechanistic system in defining complex GPCR responses. Despite being studied for nearly 20 years it has, and still is, been an area of controversy. Although technology has developed to quantitatively measure these associations in real time, identify the structural interfaces and even systems to understand the physiological significance of di/oligomerization, key questions remain outstanding including the role of each individual complex from the monomer to the higher-order oligomer, in their native system. Recently, single-molecule microscopy approaches have provided the tools to directly visualize individual GPCRs in dimers and oligomers, though as with any technological development each have their advantages and limitations. This chapter will describe these recent developments in single-molecule fluorescent microscopy, focusing on our recent application of super-resolution imaging of the GPCR for the luteinizing hormone/chorionic gonadotropin to quantify GPCR monomers and formation of protomers in to dimers and distinct oligomeric forms. We present our approach, considerations, strategy, and challenges to visualize this receptor beyond the light diffraction limit via photoactivated localization microscopy with photoactivatable dyes. The addition of super-resolution approaches to the GPCR "nano-tool kit" will pave the way for novel avenues to answer outstanding questions regarding the existence and significance of these complexes to GPCR signaling.

  10. Photophysical characterization of fluorescent metal nanoclusters synthesized using oligonucleotides, proteins and small molecule ligands

    NASA Astrophysics Data System (ADS)

    Yeh, Hsin-Chih; Sharma, Jaswinder; Yoo, Hyojong; Martinez, Jennifer S.; Werner, James H.

    2010-02-01

    The size transition from bulk conducting metals to insulating nanoparticles and eventually to single atoms passes through the relatively unexplored few-atom nanocluster region. With dimensions close to the Fermi wavelength, these nanoclusters demonstrate molecule-like properties distinct from bulk metals or atoms, such as discrete and size-tunable electronic transitions which lead to photoluminescence. Current research aims to elucidate the fundamental photophysical properties of metal nanoclusters made by different means and based on different encapsulation agents. Here, we report the study of the photophysical properties, including quantum yields, lifetimes, extinction coefficients, blinking dynamics and sizes, of silver and gold nanoclusters synthesized using oligonucleotides, a protein (bovine serum albumin) and a Good's buffer molecule (MES, 2-(N-morpholino) ethanesulfonic acid) as encapsulation agents. We also investigate the change of photoluminescence as a function of temperature. Furthermore, we show that the fluorescent metal clusters can be used as a donor in forming a resonance energy transfer pair with a commercial organic quencher. These new fluorophores have great potential as versatile tools for a broad range of applications in biological and chemical detection.

  11. Quantitative single-molecule detection of protein based on DNA tetrahedron fluorescent nanolabels.

    PubMed

    Ding, Yongshun; Liu, Xingti; Zhu, Jing; Wang, Lei; Jiang, Wei

    2014-07-01

    A highly sensitive method for single-molecule quantitative detection of human IgG is presented by the employment of a new fluorescent nanolabel. In this method, fluorescent nanolabels were assembled by inserting SYBR Green I into DNA tetrahedron nanostructure. The bio-nanolabels were attached to the streptavidin-antihuman antibody by a specific reaction between biotin and streptavidin. The antibody was combined with the target antigen, human IgG, which was immobilized on the silanized glass subtrate surface. Finally, epi-fluorescence microscopy (EFM) coupled with an electron multiplying charge-coupled device was employed for fluorescence imaging. The fluorescent spots corresponding to single protein molecule on images were counted and further used for the quantitative detection. It was found that the new nanolabel shows good photostability, biocompatiblity and exhibits no blinking compared to traditional labels like fluorescence dyes and quantum dot (QDs). In addition, the number of fluorescence spots on the images has a linear relationship with the concentration of human IgG in the range of 3.0×10(-14) to 1.0×10(-12)mol L(-1). What is more, this method showed an excellent specificity and a low matrix effect.

  12. Direct and Propagated Effects of Small Molecules on Protein–Protein Interaction Networks

    PubMed Central

    Cesa, Laura C.; Mapp, Anna K.; Gestwicki, Jason E.

    2015-01-01

    Networks of protein–protein interactions (PPIs) link all aspects of cellular biology. Dysfunction in the assembly or dynamics of PPI networks is a hallmark of human disease, and as such, there is growing interest in the discovery of small molecules that either promote or inhibit PPIs. PPIs were once considered undruggable because of their relatively large buried surface areas and difficult topologies. Despite these challenges, recent advances in chemical screening methodologies, combined with improvements in structural and computational biology have made some of these targets more tractable. In this review, we highlight developments that have opened the door to potent chemical modulators. We focus on how allostery is being used to produce surprisingly robust changes in PPIs, even for the most challenging targets. We also discuss how interfering with one PPI can propagate changes through the broader web of interactions. Through this analysis, it is becoming clear that a combination of direct and propagated effects on PPI networks is ultimately how small molecules re-shape biology. PMID:26380257

  13. Selector function of MHC I molecules is determined by protein plasticity

    NASA Astrophysics Data System (ADS)

    Bailey, Alistair; Dalchau, Neil; Carter, Rachel; Emmott, Stephen; Phillips, Andrew; Werner, Jörn M.; Elliott, Tim

    2015-10-01

    The selection of peptides for presentation at the surface of most nucleated cells by major histocompatibility complex class I molecules (MHC I) is crucial to the immune response in vertebrates. However, the mechanisms of the rapid selection of high affinity peptides by MHC I from amongst thousands of mostly low affinity peptides are not well understood. We developed computational systems models encoding distinct mechanistic hypotheses for two molecules, HLA-B*44:02 (B*4402) and HLA-B*44:05 (B*4405), which differ by a single residue yet lie at opposite ends of the spectrum in their intrinsic ability to select high affinity peptides. We used in vivo biochemical data to infer that a conformational intermediate of MHC I is significant for peptide selection. We used molecular dynamics simulations to show that peptide selector function correlates with protein plasticity, and confirmed this experimentally by altering the plasticity of MHC I with a single point mutation, which altered in vivo selector function in a predictable way. Finally, we investigated the mechanisms by which the co-factor tapasin influences MHC I plasticity. We propose that tapasin modulates MHC I plasticity by dynamically coupling the peptide binding region and α3 domain of MHC I allosterically, resulting in enhanced peptide selector function.

  14. Small molecule inhibitor of the bone morphogenetic protein pathway DMH1 reduces ovarian cancer cell growth.

    PubMed

    Hover, Laura D; Young, Christian D; Bhola, Neil E; Wilson, Andrew J; Khabele, Dineo; Hong, Charles C; Moses, Harold L; Owens, Philip

    2015-11-01

    The bone morphogenetic protein (BMP) pathway belonging to the Transforming Growth Factor beta (TGFβ) family of secreted cytokines/growth factors is an important regulator of cancer. BMP ligands have been shown to play both tumor suppressive and promoting roles in human cancers. We have found that BMP ligands are amplified in human ovarian cancers and that BMP receptor expression correlates with poor progression-free-survival (PFS). Furthermore, active BMP signaling has been observed in human ovarian cancer tissue. We also determined that ovarian cancer cell lines have active BMP signaling in a cell autonomous fashion. Inhibition of BMP signaling with a small molecule receptor kinase antagonist is effective at reducing ovarian tumor sphere growth. Furthermore, BMP inhibition can enhance sensitivity to Cisplatin treatment and regulates gene expression involved in platinum resistance in ovarian cancer. Overall, these studies suggest targeting the BMP pathway as a novel source to enhance chemo-sensitivity in ovarian cancer.

  15. A fully integrated protein crystallization platform for small-molecule drug discovery.

    PubMed

    Hosfield, David; Palan, John; Hilgers, Mark; Scheibe, Daniel; McRee, Duncan E; Stevens, Raymond C

    2003-04-01

    Structure-based drug discovery in the pharmaceutical industry benefits from cost-efficient methodologies that quickly assess the feasibility of specific, often refractory, protein targets to form well-diffracting crystals. By tightly coupling construct and purification diversity with nanovolume crystallization, the Structural Biology Group at Syrrx has developed such a platform to support its small-molecule drug-discovery program. During the past 18 months of operation at Syrrx, the Structural Biology Group has executed several million crystallization and imaging trials on over 400 unique drug-discovery targets. Here, key components of the platform, as well as an analysis of some experimental results that allowed for platform optimization, will be described.

  16. Single-Molecule Imaging of Individual Amyloid Protein Aggregates in Human Biofluids

    PubMed Central

    2016-01-01

    The misfolding and aggregation of proteins into amyloid fibrils characterizes many neurodegenerative disorders such as Parkinson’s and Alzheimer’s diseases. We report here a method, termed SAVE (single aggregate visualization by enhancement) imaging, for the ultrasensitive detection of individual amyloid fibrils and oligomers using single-molecule fluorescence microscopy. We demonstrate that this method is able to detect the presence of amyloid aggregates of α-synuclein, tau, and amyloid-β. In addition, we show that aggregates can also be identified in human cerebrospinal fluid (CSF). Significantly, we see a twofold increase in the average aggregate concentration in CSF from Parkinson’s disease patients compared to age-matched controls. Taken together, we conclude that this method provides an opportunity to characterize the structural nature of amyloid aggregates in a key biofluid, and therefore has the potential to study disease progression in both animal models and humans to enhance our understanding of neurodegenerative disorders. PMID:26800462

  17. Diffracted X-ray tracking for monitoring intramolecular motion in individual protein molecules using broad band X-ray

    SciTech Connect

    Ichiyanagi, Kouhei; Sasaki, Yuji C.; Sekiguchi, Hiroshi; Hoshino, Masato; Kajiwara, Kentaro; Senba, Yasunori; Ohashi, Haruhiko; Ohta, Noboru; Hoshisashi, Kentaro; Jae-won, Chang; Tokue, Maki; Matsushita, Yufuku; Nishijima, Masaki; Inoue, Yoshihisa; Yagi, Naoto

    2013-10-15

    Diffracted X-ray tracking (DXT) enables the tilting and twisting motions of single protein molecules to be monitored with micro- to milliradian resolution using a highly brilliant X-ray source with a wide energy bandwidth. We have developed a technique to monitor single molecules using gold nanocrystals attached to individual protein molecules using the BL28B2 beamline at SPring-8. In this paper we present the installation of a single toroidal X-ray mirror at BL28B2 to focus X-rays in an energy range of 10–20 keV (△E/E = 82% for an X-ray with a wide energy bandwidth). With this beamline we tracked diffraction spots from gold nanocrystals over a wide angle range than that using quasi-monochromatic X-rays. Application of the wide angle DXT technique to biological systems enabled us to observe the on-site motions of single protein molecules that have been functionalized in vivo. We further extend the capability of DXT by observing the fractional tilting and twisting motions of inner proteins under various conditions. As a proof of this methodology and to determine instrumental performance the intramolecular motions of a human serum albumin complex with 2-anthracenecarboxylic acid was investigated using the BL28B2 beamline. The random tilting and twisting intramolecular motions are shown to be directly linked to the movement of individual protein molecules in the buffer solution.

  18. Diffracted X-ray tracking for monitoring intramolecular motion in individual protein molecules using broad band X-ray.

    PubMed

    Ichiyanagi, Kouhei; Sekiguchi, Hiroshi; Hoshino, Masato; Kajiwara, Kentaro; Hoshisashi, Kentaro; Chang, Jae-won; Tokue, Maki; Matsushita, Yufuku; Nishijima, Masaki; Inoue, Yoshihisa; Senba, Yasunori; Ohashi, Haruhiko; Ohta, Noboru; Yagi, Naoto; Sasaki, Yuji C

    2013-10-01

    Diffracted X-ray tracking (DXT) enables the tilting and twisting motions of single protein molecules to be monitored with micro- to milliradian resolution using a highly brilliant X-ray source with a wide energy bandwidth. We have developed a technique to monitor single molecules using gold nanocrystals attached to individual protein molecules using the BL28B2 beamline at SPring-8. In this paper we present the installation of a single toroidal X-ray mirror at BL28B2 to focus X-rays in an energy range of 10-20 keV (ΔE/E = 82% for an X-ray with a wide energy bandwidth). With this beamline we tracked diffraction spots from gold nanocrystals over a wide angle range than that using quasi-monochromatic X-rays. Application of the wide angle DXT technique to biological systems enabled us to observe the on-site motions of single protein molecules that have been functionalized in vivo. We further extend the capability of DXT by observing the fractional tilting and twisting motions of inner proteins under various conditions. As a proof of this methodology and to determine instrumental performance the intramolecular motions of a human serum albumin complex with 2-anthracenecarboxylic acid was investigated using the BL28B2 beamline. The random tilting and twisting intramolecular motions are shown to be directly linked to the movement of individual protein molecules in the buffer solution.

  19. Geometrically centered region: a "wet" model of protein binding hot spots not excluding water molecules.

    PubMed

    Li, Zhenhua; Li, Jinyan

    2010-12-01

    A protein interface can be as "wet" as a protein surface in terms of the number of immobilized water molecules. This important water information has not been explicitly taken by computational methods to model and identify protein binding hot spots, overlooking the water role in forming interface hydrogen bonds and in filing cavities. Hot spot residues are usually clustered at the core of the protein binding interfaces. However, traditional machine learning methods often identify the hot spot residues individually, breaking the cooperativity of the energetic contribution. Our idea in this work is to explore the role of immobilized water and meanwhile to capture two essential properties of hot spots: the compactness in contact and the far distance from bulk solvent. Our model is named geometrically centered region (GCR). The detection of GCRs is based on novel tripartite graphs, and atom burial levels which are a concept more intuitive than SASA. Applying to a data set containing 355 mutations, we achieved an F measure of 0.6414 when ΔΔG ≥ 1.0 kcal/mol was used to define hot spots. This performance is better than Robetta, a benchmark method in the field. We found that all but only one of the GCRs contain water to a certain degree, and most of the outstanding hot spot residues have water-mediated contacts. If the water is excluded, the burial level values are poorly related to the ΔΔG, and the model loses its performance remarkably. We also presented a definition for the O-ring of a GCR as the set of immediate neighbors of the residues in the GCR. Comparative analysis between the O-rings and GCRs reveals that the newly defined O-ring is indeed energetically less important than the GCR hot spot, confirming a long-standing hypothesis. PMID:20818601

  20. Generic inhibition of amyloidogenic proteins by two naphthoquinone-tryptophan hybrid molecules.

    PubMed

    Scherzer-Attali, Roni; Shaltiel-Karyo, Ronit; Adalist, Yonatan H; Segal, Daniel; Gazit, Ehud

    2012-08-01

    Amyloid formation is associated with several human diseases including Alzheimer's disease (AD), Parkinson's disease, Type 2 Diabetes, and so forth, no disease modifying therapeutics are available for them. Because of the structural similarities between the amyloid species characterizing these diseases, (despite the lack of amino acid homology) it is believed that there might be a common mechanism of toxicity for these conditions. Thus, inhibition of amyloid formation could be a promising disease-modifying therapeutic strategy for them. Aromatic residues have been identified as crucial in formation and stabilization of amyloid structures. This finding was corroborated by high-resolution structural studies, theoretical analysis, and molecular dynamics simulations. Amongst the aromatic entities, tryptophan was found to possess the most amyloidogenic potential. We therefore postulate that targeting aromatic recognition interfaces by tryptophan could be a useful approach for inhibiting the formation of amyloids. Quinones are known as inhibitors of cellular metabolic pathways, to have anti- cancer, anti-viral and anti-bacterial properties and were shown to inhibit aggregation of several amyloidogenic proteins in vitro. We have previously described two quinone-tryptophan hybrids which are capable of inhibiting amyloid-beta, the protein associated with AD pathology, both in vitro and in vivo. Here we tested their generic properties and their ability to inhibit other amyloidogenic proteins including α-synuclein, islet amyloid polypeptide, lysozyme, calcitonin, and insulin. Both compounds showed efficient inhibition of all five proteins examined both by ThT fluorescence analysis and by electron microscope imaging. If verified in vivo, these small molecules could serve as leads for developing generic anti-amyloid drugs.

