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Sample records for adsorbed protein molecules

  1. 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.

  2. 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.

  3. Conformational changes of adsorbed proteins

    NASA Astrophysics Data System (ADS)

    Allen, Scott

    2005-03-01

    The adsorption of bovine serum albumin (BSA) and pepsin to gold surfaces has been studied using surface plasmon resonance (SPR). Proteins are adsorbed from solution onto a gold surface and changes in the conformation of the adsorbed proteins are induced by changing the buffer solution. We selected pH and ionic strength values for the buffer solutions that are known from our circular dichroism measurements to cause conformational changes of the proteins in bulk solution. We find that for both BSA and pepsin the changes in conformation are impeded by the interaction of the protein with the gold surface.

  4. Imaging the wave functions of adsorbed molecules

    PubMed Central

    Lüftner, Daniel; Ules, Thomas; Reinisch, Eva Maria; Koller, Georg; Soubatch, Serguei; Tautz, F. Stefan; Ramsey, Michael G.; Puschnig, Peter

    2014-01-01

    The basis for a quantum-mechanical description of matter is electron wave functions. For atoms and molecules, their spatial distributions and phases are known as orbitals. Although orbitals are very powerful concepts, experimentally only the electron densities and -energy levels are directly observable. Regardless whether orbitals are observed in real space with scanning probe experiments, or in reciprocal space by photoemission, the phase information of the orbital is lost. Here, we show that the experimental momentum maps of angle-resolved photoemission from molecular orbitals can be transformed to real-space orbitals via an iterative procedure which also retrieves the lost phase information. This is demonstrated with images obtained of a number of orbitals of the molecules pentacene (C22H14) and perylene-3,4,9,10-tetracarboxylic dianhydride (C24H8O6), adsorbed on silver, which are in excellent agreement with ab initio calculations. The procedure requires no a priori knowledge of the orbitals and is shown to be simple and robust. PMID:24344291

  5. SPR-MS: from identifying adsorbed molecules to image tissues

    NASA Astrophysics Data System (ADS)

    Masson, Jean-François; Breault-Turcot, Julien; Forest, Simon; Chaurand, Pierre

    2015-03-01

    Surface plasmon resonance (SPR) sensors have become valuable analytical sensors for biomolecule detection. While SPR is heralded with high sensitivity, label-free and real-time detection, nonspecific adsorption and detection of ultralow concentrations remain issues. Nonspecific adsorption can be minimized using adequate surface chemistry. For example, we have employed peptide monolayers to reduce nonspecific adsorption of crude serum or cell lysate. It is important to uncover the nature of molecules nonspecifically adsorbing to surfaces in these biofluids, to further improve understanding of the nonspecific adsorption processes. Mass spectrometry (MS) provides a complementary tool to SPR to identify biomolecule adsorbed to surface. Trypsic digestion of the proteins adsorbed to surfaces led to identification of characteristic peptides from the proteins involved in nonspecific adsorption. Nonspecific adsorption in crude cell lysate results mainly from lipids, as confirmed with SPR and MS but proteins were observed on some surfaces. In another application of SPR and MS, imaging SPR can be used in combination to imaging MS to image tissue sections. Thin sections of mouse liver were inserted in the fluidic chamber of a SPRi instrument and proteins were transferred to the SPRi chip. The SPR chip was then imaged using MALDI imaging MS to identify the biomolecules that were transferred to the SPRi chip.

  6. The density and refractive index of adsorbing protein layers.

    PubMed

    Vörös, Janos

    2004-07-01

    The structure of the adsorbing layers of native and denatured proteins (fibrinogen, gamma-immunoglobulin, albumin, and lysozyme) was studied on hydrophilic TiO(2) and hydrophobic Teflon-AF surfaces using the quartz crystal microbalance with dissipation and optical waveguide lightmode spectroscopy techniques. The density and the refractive index of the adsorbing protein layers could be determined from the complementary information provided by the two in situ instruments. The observed density and refractive index changes during the protein-adsorption process indicated the presence of conformational changes (e.g., partial unfolding) in general, especially upon contact with the hydrophobic surface. The structure of the formed layers was found to depend on the size of the proteins and on the experimental conditions. On the TiO(2) surface smaller proteins formed a denser layer than larger ones and the layer of unfolded proteins was less dense than that adsorbed from the native conformation. The hydrophobic surface induced denaturation and resulted in the formation of thin compact protein films of albumin and lysozyme. A linear correlation was found between the quartz crystal microbalance measured dissipation factor and the total water content of the layer, suggesting the existence of a dissipative process that is related to the solvent molecules present inside the adsorbed protein layer. Our measurements indicated that water and solvent molecules not only influence the 3D structure of proteins in solution but also play a crucial role in their adsorption onto surfaces. PMID:15240488

  7. 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

  8. Conformation Distributions in Adsorbed Proteins.

    NASA Astrophysics Data System (ADS)

    Meuse, Curtis W.; Hubbard, Joseph B.; Vrettos, John S.; Smith, Jackson R.; Cicerone, Marcus T.

    2007-03-01

    While the structural basis of protein function is well understood in the biopharmaceutical and biotechnology industries, few methods for the characterization and comparison of protein conformation distributions are available. New methods capable of measuring the stability of protein conformations and the integrity of protein-protein, protein-ligand and protein-surface interactions both in solution and on surfaces are needed to help the development of protein-based products. We are developing infrared spectroscopy methods for the characterization and comparison of molecular conformation distributions in monolayers and in solutions. We have extracted an order parameter describing the orientational and conformational variations of protein functional groups around the average molecular values from a single polarized spectrum. We will discuss the development of these methods and compare them to amide hydrogen/deuterium exchange methods for albumin in solution and on different polymer surfaces to show that our order parameter is related to protein stability.

  9. 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.

  10. Probing atomic positions of adsorbed ammonia molecules in zeolite.

    PubMed

    Ye, Lin; Lo, Benedict T W; Qu, Jin; Wilkinson, Ian; Hughes, Tim; Murray, Claire A; Tang, Chiu C; Tsang, Shik Chi Edman

    2016-02-25

    Atomic positions and interactions between adsorbed guest molecules, such as ammonia in H-ZSM-5 microporous solids, are for the first time revealed by making use of the change in the periodical scattering parameter using in situ synchrotron powder X-ray diffraction combined with refinement within experimental errors. PMID:26833032

  11. 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.

  12. Candidate Source of Flux Noise in SQUIDs: Adsorbed Oxygen Molecules.

    PubMed

    Wang, Hui; Shi, Chuntai; Hu, Jun; Han, Sungho; Yu, Clare C; Wu, R Q

    2015-08-14

    A major obstacle to using superconducting quantum interference devices (SQUIDs) as qubits is flux noise. We propose that the heretofore mysterious spins producing flux noise could be O_{2} molecules adsorbed on the surface. Using density functional theory calculations, we find that an O_{2} molecule adsorbed on an α-alumina surface has a magnetic moment of ~1.8 μ_{B}. The spin is oriented perpendicular to the axis of the O-O bond, the barrier to spin rotations is about 10 mK. Monte Carlo simulations of ferromagnetically coupled, anisotropic XY spins on a square lattice find 1/f magnetization noise, consistent with flux noise in Al SQUIDs. PMID:26317742

  13. Theory of raman scattering from molecules adsorbed at semiconductor surfaces

    NASA Astrophysics Data System (ADS)

    Ueba, H.

    1983-09-01

    A theory is presented to calculate the Raman polarizability of an adsorbed molecule at a semiconductor surface, where the electronic excitation in the molecular site interacts with excitons (elementary excitations in the semiconductor) through non-radiative energy transfer between them, in an intermediate state in the Raman scattering process. The Raman polarizability thus calculated is found to exhibit a peak at the energy corresponding to a resonant excitation of excitons, thereby suggesting the possibility of surface enhanced Raman scattering on semiconductor surfaces. The mechanism studied here can also give an explanation of a recent observation of the Raman excitation profiles of p-NDMA and p-DMAAB adsorbed on ZnO or TiO 2, where those profiles were best described by assuming a resonant intermediate state of the exciton transition in the semiconductors. It is also demonstrated that in addition to vibrational Raman scattering, excitonic Raman scattering of adsorbed molecules will occur in the coupled molecule-semiconductor system, where the molecular returns to its ground electronic state by leaving an exciton in the semiconductor. A spectrum of the excitonic Raman scattering is expected to appear in the background of the vibrational Raman band and to be characterized by the electronic structure of excitons. A desirable experiment is suggested for an examination of the theory.

  14. A simple model for electronic properties of surface adsorbed molecules

    NASA Astrophysics Data System (ADS)

    Dhakal, Rajesh; Schwalm, William

    We adapt a minimal approximation to one electron quantum theory of molecules referred as Fast Accurate Kinetic Energy method. This in principle handles large complex molecular structures with less computational effort to compute electronic properties of adsorbed molecules. Kinetic energy integrals are calculated accurately but multi-electron potential energy integrals are approximated. The neighboring atom interactions are included also. For layers of isopthalic acids formed on pyrolytic graphite the configuration changes as a function of length of hydrocarbon tails. We study properties of this system as a function of tail length.