  1. Stretching and immobilization of DNA for studies of protein-DNA interactions at the single-molecule level

    NASA Astrophysics Data System (ADS)

    Kim, Ji Hoon; Dukkipati, Venkat Ram; Pang, Stella W.; Larson, Ronald G.

    2007-04-01

    Single-molecule studies of the interactions of DNA and proteins are important in a variety of biological or biotechnology processes ranging from the protein’s search for its DNA target site, DNA replication, transcription, or repair, and genome sequencing. A critical requirement for single-molecule studies is the stretching and immobilization of otherwise randomly coiled DNA molecules. Several methods for doing so have been developed over the last two decades, including the use of forces derived from light, magnetic and electric fields, and hydrodynamic flow. Here we review the immobilization and stretching mechanisms for several of these techniques along with examples of single-molecule DNA-protein interaction assays that can be performed with each of them.

  2. Cardiac action of the first G protein biased small molecule apelin agonist.

    PubMed

    Read, Cai; Fitzpatrick, Christopher M; Yang, Peiran; Kuc, Rhoda E; Maguire, Janet J; Glen, Robert C; Foster, Richard E; Davenport, Anthony P

    2016-09-15

    Apelin peptide analogues displaying bias towards G protein signalling pathways have beneficial cardiovascular actions compared with the native peptide in humans in vivo. Our aim was to determine whether small molecule agonists could retain G protein bias. We have identified a biased small molecule, CMF-019, and characterised it in vitro and in vivo. In competition radioligand binding experiments in heart homogenates, CMF-019 bound to the human, rat and mouse apelin receptor with high affinity (pKi=8.58±0.04, 8.49±0.04 and 8.71±0.06 respectively). In cell-based functional assays, whereas, CMF-019 showed similar potency for the Gαi pathway to the endogenous agonist [Pyr(1)]apelin-13 (pD2=10.00±0.13 vs 9.34±0.15), in β-arrestin and internalisation assays it was less potent (pD2=6.65±0.15 vs 8.65±0.10 and pD2=6.16±0.21 vs 9.28±0.10 respectively). Analysis of these data demonstrated a bias of ∼400 for the Gαi over the β-arrestin pathway and ∼6000 over receptor internalisation. CMF-019 was tested for in vivo activity using intravenous injections into anaesthetised male Sprague-Dawley rats fitted with a pressure-volume catheter in the left ventricle. CMF-019 caused a significant increase in cardiac contractility of 606±112mmHg/s (p<0.001) at 500nmol. CMF-019 is the first biased small molecule identified at the apelin receptor and increases cardiac contractility in vivo. We have demonstrated that Gαi over β-arrestin/internalisation bias can be retained in a non-peptide analogue and predict that such bias will have the therapeutic benefit following chronic use. CMF-019 is suitable as a tool compound and provides the basis for design of biased agonists with improved pharmacokinetics for treatment of cardiovascular conditions such as pulmonary arterial hypertension. PMID:27475715

  3. Interactions of DNA binding proteins with G-Quadruplex structures at the single molecule level

    NASA Astrophysics Data System (ADS)

    Ray, Sujay

    Guanine-rich nucleic acid (DNA/RNA) sequences can form non-canonical secondary structures, known as G-quadruplex (GQ). Numerous in vivo and in vitro studies have demonstrated formation of these structures in telomeric and non-telomeric regions of the genome. Telomeric GQs protect the chromosome ends whereas non-telomeric GQs either act as road blocks or recognition sites for DNA metabolic machinery. These observations suggest the significance of these structures in regulation of different metabolic processes, such as replication and repair. GQs are typically thermodynamically more stable than the corresponding Watson-Crick base pairing formed by G-rich and C-rich strands, making protein activity a crucial factor for their destabilization. Inside the cell, GQs interact with different proteins and their enzymatic activity is the determining factor for their stability. We studied interactions of several proteins with GQs to understand the underlying principles of protein-GQ interactions using single-molecule FRET and other biophysical techniques. Replication Protein-A (RPA), a single stranded DNA (ssDNA) binding protein, is known to posses GQ unfolding activity. First, we compared the thermal stability of three potentially GQ-forming DNA sequences (PQS) to their stability against RPA-mediated unfolding. One of these sequences is the human telomeric repeat and the other two, located in the promoter region of tyrosine hydroxylase gene, are highly heterogeneous sequences that better represent PQS in the genome. The thermal stability of these structures do not necessarily correlate with their stability against protein-mediated unfolding. We conclude that thermal stability is not necessarily an adequate criterion for predicting the physiological viability of GQ structures. To determine the critical structural factors that influence protein-GQ interactions we studied two groups of GQ structures that have systematically varying loop lengths and number of G-tetrad layers. We

  4. Physical manipulation of single-molecule DNA using microbead and its application to analysis of DNA-protein interaction

    NASA Astrophysics Data System (ADS)

    Kurita, Hirofumi; Yasuda, Hachiro; Takashima, Kazunori; Katsura, Shinji; Mizuno, Akira

    2009-04-01

    We carried out an individual DNA manipulation using an optical trapping for a microbead. This manipulation system is based on a fluorescent microscopy equipped with an IR laser. Both ends of linear DNA molecule were labeled with a biotin and a thiol group, respectively. Then the biotinylated end was attached to a microbead, and the other was immobilized on a thiol-linkable glass surface. We controlled the form of an individual DNA molecule by moving the focal point of IR laser, which trapped the microbead. In addition, we applied single-molecule approach to analyze DNA hydrolysis. We also used microchannel for single-molecule observation of DNA hydrolysis. The shortening of DNA in length caused by enzymatic hydrolysis was observed in real-time. The single-molecule DNA manipulation should contribute to elucidate detailed mechanisms of DNA-protein interactions.

  5. A noncanonical function of sortase enables site-specific conjugation of small molecules to lysine residues in proteins.

    PubMed

    Bellucci, Joseph J; Bhattacharyya, Jayanta; Chilkoti, Ashutosh

    2015-01-01

    We provide the first demonstration that isopeptide ligation, a noncanonical activity of the enzyme sortase A, can be used to modify recombinant proteins. This reaction was used in vitro to conjugate small molecules to a peptide, an engineered targeting protein, and a full-length monoclonal antibody with an exquisite level of control over the site of conjugation. Attachment to the protein substrate occurred exclusively through isopeptide bonds at a lysine ε-amino group within a specific amino acid sequence. This reaction allows more than one molecule to be site-specifically conjugated to a protein at internal sites, thereby overcoming significant limitations of the canonical native peptide ligation reaction catalyzed by sortase A. Our method provides a unique chemical ligation procedure that is orthogonal to existing methods, supplying a new method to site-specifically modify lysine residues that will be a valuable addition to the protein conjugation toolbox. PMID:25363491

  6. A non-canonical function of sortase enables site-specific conjugation of small molecules to lysine residues in proteins**

    PubMed Central

    Bellucci, Joseph J.; Bhattacharyya, Jayanta

    2014-01-01

    We provide the first demonstration that isopeptide ligation, a non-canonical activity of the enzyme sortase A, can be used to modify recombinant proteins. This reaction was used in vitro to conjugate small molecules to a peptide, an engineered targeting protein, and a full-length monoclonal antibody with an exquisite level of control over the site of conjugation. Attachment to the protein substrate occurred exclusively through isopeptide bonds at a lysine ε-amino group within a specific amino acid sequence. This reaction allows more than one molecule to be site-specifically conjugated to a protein at internal sites, thereby overcoming significant limitations of the canonical native peptide ligation reaction catalyzed by sortase A. Our method provides a unique chemical ligation procedure that is orthogonal to existing methods, supplying a new method to site-specifically modify lysine residues that will be a valuable addition to the protein conjugation toolbox. PMID:25363491

  7. A Small Molecule RAS-Mimetic Disrupts RAS Association with Effector Proteins to Block Signaling.

    PubMed

    Athuluri-Divakar, Sai Krishna; Vasquez-Del Carpio, Rodrigo; Dutta, Kaushik; Baker, Stacey J; Cosenza, Stephen C; Basu, Indranil; Gupta, Yogesh K; Reddy, M V Ramana; Ueno, Lynn; Hart, Jonathan R; Vogt, Peter K; Mulholland, David; Guha, Chandan; Aggarwal, Aneel K; Reddy, E Premkumar

    2016-04-21

    Oncogenic activation of RAS genes via point mutations occurs in 20%-30% of human cancers. The development of effective RAS inhibitors has been challenging, necessitating new approaches to inhibit this oncogenic protein. Functional studies have shown that the switch region of RAS interacts with a large number of effector proteins containing a common RAS-binding domain (RBD). Because RBD-mediated interactions are essential for RAS signaling, blocking RBD association with small molecules constitutes an attractive therapeutic approach. Here, we present evidence that rigosertib, a styryl-benzyl sulfone, acts as a RAS-mimetic and interacts with the RBDs of RAF kinases, resulting in their inability to bind to RAS, disruption of RAF activation, and inhibition of the RAS-RAF-MEK pathway. We also find that ribosertib binds to the RBDs of Ral-GDS and PI3Ks. These results suggest that targeting of RBDs across multiple signaling pathways by rigosertib may represent an effective strategy for inactivation of RAS signaling. PMID:27104980

  8. A proactive role of water molecules in acceptor recognition by Protein-O-fucosyltransferase 2

    PubMed Central

    Valero-González, Jessika; Leonhard-Melief, Christina; Lira-Navarrete, Erandi; Jiménez-Osés, Gonzalo; Hernández-Ruiz, Cristina; Pallarés, María Carmen; Yruela, Inmaculada; Vasudevan, Deepika; Lostao, Anabel; Corzana, Francisco; Takeuchi, Hideyuki; Haltiwanger, Robert S.; Hurtado-Guerrero, Ramon

    2016-01-01

    Protein O-fucosyltransferase 2 (POFUT2) is an essential enzyme that fucosylates serine/threonine residues of folded thrombospondin type 1 repeats (TSRs). To date, the mechanism by which this enzyme recognizes very dissimilar TSRs remained unclear. By engineering of a fusion protein, we report the crystal structure of Caenorhabditis elegans POFUT2 (CePOFUT2) in complex with GDP and human TSR1 that suggests an inverting mechanism for fucose transfer assisted by a catalytic base, and shows that nearly half of the TSR1 is embraced by CePOFUT2. A small number of direct interactions and a large network of water molecules maintain the complex. Site-directed mutagenesis demonstrates that POFUT2 fucosylates threonine preferentially over serine and relies on folded TSRs containing the minimal consensus sequence CXX(S/T)C. Crystallographic and mutagenesis data together with atomic-level simulations uncover an unprecedented binding mechanism by which POFUT2 promiscuously recognizes the structural fingerprint of poorly homologous TSRs through a dynamic network of water-mediated interactions. PMID:26854667

  9. Repulsive Guidance Molecule is a structural bridge between Neogenin and Bone Morphogenetic Protein

    PubMed Central

    Healey, Eleanor G.; Bishop, Benjamin; Elegheert, Jonathan; Bell, Christian H.; Padilla-Parra, Sergi; Siebold, Christian

    2015-01-01

    Repulsive guidance molecules (RGMs) control crucial processes spanning cell motility, adhesion, immune cell regulation and systemic iron metabolism. RGMs signal via two fundamental signaling cascades: the Neogenin (NEO1) and the Bone Morphogenetic Protein (BMP) pathways. Here, we report crystal structures of the N-terminal domains of all human RGM family members in complex with the BMP ligand BMP2, revealing a novel protein fold and a conserved BMP-binding mode. Our structural and functional data suggest a pH-linked mechanism for RGM-activated BMP signaling and offer a rationale for RGM mutations causing juvenile hemochromatosis. We also determined the ternary BMP2–RGM–NEO1 complex crystal structure, which combined with solution scattering and live-cell super-resolution fluorescence microscopy, indicates BMP-induced clustering of the RGM–NEO1 complex. Our results show how RGM acts as the central hub linking BMP and NEO1 and physically connecting these fundamental signaling pathways. PMID:25938661

  10. Identification of an Allosteric Small Molecule Inhibitor Selective for Inducible Form of Heat Shock Protein 70

    PubMed Central

    Howe, Matthew K.; Bodoor, Khaldon; Carlson, David A.; Hughes, Philip F.; Alwarawrah, Yazan; Loiselle, David R.; Jaeger, Alex M.; Darr, David B.; Jordan, Jamie L.; Hunter, Lucas M.; Molzberger, Eileen T.; Gobillot, Theodore A.; Thiele, Dennis J.; Brodsky, Jeffrey L.; Spector, Neil L.; Haystead, Timothy A. J.

    2014-01-01

    Summary Inducible Hsp70 (Hsp70i) is overexpressed in a wide spectrum of human tumors and its expression correlates with metastasis, poor outcomes, and resistance to chemotherapy in patients. Identification of small molecule inhibitors selective for Hsp70i could provide new therapeutic tools for cancer treatment. In this work, we used fluorescence-linked enzyme chemoproteomic strategy (FLECS) to identify HS-72, an allosteric inhibitor selective for Hsp70i. HS-72 displays the hallmarks of Hsp70 inhibition in cells, promoting substrate protein degradation and growth inhibition. Importantly, HS-72 is selective for Hsp70i over the closely related constitutively active Hsc70. Studies with purified protein show HS-72 acts as an allosteric inhibitor, reducing ATP affinity. In vivo HS-72 is well-tolerated, showing bioavailability and efficacy, inhibiting tumor growth and promoting survival in a HER2+ model of breast cancer. The HS-72 scaffold is amenable to resynthesis and iteration, suggesting an ideal starting point for a new generation of anticancer therapeutics targeting Hsp70i. PMID:25500222

  11. A curated census of autophagy-modulating proteins and small molecules: candidate targets for cancer therapy.

    PubMed

    Lorenzi, Philip L; Claerhout, Sofie; Mills, Gordon B; Weinstein, John N

    2014-07-01

    Autophagy, a programmed process in which cell contents are delivered to lysosomes for degradation, appears to have both tumor-suppressive and tumor-promoting functions; both stimulation and inhibition of autophagy have been reported to induce cancer cell death, and particular genes and proteins have been associated both positively and negatively with autophagy. To provide a basis for incisive analysis of those complexities and ambiguities and to guide development of new autophagy-targeted treatments for cancer, we have compiled a comprehensive, curated inventory of autophagy modulators by integrating information from published siRNA screens, multiple pathway analysis algorithms, and extensive, manually curated text-mining of the literature. The resulting inventory includes 739 proteins and 385 chemicals (including drugs, small molecules, and metabolites). Because autophagy is still at an early stage of investigation, we provide extensive analysis of our sources of information and their complex relationships with each other. We conclude with a discussion of novel strategies that could potentially be used to target autophagy for cancer therapy.