  15. Mobility of adsorbed proteins: a Brownian dynamics study.

    PubMed

    Ravichandran, S; Talbot, J

    2000-01-01

    We simulate the adsorption of lysozyme on a solid surface, using Brownian dynamics simulations. A protein molecule is represented as a uniformly charged sphere and interacts with other molecules through screened Coulombic and double-layer forces. The simulation starts from an empty surface and attempts are made to introduce additional proteins at a fixed time interval that is inversely proportional to the bulk protein concentration. We examine the effect of ionic strength and bulk protein concentration on the adsorption kinetics over a range of surface coverages. The structure of the adsorbed layer is examined through snapshots of the configurations and quantitatively with the radial distribution function. We extract the surface diffusion coefficient from the mean square displacement. At high ionic strengths the Coulombic interaction is effectively shielded, leading to increased surface coverage. This effect is quantified with an effective particle radius. Clustering of the adsorbed molecules is promoted by high ionic strength and low bulk concentrations. We find that lateral protein mobility decreases with increasing surface coverage. The observed trends are consistent with previous theoretical and experimental studies. PMID:10620278

  16. 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

  17. 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

  18. Controlling spins in adsorbed molecules by a chemical switch

    PubMed Central

    Wäckerlin, Christian; Chylarecka, Dorota; Kleibert, Armin; Müller, Kathrin; Iacovita, Cristian; Nolting, Frithjof; Jung, Thomas A.; Ballav, Nirmalya

    2010-01-01

    The development of chemical systems with switchable molecular spins could lead to the architecture of materials with controllable magnetic or spintronic properties. Here, we present conclusive evidence that the spin of an organometallic molecule coupled to a ferromagnetic substrate can be switched between magnetic off and on states by a chemical stimulus. This is achieved by nitric oxide (NO) functioning as an axial ligand of cobalt(II)tetraphenylporphyrin (CoTPP) ferromagnetically coupled to nickel thin-film (Ni(001)). On NO addition, the coordination sphere of Co2+ is modified and a NO–CoTPP nitrosyl complex is formed, which corresponds to an off state of the Co spin. Thermal dissociation of NO from the nitrosyl complex restores the on state of the Co spin. The NO-induced reversible off–on switching of surface-adsorbed molecular spins observed here is attributed to a spin trans effect. PMID:20975713

  19. Dynamics of photoinduced electron transfer from adsorbed molecules into solids

    NASA Astrophysics Data System (ADS)

    Gundlach, L.; Ernstorfer, R.; Willig, F.

    2007-08-01

    Ultrafast interfacial electron transfer from the donor orbital of organic chromophores into empty electronic acceptor states of a semiconductor and of a metal was investigated by two-photon photoemission spectroscopy (2PPE). Experimental tools and procedures have been developed for carrying out wet-chemistry preparation of the molecule/solid interface. The organic chromophore perylene was investigated with several different bridge/anchor groups on TiO2(110). One perylene compound was investigated for comparison on Ag(110). Angle and polarization dependent 2PPE measurements revealed the orientation of the perylene chromophore on the surface as controlled by the adsorption geometry of the respective anchor group on TiO2. UPS measurements gave the position of the HOMO level of the chromophore with respect to the Fermi level of the solid. The donor level of each molecule was found high enough to fulfill the “wide band limit” of heterogeneous electron transfer dynamics. Time constants for heterogeneous electron transfer were extracted from 2PPE transients. A difference by a factor of four was found, 13 fs against 47 fs, when a conjugated bond was exchanged for a saturated bond in the otherwise identical bridge group. The two different contributions to the 2PPE transients arising firstly from the excited state of the chromophore and secondly from the injected electrons were separated by measuring the latter contribution separately in the case of instantaneous interfacial electron transfer realized with catechol as adsorbate. The time scales measured for the electron transfer step and for the subsequent electron escape process from the surface into the bulk of TiO2 showed both good agreement with recent theoretical predictions of other groups for these systems.

  20. Alignment and assembly of adsorbed collagen molecules induced by anisotropic chemical nanopatterns.

    PubMed

    Denis, Frédéric A; Pallandre, Antoine; Nysten, Bernard; Jonas, Alain M; Dupont-Gillain, Christine C

    2005-10-01

    Collagen, a protein widely used to control cell-material interactions, is known to self-assemble in solution. Supramolecular structures also form on material surfaces following collagen adsorption. Herein, we report the use of anisotropic, flat, surface chemical nanopatterns, which consist of alkyl-terminated tracks drawn in an oligo(ethylene glycol)-terminated matrix, to direct collagen adsorption. As revealed by atomic force microscopy, the spontaneous collagen adsorption performed on such patterned substrates results in the accumulation of collagen on the hydrophobic tracks. Moreover, the width of the tracks (30-90 nm), which is much smaller than the length of the collagen molecule (approximately 300 nm), is the origin of preferential alignment of the molecules and of their assembly into continuous bundles of adsorbed collagen. This chemical guidance effect due to self-confinement of proteins upon adsorption may bring novel and valuable applications, specifically in biomaterials science and cell growth control. PMID:17193383

  1. Polarizabilities of Adsorbed and Assembled Molecules: Measuring the Conductance through Buried Contacts

    PubMed Central

    2010-01-01

    We have measured the polarizabilities of four families of molecules adsorbed to Au{111} surfaces, with structures ranging from fully saturated to fully conjugated, including single-molecule switches. Measured polarizabilities increase with increasing length and conjugation in the adsorbed molecules and are consistent with theoretical calculations. For single-molecule switches, the polarizability reflects the difference in substrate−molecule electronic coupling in the ON and OFF conductance states. Calculations suggest that the switch between the two conductance states is correlated with an oxidation state change in a nitro functional group in the switch molecules. PMID:21077677

  2. Anomalous thermal denaturing of proteins adsorbed to nanoparticles

    NASA Astrophysics Data System (ADS)

    Teichroeb, J. H.; Forrest, J. A.; Ngai, V.; Jones, L. W.

    2006-09-01

    We have used localized surface plasmon resonance (LSPR) to monitor the structural changes that accompany thermal denaturing of bovine serum albumin (BSA) adsorbed onto gold nanospheres of size 5nm-60nm. The effect of the protein on the LSPR was monitored by visible extinction spectroscopy. The position of the resonance is affected by the conformation of the adsorbed protein layer, and as such can be used as a very sensitive probe of thermal denaturing that is specific to the adsorbed protein. The results are compared to detailed calculations and show that full calculations can lead to significant increases in knowledge where gold nanospheres are used as biosensors. Thermal denaturing on spheres with diameter > 20 nm show strong similarity to bulk calorimetric studies of BSA in solution. BSA adsorbed on nanospheres with d ⩽ 15nm shows a qualitative difference in behavior, suggesting a sensitivity of denaturing characteristics on local surface curvature. This may have important implications for other protein-nanoparticle interactions.

  3. Excitation energy migration in yellow fluorescent protein (citrine) layers adsorbed on modified gold surfaces

    NASA Astrophysics Data System (ADS)

    Yusoff, Hanis Mohd; Rzeźnicka, Izabela I.; Hoshi, Hirotaka; Kajimoto, Shinji; Horimoto, Noriko Nishizawa; Sogawa, Kazuhiro; Fukumura, Hiroshi

    2013-09-01

    The nature of functional proteins adsorbed on solid surfaces is interesting from the perspective of developing of bioelectronics and biomaterials. Here we present evidence that citrine (one of yellow fluorescent protein variants) adsorbed on modified gold surfaces would not undergo denaturation and energy transfer among the adsorbed citrine molecules would occur. Gold substrates were chemically modified with 3-mercaptopropionic acid and tert-butyl mercaptan for the preparation of hydrophilic and hydrophobic surfaces, respectively. A pure solution of citrine was dropped and dried on the modified gold substrates and their surface morphology was studied with scanning tunnelling microscopy (STM). The obtained STM images showed multilayers of citrine adsorbed on the modified surfaces. On hydrophobic surfaces, citrine was adsorbed more randomly, formed various non-uniform aggregates, while on hydrophilic surfaces, citrine appeared more aligned and isolated uniform protein clusters were observed. Fluorescence lifetime and anisotropy decay of these dried citrine layers were also measured using the time correlated single photon counting method. Fluorescence anisotropy of citrine on the hydrophobic surface decayed faster than citrine on the hydrophilic surface. From these results we concluded that fluorescence energy migration occurred faster among citrine molecules which were randomly adsorbed on the hydrophobic surface to compare with the hydrophilic surface.

  4. Quasielastic neutron scattering from adsorbed water molecules on pyrogenic silica surfaces

    NASA Astrophysics Data System (ADS)

    Tumanov, A. A.; Zarko, V. I.

    1994-04-01

    Quasielastic neutron scattering (QNS) from hydrated samples of high dispersion (Aerosil) and of porous dioxide silicon was investigated. The broadening of the QNS peak analysis permits one to obtain the effective diffusion coefficient D of adsorbed water molecules. It was obtained that the D-value increases with silica hydration. The mean square displacement of the water molecules from equilibrium < x2> equals approximately 0.1 Å 2 and does not depend on the quantity of adsorbed water.

  5. 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

  6. 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

  7. Relaxation dynamics of surface-adsorbed water molecules in nanoporous silica probed by terahertz spectroscopy

    NASA Astrophysics Data System (ADS)

    Huang, Yu-Ru; Liu, Kao-Hsiang; Mou, Chung-Yuan; Sun, Chi-Kuang

    2015-08-01

    Relaxation dynamics of an exclusively adsorbed water molecule in mesoporous silica MCM-41-S was studied by using terahertz spectroscopy. With the temperature controlled from 0 to 50 °C, we observed strongly frequency- and temperature-dependent dielectric relaxation responses, implying that, unlike ice, surface-adsorbed water molecules retained flourishing picosecond dynamics. Based on the Debye relaxation model, a relaxation time constant was found to increase from 1.77 to 4.83 ps when the water molecule was cooled from 50 to 0 °C. An activation energy of ˜15 kJ/mol, which was in close agreement with a hydrogen-bonding energy, was further extracted from the Arrhenius analysis. Combined with previous molecular dynamics simulations, our results indicate that the reorientation relaxation originated from the "flip-flop" rotation of a three hydrogen-bonded surface-adsorbed water molecule.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. Transient magnetization of core excited organic molecules adsorbed on graphene

    NASA Astrophysics Data System (ADS)

    Ravikumar, Abhilash; Baby, Anu; Lin, He; Brivio, Gian Paolo; Fratesi, Guido

    This work presents a density functional theory based computational investigation of electronic and magnetic properties of physisorbed and chemisorbed organic molecules on graphene in the ground state and core excited one at low molecular coverage. For physisorbed molecules, where the interaction with graphene is dominated by van der Waals forces and the system is non-magnetic in the ground state, it is found that the valence electrons relax towards a spin polarized configuration upon excitation of a core-level electron. The magnetism depends on efficient electron transfer from graphene on the femtosecond time scale. On the contrary, when graphene is covalently functionalized, the system is magnetic in the ground state presenting two spin dependent mid gap states localized around the adsorption site. At variance with the physisorbed case upon core-level excitation, the LUMO of the molecule and the mid gap states of graphene hybridize and the relaxed valence shell is not magnetic anymore. This project has received funding from the European Union Seventh Framework Programme under grant agreement n∘ 607232 [THINFACE].