  12. A proactive role of water molecules in acceptor recognition by protein O-fucosyltransferase 2.

    PubMed

    Valero-González, Jessika; Leonhard-Melief, Christina; Lira-Navarrete, Erandi; Jiménez-Osés, Gonzalo; Hernández-Ruiz, Cristina; Pallarés, María Carmen; Yruela, Inmaculada; Vasudevan, Deepika; Lostao, Anabel; Corzana, Francisco; Takeuchi, Hideyuki; Haltiwanger, Robert S; Hurtado-Guerrero, Ramon

    2016-04-01

    Protein O-fucosyltransferase 2 (POFUT2) is an essential enzyme that fucosylates serine and threonine residues of folded thrombospondin type 1 repeats (TSRs). To date, the mechanism by which this enzyme recognizes very dissimilar TSRs has been unclear. By engineering a fusion protein, we report the crystal structure of Caenorhabditis elegans POFUT2 (CePOFUT2) in complex with GDP and human TSR1 that suggests an inverting mechanism for fucose transfer assisted by a catalytic base and shows that nearly half of the TSR1 is embraced by CePOFUT2. A small number of direct interactions and a large network of water molecules maintain the complex. Site-directed mutagenesis demonstrates that POFUT2 fucosylates threonine preferentially over serine and relies on folded TSRs containing the minimal consensus sequence C-X-X-S/T-C. Crystallographic and mutagenesis data, together with atomic-level simulations, uncover a binding mechanism by which POFUT2 promiscuously recognizes the structural fingerprint of poorly homologous TSRs through a dynamic network of water-mediated interactions. PMID:26854667

  13. A proactive role of water molecules in acceptor recognition by protein O-fucosyltransferase 2.

    PubMed

    Valero-González, Jessika; Leonhard-Melief, Christina; Lira-Navarrete, Erandi; Jiménez-Osés, Gonzalo; Hernández-Ruiz, Cristina; Pallarés, María Carmen; Yruela, Inmaculada; Vasudevan, Deepika; Lostao, Anabel; Corzana, Francisco; Takeuchi, Hideyuki; Haltiwanger, Robert S; Hurtado-Guerrero, Ramon

    2016-04-01

    Protein O-fucosyltransferase 2 (POFUT2) is an essential enzyme that fucosylates serine and threonine residues of folded thrombospondin type 1 repeats (TSRs). To date, the mechanism by which this enzyme recognizes very dissimilar TSRs has been unclear. By engineering a fusion protein, we report the crystal structure of Caenorhabditis elegans POFUT2 (CePOFUT2) in complex with GDP and human TSR1 that suggests an inverting mechanism for fucose transfer assisted by a catalytic base and shows that nearly half of the TSR1 is embraced by CePOFUT2. A small number of direct interactions and a large network of water molecules maintain the complex. Site-directed mutagenesis demonstrates that POFUT2 fucosylates threonine preferentially over serine and relies on folded TSRs containing the minimal consensus sequence C-X-X-S/T-C. Crystallographic and mutagenesis data, together with atomic-level simulations, uncover a binding mechanism by which POFUT2 promiscuously recognizes the structural fingerprint of poorly homologous TSRs through a dynamic network of water-mediated interactions.

  14. Design and testing of aptamer-based electrochemical biosensors for proteins and small molecules.

    PubMed

    Cheng, Alan K H; Sen, Dipankar; Yu, Hua-Zhong

    2009-11-01

    The fabrication of aptamer-based electrochemical biosensors as an emerging technology has made the detection of small and macromolecular analytes easier, faster, and more suited for the ongoing transition from fundamental analytical science to the early detection of protein biomarkers. Aptamers are synthetic oligonucleotides that have undergone iterative rounds of in vitro selection for binding with high affinity to specific analytes of choice; a sensitive yet simple method to utilize aptamers as recognition entities for the development of biosensors is to transduce the signal electrochemically. In this review article, we attempt to summarize the state-of-the-art research progresses that have been published in recent years; in particular, we focus on the electrochemical biosensors that incorporate aptamers for sensing small organic molecules and proteins. Based on differences in the design of the DNA/RNA-modified electrodes, we classify aptamer-based electrochemical sensors into three categories, for which the analyte detection relies on: (a) configurational change, i.e., the analyte binding induces either an assembly or dissociation of the sensor construct; (b) conformational change, i.e., the analyte binding induces an alteration in the conformation (folding) of the surface immobilized aptamer strands; and (c) conductivity change, i.e., the analyte binding "switches on" the conductivity of the surface-bound aptamer-DNA constructs. In each section, we will discuss the performance of these novel biosensors with representative examples reported in recent literature.

  15. Myelin basic protein cleaves cell adhesion molecule L1 and promotes neuritogenesis and cell survival.

    PubMed

    Lutz, David; Loers, Gabriele; Kleene, Ralf; Oezen, Iris; Kataria, Hardeep; Katagihallimath, Nainesh; Braren, Ingke; Harauz, George; Schachner, Melitta

    2014-05-01

    The cell adhesion molecule L1 is a Lewis(x)-carrying glycoprotein that plays important roles in the developing and adult nervous system. Here we show that myelin basic protein (MBP) binds to L1 in a Lewis(x)-dependent manner. Furthermore, we demonstrate that MBP is released by murine cerebellar neurons as a sumoylated dynamin-containing protein upon L1 stimulation and that this MBP cleaves L1 as a serine protease in the L1 extracellular domain at Arg(687) yielding a transmembrane fragment that promotes neurite outgrowth and neuronal survival in cell culture. L1-induced neurite outgrowth and neuronal survival are reduced in MBP-deficient cerebellar neurons and in wild-type cerebellar neurons in the presence of an MBP antibody or L1 peptide containing the MBP cleavage site. Genetic ablation of MBP in shiverer mice and mutagenesis of the proteolytically active site in MBP or of the MBP cleavage site within L1 as well as serine protease inhibitors and an L1 peptide containing the MBP cleavage site abolish generation of the L1 fragment. Our findings provide evidence for novel functions of MBP in the nervous system. PMID:24671420

  16. Label-free detection of protein molecules secreted from an organ-on-a-chip model for drug toxicity assays

    NASA Astrophysics Data System (ADS)

    Morales, Andres W.; Zhang, Yu S.; Aleman, Julio; Alerasool, Parissa; Dokmeci, Mehmet R.; Khademhosseini, Ali; Ye, Jing Yong

    2016-03-01

    Clinical attrition is about 30% from failure of drug candidates due to toxic side effects, increasing the drug development costs significantly and slowing down the drug discovery process. This partly originates from the fact that the animal models do not accurately represent human physiology. Hence there is a clear unmet need for developing drug toxicity assays using human-based models that are complementary to traditional animal models before starting expensive clinical trials. Organ-on-a-chip techniques developed in recent years have generated a variety of human organ models mimicking different human physiological conditions. However, it is extremely challenging to monitor the transient and long-term response of the organ models to drug treatments during drug toxicity tests. First, when an organ-on-a-chip model interacts with drugs, a certain amount of protein molecules may be released into the medium due to certain drug effects, but the amount of the protein molecules is limited, since the organ tissue grown inside microfluidic bioreactors have minimum volume. Second, traditional fluorescence techniques cannot be utilized for real-time monitoring of the concentration of the protein molecules, because the protein molecules are continuously secreted from the tissue and it is practically impossible to achieve fluorescence labeling in the dynamically changing environment. Therefore, direct measurements of the secreted protein molecules with a label-free approach is strongly desired for organs-on-a-chip applications. In this paper, we report the development of a photonic crystal-based biosensor for label-free assays of secreted protein molecules from a liver-on-a-chip model. Ultrahigh detection sensitivity and specificity have been demonstrated.

  17. Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling.

    PubMed

    Head, Sarah A; Liu, Jun O

    2016-01-01

    Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several target identification methods have been developed and used to successfully elucidate the binding proteins of a variety of small molecules, these techniques have drawbacks that make them unsuitable for detecting certain types of small molecule-target interactions. In particular, non-covalent interactions that depend on native cellular conditions, such as those of membrane proteins whose structures may be perturbed upon cell lysis, are often not amenable to affinity-based target identification methods. Here, we demonstrate a method wherein a probe containing a photolabile group is used to covalently crosslink to the small molecule binding protein within the environment of the live cell, allowing the detection and isolation of the target protein without the need for maintenance of the interaction after cell lysis. This technique is a valuable tool for studying biologically interesting small molecules with unknown mechanisms, both in the context of basic biology as well as drug discovery. PMID:27684515

  18. Interleukin-1 receptor accessory protein organizes neuronal synaptogenesis as a cell adhesion molecule.

    PubMed

    Yoshida, Tomoyuki; Shiroshima, Tomoko; Lee, Sung-Jin; Yasumura, Misato; Uemura, Takeshi; Chen, Xigui; Iwakura, Yoichiro; Mishina, Masayoshi

    2012-02-22

    Interleukin-1 receptor accessory protein (IL-1RAcP) is the essential component of receptor complexes mediating immune responses to interleukin-1 family cytokines. IL-1RAcP in the brain exists in two isoforms, IL-1RAcP and IL-1RAcPb, differing only in the C-terminal region. Here, we found robust synaptogenic activities of IL-1RAcP in cultured cortical neurons. Knockdown of IL-1RAcP isoforms in cultured cortical neurons suppressed synapse formation as indicated by decreases of active zone protein Bassoon puncta and dendritic protrusions. IL-1RAcP recovered the accumulation of presynaptic Bassoon puncta, while IL-1RAcPb rescued both Bassoon puncta and dendritic protrusions. Consistently, the expression of IL-1RAcP in cortical neurons enhances the accumulation of Bassoon puncta and that of IL-1RAcPb stimulated both Bassoon puncta accumulation and spinogenesis. IL-1RAcP interacted with protein tyrosine phosphatase (PTP) δ through the extracellular domain. Mini-exon peptides in the Ig-like domains of PTPδ splice variants were critical for their efficient binding to IL-1RAcP. The synaptogenic activities of IL-1RAcP isoforms were diminished in cortical neurons from PTPδ knock-out mice. Correspondingly, PTPδ required IL-1RAcPb to induce postsynaptic differentiation. Thus, IL-1RAcPb bidirectionally regulated synapse formation of cortical neurons. Furthermore, the spine densities of cortical and hippocampal pyramidal neurons were reduced in IL-1RAcP knock-out mice lacking both isoforms. These results suggest that IL-1RAcP isoforms function as trans-synaptic cell adhesion molecules in the brain and organize synapse formation. Thus, IL-1RAcP represents an interesting molecular link between immune systems and synapse formation in the brain.

  19. Protein kinase Calpha and epsilon small-molecule targeted therapeutics: a new roadmap to two Holy Grails in drug discovery?

    PubMed

    O'Brian, Catherine A; Chu, Feng; Bornmann, William G; Maxwell, David S

    2006-02-01

    Protein kinase (PK)Calpha and epsilon are rational targets for cancer therapy. However, targeted experimental therapeutics that inhibit PKCalpha or epsilon are unavailable. The authors established recently that covalent modification of an active-site cysteine in human PKCepsilon, Cys452, by small molecules, for example 2-mercaptoethanolamine, is necessary and sufficient to render PKCepsilon kinase-dead. Cys452 is conserved in only eleven human protein kinase genes, including PKCalpha. Therefore, the design of small molecules that bind PKC active sites with an electrophile substituent positioned proximal to the Cys452 side chain may lead to targeted therapeutics that selectively inhibit PKCepsilon, PKCalpha or other PKC isozymes.

  20. Life in extreme environments: single molecule force spectroscopy as a tool to explore proteins from extremophilic organisms.

    PubMed

    Tych, Katarzyna M; Hoffmann, Toni; Batchelor, Matthew; Hughes, Megan L; Kendrick, Katherine E; Walsh, Danielle L; Wilson, Michael; Brockwell, David J; Dougan, Lorna

    2015-04-01

    Extremophiles are organisms which survive and thrive in extreme environments. The proteins from extremophilic single-celled organisms have received considerable attention as they are structurally stable and functionally active under extreme physical and chemical conditions. In this short article, we provide an introduction to extremophiles, the structural adaptations of proteins from extremophilic organisms and the exploitation of these proteins in industrial applications. We provide a review of recent developments which have utilized single molecule force spectroscopy to mechanically manipulate proteins from extremophilic organisms and the information which has been gained about their stability, flexibility and underlying energy landscapes.

  1. Distribution patterns of small-molecule ligands in the protein universe and implications for origin of life and drug discovery

    PubMed Central

    Ji, Hong-Fang; Kong, De-Xin; Shen, Liang; Chen, Ling-Ling; Ma, Bin-Guang; Zhang, Hong-Yu

    2007-01-01

    Background Extant life depends greatly on the binding of small molecules (such as ligands) with macromolecules (such as proteins), and one ligand can bind multiple proteins. However, little is known about the global patterns of ligand-protein mapping. Results By examining 2,186 well-defined small-molecule ligands and thousands of protein domains derived from a database of druggable binding sites, we show that a few ligands bind tens of protein domains or folds, whereas most ligands bind only one, which indicates that ligand-protein mapping follows a power law. Through assigning the protein-binding orders (early or late) for bio-ligands, we demonstrate that the preferential attachment principle still holds for the power-law relation between ligands and proteins. We also found that polar molecular surface area, H-bond acceptor counts, H-bond donor counts and partition coefficient are potential factors to discriminate ligands from ordinary molecules and to differentiate super ligands (shared by three or more folds) from others. Conclusion These findings have significant implications for evolution and drug discovery. First, the chronology of ligand-protein binding can be inferred by the power-law feature of ligand-protein mapping. Some nucleotide-containing ligands, such as ATP, ADP, GDP, NAD, FAD, dihydro-nicotinamide-adenine-dinucleotide phosphate (NDP), nicotinamide-adenine-dinucleotide phosphate (NAP), flavin mononucleotide (FMN) and AMP, are found to be the earliest cofactors bound to proteins, agreeing with the current understanding of evolutionary history. Second, the finding that about 30% of ligands are shared by two or more domains will help with drug discovery, such as in finding new functions from old drugs, developing promiscuous drugs and depending more on natural products. PMID:17727706

  2. Dual Screening of BPTF and Brd4 Using Protein-Observed Fluorine NMR Uncovers New Bromodomain Probe Molecules.

    PubMed

    Urick, Andrew K; Hawk, Laura M L; Cassel, Melissa K; Mishra, Neeraj K; Liu, Shuai; Adhikari, Neeta; Zhang, Wei; dos Santos, Camila O; Hall, Jennifer L; Pomerantz, William C K

    2015-10-16

    Bromodomain-containing protein dysregulation is linked to cancer, diabetes, and inflammation. Selective inhibition of bromodomain function is a newly proposed therapeutic strategy. We describe a (19)F NMR dual screening method for small molecule discovery using fluorinated tryptophan resonances on two bromodomain-containing proteins. The chemical shift dispersion of (19)F resonances within fluorine-labeled proteins enables the simultaneous analysis of two fluorinated bromodomains by NMR. A library of 229 small molecules was screened against the first bromodomain of Brd4 and the BPTF bromodomain. We report the first small molecule selective for BPTF over Brd4, termed AU1. The Kd = 2.8 μM for AU1, which is active in a cell-based reporter assay. No binding is detected with Brd4. Three new Brd4 inhibitors with submicromolar affinity were also discovered. Brd4 hits were validated in a thermal stability assay and potency determined via fluorescence anisotropy. The speed, ease of interpretation, and low protein concentration needed for protein-observed (19)F NMR experiments in a multiprotein format offers a new method to discover and characterize selective ligands for bromodomain-containing proteins.