  13. Rotational Spectromicroscopy: Imaging the Orbital Interaction between Molecular Hydrogen and an Adsorbed Molecule

    NASA Astrophysics Data System (ADS)

    Li, Shaowei; Yuan, Dingwang; Yu, Arthur; Czap, Gregory; Wu, Ruqian; Ho, W.

    2015-05-01

    A hydrogen molecule can diffuse freely on the surface and be trapped above an adsorbed molecule within the junction of a scanning tunneling microscope. The trapped dihydrogen exhibits the properties of a free rotor. Here we show that the intermolecular interaction between dihydrogen and Mg-porphyrin (MgP) can be visualized by imaging j =0 to 2 rotational excitation of dihydrogen. The interaction leads to a weakened H-H bond and modest electron donation from the dihydrogen to the lowest unoccupied molecular orbital of MgP, a process similarly observed for the interaction between dihydrogen and an adsorbed Au atom.

  14. Rotational Spectromicroscopy: Imaging the Orbital Interaction between Molecular Hydrogen and an Adsorbed Molecule.

    PubMed

    Li, Shaowei; Yuan, Dingwang; Yu, Arthur; Czap, Gregory; Wu, Ruqian; Ho, W

    2015-05-22

    A hydrogen molecule can diffuse freely on the surface and be trapped above an adsorbed molecule within the junction of a scanning tunneling microscope. The trapped dihydrogen exhibits the properties of a free rotor. Here we show that the intermolecular interaction between dihydrogen and Mg-porphyrin (MgP) can be visualized by imaging j=0 to 2 rotational excitation of dihydrogen. The interaction leads to a weakened H-H bond and modest electron donation from the dihydrogen to the lowest unoccupied molecular orbital of MgP, a process similarly observed for the interaction between dihydrogen and an adsorbed Au atom. PMID:26047242

  15. Dynamics of Molecules Adsorbed in Zeolitic Systems: Neutron Scattering and MD Simulation Studies

    SciTech Connect

    Mitra, S.; Sharma, V. K.; Mukhopadhyay, R.

    2011-07-15

    Zeolites represent a class of technologically important materials because of their characteristic properties of molecular sieving and catalysis, which makes them indispensable in the petroleum industries. While the catalytic properties depend upon many factors, a major role is played by the dynamics of hydrocarbon gases. In order to be able to tailor make these materials for use in industry for catalytic and sieving purposes, it is important to understand the dynamical properties of the guest molecules adsorbed in the zeolitic materials. It is of interest to study the effects of size and shape of guest molecules and also the host zeolitic structure, governing the diffusion mechanism of the adsorbed species. Here we report the results of Quasielastic Neutron Scattering (QENS) and classical molecular dynamics (MD) simulation studies of two hydrocarbons namely acetylene and propylene adsorbed in two structurally different zeolites Na-Y and ZSM-5.

  16. 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

  17. Dynamics of water molecules adsorbed by silica and resin SGK-7

    NASA Astrophysics Data System (ADS)

    Lisichkin, Yu. V.; Sakharova, L. A.; Tumanov, A. A.

    2014-01-01

    This paper has presented neutron spectroscopy data on the dynamics of light water molecules adsorbed in the cation exchanger (ion-exchange resin) SGK-7 and on the surface of aerosils (highly dispersed pyrogenic silica) with different levels of hydration. The measurements have been performed on a DIN-2PI spectrometer (Frank Laboratory of Neutron Physics of the Joint Institute for Nuclear Research, Dubna, Russia). The characteristics of the diffusive and vibrational motions of adsorbed water molecules have been determined from the experimental neutron scattering spectra. The data obtained in the quasi-elastic neutron scattering region have been analyzed using a model accounting for the effects of restricted translational and rotational diffusion. The results have demonstrated a significant decrease in the diffusion mobility of adsorbed water molecules as compared to conventional (bulk) water. In particular, the self-diffusion coefficient decreases several times, and the diffusion rate is the lower, the smaller is the thickness of the hydration layer. The dependences of the intensity and half-width of the quasi-elastic scattering peak on the magnitude of the neutron momentum transfer q in the scattering process exhibit a nonmonotonic character. This indicates manifestation of the effects of restricted translational diffusion, rotational diffusion, and jump diffusion. The partial distributions of vibrational frequencies of hydrogen atoms of water molecules adsorbed by the cation exchanger and aerosils have been obtained from the inelastic neutron scattering data.

  18. Candida albicans binds to saliva proteins selectively adsorbed to silicone.

    PubMed

    Holmes, Ann R; van der Wielen, Pauline; Cannon, Richard D; Ruske, Dean; Dawes, Patrick

    2006-10-01

    Explanted voice prostheses obtained from 5 patients at the time of prosthesis replacement were consistently colonized by yeast, in particular Candida albicans. A simple, reproducible, in vitro model of C. albicans adherence to saliva-coated voice prosthesis silicone was developed. Whole saliva promoted adherence of C. albicans to silicone in a dose-dependent manner. Saliva rinses from voice prosthesis patients also promoted binding of C. albicans to silicone in vitro (mean adherence 14.9% +/- 2.8% of input C. albicans cells). This was significantly higher than C. albicans adherence to silicone in the absence of saliva (P < .001) or adherence promoted by saliva rinses from healthy volunteers (P < .005). Polyacrylamide gel electrophoresis analysis and a blot overlay adherence assay revealed that certain salivary proteins were selectively adsorbed to silicone and that C. albicans yeast cells adhered specifically to the adsorbed salivary proteins. PMID:16997116

  19. Confocal Raman microscopy of protein adsorbed in chromatographic particles.

    PubMed

    Xiao, Yuewu; Stone, Thomas; Bell, David; Gillespie, Christopher; Portoles, Marta

    2012-09-01

    Confocal Raman microscopy is a nondestructive analytical technique that combines the chemical information from vibrational spectroscopy with the spatial resolution of confocal microscopy. It was applied, for the first time, to measure conformation and distribution of protein adsorbed in wetted chromatographic particles. Monoclonal antibody was loaded into the Fractogel EMD SO(3) (M) cation exchanger at 2 mS/cm or 10 mS/cm. Amide I and III frequencies in the Raman spectrum of the adsorbed protein suggest that there are no detectable changes of the original β-sheet conformation in the chromatographic particles. Protein depth profile measurements indicate that, when the conductivity is increased from 2 mS/cm to 10 mS/cm, there is a change in mass transport mechanism for protein adsorption, from the shrinking-core model to the homogeneous-diffusion model. In this study, the use of confocal Raman microscopy to measure protein distribution in chromatographic particles fundamentally agrees with previous confocal laser scanning microscopic investigations, but confocal Raman spectroscopy enjoys additional advantages: use of unlabeled protein to eliminate fluorescent labeling, ability for characterization of protein secondary structure, and ability for spectral normalization to provide a nondestructive experimental approach to correct light attenuation effects caused by refractive index (RI) mismatching in semiopaque chromatographic particles. PMID:22803776

  20. 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.

  1. Rod-like cyanophenyl probe molecules nanoconfined to oxide particles: Density of adsorbed surface species

    NASA Astrophysics Data System (ADS)

    Frunza, Stefan; Frunza, Ligia; Ganea, Constantin Paul; Zgura, Irina; Brás, Ana Rita; Schönhals, Andreas

    2016-02-01

    Surface layers have already been observed by broadband dielectric spectroscopy for composite systems formed by adsorption of rod-like cyanophenyl derivates as probe molecules on the surface of oxide particles. In this work, features of the surface layer are reported; samples with different amounts of the probe molecules adsorbed onto oxide (nano) particles were prepared in order to study their interactions with the surface. Thermogravimetric analysis (TGA) was applied to analyze the amount of loaded probe molecules. The density of the surface species ns was introduced and its values were estimated from quantitative Fourier transform infrared spectroscopy (FTIR) coupled with TGA. This parameter allows discriminating the composites into several groups assuming a similar interaction of the probe molecules with the hosts of a given group. An influence factor H is further proposed as the ratio of the number of molecules in the surface layer showing a glassy dynamics and the number of molecules adsorbed tightly on the surface of the support: It was found for aerosil composites and used for calculating the maximum filling degree of partially filled silica MCM-41 composites showing only one dielectric process characteristic for glass-forming liquids and a bulk behavior for higher filling degrees.

  2. Modeling the binding of fulvic acid by goethite: the speciation of adsorbed FA molecules

    NASA Astrophysics Data System (ADS)

    Filius, Jeroen D.; Meeussen, Johannes C. L.; Lumsdon, David G.; Hiemstra, Tjisse; van Riemsdijk, Willem H.

    2003-04-01

    Under natural conditions, the adsorption of ions at the solid-water interface may be strongly influenced by the adsorption of organic matter. In this paper, we describe the adsorption of fulvic acid (FA) by metal(hydr)oxide surfaces with a heterogeneous surface complexation model, the ligand and charge distribution (LCD) model. The model is a self-consistent combination of the nonideal competitive adsorption (NICA) equation and the CD-MUSIC model. The LCD model can describe simultaneously the concentration, pH, and salt dependency of the adsorption with a minimum of only three adjustable parameters. Furthermore, the model predicts the coadsorption of protons accurately for an extended range of conditions. Surface speciation calculations show that almost all hydroxyl groups of the adsorbed FA molecules are involved in outer sphere complexation reactions. The carboxylic groups of the adsorbed FA molecule form inner and outer sphere complexes. Furthermore, part of the carboxylate groups remain noncoordinated and deprotonated.

  3. Adsorbed Oxygen Molecules as a Possible Source of Flux Noise in SQUIDs

    NASA Astrophysics Data System (ADS)

    Shi, Chuntai; Wang, Hui; Hu, Jun; Yu, Clare; Wu, Ruqian

    2015-03-01

    One of the dominant source of flux noise in SQUIDs is flux noise which has been attributed to mysterious fluctuating magnetic spins on the surface. We propose that the spins producing flux noise could be adsorbed O2 molecules that have a magnetic moment of about 2 μB. Using density functional calculations, we studied O2 molecules adsorbed on a sapphire surface. We find that the barrier for spin rotation is small enough to allow almost free spin reorientation due to thermal excitations at low temperatures. Monte Carlo simulations of a 2D XY spin model yields 1 / f noise where f is frequency. This work was supported by 1000 Talent Program of China through Fudan University. Work at UCI was supported by DOE-BES (Grant No. DE-FG02-05ER46237) and the Army Research Office (Grant No. W911NF-10-1-0494).

  4. Stability, structural and electronic properties of benzene molecule adsorbed on free standing Au layer

    NASA Astrophysics Data System (ADS)

    Katoch, Neha; Kapoor, Pooja; Sharma, Munish; Kumar, Ashok; Ahluwalia, P. K.

    2016-05-01

    We report stability and electronic properties of benzene molecule adsorbed on the Au atomic layer within the framework of density function theory (DFT). Horizontal configuration of benzene on the top site of Au monolayer prefers energetically over other studied configurations. On the adsorption of benzene, the ballistic conductance of Au monolayer is found to decrease from 4G0 to 2G0 suggesting its applications for the fabrications of organic sensor devices based on the Au atomic layers.

  5. 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.

  6. Preparation and performance of chitosan encapsulated activated charcoal (ACCB) adsorbents for small molecules.

    PubMed

    Chandy, T; Sharma, C P

    1993-01-01

    A technique is described to encapsulate activated charcoal for haemoperfusion to be used in an artificial liver support. Activated charcoal was encapsulated within chitosan matrix (ACCB) in different concentrations, and was used as the supports for perfusion of a mixture of solutes, having molecular weight ranges from 60 to 69,000; under a flow rate of 8 ml/min. It seems the ACCB may be a good adsorbent system for the removal of toxic uric acid, creatinine, bilirubin, etc., from solutions; while larger molecules such as albumin are adsorbed less. The encapsulated charcoal did not leach out from the matrix during perfusion, and the system may be useful for detoxification of blood. The haemolytic potential of ACCB has been compatible with polystyrene control tubes. However, further studies are needed to determine their behaviour under clinical conditions. PMID:8263676

  7. 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

  8. 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.

  9. Ultra-sensitive fluorescence spectroscopy of isolated surface-adsorbed molecules using an optical nanofiber.

    PubMed

    Stiebeiner, A; Rehband, O; Garcia-Fernandez, R; Rauschenbeutel, A

    2009-11-23

    The strong radial confinement and the pronounced evanescent field of the guided light in optical nanofibers yield favorable conditions for ultra-sensitive surface spectroscopy of molecules deposited on the fiber. Using the guided mode of the nanofiber for both excitation and fluorescence collection, we present spectroscopic measurements on 3,4,9,10-perylenetetracarboxylic dianhydride molecules (PTCDA) at ambient conditions. Surface coverages as small as 1 per thousand of a compact monolayer still give rise to fluorescence spectra with a good signal to noise ratio. Moreover, we analyze and quantify the self-absorption effects due to reabsorption of the emitted fluorescence light by circumjacent surface-adsorbed molecules distributed along the fiber waist. PMID:19997412

  10. 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.

  11. Structure formation in adsorbed overlayers comprising functional cross-shaped molecules: A Monte Carlo study

    NASA Astrophysics Data System (ADS)

    Kasperski, Adam; Nieckarz, Damian; Szabelski, Paweł

    2015-11-01

    Surface confined self-assembly of functional star-shaped organic molecules is a promising method to create nanoporous networks with tailorable structure and functions. In this work we use the Monte Carlo simulation method to demonstrate how the morphology of these supramolecular assemblies can be tuned by manipulating intrinsic parameters of the building blocks and modified by the presence of co-adsorbed metal atoms. To that purpose we study the 2D self-assembly of planar cruciform molecules modeled as collections of interconnected segments, some of which were activated to represent discrete interaction centers. We consider a few exemplary adsorbed systems in which the molecules with different size, aspect ratio and intramolecular distribution of active centers form superstructures stabilized by short-range segment-segment interactions or by metal-segment interactions. These two situations correspond to supramolecular assemblies sustained by, for example, hydrogen bonding and metal-organic ligand coordination, respectively. The simulated results show that proper encoding of intramolecular interactions into the cruciform building bricks allows for directing the self-assembly towards largely diversified structures ranging from nanoclusters to porous grids. The obtained findings can facilitate designing and optimization of molecular networks comprising cross-shaped units including functionalized porphyrins and phthalocyanines and they can be helpful in preliminary selection of these building blocks.

  12. Modeling colloid deposition on a protein layer adsorbed to iron-oxide-coated sand

    NASA Astrophysics Data System (ADS)

    Yang, X.; Flynn, R.; von der Kammer, F.; Hofmann, T.

    2012-11-01

    Our recent study reported that conformation change of granule-associated Bovine Serum Albumin (BSA) may influence the role of the protein controlling colloid deposition in porous media (Flynn et al., 2012). The present study conceptualized the observed phenomena with an ellipsoid morphology model, describing BSA as an ellipsoid taking a side-on or end-on conformation on granular surface, and identified the following processes: (1) at low adsorbed concentrations, BSA exhibited a side-on conformation blocking colloid deposition; (2) at high adsorbed concentrations, BSA adapted to an end-on conformation promoted colloid deposition; and (3) colloid deposition on the BSA layer may progressively generate end-on molecules (sites) by conformation change of side-on BSA, resulting in sustained increasing deposition rates. Generally, the protein layer lowered colloid attenuation by the porous medium, suggesting the overall effect of BSA was inhibitory at the experimental time scale. A mathematical model was developed to interpret the ripening curves. Modeling analysis identified the site generation efficiency of colloid as a control on the ripening rate (declining rate in colloid concentrations), and this efficiency was higher for BSA adsorbed from a more dilute BSA solution.

  13. 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

  14. Thermal and Electronic Fluctuations of Flexible Adsorbed Molecules: Azobenzene on Ag(111)

    NASA Astrophysics Data System (ADS)

    Maurer, Reinhard J.; Liu, Wei; Poltavsky, Igor; Stecher, Thomas; Oberhofer, Harald; Reuter, Karsten; Tkatchenko, Alexandre

    2016-04-01

    We investigate the thermal and electronic collective fluctuations that contribute to the finite-temperature adsorption properties of flexible adsorbates on surfaces on the example of the molecular switch azobenzene C12 H10 N2 on the Ag(111) surface. Using first-principles molecular dynamics simulations, we obtain the free energy of adsorption that accurately accounts for entropic contributions, whereas the inclusion of many-body dispersion interactions accounts for the electronic correlations that govern the adsorbate binding. We find the adsorbate properties to be strongly entropy driven, as can be judged by a kinetic molecular desorption prefactor of 1024 s-1 that largely exceeds previously reported estimates. We relate this effect to sizable fluctuations across structural and electronic observables. A comparison of our calculations to temperature-programed desorption measurements demonstrates that finite-temperature effects play a dominant role for flexible molecules in contact with polarizable surfaces, and that recently developed first-principles methods offer an optimal tool to reveal novel collective behavior in such complex systems.

  15. Thermal and Electronic Fluctuations of Flexible Adsorbed Molecules: Azobenzene on Ag(111).

    PubMed

    Maurer, Reinhard J; Liu, Wei; Poltavsky, Igor; Stecher, Thomas; Oberhofer, Harald; Reuter, Karsten; Tkatchenko, Alexandre

    2016-04-01

    We investigate the thermal and electronic collective fluctuations that contribute to the finite-temperature adsorption properties of flexible adsorbates on surfaces on the example of the molecular switch azobenzene C_{12}H_{10}N_{2} on the Ag(111) surface. Using first-principles molecular dynamics simulations, we obtain the free energy of adsorption that accurately accounts for entropic contributions, whereas the inclusion of many-body dispersion interactions accounts for the electronic correlations that govern the adsorbate binding. We find the adsorbate properties to be strongly entropy driven, as can be judged by a kinetic molecular desorption prefactor of 10^{24}  s^{-1} that largely exceeds previously reported estimates. We relate this effect to sizable fluctuations across structural and electronic observables. A comparison of our calculations to temperature-programed desorption measurements demonstrates that finite-temperature effects play a dominant role for flexible molecules in contact with polarizable surfaces, and that recently developed first-principles methods offer an optimal tool to reveal novel collective behavior in such complex systems. PMID:27104719

  16. 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

  17. Oncogenic protein interfaces: small molecules, big challenges.

    PubMed

    Nero, Tracy L; Morton, Craig J; Holien, Jessica K; Wielens, Jerome; Parker, Michael W

    2014-04-01

    Historically, targeting protein-protein interactions with small molecules was not thought possible because the corresponding interfaces were considered mostly flat and featureless and therefore 'undruggable'. Instead, such interactions were targeted with larger molecules, such as peptides and antibodies. However, the past decade has seen encouraging breakthroughs through the refinement of existing techniques and the development of new ones, together with the identification and exploitation of unexpected aspects of protein-protein interaction surfaces. In this Review, we describe some of the latest techniques to discover modulators of protein-protein interactions and how current drug discovery approaches have been adapted to successfully target these interfaces. PMID:24622521

  18. Mechanism of charge transfer and its impacts on Fermi-level pinning for gas molecules adsorbed on monolayer WS2.

    PubMed

    Zhou, Changjie; Yang, Weihuang; Zhu, Huili

    2015-06-01

    Density functional theory calculations were performed to assess changes in the geometric and electronic structures of monolayer WS2 upon adsorption of various gas molecules (H2, O2, H2O, NH3, NO, NO2, 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 WS2 with a low degree of charge transfer and accept charge from the monolayer, except for NH3, which is a charge donor. Band structure calculations showed that the valence and conduction bands of monolayer WS2 are not significantly altered upon adsorption of H2, H2O, NH3, and CO, whereas the lowest unoccupied molecular orbitals of O2, NO, and NO2 are pinned around the Fermi-level when these molecules are adsorbed on monolayer WS2. 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 WS2. 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. PMID:26049513