  3. Applications of Engineered DNA-Binding Molecules Such as TAL Proteins and the CRISPR/Cas System in Biology Research

    PubMed Central

    Fujita, Toshitsugu; Fujii, Hodaka

    2015-01-01

    Engineered DNA-binding molecules such as transcription activator-like effector (TAL or TALE) proteins and the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) (CRISPR/Cas) system have been used extensively for genome editing in cells of various types and species. The sequence-specific DNA-binding activities of these engineered DNA-binding molecules can also be utilized for other purposes, such as transcriptional activation, transcriptional repression, chromatin modification, visualization of genomic regions, and isolation of chromatin in a locus-specific manner. In this review, we describe applications of these engineered DNA-binding molecules for biological purposes other than genome editing. PMID:26404236

  4. Single-molecule detection of proteins with antigen-antibody interaction using resistive-pulse sensing of submicron latex particles

    NASA Astrophysics Data System (ADS)

    Takakura, T.; Yanagi, I.; Goto, Y.; Ishige, Y.; Kohara, Y.

    2016-03-01

    We developed a resistive-pulse sensor with a solid-state pore and measured the latex agglutination of submicron particles induced by antigen-antibody interaction for single-molecule detection of proteins. We fabricated the pore based on numerical simulation to clearly distinguish between monomer and dimer latex particles. By measuring single dimers agglutinated in the single-molecule regime, we detected single human alpha-fetoprotein molecules. Adjusting the initial particle concentration improves the limit of detection (LOD) to 95 fmol/l. We established a theoretical model of the LOD by combining the reaction kinetics and the counting statistics to explain the effect of initial particle concentration on the LOD. The theoretical model shows how to improve the LOD quantitatively. The single-molecule detection studied here indicates the feasibility of implementing a highly sensitive immunoassay by a simple measurement method using resistive-pulse sensing.

  5. Analysis of all-optical light modulation in proteorhodopsin protein molecules

    NASA Astrophysics Data System (ADS)

    Roy, Sukhdev; Sharma, Parag

    2008-03-01

    We present a detailed steady-state and time-dependent theoretical analysis of all-optical light modulation in the recently discovered, wild-type proteorhodopsin (WTpR) protein molecules based on excited-state absorption. Amplitude modulation of cw probe laser beam transmissions at 520, 405, 555 and 560 nm, corresponding to the peak absorption of pR, pRM, pRK and pRN intermediate states of pR photocycle, respectively, by cw and pulsed modulating pump laser beam at 520 nm have been analyzed. The effect of various spectral and kinetic parameters on modulation characteristics has been studied. There is an optimum value of concentration for a given pump intensity value for which maximum modulation of the probe beam can be achieved. The switching characteristics of probe beam at 405 and 520 nm exhibit dip and peak, respectively, which can be removed by decreasing the absorption of pRM state at 520 nm. The modulation in WTpR is at lower pump powers with smaller contrast in comparison to WT bacteriorhodopsin (bR) and WT pharaonis phoborhodopsin (ppR). The modulation characteristics exhibit unique features compared to bR and ppR.

  6. Sensitive bifunctional aptamer-based electrochemical biosensor for small molecules and protein.

    PubMed

    Deng, Chunyan; Chen, Jinhua; Nie, Lihua; Nie, Zhou; Yao, Shouzhuo

    2009-12-15

    In this paper, a bifunctional electrochemical biosensor for highly sensitive detection of small molecule (adenosine) or protein (lysozyme) was developed. Two aptamer units for adenosine and lysozyme were immobilized on the gold electrode by the formation of DNA/DNA duplex. The detection of adenosine or lysozyme could be carried out by virtue of switching structures of aptamers from DNA/DNA duplex to DNA/target complex. The change of the interfacial feature of the electrode was characterized by cyclic voltammertic (CV) response of surface-bound [Ru(NH(3))(6)](3+). On the other hand, DNA functionalized Au nanoparticles (DNA-AuNPs) were used to enhance the sensitivity of the aptasensor because DNA-AuNPs modified interface could load more [Ru(NH(3))(6)](3+) cations. Thus, the assembly of two aptamer-contained DNA strands integrated with the DNA-AuNPs amplification not only improves the sensitivity of the electrochemical aptasensor but also presents a simple and general model for bifunctional aptasensor. The proposed aptasensor has low detection limit (0.02 nM for adenosine and 0.01 microg mL(-1) for lysozyme) and exhibits several advantages such as high sensitivity and bifunctional recognition.

  7. Myelin Basic Protein Cleaves Cell Adhesion Molecule L1 and Improves Regeneration After Injury.

    PubMed

    Lutz, David; Kataria, Hardeep; Kleene, Ralf; Loers, Gabriele; Chaudhary, Harshita; Guseva, Daria; Wu, Bin; Jakovcevski, Igor; Schachner, Melitta

    2016-07-01

    Myelin basic protein (MBP) is a serine protease that cleaves neural cell adhesion molecule L1 and generates a transmembrane L1 fragment which facilitates L1-dependent functions in vitro, such as neurite outgrowth, neuronal cell migration and survival, myelination by Schwann cells as well as Schwann cell proliferation, migration, and process formation. Ablation and blocking of MBP or disruption of its proteolytic activity by mutation of a proteolytically active serine residue abolish L1-dependent cellular responses. In utero injection of adeno-associated virus encoding proteolytically active MBP into MBP-deficient shiverer mice normalizes differentiation, myelination, and synaptogenesis in the developing postnatal spinal cord, in contrast to proteolytically inactive MBP. Application of active MBP to the injured wild-type spinal cord and femoral nerve augments levels of a transmembrane L1 fragment, promotes remyelination, and improves functional recovery after injury. Application of MBP antibody impairs recovery. Virus-mediated expression of active MBP in the lesion site after spinal cord injury results in improved functional recovery, whereas injection of virus encoding proteolytically inactive MBP fails to do so. The present study provides evidence for a novel L1-mediated function of MBP in the developing spinal cord and in the injured adult mammalian nervous system that leads to enhanced recovery after acute trauma.

  8. The consequences of translational and rotational entropy lost by small molecules on binding to proteins.

    PubMed

    Murray, Christopher W; Verdonk, Marcel L

    2002-10-01

    When a small molecule binds to a protein, it loses a significant amount of rigid body translational and rotational entropy. Estimates of the associated energy barrier vary widely in the literature yet accurate estimates are important in the interpretation of results from fragment-based drug discovery techniques. This paper describes an analysis that allows the estimation of the rigid body entropy barrier from the increase in binding affinities that results when two fragments of known affinity and known binding mode are joined together. The paper reviews the relatively rare number of examples where good quality data is available. From the analysis of this data, we estimate that the barrier to binding, due to the loss of rigid-body entropy, is 15-20 kJ/mol, i.e. around 3 orders of magnitude in affinity at 298 K. This large barrier explains why it is comparatively rare to observe multiple fragments binding to non-overlapping adjacent sites in enzymes. The barrier is also consistent with medicinal chemistry experience where small changes in the critical binding regions of ligands are often poorly tolerated by enzymes. PMID:12650591

  9. The Prion Protein Controls Polysialylation of Neural Cell Adhesion Molecule 1 during Cellular Morphogenesis

    PubMed Central

    Mehrabian, Mohadeseh; Brethour, Dylan; Wang, Hansen; Xi, Zhengrui; Rogaeva, Ekaterina; Schmitt-Ulms, Gerold

    2015-01-01

    Despite its multi-faceted role in neurodegenerative diseases, the physiological function of the prion protein (PrP) has remained elusive. On the basis of its evolutionary relationship to ZIP metal ion transporters, we considered that PrP may contribute to the morphogenetic reprogramming of cells underlying epithelial-to-mesenchymal transitions (EMT). Consistent with this hypothesis, PrP transcription increased more than tenfold during EMT, and stable PrP-deficient cells failed to complete EMT in a mammalian cell model. A global comparative proteomics analysis identified the neural cell adhesion molecule 1 (NCAM1) as a candidate mediator of this impairment, which led to the observation that PrP-deficient cells fail to undergo NCAM1 polysialylation during EMT. Surprisingly, this defect was caused by a perturbed transcription of the polysialyltransferase ST8SIA2 gene. Proteomics data pointed toward β-catenin as a transcriptional regulator affected in PrP-deficient cells. Indeed, pharmacological blockade or siRNA-based knockdown of β-catenin mimicked PrP-deficiency in regards to NCAM1 polysialylation. Our data established the existence of a PrP-ST8SIA2-NCAM signaling loop, merged two mature fields of investigation and offer a simple model for explaining phenotypes linked to PrP. PMID:26288071

  10. Myelin Basic Protein Cleaves Cell Adhesion Molecule L1 and Improves Regeneration After Injury.

    PubMed

    Lutz, David; Kataria, Hardeep; Kleene, Ralf; Loers, Gabriele; Chaudhary, Harshita; Guseva, Daria; Wu, Bin; Jakovcevski, Igor; Schachner, Melitta

    2016-07-01

    Myelin basic protein (MBP) is a serine protease that cleaves neural cell adhesion molecule L1 and generates a transmembrane L1 fragment which facilitates L1-dependent functions in vitro, such as neurite outgrowth, neuronal cell migration and survival, myelination by Schwann cells as well as Schwann cell proliferation, migration, and process formation. Ablation and blocking of MBP or disruption of its proteolytic activity by mutation of a proteolytically active serine residue abolish L1-dependent cellular responses. In utero injection of adeno-associated virus encoding proteolytically active MBP into MBP-deficient shiverer mice normalizes differentiation, myelination, and synaptogenesis in the developing postnatal spinal cord, in contrast to proteolytically inactive MBP. Application of active MBP to the injured wild-type spinal cord and femoral nerve augments levels of a transmembrane L1 fragment, promotes remyelination, and improves functional recovery after injury. Application of MBP antibody impairs recovery. Virus-mediated expression of active MBP in the lesion site after spinal cord injury results in improved functional recovery, whereas injection of virus encoding proteolytically inactive MBP fails to do so. The present study provides evidence for a novel L1-mediated function of MBP in the developing spinal cord and in the injured adult mammalian nervous system that leads to enhanced recovery after acute trauma. PMID:26081148

  11. The Prion Protein Controls Polysialylation of Neural Cell Adhesion Molecule 1 during Cellular Morphogenesis.

    PubMed

    Mehrabian, Mohadeseh; Brethour, Dylan; Wang, Hansen; Xi, Zhengrui; Rogaeva, Ekaterina; Schmitt-Ulms, Gerold

    2015-01-01

    Despite its multi-faceted role in neurodegenerative diseases, the physiological function of the prion protein (PrP) has remained elusive. On the basis of its evolutionary relationship to ZIP metal ion transporters, we considered that PrP may contribute to the morphogenetic reprogramming of cells underlying epithelial-to-mesenchymal transitions (EMT). Consistent with this hypothesis, PrP transcription increased more than tenfold during EMT, and stable PrP-deficient cells failed to complete EMT in a mammalian cell model. A global comparative proteomics analysis identified the neural cell adhesion molecule 1 (NCAM1) as a candidate mediator of this impairment, which led to the observation that PrP-deficient cells fail to undergo NCAM1 polysialylation during EMT. Surprisingly, this defect was caused by a perturbed transcription of the polysialyltransferase ST8SIA2 gene. Proteomics data pointed toward β-catenin as a transcriptional regulator affected in PrP-deficient cells. Indeed, pharmacological blockade or siRNA-based knockdown of β-catenin mimicked PrP-deficiency in regards to NCAM1 polysialylation. Our data established the existence of a PrP-ST8SIA2-NCAM signaling loop, merged two mature fields of investigation and offer a simple model for explaining phenotypes linked to PrP. PMID:26288071

  12. Anion Effects on Sodium Ion and Acid Molecule Adduction to Protein Ions in Electrospray Ionization Mass Spectrometry

    PubMed Central

    Flick, Tawnya G.; Merenbloom, Samuel I.; Williams, Evan R.

    2012-01-01

    Gaseous protein–metal ion and protein–molecule complexes can be readily formed by electrospray ionization (ESI) from aqueous solutions containing proteins and millimolar concentrations of sodium salts of various anions. The extent of sodium and acid molecule adduction to multiply charged protein ions is inversely related and depends strongly on the proton affinity (PA) of the anion, with extensive sodium adduction occurring for anions with PA values greater than ~300 kcal·mol−1 and extensive acid molecule adduction occurring for anions with PA values less than 315 kcal·mol−1. The role of the anion on the extent of sodium and acid molecule adduction does not directly follow the Hofmeister series, suggesting that direct protein–ion interactions may not play a significant role in the observed effect of anions on protein structure in solution. These results indicate that salts with anions that have low PA values may be useful solution-phase additives to minimize nonspecific metal ion adduction in ESI experiments designed to identify specific protein-metal ion interactions. PMID:21952761

  13. The mechanism and properties of bio-photon emission and absorption in protein molecules in living systems

    NASA Astrophysics Data System (ADS)

    Pang, Xiao-feng

    2012-05-01

    The mechanism and properties of bio-photon emission and absorption in bio-tissues were studied using Pang's theory of bio-energy transport, in which the energy spectra of protein molecules are obtained from the discrete dynamic equation. From the energy spectra, it was determined that the protein molecules could both radiate and absorb bio-photons with wavelengths of <3 μm and 5-7 μm, consistent with the energy level transitions of the excitons. These results were consistent with the experimental data; this consisted of infrared absorption data from collagen, bovine serum albumin, the protein-like molecule acetanilide, plasma, and a person's finger, and the laser-Raman spectra of acidity I-type collagen in the lungs of a mouse, and metabolically active Escherichia coli. We further elucidated the mechanism responsible for the non-thermal biological effects produced by the infrared light absorbed by the bio-tissues, using the above results. No temperature rise was observed; instead, the absorbed infrared light promoted the vibrations of amides as well the transport of the bio-energy from one place to other in the protein molecules, which changed their conformations. These experimental results, therefore, not only confirmed the validity of the mechanism of bio-photon emission, and the newly developed theory of bio-energy transport mentioned above, but also explained the mechanism and properties of the non-thermal biological effects produced by the absorption of infrared light by the living systems.

  14. Probing Nanosecond Protein Motions of Calmodulin by Single-Molecule Fluorescence Anisotropy

    SciTech Connect

    Tan, Xin; Hu, Dehong; Squier, Thomas C.; Lu, H PETER.

    2004-01-01

    Proteins operate as part of molecular networks that perform specific cellular functions. To understand the complex molecular network, it is important to study individual processes within the network such as protein motions and protein-protein interactions.

  15. Probing Nanosecond Protein Motions of Calmodulin by Single-Molecule Fluorescence Anisotropy

    SciTech Connect

    Tan, Xin; Hu, Dehong; Squier, Thomas C.; Lu, H PETER.