  19. Aggregated Gas Molecules: Toxic to Protein?

    PubMed Central

    Zhang, Meng; Zuo, Guanghong; Chen, Jixiu; Gao, Yi; Fang, Haiping

    2013-01-01

    The biological toxicity of high levels of breathing gases has been known for centuries, but the mechanism remains elusive. Earlier work mainly focused on the influences of dispersed gas molecules dissolved in water on biomolecules. However, recent studies confirmed the existence of aggregated gas molecules at the water-solid interface. In this paper, we have investigated the binding preference of aggregated gas molecules on proteins with molecular dynamics simulations, using nitrogen (N2) gas and the Src-homology 3 (SH3) domain as the model system. Aggregated N2 molecules were strongly bound by the active sites of the SH3 domain, which could impair the activity of the protein. In contrast, dispersed N2 molecules did not specifically interact with the SH3 domain. These observations extend our understanding of the possible toxicity of aggregates of gas molecules in the function of proteins. PMID:23588597

  20. 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

  1. Imaging the wave functions of adsorbed molecules using angle-resolved photoemmision data

    NASA Astrophysics Data System (ADS)

    Lüftner, Daniel; Ules, Thomas; Reinisch, Eva Maria; Koller, Georg; Soubatch, Serguei; Tautz, F. Stefan; Ramsey, Michael G.; Puschnig, Peter

    2014-03-01

    The frontier electronic orbitals of molecules are the prime determinants of the respective compounds' chemical, electronic, and optical properties. Although orbitals are very powerful concepts, experimentally only the electron densities and energy levels are directly observable. As has been shown in recent publications, angle-resolved photoemission (ARPES) intensity maps of organic molecular layers are related to the absolute value of the Fourier transform of the initial state molecular orbital. However, the lost phase information impedes the back-transformation of the orbital into real space. Here, we show how molecular orbital images as well as the absent phase information can be retrieved by applying an iterative procedure which takes experimental ARPES maps as input and only assumes spatial confinement of the orbital. The method is demonstrated for several molecular orbitals of two proto-typical pi-conjugated molecules: the LUMO, HOMO, and HOMO-1 of pentacene, and the LUMO and HOMO of PTCDA. The technique is simple and robust and further emphasizes the capabilities of ARPES looking at spatial distributions of wave functions of adsorbed molecules thereby complementing data obtained from scanning probe methods.

  2. 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

  3. Effect of resonance dipole-dipole interaction on spectra of adsorbed SF6 molecules.

    PubMed

    Dobrotvorskaia, Anna N; Kolomiitsova, Tatiana D; Petrov, Sergey N; Shchepkin, Dmitriy N; Smirnov, Konstantin S; Tsyganenko, Alexey A

    2015-09-01

    Adsorption of SF6 on zinc oxide and on silicalite-1 was investigated by a combination of IR spectroscopy with the calculations of spectra by means of a modernized model, developed previously for liquids. Comparison of the experimental spectra and the results of modeling shows that the complex band shapes in spectra of adsorbed molecules with extremely high absorbance are due to the strong resonance dipole-dipole interaction (RDDI) rather that the surface heterogeneity or the presence of specific surface sites. Perfect agreement between calculated and observed spectra was found for ZnO, while some dissimilarity in band intensities for silicalite-1 was attributed to complicated geometry of molecular arrangement in the channels. PMID:25897721

  4. Protein folding at single-molecule resolution

    PubMed Central

    Ferreon, Allan Chris M.; Deniz, Ashok A.

    2011-01-01

    The protein folding reaction carries great significance for cellular function and hence continues to be the research focus of a large interdisciplinary protein science community. Single-molecule methods are providing new and powerful tools for dissecting the mechanisms of this complex process by virtue of their ability to provide views of protein structure and dynamics without associated ensemble averaging. This review briefly introduces common FRET and force methods, and then explores several areas of protein folding where single-molecule experiments have yielded insights. These include exciting new information about folding landscapes, dynamics, intermediates, unfolded ensembles, intrinsically disordered proteins, assisted folding and biomechanical unfolding. Emerging and future work is expected to include advances in single-molecule techniques aimed at such investigations, and increasing work on more complex systems from both the physics and biology standpoints, including folding and dynamics of systems of interacting proteins and of proteins in cells and organisms. PMID:21303706

  5. Quantitative surface studies of protein adsorption by infrared spectroscopy. II. Quantification of adsorbed and bulk proteins

    SciTech Connect

    Fink, D.J.; Hutson, T.B.; Chittur, K.K.; Gendreau, R.M.

    1987-08-15

    Attenuated total reflectance Fourier transform infrared spectra of surface-adsorbed proteins are correlated with concentration measurements determined by /sup 125/I-labeled proteins. This paper demonstrates that linear correlations between the intensity of the major bands of proteins and the quantity of proteins can be obtained for human albumin and immunoglobulin G up to surface concentrations of approximately 0.25 microgram/cm2. A poorer correlation was observed for human fibrinogen. A linear correlation was also observed between the concentration in the bulk solution and the major bands of albumin up to a concentration of 60 mg/ml.

  6. 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).

  7. 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

  8. The analysis of surface-adsorbed organic molecules by alkali-assisted MIES combined with UPS(He I)

    NASA Astrophysics Data System (ADS)

    Günster, J.; Ochs, D.; Dieckhoff, S.; Kempter, V.

    1996-12-01

    Metastable impact electron spectroscopy (MIES) in combination with UPS(He I) is applied to the study of s-triazine and triethoxytriazine molecules adsorbed on Si(100) either alone or in combination with cesium atoms. It is demonstrated that the presence of the Cs atoms facilitates the identification of the adsorbed species considerably. It is concluded that (i) non-dissociative adsorption of the studied organic molecules occurs whereby the basal rings of the molecules lie flat on the silicon surface, in accordance with previous studies by Bu and Lin, (ii) the binding of the molecules to the surface is mainly via the lone pair orbitals of the nitrogen in the ring, and (iii) s-triazine reacts strongly with oxygen which bonds to the carbon atoms of the triazine ring.

  9. 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.

  10. 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.

  11. Plasma protein adsorbed biomedical polymers: activation of human monocytes and induction of interleukin 1.

    PubMed

    Bonfield, T L; Colton, E; Anderson, J M

    1989-06-01

    These studies involved the evaluation of human monocyte/macrophage activation by biomedical polymers coated with human blood proteins. The biomedical polymers were polyethylene, polydimethylsiloxane, woven Dacron fabric, expanded polytetrafluoroethylene, Biomer, and tissue culture treated polystyrene as the control. They were adsorbed with human blood proteins: albumin, fibrinogen, fibronectin, hemoglobin, and gamma globulin. The protein adsorbed polymers were evaluated for their potential to activate the monocyte/macrophage cellular population in vitro as assessed by the induction of the monocyte/macrophage inflammatory mediator, Interleukin 1 (IL1). Suppression of IL1 was observed when protein adsorbed polymers were compared to the appropriate protein adsorbed control. Protein adsorbed polymers, when compared to polymers without protein adsorption, stimulated IL1 production. The data presented in this manuscript show the level of induction and secretion of IL1 was dependent on the biomedical polymer and the protein adsorbed, as well as the requirement of lipopolysaccharide. These results show differential interactions occur between the proteins, monocytes/macrophages, and biomedical polymers which alter activation and induction of IL1. PMID:2786877

  12. 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.

  13. Copper iodide staining and determination of proteins adsorbed to microtiter plates.

    PubMed

    Root, D D; Reisler, E

    1990-04-01

    Copper iodide staining and determination of proteins adsorbed to polystyrene microtiter plates are described. The minimum amount of copper iodide-stained protein detected in densitometric measurements is approximately 20 pg/mm2. Enzyme immunoassay readers may also be used for the determination of copper iodide-stained proteins, but are less sensitive than densitometers. The densitometric readings of copper iodide-stained proteins vary linearly with the amount of protein present as verified by enzymatic and radioactive probes. Staining is complete in 2-3 min and may be removed by a 30-min treatment with EDTA without loss of adsorbed protein or immunoreactivity. The exact amount of protein adsorbed to microtiter plate wells can be measured by using protein bound and stained on nitrocellulose as a calibration curve. Copper iodide staining is a rapid, convenient, and inexpensive alternative to radioactive measurements of similar parameters. PMID:1694063

  14. Barriers to intramolecular rotation determined from the temperature dependence of the Henry constant in the region of adsorbed molecule rigidity failure

    NASA Astrophysics Data System (ADS)

    Dolgonosov, A. M.; Prudkovskii, A. G.

    2008-05-01

    A distribution for the rigid and nonrigid adsorbed molecule forms was found. Adsorbed molecule rigidity failure was shown to be accompanied by a weak nonlinear effect, which manifested itself as a temperature dependence of the Henry constant. A method for the determination of the barrier to intramolecular rotation from the temperature dependence of the molecule adsorption constant was suggested. Barriers to rotation about the C-C and C-O bonds were determined for several molecules.

  15. 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.

  16. 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.

  17. Identification of vitronectin as a major plasma protein adsorbed on polymer surfaces of different copolymer composition.