    2004-09-20

    Proteins operate as part of molecular networks that perform specific cellular functions. To understand the complex molecular network, it is important to study individual processes within the network such as protein motions and protein-protein interactions.

  16. Cancer cell death induced by novel small molecules degrading the TACC3 protein via the ubiquitin-proteasome pathway.

    PubMed

    Ohoka, N; Nagai, K; Hattori, T; Okuhira, K; Shibata, N; Cho, N; Naito, M

    2014-11-06

    The selective degradation of target proteins with small molecules is a novel approach to the treatment of various diseases, including cancer. We have developed a protein knockdown system with a series of hybrid small compounds that induce the selective degradation of target proteins via the ubiquitin-proteasome pathway. In this study, we designed and synthesized novel small molecules called SNIPER(TACC3)s, which target the spindle regulatory protein transforming acidic coiled-coil-3 (TACC3). SNIPER(TACC3)s induce poly-ubiquitylation and proteasomal degradation of TACC3 and reduce the TACC3 protein level in cells. Mechanistic analysis indicated that the ubiquitin ligase APC/C(CDH1) mediates the SNIPER(TACC3)-induced degradation of TACC3. Intriguingly, SNIPER(TACC3) selectively induced cell death in cancer cells expressing a larger amount of TACC3 protein than normal cells. These results suggest that protein knockdown of TACC3 by SNIPER(TACC3) is a potential strategy for treating cancers overexpressing the TACC3 protein.

  17. Human perivascular stem cells show enhanced osteogenesis and vasculogenesis with Nel-like molecule I protein.

    PubMed

    Askarinam, Asal; James, Aaron W; Zara, Janette N; Goyal, Raghav; Corselli, Mirko; Pan, Angel; Liang, Pei; Chang, Le; Rackohn, Todd; Stoker, David; Zhang, Xinli; Ting, Kang; Péault, Bruno; Soo, Chia

    2013-06-01

    An ideal mesenchymal stem cell (MSC) source for bone tissue engineering has yet to be identified. Such an MSC population would be easily harvested in abundance, with minimal morbidity and with high purity. Our laboratories have identified perivascular stem cells (PSCs) as a candidate cell source. PSCs are readily isolatable through fluorescent-activated cell sorting from adipose tissue and have been previously shown to be indistinguishable from MSCs in the phenotype and differentiation potential. PSCs consist of two distinct cell populations: (1) pericytes (CD146+, CD34-, and CD45-), which surround capillaries and microvessels, and (2) adventitial cells (CD146-, CD34+, and CD45-), found within the tunica adventitia of large arteries and veins. We previously demonstrated the osteogenic potential of pericytes by examining pericytes derived from the human fetal pancreas, and illustrated their in vivo trophic and angiogenic effects. In the present study, we used an intramuscular ectopic bone model to develop the translational potential of our original findings using PSCs (as a combination of pericytes and adventitial cells) from human white adipose tissue. We evaluated human PSC (hPSC)-mediated bone formation and vascularization in vivo. We also examined the effects of hPSCs when combined with the novel craniosynostosis-associated protein, Nel-like molecule I (NELL-1). Implants consisting of the demineralized bone matrix putty combined with NELL-1 (3 μg/μL), hPSC (2.5×10(5) cells), or hPSC+NELL-1, were inserted in the bicep femoris of SCID mice. Bone growth was evaluated using microcomputed tomography, histology, and immunohistochemistry over 4 weeks. Results demonstrated the osteogenic potential of hPSCs and the additive effect of hPSC+NELL-1 on bone formation and vasculogenesis. Comparable osteogenesis was observed with NELL-1 as compared to the more commonly used bone morphogenetic protein-2. Next, hPSCs induced greater implant vascularization than the unsorted

  18. The cytoplasmic domain of the cell adhesion molecule uvomorulin associates with three independent proteins structurally related in different species.

    PubMed Central

    Ozawa, M; Baribault, H; Kemler, R

    1989-01-01

    Uvomorulin belongs to the group of Ca2+-dependent cell adhesion molecules, which are integral membrane proteins with several structural features in common. In particular, the cytoplasmic part of these proteins is highly conserved in different species, suggesting a common biological function. To test this assumption we transfected a uvomorulin full-length cDNA into uvomorulin-negative mouse NIH 3T3 and L cells. Immunoprecipitations with anti-uvomorulin antibodies detected, in addition to uvomorulin, three independent proteins of 102, 88 and 80 kd which are of host origin and which form complexes with uvomorulin. Using cDNA constructs coding for uvomorulin with cytoplasmic or extracellular deletions it is shown that the 102, 88 and 80 kd proteins complex with the cytoplasmic domain of uvomorulin. Peptide pattern analysis revealed that these three proteins are identical in different mouse cells. When uvomorulin cDNA was introduced into cell lines from other species, such as human HeLa and avian fibroblasts, the expressed uvomorulin was also associated with endogenous 102, 88 and 80 kd proteins and, moreover, each of these proteins showed structural similarities to the respective mouse molecule. A panel of antibodies specific for known cytoplasmic proteins of mol. wts similar to those of the three proteins did not react with any of the described components. This suggests that the 102, 88 and 80 kd proteins constitute a new group of proteins for which we propose the nomenclature of catenin alpha, beta and gamma respectively. The characterization of these proteins provides a first molecular basis for a possible cytoplasmic anchorage of uvomorulin to the cytoskeleton. Images PMID:2788574

  19. Recent Developments in the Use of Differential Scanning Fluorometry in Protein and Small Molecule Discovery and Characterization

    PubMed Central

    Simeonov, Anton

    2016-01-01

    Introduction Despite tremendous advances in the application of biophysical methods in drug discovery, the preponderance of instruments and techniques still require sophisticated analyses by dedicated personnel and/or large amounts of frequently hard-to-produce proteins. A technique which carries the promise of simplicity and relatively low protein consumption is the differential scanning fluorometry (DSF), wherein protein denaturation is monitored, through the use of environmentally sensitive fluorescent dye, in a temperature-ramp regime by observing the gradual exposure to the solvent of otherwise buried hydrophobic faces of protein domains. Areas covered This review describes recent developments in the field, with a special emphasis on advances published during the 2010–2013 period. Expert Opinion There has been a significant diversification of DSF applications beyond initial small molecule discovery into areas such as protein therapeutic development, formulation studies, and various mechanistic investigations, serving as a further indication of the broad penetration of the technique. In the small molecule arena, DSF has expanded towards sophisticated co-dependency MOA tests, demonstrating the wealth of information which the technique can provide. Importantly, the first public deposition of a large screening dataset may enable the use of thermal stabilization data in refining in silico models for small molecule binding. PMID:23738712

  20. Inhibition of Inducible Nitric Oxide Synthase Expression by a Novel Small Molecule Activator of the Unfolded Protein Response

    PubMed Central

    Symons, Kent T; Massari, Mark E; Dozier, Sara J; Nguyen, Phan M; Jenkins, David; Herbert, Mark; Gahman, Timothy C; Noble, Stewart A; Rozenkrants, Natasha; Zhang, Yan; Rao, Tadimeti S; Shiau, Andrew K; Hassig, Christian A

    2008-01-01

    The transcription of inducible nitric oxide synthase (iNOS) is activated by a network of proinflammatory signaling pathways. Here we describe the identification of a small molecule that downregulates the expression of iNOS mRNA and protein in cytokine-activated cells and suppresses nitric oxide production in vivo. Mechanistic analysis suggests that this small molecule, erstressin, also activates the unfolded protein response (UPR), a signaling pathway triggered by endoplasmic reticulum stress. Erstressin induces rapid phosphorylation of eIF2α and the alternative splicing of XBP-1, hallmark initiating events of the UPR. Further, erstressin activates the transcription of multiple genes involved in the UPR. These data suggest an inverse relationship between UPR activation and iNOS mRNA and protein expression under proinflammatory conditions. PMID:20161838

  1. DNA curtains: novel tools for imaging protein-nucleic acid interactions at the single-molecule level.

    PubMed

    Collins, Bridget E; Ye, Ling F; Duzdevich, Daniel; Greene, Eric C

    2014-01-01

    Interactions between proteins and nucleic acids are at the molecular foundations of most key biological processes, including DNA replication, genome maintenance, the regulation of gene expression, and chromosome segregation. A complete understanding of these types of biological processes requires tackling questions with a range of different techniques, such as genetics, cell biology, molecular biology, biochemistry, and structural biology. Here, we describe a novel experimental approach called "DNA curtains" that can be used to complement and extend these more traditional techniques by providing real-time information about protein-nucleic acid interactions at the level of single molecules. We describe general features of the DNA curtain technology and its application to the study of protein-nucleic acid interactions in vitro. We also discuss some future developments that will help address crucial challenges to the field of single-molecule biology.

  2. Protein electrochemistry using graphene-based nano-assembly: an ultrasensitive electrochemical detection of protein molecules via nanoparticle-electrode collisions.

    PubMed

    Li, Da; Liu, Jingquan; Barrow, Colin J; Yang, Wenrong

    2014-08-01

    We describe a new electrochemical detection approach towards single protein molecules (microperoxidase-11, MP-11), which are attached to the surface of graphene nanosheets. The non-covalently functionalized graphene nanosheets exhibit enhanced electroactive surface area, where amplified redox current is produced when graphene nanosheets collide with the electrode.

  3. Measurement of Small Molecule Binding Kinetics on a Protein Microarray by Plasmonic-Based Electrochemical Impedance Imaging

    PubMed Central

    2015-01-01

    We report on a quantitative study of small molecule binding kinetics on protein microarrays with plasmonic-based electrochemical impedance microscopy (P-EIM). P-EIM measures electrical impedance optically with high spatial resolution by converting a surface charge change to a surface plasmon resonance (SPR) image intensity change, and the signal is not scaled to the mass of the analyte. Using P-EIM, we measured binding kinetics and affinity between small molecule drugs (imatinib and SB202190) and their target proteins (kinases Abl1 and p38-α). The measured affinity values are consistent with reported values measured by an indirect competitive binding assay. We also found that SB202190 has weak bindings to ABL1 with KD > 10 μM, which is not reported in the literature. Furthermore, we found that P-EIM is less prone to nonspecific binding, a long-standing issue in SPR. Our results show that P-EIM is a novel method for high-throughput measurement of small molecule binding kinetics and affinity, which is critical to the understanding of small molecules in biological systems and discovery of small molecule drugs. PMID:25153794

  4. Screening for Small-Molecule Modulators of Long Noncoding RNA-Protein Interactions Using AlphaScreen

    PubMed Central

    Pedram Fatemi, Roya; Salah-Uddin, Sultan; Modarresi, Farzaneh; Khoury, Nathalie; Wahlestedt, Claes

    2015-01-01

    Long non–protein coding RNAs (lncRNAs) are an important class of molecules that help orchestrate key cellular events. Although their functional roles in cells are not well understood, thousands of lncRNAs and a number of possible mechanisms by which they act have been reported. LncRNAs can exert their regulatory function in cells by interacting with epigenetic enzymes. In this study, we developed a tool to study lncRNA-protein interactions for high-throughput screening of small-molecule modulators using AlphaScreen technology. We tested the interaction of two lncRNAs: brain-derived neurotrophic factor antisense (BDNF-AS) and Hox transcript antisense RNA (HOTAIR), with Enhancer of zeste homolog 2 (EZH2), a histone methyltransferase against a phytochemical library, to look for small-molecule inhibitors that can alter the expression of downstream target genes. We identified ellipticine, a compound that up-regulates BDNF transcription. Our study shows the feasibility of using high-throughput screening to identify modulators of lncRNA-protein interactions and paves the road for targeting lncRNAs that are dysregulated in human disorders using small-molecule therapies. PMID:26173710

  5. Control of Transmembrane Protein Diffusion within the Postsynaptic Density Assessed by Simultaneous Single-Molecule Tracking and Localization Microscopy

    PubMed Central

    Li, Tuo P.; Blanpied, Thomas A.

    2016-01-01

    Postsynaptic transmembrane proteins are critical elements of synapses, mediating trans-cellular contact, sensitivity to neurotransmitters and other signaling molecules, and flux of Ca and other ions. Positioning and mobility of each member of this large class of proteins is critical to their individual function at the synapse. One critical example is that the position of glutamate receptors within the postsynaptic density (PSD) strongly modulates their function by aligning or misaligning them with sites of presynaptic vesicle fusion. In addition, the regulated ability of receptors to move in or out of the synapse is critical for activity-dependent plasticity. However, factors that control receptor mobility within the boundaries of the synapse are not well understood. Notably, PSD scaffold molecules accumulate in domains much smaller than the synapse. Within these nanodomains, the density of proteins is considerably higher than that of the synapse as a whole, so high that steric hindrance is expected to reduce receptor mobility substantially. However, while numerical modeling has demonstrated several features of how the varying protein density across the face of a single PSD may modulate receptor motion, there is little experimental information about the extent of this influence. To address this critical aspect of synaptic organizational dynamics, we performed single-molecule tracking of transmembrane proteins using universal point accumulation-for-imaging-in-nanoscale-topography (uPAINT) over PSDs whose internal structure was simultaneously resolved using photoactivated localization microscopy (PALM). The results provide important experimental confirmation that PSD scaffold protein density strongly influences the mobility of transmembrane proteins. A protein with a cytosolic domain that does not bind PSD-95 was still slowed in regions of high PSD-95 density, suggesting that crowding by scaffold molecules and perhaps other proteins is sufficient to stabilize

  6. Control of Transmembrane Protein Diffusion within the Postsynaptic Density Assessed by Simultaneous Single-Molecule Tracking and Localization Microscopy.

    PubMed

    Li, Tuo P; Blanpied, Thomas A

    2016-01-01

    Postsynaptic transmembrane proteins are critical elements of synapses, mediating trans-cellular contact, sensitivity to neurotransmitters and other signaling molecules, and flux of Ca and other ions. Positioning and mobility of each member of this large class of proteins is critical to their individual function at the synapse. One critical example is that the position of glutamate receptors within the postsynaptic density (PSD) strongly modulates their function by aligning or misaligning them with sites of presynaptic vesicle fusion. In addition, the regulated ability of receptors to move in or out of the synapse is critical for activity-dependent plasticity. However, factors that control receptor mobility within the boundaries of the synapse are not well understood. Notably, PSD scaffold molecules accumulate in domains much smaller than the synapse. Within these nanodomains, the density of proteins is considerably higher than that of the synapse as a whole, so high that steric hindrance is expected to reduce receptor mobility substantially. However, while numerical modeling has demonstrated several features of how the varying protein density across the face of a single PSD may modulate receptor motion, there is little experimental information about the extent of this influence. To address this critical aspect of synaptic organizational dynamics, we performed single-molecule tracking of transmembrane proteins using universal point accumulation-for-imaging-in-nanoscale-topography (uPAINT) over PSDs whose internal structure was simultaneously resolved using photoactivated localization microscopy (PALM). The results provide important experimental confirmation that PSD scaffold protein density strongly influences the mobility of transmembrane proteins. A protein with a cytosolic domain that does not bind PSD-95 was still slowed in regions of high PSD-95 density, suggesting that crowding by scaffold molecules and perhaps other proteins is sufficient to stabilize

  7. Site-specifically arraying small molecules or proteins on DNA using an expanded genetic alphabet

    PubMed Central

    Zimmermann, Jörg; Adhikary, Ramkrishna; Dhami, Kirandeep; Ordoukhanian, Phillip; Sun, Zhelin; Xiang, Jie; Romesberg, Floyd E.

    2014-01-01

    We have developed a class of replicable unnatural DNA base pairs formed between d5SICS and either dMMO2, dDMO, or dNaM. To explore the use of these pairs to produce site-specifically labeled DNA, we report the synthesis of a variety of derivatives bearing propynyl groups, an analysis of their polymerase-mediated replication, and subsequent site-specific modification of the amplified DNA via Click chemistry. We find that with the d5SICS scaffold, a propynyl ether linker is accommodated better than its aliphatic analog, but not as well as the protected propargyl amine linker explored previously. We also find that with the dMMO2 and dDMO analogs, the dMMO2 position para to the glycosidic linkage is best suited for linker attachment, and that while aliphatic and ether-based linkers are similarly accommodated, the direct attachment of an ethynyl group to the nucleobase core is most well tolerated. To demonstrate the utility of these analogs, a variety of them are used to site-selectively attach a biotin tag to the amplified DNA. Finally, we use d5SICSCO-dNaM to couple one or two proteins to amplified DNA, with the double labeled product visualized by atomic force microscopy. The ability to encode the spatial relationships of arrayed molecules in PCR amplifiable DNA should have important applications, ranging from SELEX with functionalities not naturally present in DNA to the production, and perhaps “evolution” of nanomaterials. PMID:24026962

  8. Induction of the neural cell adhesion molecule and neuronal aggregation by osteogenic protein 1.

    PubMed Central

    Perides, G; Safran, R M; Rueger, D C; Charness, M E

    1992-01-01

    The neural cell adhesion molecule (N-CAM) plays a fundamental role in nervous system development and regeneration, yet the regulation of the expression of N-CAM in different brain regions has remained poorly understood. Osteogenic protein 1 (OP-1) is a member of the transforming growth factor beta superfamily that is expressed in the nervous system. Treatment of the neuroblastoma-glioma hybrid cell line NG108-15 for 1-4 days with recombinant human OP-1 (hOP-1) induced alterations in cell shape, formation of epithelioid sheets, and aggregation of cells into multilayered clusters. Immunofluorescence studies and Western blots demonstrated a striking differential induction of the three N-CAM isoforms in hOP-1-treated cells. hOP-1 caused a 6-fold up-regulation of the 140-kDa N-CAM, the isoform showing the highest constitutive expression, and a 29-fold up-regulation of the 180-kDa isoform. The 120-kDa isoform was not detected in control NG108-15 cells but was readily identified in hOP-1-treated cells. Incubation of NG108-15 cells with an antisense N-CAM oligonucleotide reduced the induction of N-CAM by hOP-1 and decreased the formation of multilayered cell aggregates. Anti-N-CAM monoclonal antibodies also diminished the formation of multilayered cell aggregates by hOP-1 and decreased cell-cell adhesion when hOP-1-treated NG108-15 cells were dispersed and replated. Thus, hOP-1 produces morphologic changes in NG108-15 cells, at least in part, by inducing N-CAM. These observations suggest that OP-1 or a homologue may participate in the regulation of N-CAM during nervous system development and regeneration. Images PMID:1438217

  9. Site-specifically arraying small molecules or proteins on DNA using an expanded genetic alphabet.

    PubMed

    Li, Zhengtao; Lavergne, Thomas; Malyshev, Denis A; Zimmermann, Jörg; Adhikary, Ramkrishna; Dhami, Kirandeep; Ordoukhanian, Phillip; Sun, Zhelin; Xiang, Jie; Romesberg, Floyd E

    2013-10-11

    A class of replicable unnatural DNA base pairs formed between d5SICS and either dMMO2, dDMO, or dNaM were developed. To explore the use of these pairs to produce site-specifically labeled DNA, the synthesis of a variety of derivatives bearing propynyl groups, an analysis of their polymerase-mediated replication, and subsequent site-specific modification of the amplified DNA by Click chemistry is reported. With the d5SICS scaffold a propynyl ether linker is accommodated better than its aliphatic analogue, but not as well as the protected propargyl amine linker explored previously. It was also found that with the dMMO2 and dDMO analogues, the dMMO2 position para to the glycosidic linkage is best suited for linker attachment and that although aliphatic and ether-based linkers are similarly accommodated, the direct attachment of an ethynyl group to the nucleobase core is most well tolerated. To demonstrate the utility of these analogues, a variety of them were used to site-selectively attach a biotin tag to the amplified DNA. Finally, we use d5SICS(CO) -dNaM to couple one or two proteins to amplified DNA, with the double labeled product visualized by atomic force microscopy. The ability to encode the spatial relationships of arrayed molecules in PCR amplifiable DNA should have important applications, ranging from SELEX with functionalities not naturally present in DNA to the production, and perhaps "evolution" of nanomaterials. PMID:24026962

  10. Site-specifically arraying small molecules or proteins on DNA using an expanded genetic alphabet.

    PubMed

    Li, Zhengtao; Lavergne, Thomas; Malyshev, Denis A; Zimmermann, Jörg; Adhikary, Ramkrishna; Dhami, Kirandeep; Ordoukhanian, Phillip; Sun, Zhelin; Xiang, Jie; Romesberg, Floyd E

    2013-10-11

    A class of replicable unnatural DNA base pairs formed between d5SICS and either dMMO2, dDMO, or dNaM were developed. To explore the use of these pairs to produce site-specifically labeled DNA, the synthesis of a variety of derivatives bearing propynyl groups, an analysis of their polymerase-mediated replication, and subsequent site-specific modification of the amplified DNA by Click chemistry is reported. With the d5SICS scaffold a propynyl ether linker is accommodated better than its aliphatic analogue, but not as well as the protected propargyl amine linker explored previously. It was also found that with the dMMO2 and dDMO analogues, the dMMO2 position para to the glycosidic linkage is best suited for linker attachment and that although aliphatic and ether-based linkers are similarly accommodated, the direct attachment of an ethynyl group to the nucleobase core is most well tolerated. To demonstrate the utility of these analogues, a variety of them were used to site-selectively attach a biotin tag to the amplified DNA. Finally, we use d5SICS(CO) -dNaM to couple one or two proteins to amplified DNA, with the double labeled product visualized by atomic force microscopy. The ability to encode the spatial relationships of arrayed molecules in PCR amplifiable DNA should have important applications, ranging from SELEX with functionalities not naturally present in DNA to the production, and perhaps "evolution" of nanomaterials.

  11. Small molecule targeting malaria merozoite surface protein-1 (MSP-1) prevents host invasion of divergent plasmodial species.

    PubMed

    Chandramohanadas, Rajesh; Basappa; Russell, Bruce; Liew, Kingsley; Yau, Yin Hoe; Chong, Alvin; Liu, Min; Gunalan, Karthigayan; Raman, Rahul; Renia, Laurent; Nosten, Francois; Shochat, Susana Geifman; Dao, Ming; Sasisekharan, Ram; Suresh, Subra; Preiser, Peter

    2014-11-15

    Malaria causes nearly 1 million deaths annually. Recent emergence of multidrug resistance highlights the need to develop novel therapeutic interventions against human malaria. Given the involvement of sugar binding plasmodial proteins in host invasion, we set out to identify such proteins as targets of small glycans. Combining multidisciplinary approaches, we report the discovery of a small molecule inhibitor, NIC, capable of inhibiting host invasion through interacting with a major invasion-related protein, merozoite surface protein-1 (MSP-1). This interaction was validated through computational, biochemical, and biophysical tools. Importantly, treatment with NIC prevented host invasion by Plasmodium falciparum and Plasmodium vivax--major causative organisms of human malaria. MSP-1, an indispensable antigen critical for invasion and suitably localized in abundance on the merozoite surface represents an ideal target for antimalarial development. The ability to target merozoite invasion proteins with specific small inhibitors opens up a new avenue to target this important pathogen.

  12. Anisotropic solvent model of the lipid bilayer. 2. Energetics of insertion of small molecules, peptides, and proteins in membranes.

    PubMed

    Lomize, Andrei L; Pogozheva, Irina D; Mosberg, Henry I

    2011-04-25

    A new computational approach to calculating binding energies and spatial positions of small molecules, peptides, and proteins in the lipid bilayer has been developed. The method combines an anisotropic solvent representation of the lipid bilayer and universal solvation model, which predicts transfer energies of molecules from water to an arbitrary medium with defined polarity properties. The universal solvation model accounts for hydrophobic, van der Waals, hydrogen-bonding, and electrostatic solute-solvent interactions. The lipid bilayer is represented as a fluid anisotropic environment described by profiles of dielectric constant (ε), solvatochromic dipolarity parameter (π*), and hydrogen bonding acidity and basicity parameters (α and β). The polarity profiles were calculated using published distributions of quasi-molecular segments of lipids determined by neutron and X-ray scattering for DOPC bilayer and spin-labeling data that define concentration of water in the lipid acyl chain region. The model also accounts for the preferential solvation of charges and polar groups by water and includes the effect of the hydrophobic mismatch for transmembrane proteins. The method was tested on calculations of binding energies and preferential positions in membranes for small-molecules, peptides and peripheral membrane proteins that have been experimentally studied. The new theoretical approach was implemented in a new version (2.0) of our PPM program and applied for the large-scale calculations of spatial positions in membranes of more than 1000 peripheral and integral proteins. The results of calculations are deposited in the updated OPM database ( http://opm.phar.umich.edu ).

  13. Fusion Molecules of Heat Shock Protein HSPX with Other Antigens of Mycobacterium tuberculosis Show High Potential in Serodiagnosis of Tuberculosis

    PubMed Central

    Khalid, Ruqyya; Afzal, Madeeha; Khurshid, Sana; Paracha, Rehan Zafar; Khan, Imran H.

    2016-01-01

    Variable individual response against the antigens of Mycobacterium tuberculosis necessitates detection of multiple antibodies for enhancing reliability of serodiagnosis of tuberculosis. Fusion molecules consisting of two or more antigens showing high sensitivity would be helpful in achieving this objective. Antigens of M. tuberculosis HSPX and PE35 were expressed in a soluble form whereas tnPstS1 and FbpC1 were expressed as inclusion bodies at 37°C. Heat shock protein HSPX when attached to the N-termini of the antigens PE35, tnPstS1 and FbpC1, all the fusion molecules were expressed at high levels in E. coli in a soluble form. ELISA analysis of the plasma samples of TB patients against HSPX-tnPstS1 showed 57.7% sensitivity which is nearly the same as the expected combined value obtained after deducting the number of plasma samples (32) containing the antibodies against both the individual antigens. Likewise, the 54.4% sensitivity of HSPX-PE35 was nearly the same as that expected from the combined values of the contributing antigens. Structural analysis of all the fusion molecules by CD spectroscopy showed that α-helical and β-sheet contents were found close to those obtained through molecular modeling. Molecular modeling studies of HSPX-tnPstS1 and HSPX-PE35 support the analytical results as most of the epitopes of the contributing antigens were found to be available for binding to the corresponding antibodies. Using these fusion molecules in combination with other antigenic molecules should reduce the number of antigenic proteins required for a more reliable and economical serodiagnosis of tuberculosis. Also, HSPX seems to have potential application in soluble expression of heterologous proteins in E. coli. PMID:27654048

  14. Molecular Adsorber Coating

    NASA Technical Reports Server (NTRS)

    Straka, Sharon; Peters, Wanda; Hasegawa, Mark; Hedgeland, Randy; Petro, John; Novo-Gradac, Kevin; Wong, Alfred; Triolo, Jack; Miller, Cory

    2011-01-01

    A document discusses a zeolite-based sprayable molecular adsorber coating that has been developed to alleviate the size and weight issues of current ceramic puck-based technology, while providing a configuration that more projects can use to protect against degradation from outgassed materials within a spacecraft, particularly contamination-sensitive instruments. This coating system demonstrates five times the adsorption capacity of previously developed adsorber coating slurries. The molecular adsorber formulation was developed and refined, and a procedure for spray application was developed. Samples were spray-coated and tested for capacity, thermal optical/radiative properties, coating adhesion, and thermal cycling. Work performed during this study indicates that the molecular adsorber formulation can be applied to aluminum, stainless steel, or other metal substrates that can accept silicate-based coatings. The coating can also function as a thermal- control coating. This adsorber will dramatically reduce the mass and volume restrictions, and is less expensive than the currently used molecular adsorber puck design.

  15. Characterization of the surface-active components of sugar beet pectin and the hydrodynamic thickness of the adsorbed pectin layer.

    PubMed

    Siew, Chee Kiong; Williams, Peter A; Cui, Steve W; Wang, Qi

    2008-09-10

    The fraction of sugar beet pectin (SBP) adsorbed onto limonene oil droplets during emulsification has been isolated, and its chemical and physicochemical characteristics have been determined. While the SBP sample itself was found to contain 2.67 and 1.06% protein and ferulic acid, respectively, the adsorbed fraction contained 11.10% protein and 2.16% ferulic acid. The adsorbed fraction was also found to have a higher degree of acetylation, notably at the C2 position on the galacturonic acid residues, and was also found to contain a higher proportion of neutral sugars, which are present in the ramified side chains of the pectin molecules. The thickness of the layer of SBP adsorbed onto polystyrene latex particles was studied by dynamic light scattering and was found to increase with increasing surface coverage. It was found to have a value of approximately 140 nm at plateau coverage, which closely corresponded to the hydrodynamic diameter of the pectin chains. The adsorbed layer thickness was found to be sensitive to pH and the presence of electrolyte. The thickness at a surface coverage of approximately 20 mg/m(2) in the absence of electrolyte at pH approximately 4 was 107 nm and at pH 8.8 was 70 nm, while at pH approximately 4 in the presence of 10 mM NaCl the thickness was found to be 70 nm. It was concluded that the SBP molecules form multilayers at the surface due to electrostatic interaction between the positively charged protein moieties and the galacturonic acid residues. The removal of calcium from the SBP had no effect on the adsorbed layer thickness; hence, multilayer formation due to calcium ion cross-linking was considered unlikely.

  16. Free Energy Calculations of Mutations Involving a Tightly Bound Water Molecule and Ligand Substitutions in a Ligand-Protein Complex.

    PubMed

    García-Sosa, Alfonso T; Mancera, Ricardo L

    2010-09-17

    The accurate calculation of the free energy of interaction of protein-water-ligand systems has an important role in molecular recognition and drug design that is often not fully considered. We report free energy thermodynamic integration calculations used to evaluate the effects of inclusion, neglect, and targeting and removal (i.e., systematic substitution by ligand functional groups) of an important, tightly bound, water molecule in the SH3 domain of Abl tyrosine kinase. The effects of this water molecule on the free energies of interaction of several Abl-SH3 domain-ligand systems reveal that there is an unfavourable free energy change associated with its removal into the bulk solvent. Only three substitutions by an additional functional group (out of methyl, ethyl, hydroxyl, amino, and amide groups) in the phenyl ring of a tyrosine in the peptide ligand resulted in a favourable change in the free energy of binding upon replacement of the ordered water molecule. This computational approach provides a direct route to the systematic and rigorous prediction of the thermodynamic influence of ordered, structural water molecules on ligand modification and optimization in drug design by calculating free energy changes in protein-water-ligand systems. PMID:27463454

  17. Amplification and detection of single-molecule conformational fluctuation through a protein interaction network with bimodal distributions.