    PubMed

    Bale, M D; Wohlfahrt, L A; Mosher, D F; Tomasini, B; Sutton, R C

    1989-12-01

    The arrays of proteins adsorbed from plasma onto a series of polystyrene copolymeric latexes were analyzed by enzyme-linked immunosorbent assay (ELISA) of washed beads and immunoblotting of proteins desorbed from the beads and separated by polyacrylamide gel electrophoresis (PAGE). Beads were prepared by continuous emulsion polymerization in the absence of surfactant. Coomassie brilliant blue staining of gel electropherograms of desorbed proteins indicated that the presence of small amounts of comonomers (1 to 10 mole %) significantly influenced the composition of the adsorbed protein layer. Immunoblotting revealed that fibrinogen, fibronectin, and vitronectin were adsorbed by all surfaces investigated. C3 and Clq adsorption varied significantly with copolymer composition. The ELISAs revealed that although the concentrations of vitronectin and fibronectin in plasma are similar, the extent of vitronectin adsorption from 70% to 85% plasma was greater by two orders of magnitude than fibronectin adsorption. Vitronectin adsorbed on carboxylic acid-containing copolymers reacted more strongly with a conformationally sensitive antivitronectin monoclonal antibody (MoAb) than vitronectin adsorbed to polystyrene and was more susceptible to cleavage by plasma proteases(s). The results show that vitronectin is a major protein adsorbed from concentrated plasma and that small changes in the chemical composition of a copolymer profoundly affects the extent and nature of protein adsorption from complex mixtures such as plasma. PMID:2479428

  18. 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

  19. 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. PMID:24450578

  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. 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.

  2. Charge transfer interactions of a Ru(II) dye complex and related ligand molecules adsorbed on Au(111)

    SciTech Connect

    Britton, Andrew J.; Weston, Matthew; O'Shea, James N.; Taylor, J. Ben; Rienzo, Anna; Mayor, Louise C.

    2011-10-28

    The interaction of the dye molecule, N3 (cis-bis(isothiocyanato)bis(2,2{sup '}-bipyridyl-4,4{sup '}-dicarboxylato) -ruthenium(II)), and related ligand molecules with a Au(111) surface has been studied using synchrotron radiation-based electron spectroscopy. Resonant photoemission spectroscopy (RPES) and autoionization of the adsorbed molecules have been used to probe the coupling between the molecules and the substrate. Evidence of charge transfer from the states near the Fermi level of the gold substrate into the lowest unoccupied molecular orbital (LUMO) of the molecules is found in the monolayer RPES spectra of both isonicotinic acid and bi-isonicotinic acid (a ligand of N3), but not for the N3 molecule itself. Calibrated x-ray absorption spectroscopy and valence band spectra of the monolayers reveals that the LUMO crosses the Fermi level of the surface in all cases, showing that charge transfer is energetically possible both from and to the molecule. A core-hole clock analysis of the resonant photoemission reveals a charge transfer time of around 4 fs from the LUMO of the N3 dye molecule to the surface. The lack of charge transfer in the opposite direction is understood in terms of the lack of spatial overlap between the {pi}*-orbitals in the aromatic rings of the bi-isonicotinic acid ligands of N3 and the gold surface.

  3. 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

  4. 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.

  5. Kinetic silver staining and quantification of proteins adsorbed to microtiter plates.

    PubMed

    Root, D D; Wang, K

    1993-03-01

    A silver stain was used to detect and quantitate proteins adsorbed to microtiter plate wells. The kinetics of the development of the silver stain were analyzed with an automated microtiter plate reader. The lag time for stain development was found to be a consistent indicator of the amount of protein adsorbed to a microtiter plate well. Protein which was not preadsorbed to the microtiter plate was not effectively stained by silver. Complete adsorption of protein applied to the microtiter plate was possible by drying small amounts of protein in very dilute buffers. Variations in sensitivity for different proteins were less than 30% for the panel of proteins examined. Determinations from kinetic silver staining agreed with those from copper staining for bovine albumin adsorbed to microtiter plates. The precision of kinetic silver staining assay was optimal in the range of 40 to 200 ng per microtiter plate well. In this range, the standard deviations averaged less than 5%. Even smaller amounts of protein can be detected and interpolated down to approximately 10 ng per well. The kinetic silver staining method can be used on standard microtiter plate readers without special filters and is readily adaptable to automated systems. PMID:8470810

  6. 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.

  7. 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.

  8. 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

  9. 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.

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

    PubMed

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

    2016-06-28

    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. PMID:27369529

  11. How hottest geometries and adsorptive parameters influence the SER(R)S spectra of Methylene Blue molecules adsorbed on nanocolloidal gold particles of varied sizes?

    PubMed

    Roy, Sannak Dutta; Ghosh, Manash; Chowdhury, Joydeep

    2015-12-01

    The SER(R)S spectra of Methylene Blue (MB) molecule adsorbed on gold nanocolloidal particles (AuNCs) have been investigated. The adsorptive parameters of the molecule adsorbed on AuNCs have been evaluated with the aid of Fluorescence Spectroscopy study. Fluorescence spectroscopic studies have been further applied to understand the concentration of probe molecule actually adsorbed on AuNC surfaces. Gigantic enhancements ∼10(6)-10(16) orders of magnitude have been recorded for the enhanced Raman bands in the SER(R)S spectra. Three-dimensional Finite Difference Time Domain (3D-FDTD) simulations studies have been carried out to predict the distributions of electric fields around the possible nanoaggregated hot geometries considered to be responsible for the huge enhancements of SER(R)S bands of the MB molecule. PMID:26172467

  12. 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

  13. 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.

  14. Effects of non-local exchange on core level shifts for gas-phase and adsorbed molecules

    SciTech Connect

    Van den Bossche, M.; Grönbeck, H.; Martin, N. M.; Gustafson, J.; Lundgren, E.; Hakanoglu, C.; Weaver, J. F.

    2014-07-21

    Density functional theory calculations are often used to interpret experimental shifts in core level binding energies. Calculations based on gradient-corrected (GC) exchange-correlation functionals are known to reproduce measured core level shifts (CLS) of isolated molecules and metal surfaces with reasonable accuracy. In the present study, we discuss a series of examples where the shifts calculated within a GC-functional significantly deviate from the experimental values, namely the CLS of C 1s in ethyl trifluoroacetate, Pd 3d in PdO and the O 1s shift for CO adsorbed on PdO(101). The deviations are traced to effects of the electronic self-interaction error with GC-functionals and substantially better agreements between calculated and measured CLS are obtained when a fraction of exact exchange is used in the exchange-correlation functional.

  15. Bringing single-molecule spectroscopy to macromolecular protein complexes

    PubMed Central

    Joo, Chirlmin; Fareh, Mohamed; Kim, V. Narry

    2013-01-01

    Single-molecule fluorescence spectroscopy offers real-time, nanometer-resolution information. Over the past two decades, this emerging single-molecule technique has been rapidly adopted to investigate the structural dynamics and biological functions of proteins. Despite this remarkable achievement, single-molecule fluorescence techniques must be extended to macromolecular protein complexes that are physiologically more relevant for functional studies. In this review, we present recent major breakthroughs for investigating protein complexes within cell extracts using single-molecule fluorescence. We outline the challenges, future prospects and potential applications of these new single-molecule fluorescence techniques in biological and clinical research. PMID:23200186

  16. Single-molecule protein arrays enabled by scanning probe block copolymer lithography.

    PubMed

    Chai, Jinan; Wong, Lu Shin; Giam, Louise; Mirkin, Chad A

    2011-12-01

    The ability to control the placement of individual protein molecules on surfaces could enable advances in a wide range of areas, from the development of nanoscale biomolecular devices to fundamental studies in cell biology. Such control, however, remains a challenge in nanobiotechnology due to the limitations of current lithographic techniques. Herein we report an approach that combines scanning probe block copolymer lithography with site-selective immobilization strategies to create arrays of proteins down to the single-molecule level with arbitrary pattern control. Scanning probe block copolymer lithography was used to synthesize individual sub-10-nm single crystal gold nanoparticles that can act as scaffolds for the adsorption of functionalized alkylthiol monolayers, which facilitate the immobilization of specific proteins. The number of protein molecules that adsorb onto the nanoparticles is dependent upon particle size; when the particle size approaches the dimensions of a protein molecule, each particle can support a single protein. This was demonstrated with both gold nanoparticle and quantum dot labeling coupled with transmission electron microscopy imaging experiments. The immobilized proteins remain bioactive, as evidenced by enzymatic assays and antigen-antibody binding experiments. Importantly, this approach to generate single-biomolecule arrays is, in principle, applicable to many parallelized cantilever and cantilever-free scanning probe molecular printing methods. PMID:22106270

  17. 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.}

  18. 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

  19. Adsorbed plasma proteins modulate the effects of single-walled carbon nanotubes on neutrophils in blood.

    PubMed

    Vlasova, Irina I; Mikhalchik, Elena V; Barinov, Nikolay A; Kostevich, Valeria A; Smolina, Natalia V; Klinov, Dmitry V; Sokolov, Alexey V

    2016-08-01

    Proteins adsorbed on a surface may affect the interaction of this surface with cells. Here, we studied the binding of human serum albumin (HSA), fibrinogen (FBG) and immunoglobulin G (IgG) to PEGylated single-walled carbon nanotubes (PEG-SWCNTs) and evaluated the impact of PEG-SWCNT treated by these proteins on neutrophils in whole blood samples. Measurements of adsorption parameters revealed tight binding of proteins to PEG-SWCNTs. AFM was employed to directly observe protein binding to sidewalls of PEG-SWCNTs. Fluorescein-labeled IgG was used to ascertain the stability of PEG-SWCNT-IgG complexes in plasma. In blood samples, all plasma proteins mitigated damage of neutrophils observed just after blood exposure to PEG-SWCNTs, while only treatment of PEG-SWCNTs with IgG resulted in dose- and time-dependent enhancement of CNT-induced neutrophil activation and in potentiation of oxidative stress. Our study demonstrates the ability of adsorbed plasma proteins to influence neutrophil response caused by PEG-SWCNTs in whole blood. PMID:27015767

  20. Prediction of the orientations of adsorbed protein using an empirical energy function with implicit solvation.

    PubMed

    Sun, Yu; Welsh, William J; Latour, Robert A

    2005-06-01

    When simulating protein adsorption behavior, decisions must first be made regarding how the protein should be oriented on the surface. To address this problem, we have developed a molecular simulation program that combines an empirical adsorption free energy function with an efficient configurational search method to calculate orientation-dependent adsorption free energies between proteins and functionalized surfaces. The configuration space is searched systematically using a quaternion rotation technique, and the adsorption free energy is evaluated using an empirical energy function with an efficient grid-based calculational method. In this paper, the developed method is applied to analyze the preferred orientations of a model protein, lysozyme, on various functionalized alkanethiol self-assembled monolayer (SAM) surfaces by the generation of contour graphs that relate adsorption free energy to adsorbed orientation, and the results are compared with experimental observations. As anticipated, the adsorbed orientation of lysozyme is predicted to be dependent on the discrete organization of the functional groups presented by the surface. Lysozyme, which is a positively charged protein, is predicted to adsorb on its 'side' on both hydrophobic and negatively charged surfaces. On surfaces with discrete positively charged sites, attractive interaction energies can also be obtained due to the presence of discrete local negative charges present on the lysozyme surface. In this case, 'end-on' orientations are preferred. Additionally, SAM surface models with mixed functionality suggest that the interactions between lysozyme and surfaces could be greatly enhanced if individual surface functional groups are able to access the catalytic cleft region of lysozyme, similar to ligand-receptor interactions. The contour graphs generated by this method can be used to identify low-energy orientations that can then be used as starting points for further simulations to investigate

  1. 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.