    PubMed

    Wu, Zhanghan; Elgart, Vlad; Qian, Hong; Xing, Jianhua

    2009-09-10

    A protein undergoes conformational dynamics with multiple time scales, which results in fluctuating enzyme activities. Recent studies in single-molecule enzymology have observe this "age-old" dynamic disorder phenomenon directly. However, the single-molecule technique has its limitation. To be able to observe this molecular effect with real biochemical functions in situ, we propose to couple the fluctuations in enzymatic activity to noise propagations in small protein interaction networks such as a zeroth-order ultrasensitive phosphorylation-dephosphorylation cycle. We show that enzyme fluctuations can indeed be amplified by orders of magnitude into fluctuations in the level of substrate phosphorylation, a quantity of wide interest in cellular biology. Enzyme conformational fluctuations sufficiently slower than the catalytic reaction turnover rate result in a bimodal concentration distribution of the phosphorylated substrate. In return, this network-amplified single-enzyme fluctuation can be used as a novel biochemical "reporter" for measuring single-enzyme conformational fluctuation rates.

  18. Structure and properties of water film adsorbed on mica surfaces.

    PubMed

    Zhao, Gutian; Tan, Qiyan; Xiang, Li; Cai, Di; Zeng, Hongbo; Yi, Hong; Ni, Zhonghua; Chen, Yunfei

    2015-09-14

    The structure profiles and physical properties of the adsorbed water film on a mica surface under conditions with different degrees of relative humidity are investigated by a surface force apparatus. The first layer of the adsorbed water film shows ice-like properties, including a lattice constant similar with ice crystal, a high bearing capacity that can support normal pressure as high as 4 MPa, a creep behavior under the action of even a small normal load, and a character of hydrogen bond. Adjacent to the first layer of the adsorbed water film, the water molecules in the outer layer are liquid-like that can flow freely under the action of external loads. Experimental results demonstrate that the adsorbed water layer makes the mica surface change from hydrophilic to weak hydrophobic. The weak hydrophobic surface may induce the latter adsorbed water molecules to form water islands on a mica sheet. PMID:26374054

  19. Structure and properties of water film adsorbed on mica surfaces.

    PubMed

    Zhao, Gutian; Tan, Qiyan; Xiang, Li; Cai, Di; Zeng, Hongbo; Yi, Hong; Ni, Zhonghua; Chen, Yunfei

    2015-09-14

    The structure profiles and physical properties of the adsorbed water film on a mica surface under conditions with different degrees of relative humidity are investigated by a surface force apparatus. The first layer of the adsorbed water film shows ice-like properties, including a lattice constant similar with ice crystal, a high bearing capacity that can support normal pressure as high as 4 MPa, a creep behavior under the action of even a small normal load, and a character of hydrogen bond. Adjacent to the first layer of the adsorbed water film, the water molecules in the outer layer are liquid-like that can flow freely under the action of external loads. Experimental results demonstrate that the adsorbed water layer makes the mica surface change from hydrophilic to weak hydrophobic. The weak hydrophobic surface may induce the latter adsorbed water molecules to form water islands on a mica sheet.

  20. Electronic transport in single-helical protein molecules: Effects of multiple charge conduction pathways and helical symmetry

    NASA Astrophysics Data System (ADS)

    Kundu, Sourav; Karmakar, S. N.

    2016-07-01

    We propose a tight-binding model to investigate electronic transport properties of single helical protein molecules incorporating both the helical symmetry and the possibility of multiple charge transfer pathways. Our study reveals that due to existence of both the multiple charge transfer pathways and helical symmetry, the transport properties are quite rigid under influence of environmental fluctuations which indicates that these biomolecules can serve as better alternatives in nanoelectronic devices than its other biological counterparts e.g., single-stranded DNA.

  1. Influences of Electromagnetic Energy on Bio-Energy Transport through Protein Molecules in Living Systems and Its Experimental Evidence.

    PubMed

    Pang, Xiaofeng; Chen, Shude; Wang, Xianghui; Zhong, Lisheng

    2016-07-25

    The influences of electromagnetic fields (EMFs) on bio-energy transport and its mechanism of changes are investigated through analytic and numerical simulation and experimentation. Bio-energy transport along protein molecules is performed by soliton movement caused by the dipole-dipole electric interactions between neighboring amino acid residues. As such, EMFs can affect the structure of protein molecules and change the properties of the bio-energy transported in living systems. This mechanism of biological effect from EMFs involves the amino acid residues in protein molecules. To study and reveal this mechanism, we simulated numerically the features of the movement of solitons along protein molecules with both a single chain and with three channels by using the Runge-Kutta method and Pang's soliton model under the action of EMFs with the strengths of 25,500, 51,000, 76,500, and 102,000 V/m in the single-chain protein, as well as 17,000, 25,500, and 34,000 V/m in the three-chain protein, respectively. Results indicate that electric fields (EFs) depress the binding energy of the soliton, decrease its amplitude, and change its wave form. Also, the soliton disperses at 102,000 V/m in a single-chain protein and at 25,500 and 34,000 V/m in three-chain proteins. These findings signify that the influence of EMFs on the bio-energy transport cannot be neglected; however, these variations depend on both the strength and the direction of the EF in the EMF. This direction influences the biological effects of EMF, which decrease with increases in the angle between the direction of the EF and that of the dipole moment of amino acid residues; however, randomness at the macroscopic level remains. Lastly, we experimentally confirm the existence of a soliton and the validity of our conclusion by using the infrared spectra of absorption of the collagens, which is activated by another type of EF. Thus, we can affirm that both the described mechanism and the corresponding theory are

  2. Influences of Electromagnetic Energy on Bio-Energy Transport through Protein Molecules in Living Systems and Its Experimental Evidence.

    PubMed

    Pang, Xiaofeng; Chen, Shude; Wang, Xianghui; Zhong, Lisheng

    2016-01-01

    The influences of electromagnetic fields (EMFs) on bio-energy transport and its mechanism of changes are investigated through analytic and numerical simulation and experimentation. Bio-energy transport along protein molecules is performed by soliton movement caused by the dipole-dipole electric interactions between neighboring amino acid residues. As such, EMFs can affect the structure of protein molecules and change the properties of the bio-energy transported in living systems. This mechanism of biological effect from EMFs involves the amino acid residues in protein molecules. To study and reveal this mechanism, we simulated numerically the features of the movement of solitons along protein molecules with both a single chain and with three channels by using the Runge-Kutta method and Pang's soliton model under the action of EMFs with the strengths of 25,500, 51,000, 76,500, and 102,000 V/m in the single-chain protein, as well as 17,000, 25,500, and 34,000 V/m in the three-chain protein, respectively. Results indicate that electric fields (EFs) depress the binding energy of the soliton, decrease its amplitude, and change its wave form. Also, the soliton disperses at 102,000 V/m in a single-chain protein and at 25,500 and 34,000 V/m in three-chain proteins. These findings signify that the influence of EMFs on the bio-energy transport cannot be neglected; however, these variations depend on both the strength and the direction of the EF in the EMF. This direction influences the biological effects of EMF, which decrease with increases in the angle between the direction of the EF and that of the dipole moment of amino acid residues; however, randomness at the macroscopic level remains. Lastly, we experimentally confirm the existence of a soliton and the validity of our conclusion by using the infrared spectra of absorption of the collagens, which is activated by another type of EF. Thus, we can affirm that both the described mechanism and the corresponding theory are

  3. A novel high-throughput scanning microscope for label-free detection of protein and small-molecule chemical microarrays

    PubMed Central

    Fei, Y.Y.; Landry, J.P.; Sun, Y.S.; Luo, J.T.; Wang, X.B.; Lam, K.S; Zhu, X.D.

    2009-01-01

    We describe a novel scanning optical microscope based on a polarization-modulated nulling ellipsometry. The new microscope employs a combination of scanning mirror and sample translation and thus enables high-throughput label-free detection of biomolecular microarrays with more than 10,000 protein or small molecule targets. For illustration, we show the image of a 2760-spot protein microarray on a functionalized glass slide obtained with such a microscope. The new scanning microscope is also capable of determining, in parallel, the real-time binding kinetics of multiple molecular species under aqueous conditions. PMID:18248040

  4. Influences of Electromagnetic Energy on Bio-Energy Transport through Protein Molecules in Living Systems and Its Experimental Evidence

    PubMed Central

    Pang, Xiaofeng; Chen, Shude; Wang, Xianghui; Zhong, Lisheng

    2016-01-01

    The influences of electromagnetic fields (EMFs) on bio-energy transport and its mechanism of changes are investigated through analytic and numerical simulation and experimentation. Bio-energy transport along protein molecules is performed by soliton movement caused by the dipole–dipole electric interactions between neighboring amino acid residues. As such, EMFs can affect the structure of protein molecules and change the properties of the bio-energy transported in living systems. This mechanism of biological effect from EMFs involves the amino acid residues in protein molecules. To study and reveal this mechanism, we simulated numerically the features of the movement of solitons along protein molecules with both a single chain and with three channels by using the Runge–Kutta method and Pang’s soliton model under the action of EMFs with the strengths of 25,500, 51,000, 76,500, and 102,000 V/m in the single-chain protein, as well as 17,000, 25,500, and 34,000 V/m in the three-chain protein, respectively. Results indicate that electric fields (EFs) depress the binding energy of the soliton, decrease its amplitude, and change its wave form. Also, the soliton disperses at 102,000 V/m in a single-chain protein and at 25,500 and 34,000 V/m in three-chain proteins. These findings signify that the influence of EMFs on the bio-energy transport cannot be neglected; however, these variations depend on both the strength and the direction of the EF in the EMF. This direction influences the biological effects of EMF, which decrease with increases in the angle between the direction of the EF and that of the dipole moment of amino acid residues; however, randomness at the macroscopic level remains. Lastly, we experimentally confirm the existence of a soliton and the validity of our conclusion by using the infrared spectra of absorption of the collagens, which is activated by another type of EF. Thus, we can affirm that both the described mechanism and the corresponding theory

  5. Mobility of adsorbed Cry1Aa insecticidal toxin from Bacillus thuringiensis (Bt) on montmorillonite measured by fluorescence recovery after photobleaching (FRAP)

    NASA Astrophysics Data System (ADS)

    Helassa, Nordine; Daudin, Gabrielle; Noinville, Sylvie; Janot, Jean-Marc; Déjardin, Philippe; Staunton, Siobhán; Quiquampoix, Hervé

    2010-06-01

    The insecticidal toxins produced by genetically modified Bt crops are introduced into soil through root exudates and tissue decomposition and adsorb readily on soil components, especially on clays. This immobilisation and the consequent concentration of the toxins in "hot spots" could increase the exposure of soil organisms. Whereas the effects on non-target organisms are well documented, few studies consider the migration of the toxin in soil. In this study, the residual mobility of Bt Cry1Aa insecticidal toxin adsorbed on montmorillonite was assessed using fluorescence recovery after photobleaching (FRAP). This technique, which is usually used to study dynamics of cytoplasmic and membrane molecules in live cells, was applied for the first time to a protein adsorbed on a finely divided swelling clay mineral, montmorillonite. No mobility of adsorbed toxin was observed at any pH and at different degrees of surface saturation.

  6. Activation of protein phosphatase 1 by a small molecule designed to bind to the enzyme's regulatory site.

    PubMed

    Tappan, Erin; Chamberlin, A Richard

    2008-02-01

    The activity of protein phosphatase 1 (PP1), a serine-threonine phosphatase that participates ubiquitously in cellular signaling, is controlled by a wide variety of regulatory proteins that interact with PP1 at an allosteric regulatory site that recognizes a "loose" consensus sequence (usually designated as RVXF) found in all such regulatory proteins. Peptides containing the regulatory consensus sequence have been found to recapitulate the binding and PP1 activity modulation of the regulatory proteins, suggesting that it might be possible to design small-molecule surrogates that activate PP1 rather than inhibiting it. This prospect constitutes a largely unexplored way of controlling signaling pathways that could be functionally complementary to the much more extensively explored stratagem of kinase inhibition. Based on these principles, we have designed a microcystin analog that activates PP1. PMID:18291321

  7. Myelin-specific proteins: a structurally diverse group of membrane-interacting molecules.

    PubMed

    Han, Huijong; Myllykoski, Matti; Ruskamo, Salla; Wang, Chaozhan; Kursula, Petri

    2013-01-01

    The myelin sheath is a multilayered membrane in the nervous system, which has unique biochemical properties. Myelin carries a set of specific high-abundance proteins, the structure and function of which are still poorly understood. The proteins of the myelin sheath are involved in a number of neurological diseases, including autoimmune diseases and inherited neuropathies. In this review, we briefly discuss the structural properties and functions of selected myelin-specific proteins (P0, myelin oligodendrocyte glycoprotein, myelin-associated glycoprotein, myelin basic protein, myelin-associated oligodendrocytic basic protein, P2, proteolipid protein, peripheral myelin protein of 22 kDa, 2',3'-cyclic nucleotide 3'-phosphodiesterase, and periaxin); such properties include, for example, interactions with lipid bilayers and the presence of large intrinsically disordered regions in some myelin proteins. A detailed understanding of myelin protein structure and function at the molecular level will be required to fully grasp their physiological roles in the myelin sheath.

  8. Chemical methodology as a source of small-molecule checkpoint inhibitors and heat shock protein 70 (Hsp70) modulators

    PubMed Central

    Huryn, Donna M.; Brodsky, Jeffrey L.; Brummond, Kay M.; Chambers, Peter G.; Eyer, Benjamin; Ireland, Alex W.; Kawasumi, Masaoki; LaPorte, Matthew G.; Lloyd, Kayla; Manteau, Baptiste; Nghiem, Paul; Quade, Bettina; Seguin, Sandlin P.; Wipf, Peter

    2011-01-01

    Unique chemical methodology enables the synthesis of innovative and diverse scaffolds and chemotypes and allows access to previously unexplored “chemical space.” Compound collections based on such new synthetic methods can provide small-molecule probes of proteins and/or pathways whose functions are not fully understood. We describe the identification, characterization, and evolution of two such probes. In one example, a pathway-based screen for DNA damage checkpoint inhibitors identified a compound, MARPIN (ATM and ATR pathway inhibitor) that sensitizes p53-deficient cells to DNA-damaging agents. Modification of the small molecule and generation of an immobilized probe were used to selectively bind putative protein target(s) responsible for the observed activity. The second example describes a focused library approach that relied on tandem multicomponent reaction methodologies to afford a series of modulators of the heat shock protein 70 (Hsp70) molecular chaperone. The synthesis of libraries based on the structure of MAL3-101 generated a collection of chemotypes, each modulating Hsp70 function, but exhibiting divergent pharmacological activities. For example, probes that compromise the replication of a disease-associated polyomavirus were identified. These projects highlight the importance of chemical methodology development as a source of small-molecule probes and as a drug discovery starting point. PMID:21502524

  9. Real-time investigation of protein unfolding at an air-water interface at the 1 s time scale.

    PubMed

    Yano, Yohko F; Arakawa, Etsuo; Voegeli, Wolfgang; Matsushita, Tadashi

    2013-11-01

    Protein unfolding at an air-water interface has been demonstrated such that the X-ray reflectivity can be measured with an acquisition time of 1 s using a recently developed simultaneous multiple-angle-wavelength-dispersive X-ray reflectometer. This has enabled the electron density profile of the adsorbed protein molecules to be obtained in real time. A globular protein, lysozyme, adsorbed at the air-water interface is found to unfold into a flat shape within 1 s.

  10. Using specialized adsorbents for remediation

    SciTech Connect

    Hochmuth, D.P.; Grant, A.

    1995-11-01

    This paper describes two remediation case studies in which specialized adsorbents were used. In one case, the adsorbents were used to treat effluent from a soil vapor extraction system. In the other case, the adsorbents were used to treat air from a groundwater air stripper. The specialized adsorbents effectively removed volatile organic compounds from each air stream.

  11. Molecular Probing of the HPV-16 E6 Protein Alpha Helix Binding Groove with Small Molecule Inhibitors

    PubMed Central

    Rietz, Anne; Petrov, Dino P.; Bartolowits, Matthew; DeSmet, Marsha; Davisson, V. Jo; Androphy, Elliot J.

    2016-01-01

    The human papillomavirus (HPV) HPV E6 protein has emerged as a central oncoprotein in HPV-associated cancers in which sustained expression is required for tumor progression. A majority of the E6 protein interactions within the human proteome use an alpha-helix groove interface for binding. The UBE3A/E6AP HECT domain ubiquitin ligase binds E6 at this helix-groove interface. This enables formation of a trimeric complex with p53, resulting in destruction of this tumor suppressor. While recent x-ray crystal structures are useful, examples of small molecule probes that can modulate protein interactions at this interface are limited. To develop insights useful for potential structure-based design of ligands for HPV E6, a series of 2,6-disubstituted benzopyranones were prepared and tested as competitive antagonists of E6-E6AP helix-groove interactions. These small molecule probes were used in both binding and functional assays to evaluate recognition features of the E6 protein. Evidence for an ionic functional group interaction within the helix groove was implicated by the structure-activity among the highest affinity ligands. The molecular topographies of these protein-ligand interactions were evaluated by comparing the binding and activities of single amino acid E6 mutants with the results of molecular dynamic simulations. A group of arginine residues that form a rim-cap over the E6 helix groove offer compensatory roles in binding and recognition of the small molecule probes. The flexibility and impact on the overall helix-groove shape dictated by these residues offer new insights for structure-based targeting of HPV E6. PMID:26915086

  12. Modifications of cell signalling and redox balance by targeting protein acetylation using natural and engineered molecules: implications in cancer therapy.

    PubMed

    Venkateswaran, Kavya; Verma, Amit; Bhatt, Anant N; Agrawala, Paban K; Raj, Hanumantharao G; Malhotra, Shashwat; Prasad, Ashok K; Wever, Olivier De; Bracke, Marc E; Saso, Luciano; Parmar, Virinder S; Shrivastava, Anju; Dwarakanath, B S

    2014-01-01

    appears to depend on the development of newer engineered molecules and their rational combinations that can exploit the differences in the regulation of protein acetylation between tumor and normal cells/tissues.

  13. Electrochemical detection of nucleic acids, proteins, small molecules and cells using a DNA-nanostructure-based universal biosensing platform.

    PubMed

    Lin, Meihua; Song, Ping; Zhou, Guobao; Zuo, Xiaolei; Aldalbahi, Ali; Lou, Xiaoding; Shi, Jiye; Fan, Chunhai

    2016-07-01

    The occurrence and prognosis of many complex diseases, such as cancers, is associated with the variation of various molecules, including DNA at the genetic level, RNA at the regulatory level, proteins at the functional level and small molecules at the metabolic level (defined collectively as multilevel molecules). Thus it is highly desirable to develop a single platform for detecting multilevel biomarkers for early-stage diagnosis. Here we report a protocol on DNA-nanostructure-based programmable engineering of the biomolecular recognition interface, which provides a universal electrochemical biosensing platform for the ultrasensitive detection of nucleic acids (DNA/RNA), proteins, small molecules and whole cells. The protocol starts with the synthesis of a series of differentially sized, self-assembled tetrahedral DNA nanostructures (TDNs) with site-specifically modified thiol groups that can be readily anchored on the surface of a gold electrode with high reproducibility. By exploiting the rigid structure, nanoscale addressability and versatile functionality of TDNs, one can tailor the type of biomolecular probes appended on individual TDNs for the detection of specific molecules of interest. Target binding occurring on the gold surface patterned with TDNs is quantitatively translated into electrochemical signals via a coupled enzyme-based catalytic process. This uses a sandwich assay strategy in which biotinylated reporter probes recognize TDN-bound target biomolecules, which then allow binding of horseradish-peroxidase-conjugated avidin (avidin-HRP). Hydrogen peroxide (H2O2) is then reduced by avidin-HRP in the presence of TMB (3,3',5,5'-tetramethylbenzidine) to generate a quantitative electrochemical signal. The time range for the entire protocol is ∼1 d, whereas the detection process takes ∼30 min to 3 h. PMID:27310264

  14. Conformational Memory of a Protein Revealed by Single-Molecule Spectroscopy.

    PubMed

    Schörner, Mario; Beyer, Sebastian Reinhardt; Southall, June; Cogdell, Richard J; Köhler, Jürgen

    2015-11-01

    Proteins are supramolecular machines that carry out a wide range of different functions, many of which require flexibility. Up until now spontaneous conformational fluctuations of proteins have always been assumed to reflect a stochastic random process. However, if changing between different conformational states was random, then it would be difficult to understand how conformational control of protein function could have evolved. Here we demonstrate that a single protein can show conformational memory. This is exactly the process that can facilitate the evolution of control of switching between two conformational states that can then be used to regulate protein function.

  15. Structure and thermodynamics of effector molecule binding to the nitrogen signal transduction PII protein GlnZ from Azospirillum brasilense.

    PubMed

    Truan, Daphné; Bjelić, Saša; Li, Xiao-Dan; Winkler, Fritz K

    2014-07-29

    The trimeric PII signal transduction proteins regulate the function of a variety of target proteins predominantly involved in nitrogen metabolism. ATP, ADP and 2-oxoglutarate (2-OG) are key effector molecules influencing PII binding to targets. Studies of PII proteins have established that the 20-residue T-loop plays a central role in effector sensing and target binding. However, the specific effects of effector binding on T-loop conformation have remained poorly documented. We present eight crystal structures of the Azospirillum brasilense PII protein GlnZ, six of which are cocrystallized and liganded with ADP or ATP. We find that interaction with the diphosphate moiety of bound ADP constrains the N-terminal part of the T-loop in a characteristic way that is maintained in ADP-promoted complexes with target proteins. In contrast, the interactions with the triphosphate moiety in ATP complexes are much more variable and no single predominant interaction mode is apparent except for the ternary MgATP/2-OG complex. These conclusions can be extended to most investigated PII proteins of the GlnB/GlnK subfamily. Unlike reported for other PII proteins, microcalorimetry reveals no cooperativity between the three binding sites of GlnZ trimers for any of the three effectors under carefully controlled experimental conditions.

  16. The dynamics of single protein molecules is non-equilibrium and self-similar over thirteen decades in time

    DOE PAGES

    Hu, Xiaohu; Hong, Liang; Smith, Micholas Dean; Neusius, Thomas; Cheng, Xiaolin; Smith, Jeremy C.

    2015-11-23

    Here, internal motions of proteins are essential to their function. The time dependence of protein structural fluctuations is highly complex, manifesting subdiffusive, non-exponential behavior with effective relaxation times existing over many decades in time, from ps up to ~102s (refs 1-4). Here, using molecular dynamics simulations, we show that, on timescales from 10–12 to 10–5s, motions in single proteins are self-similar, non-equilibrium and exhibit ageing. The characteristic relaxation time for a distance fluctuation, such as inter-domain motion, is observation-time-dependent, increasing in a simple, power-law fashion, arising from the fractal nature of the topology and geometry of the energy landscape explored.more » Diffusion over the energy landscape follows a non-ergodic continuous time random walk. Comparison with single-molecule experiments suggests that the non-equilibrium self-similar dynamical behavior persists up to timescales approaching the in vivo lifespan of individual protein molecules.« less

  17. Small-molecule structure correctors target abnormal protein structure and function: structure corrector rescue of apolipoprotein E4-associated neuropathology.

    PubMed

    Mahley, Robert W; Huang, Yadong

    2012-11-01

    An attractive strategy to treat proteinopathies (diseases caused by malformed or misfolded proteins) is to restore protein function by inducing proper three-dimensional structure. We hypothesized that this approach would be effective in reversing the detrimental effects of apolipoprotein (apo) E4, the major allele that significantly increases the risk of developing Alzheimer's disease and other neurodegenerative disorders. ApoE4's detrimental effects result from its altered protein conformation ("domain interaction"), making it highly susceptible to proteolytic cleavage and the generation of neurotoxic fragments. Here, we review apoE structure and function and how apoE4 causes neurotoxicity, and describe the identification of potent small-molecule-based "structure correctors" that induce proper apoE4 folding. SAR studies identified a series of small molecules that significantly reduced apoE4's neurotoxic effects in cultured neurons and a series that reduced apoE4 fragment levels in vivo, providing proof-of-concept for our approach. Structure-corrector-based therapies could prove to be highly effective for the treatment of many protein-misfolding diseases.

  18. The dynamics of single protein molecules is non-equilibrium and self-similar over thirteen decades in time

    SciTech Connect

    Hu, Xiaohu; Hong, Liang; Smith, Micholas Dean; Neusius, Thomas; Cheng, Xiaolin; Smith, Jeremy C.

    2015-11-23

    Here, internal motions of proteins are essential to their function. The time dependence of protein structural fluctuations is highly complex, manifesting subdiffusive, non-exponential behavior with effective relaxation times existing over many decades in time, from ps up to ~102s (refs 1-4). Here, using molecular dynamics simulations, we show that, on timescales from 10–12 to 10–5s, motions in single proteins are self-similar, non-equilibrium and exhibit ageing. The characteristic relaxation time for a distance fluctuation, such as inter-domain motion, is observation-time-dependent, increasing in a simple, power-law fashion, arising from the fractal nature of the topology and geometry of the energy landscape explored. Diffusion over the energy landscape follows a non-ergodic continuous time random walk. Comparison with single-molecule experiments suggests that the non-equilibrium self-similar dynamical behavior persists up to timescales approaching the in vivo lifespan of individual protein molecules.

  19. Identification of a small-molecule ligand of the epigenetic reader protein Spindlin1 via a versatile screening platform.

    PubMed

    Wagner, Tobias; Greschik, Holger; Burgahn, Teresa; Schmidtkunz, Karin; Schott, Anne-Kathrin; McMillan, Joel; Baranauskienė, Lina; Xiong, Yan; Fedorov, Oleg; Jin, Jian; Oppermann, Udo; Matulis, Daumantas; Schüle, Roland; Jung, Manfred

    2016-05-19

    Epigenetic modifications of histone tails play an essential role in the regulation of eukaryotic transcription. Writer and eraser enzymes establish and maintain the epigenetic code by creating or removing posttranslational marks. Specific binding proteins, called readers, recognize the modifications and mediate epigenetic signalling. Here, we present a versatile assay platform for the investigation of the interaction between methyl lysine readers and their ligands. This can be utilized for the screening of small-molecule inhibitors of such protein-protein interactions and the detailed characterization of the inhibition. Our platform is constructed in a modular way consisting of orthogonal in vitro binding assays for ligand screening and verification of initial hits and biophysical, label-free techniques for further kinetic characterization of confirmed ligands. A stability assay for the investigation of target engagement in a cellular context complements the platform. We applied the complete evaluation chain to the Tudor domain containing protein Spindlin1 and established the in vitro test systems for the double Tudor domain of the histone demethylase JMJD2C. We finally conducted an exploratory screen for inhibitors of the interaction between Spindlin1 and H3K4me3 and identified A366 as the first nanomolar small-molecule ligand of a Tudor domain containing methyl lysine reader.

  20. Fluorogen Activating Proteins in Flow Cytometry for the Study of Surface Molecules and Receptors

    PubMed Central

    Saunders, Matthew J.; Szent-Gyorgyi, Christopher; Fisher, Gregory W.; Jarvik, Jonathan W.; Bruchez, Marcel P.; Waggoner, Alan S.

    2012-01-01

    The use of fluorescent proteins, particularly when genetically fused to proteins of biological interest, have greatly advanced many flow cytometry research applications. However, there remains a major limitation to this methodology in that only total cellular fluorescence is measured. Commonly used fluorescent proteins (e.g. EGFP and its variants) are fluorescent whether the fusion protein exists on the surface or in sub-cellular compartments. A flow cytometer cannot distinguish between these separate sources of fluorescence. This can be of great concern when using flow cytometry, plate readers or microscopy to quantify cell surface receptors or other surface proteins genetically fused to fluorescent proteins. Recently developed fluorogen activating proteins (FAPs) solve many of these issues by allowing the selective visualization of only those cell surface proteins that are exposed to the extra cellular milieu. FAPs are GFP-sized single chain antibodies that specifically bind to and generate fluorescence from otherwise non-fluorescent dyes (‘activate the fluorogen’). Like the fluorescent proteins, FAPs can be genetically fused to proteins of interest. When exogenously added fluorogens bind FAPs, fluorescence immediately increases by as much as 20,000 fold, rendering the FAP fusion proteins highly fluorescent. Moreover, since fluorogens can be made membrane impermeant, fluorescence can be limited to only those receptors expressed on the cell surface. Using cells expressing beta-2 adrenergic receptor (β2AR) fused at its N-terminus to a FAP, flow cytometry based receptor internalization assays have been developed and characterized. The fluorogen/FAP system is ideally suited to the study of cell surface proteins by fluorescence and avoids drawbacks of using receptor/fluorescent protein fusions, such as internal accumulation. We also briefly comment on extending FAP-based technologies to the study of events occurring inside of the cell as well. PMID:22366230

  1. Black Molecular Adsorber Coatings for Spaceflight Applications

    NASA Technical Reports Server (NTRS)

    Abraham, Nithin Susan; Hasegawa, Mark Makoto; Straka, Sharon A.

    2014-01-01

    The molecular adsorber coating is a new technology that was developed to mitigate the risk of on-orbit molecular contamination on spaceflight missions. The application of this coating would be ideal near highly sensitive, interior surfaces and instruments that are negatively impacted by outgassed molecules from materials, such as plastics, adhesives, lubricants, epoxies, and other similar compounds. This current, sprayable paint technology is comprised of inorganic white materials made from highly porous zeolite. In addition to good adhesion performance, thermal stability, and adsorptive capability, the molecular adsorber coating offers favorable thermal control characteristics. However, low reflectivity properties, which are typically offered by black thermal control coatings, are desired for some spaceflight applications. For example, black coatings are used on interior surfaces, in particular, on instrument baffles for optical stray light control. Similarly, they are also used within light paths between optical systems, such as telescopes, to absorb light. Recent efforts have been made to transform the white molecular adsorber coating into a black coating with similar adsorptive properties. This result is achieved by optimizing the current formulation with black pigments, while still maintaining its adsorption capability for outgassing control. Different binder to pigment ratios, coating thi