  2. 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.

  3. 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.

  4. Isolation of calcium-binding proteins on selective adsorbents. Application to purification of bovine calmodulin.

    PubMed

    Chaga, G S; Ersson, B; Porath, J O

    1996-05-01

    We report the fractionation of calcium-binding proteins using immobilized metal ion affinity chromatography (IMAC) with hard metal ions. Various hard metal ions (Mn2+, La3+, Nd3+, Eu(3 were immobilized on cross-linked agarose substituted with Tris(carboxymethyl)ethylenediamine (TED) and used as an adsorbent. After systematic studies, europium was selected for further work on the fractionation of calcium-binding proteins. It was found that the presence of Ca2+ in the sample and the solvent strongly promoted the adsorption and selectivity. Selective elution was accomplished in stepwise mode by the addition of calcium chelators such as malonate, citrate and phosphate. Calmodulin of high purity was isolated from a crude extract. Similar behavior of other calcium-binding proteins indicates that the reported chromatographic procedure can be generally applied to such proteins. PMID:8653201

  5. 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. PMID:26948763

  6. 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.

  7. 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

  8. 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

  9. Photon-exposure-dependent photon-stimulated desorption for obtaining photolysis cross section of molecules adsorbed on surface by monochromatic soft x-ray photons.

    PubMed

    Chou, L-C; Jang, C-Y; Wu, Y-H; Tsai, W-C; Wang, S-K; Chen, J; Chang, S-C; Liu, C-C; Shai, Y; Wen, C-R

    2008-12-01

    Photon-exposure-dependent positive- and negative-ion photon-stimulated desorption (PSD) was proposed to study the photoreactions and obtain the photolysis cross sections of molecules adsorbed on a single-crystal surface by monochromatic soft x-ray photons with energy near the core level of adsorbate. The changes in the F(+) and F(-) PSD ion yields were measured from CF(3)Cl molecules adsorbed on Si(111)-7x7 at 30 K (CF(3)Cl dose=0.3x10(15) molecules/cm(2), approximately 0.75 monolayer) during irradiation of monochromatic soft x-ray photons near the F(1s) edge. The PSD ion yield data show the following characteristics: (a) The dissociation of adsorbed CF(3)Cl molecules is due to a combination of direct photodissociation via excitation of F(1s) core level and substrate-mediated dissociation [dissociative attachment and dipolar dissociation induced by the photoelectrons emitting from the silicon substrate]. (b) the F(+) ion desorption is associated with the bond breaking of the surface CF(3)Cl, CF(2)Cl, CFCl, and SiF species. (c) the F(-) yield is mainly due to DA and DD of the adsorbed CF(3)Cl molecules. (d) The surface SiF is formed by reaction of the surface Si atom with the neutral fluorine atom, F(+), or F(-) ion produced by scission of C-F bond of CF(3)Cl, CF(2)Cl, or CFCl species. A kinetic model was proposed for the explanation of the photolysis of this submonolayer CF(3)Cl-covered surface. Based on this model and the variation rates of the F(+)F(-) signals during fixed-energy monochromatic photon bombardment at 690.2 and 692.6 eV [near the F(1s) edge], the photolysis cross section was deduced as a function of energy. PMID:19063541

  10. 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. PMID:21725631

  11. Distinct Conformation of ATP Molecule in Solution and on Protein

    PubMed Central

    Kobayashi, Eri; Yura, Kei; Nagai, Yoshinori

    2013-01-01

    Adenosine triphosphate (ATP) is a versatile molecule used mainly for energy and a phosphate source. The hydrolysis of γ phosphate initiates the reactions and these reactions almost always start when ATP binds to protein. Therefore, there should be a mechanism to prevent spontaneous hydrolysis reaction and a mechanism to lead ATP to a pure energy source or to a phosphate source. To address these questions, we extensively analyzed the effect of protein to ATP conformation based on the sampling of the ATP solution conformations obtained from molecular dynamics simulation and the sampling of ATP structures bound to protein found in a protein structure database. The comparison revealed mainly the following three points; 1) The ribose ring in ATP molecule, which puckers in many ways in solution, tends to assume either C2′ exo or C2′ endo when it binds to protein. 2) The adenine ring in ATP molecule, which takes open-book motion with the two ring structures, has two distinct structures when ATP binds to protein. 3) The glycosyl-bond and the bond between phosphate and the ribose have unique torsion angles, when ATP binds to protein. The combination of torsion angles found in protein-bound forms is under-represented in ATP molecule in water. These findings suggest that ATP-binding protein exerts forces on ATP molecule to assume a conformation that is rarely found in solution, and that this conformation change should be a trigger for the reactions on ATP molecule. PMID:27493535

  12. Optical anisotropy of flagellin layers: in situ and label-free measurement of adsorbed protein orientation using OWLS.

    PubMed

    Kovacs, Noemi; Patko, Daniel; Orgovan, Norbert; Kurunczi, Sandor; Ramsden, Jeremy J; Vonderviszt, Ferenc; Horvath, Robert

    2013-06-01

    The surface adsorption of the protein flagellin was followed in situ using optical waveguide lightmode spectroscopy (OWLS). Flagellin did not show significant adsorption on a hydrophilic waveguide, but very rapidly formed a dense monolayer on a hydrophobic (silanized) surface. The homogeneous and isotropic optical layer model, which has hitherto been generally applied in OWLS data interpretation for adsorbed protein films, failed to characterize the flagellin layer, but it could be successfully modeled as an uniaxial thin film. This anisotropic modeling revealed a significant positive birefringence in the layer, suggesting oriented protein adsorption. The adsorbed flagellin orientation was further evidenced by monitoring the surface adsorption of truncated flagellin variants, in which the terminal protein regions or the central (D3) domain were removed. Without the terminal regions the protein adsorption was much slower and the resulting films were significantly less birefringent, implying that intact flagellin adsorbs on the hydrophobic surface via its terminal regions. PMID:23631669

  13. Study of the conformational change of adsorbed proteins on biomaterial surfaces using hydrogen-deuterium exchange with mass spectroscopy.

    PubMed

    Kim, Jinku

    2016-05-01

    There is no doubt that protein adsorption plays a crucial role in determining biocompatibility of biomaterials. Despite the information of the identity and composition of blood plasma/serum proteins adsorbed on surfaces of biomaterials to understand which proteins are involved in blood/biomaterial interactions, it still does not provide information about the conformations and orientations of adsorbed protein, which are very important in determining biological responses to biomaterials. Therefore, our laboratory has developed an experimental technology to probe protein conformations on materials that is applicable to mixtures of proteins. Herein, the new application of hydrogen/deuterium (H/D) exchange combined with mass spectrometry was applied to determine conformational changes of adsorbed proteins at biomaterial surfaces. The results suggest that there may be a significant conformational change in adsorbed proteins at 'low' bulk concentrations that leads to a large change in the kinetics of H/D exchange as compared to 'high' bulk concentrations. This technique may eventually be useful for the study of the kinetics of protein conformational changes. PMID:26896658

  14. 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. PMID:26905881

  15. Orientation Difference of Chemically Immobilized and Physically Adsorbed Biological Molecules on Polymers Detected at the Solid/Liquid Interfaces in Situ

    PubMed Central

    Ye, Shuji; Nguyen, Khoi Tan; Boughton, Andrew P.; Mello, Charlene M.; Chen, Zhan

    2009-01-01

    A surface sensitive second order nonlinear optical technique, sum frequency generation (SFG) vibrational spectroscopy, was applied to study peptide orientation on polymer surfaces, supplemented by a linear vibrational spectroscopy, attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). Using the antimicrobial peptide Cecropin P1 as a model system, we have quantitatively demonstrated that chemically immobilized peptides on polymers adopt a more ordered orientation than less tightly bound physically adsorbed peptides. These differences were also observed in different chemical environments, e.g., air versus water. Although numerous studies have reported a direct correlation between the choice of immobilization method and the performance of an attached biological molecule, the lack of direct biomolecular structure and orientation data has made it difficult to elucidate the relationship between structure, orientation and function at a surface. In this work, we directly studied the effect of chemical immobilization method on biomolecular orientation/ordering, an important step for future studies of biomolecular activity. The methods for orientation analysis described within are also of relevance to understanding biosensors, biocompatibility, marine-antifouling, membrane protein functions, and antimicrobial peptide activities. PMID:19961170

  16. Computational evaluation of protein – small molecule binding

    PubMed Central

    Guvench, Olgun; MacKerell, Alexander D.

    2009-01-01

    Determining protein – small molecule binding affinity is a key component of present-day rational drug discovery. To circumvent the time, labor, and materials costs associated with experimental protein – small molecule binding assays, a variety of structure-based computational methods have been developed for determining protein – small molecule binding affinities. These methods can be placed in one of two classes: accurate but slow (Class 1), and fast but approximate (Class 2). Class 1 methods, which explicitly take into account protein flexibility and include an atomic-level description of solvation, are capable of quantitatively reproducing experimental protein – small molecule absolute binding free energies. However, Class 1 computational requirements make screening thousands to millions of small molecules against a protein, as required for rational drug design, infeasible for the foreseeable future. Class 2 methods, on the other hand, are sufficiently fast to perform such inhibitor screening, yet they suffer from limited descriptions of protein flexibility and solvation, which in turn limit their ability to select and rank-order small molecules by computed binding affinities. This review presents an overview of Class 1 and Class 2 methods, avenues of research in Class 2 methods aimed at bringing them closer to Class 1 accuracy, and intermediate approaches that incorporate features of both Class 1 and Class 2 methods. PMID:19162472

  17. 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. PMID:21244507

  18. Dry powder pulmonary delivery of cationic PGA-co-PDL nanoparticles with surface adsorbed model protein.

    PubMed

    Kunda, Nitesh K; Alfagih, Iman M; Dennison, Sarah R; Somavarapu, Satyanarayana; Merchant, Zahra; Hutcheon, Gillian A; Saleem, Imran Y

    2015-08-15

    Pulmonary delivery of macromolecules has been the focus of attention as an alternate route of delivery with benefits such as; large surface area, thin alveolar epithelium, rapid absorption and extensive vasculature. In this study, a model protein, bovine serum albumin (BSA) was adsorbed onto cationic PGA-co-PDL polymeric nanoparticles (NPs) prepared by a single emulsion solvent evaporation method using a cationic surfactant didodecyldimethylammonium bromide (DMAB) at 2% w/w (particle size: 128.64±06.01 nm and zeta-potential: +42.32±02.70 mV). The optimum cationic NPs were then surface adsorbed with BSA, NP:BSA (100:4) ratio yielded 10.01±1.19 μg of BSA per mg of NPs. The BSA adsorbed NPs (5 mg/ml) were then spray-dried in an aqueous suspension of L-leucine (7.5 mg/ml, corresponding to a ratio of 1:1.5/NP:L-leu) using a Büchi-290 mini-spray dryer to produce nanocomposite microparticles (NCMPs) containing cationic NPs. The aerosol properties showed a fine particle fraction (FPF, dae<4.46 μm) of 70.67±4.07% and mass median aerodynamic diameter (MMAD) of 2.80±0.21 μm suggesting a deposition in the respiratory bronchiolar region of the lungs.The cell viability was 75.76±03.55% (A549 cell line) at 156.25 μg/ml concentration after 24 h exposure. SDS-PAGE and circular dichroism (CD) confirmed that the primary and secondary structure of the released BSA was maintained. Moreover, the released BSA showed 78.76±1.54% relative esterolytic activity compared to standard BSA. PMID:26169146

  19. 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

  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. 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

  2. Efficient nanoplasmonic antennas for fabricating single protein molecule detector

    NASA Astrophysics Data System (ADS)

    Chatterjee, Sharmistha; Dantham, Venkata Ramanaiah; Hussain, Sahid

    2015-06-01

    Real time (label-free) detection and sizing of single protein molecule at its natural state is "holy grail" in biosensing field. This non-destructive technique is useful for predicting the dangerous diseases at very early-stage. Herein, we report the synthesis and characterization of efficient nanoplasmonic antennas, which could be useful to fabricate an ultrasensitive nanoplasmonic-whispering gallery mode hybrid microresonator for the real time detection and sizing of single protein molecule. This hybrid microresonator could be easily converted as an ultrasensitive single molecule biosensor by anchoring suitable anti-bodies on the surface of the plasmonic nanoantenna.

  3. 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

  4. Single molecule techniques for the study of membrane proteins.

    PubMed

    García-Sáez, Ana J; Schwille, Petra

    2007-08-01

    Single molecule techniques promise novel information about the properties and behavior of individual particles, thus enabling access to molecular heterogeneities in biological systems. Their recent developments to accommodate membrane studies have significantly deepened the understanding of membrane proteins. In this short review, we will describe the basics of the three most common single-molecule techniques used on membrane proteins: fluorescence correlation spectroscopy, single particle tracking, and atomic force microscopy. We will discuss the most relevant findings made during the recent years and their contribution to the membrane protein field. PMID:17497147

  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. Probing small molecule microarrays with tagged proteins in cell lysates

    PubMed Central

    Pop, Marius S.; Wassaf, Dina; Koehler, Angela N.

    2014-01-01

    The technique of small-molecule microarray (SMM) screening is based on the ability of small molecules to bind to various soluble proteins. This type of interaction is easily detected by the presence of a fluorescence signal produced by labeled antibodies that specifically recognize a unique sequence (tag) present on the target protein. The fluorescent signal intensity values are determined based on signal-to-noise ratios (SNR). SMM screening is a high throughput, unbiased method that can rapidly identify novel direct ligands for various protein targets. This binding-based assay format is generally applicable to most proteins, but it is especially useful for protein targets that do not possess an enzymatic activity. SMMs enable screening a protein in a purified form or in the context of a cellular lysate, likely providing a more physiologically relevant screening environment. PMID:25445177

  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. Photon-exposure-dependent photon-stimulated desorption for obtaining photolysis cross section of molecules adsorbed on surface by monochromatic soft x-ray photons

    SciTech Connect

    Chou, L.-C.; Jang, C.-Y.; Wu, Y.-H.; Tsai, W.-C.; Wang, S.-K.; Chen, J.; Chang, S.-C.; Liu, C.-C.; Shai, Y.; Wen, C.-R.

    2008-12-07

    Photon-exposure-dependent positive- and negative-ion photon-stimulated desorption (PSD) was proposed to study the photoreactions and obtain the photolysis cross sections of molecules adsorbed on a single-crystal surface by monochromatic soft x-ray photons with energy near the core level of adsorbate. The changes in the F{sup +} and F{sup -} PSD ion yields were measured from CF{sub 3}Cl molecules adsorbed on Si(111)-7x7 at 30 K (CF{sub 3}Cl dose=0.3x10{sup 15} molecules/cm{sup 2}, {approx}0.75 monolayer) during irradiation of monochromatic soft x-ray photons near the F(1s) edge. The PSD ion yield data show the following characteristics: (a) The dissociation of adsorbed CF{sub 3}Cl molecules is due to a combination of direct photodissociation via excitation of F(1s) core level and substrate-mediated dissociation [dissociative attachment and dipolar dissociation induced by the photoelectrons emitting from the silicon substrate]. (b) the F{sup +} ion desorption is associated with the bond breaking of the surface CF{sub 3}Cl, CF{sub 2}Cl, CFCl, and SiF species. (c) the F{sup -} yield is mainly due to DA and DD of the adsorbed CF{sub 3}Cl molecules. (d) The surface SiF is formed by reaction of the surface Si atom with the neutral fluorine atom, F{sup +}, or F{sup -} ion produced by scission of C-F bond of CF{sub 3}Cl, CF{sub 2}Cl, or CFCl species. A kinetic model was proposed for the explanation of the photolysis of this submonolayer CF{sub 3}Cl-covered surface. Based on this model and the variation rates of the F{sup +}/F{sup -} signals during fixed-energy monochromatic photon bombardment at 690.2 and 692.6 eV[near the F(1s) edge], the photolysis cross section was deduced as a function of energy.

  10. Continuous-time core-level photon-stimulated desorption spectroscopy for monitoring soft x-ray-induced reactions of molecules adsorbed on a single-crystal surface

    SciTech Connect

    Chou, L.-C.; Wen, C.-R.

    2006-05-15

    Continuous-time core-level photon-stimulated desorption (PSD) spectroscopy was proposed for monitoring the soft x-ray-induced reactions of molecules adsorbed on a single-crystal surface. Monochromatic synchrotron radiation was used as a soft x-ray light source in the photon-induced reactions of CF{sub 3}Cl adsorbed on a Si(111)-7x7 surface at 30 K and also as a probe for studying the produced fluorination states of the bonding surface Si atom in the positive-ion PSD spectroscopy. The F{sup +} PSD spectrum was obtained by monitoring the F{sup +} signal as a function of incident photon energy near the Si(2p) edge (98-110 eV). Sequential F{sup +} PSD spectra were measured as a function of photon exposure at four adsorbate coverages (the first dose=0.3x10{sup 15} molecules/cm{sup 2}, the second dose=0.8x10{sup 15} molecules/cm{sup 2}, the third dose=2.2x10{sup 15} molecules/cm{sup 2}, and the fourth dose=3.2x10{sup 15} molecules/cm{sup 2}). For the first and second CF{sub 3}Cl-dosed surfaces, the sequential F{sup +} PSD spectra show the variation of their shapes with photon exposure and indicate the formation of surface SiF species. The sequential F{sup +} PSD spectra for the third and fourth CF{sub 3}Cl-dosed surfaces also show the variation of their shapes with photon exposure and depict the production of surface SiF and SiF{sub 3} species.

  11. 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.

  12. Surface Passivation for Single-molecule Protein Studies

    PubMed Central

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

    2014-01-01

    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. PMID:24797261

  13. 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

  14. Single Molecule Approaches in RNA-Protein Interactions.

    PubMed

    Serebrov, Victor; Moore, Melissa J

    2016-01-01

    RNA-protein interactions govern every aspect of RNA metabolism, and aberrant RNA-binding proteins are the cause of hundreds of genetic diseases. Quantitative measurements of these interactions are necessary in order to understand mechanisms leading to diseases and to develop efficient therapies. Existing methods of RNA-protein interactome capture can afford a comprehensive snapshot of RNA-protein interaction networks but lack the ability to characterize the dynamics of these interactions. As all ensemble methods, their resolution is also limited by statistical averaging. Here we discuss recent advances in single molecule techniques that have the potential to tackle these challenges. We also provide a thorough overview of single molecule colocalization microscopy and the essential protein and RNA tagging and detection techniques. PMID:27256383

  15. Multi-technique Characterization of Adsorbed Peptide and Protein Orientation: LK310 and Protein G B1

    SciTech Connect

    Baio, J.; Weidner, T; Samuel, N; McCrea, K; Baugh, L; Stayton, P; Castner, D

    2010-01-01

    The ability to orient biologically active proteins on surfaces is a major challenge in the design, construction, and successful deployment of many medical technologies. As methods to orient biomolecules are developed, it is also essential to develop techniques that can accurately determine the orientation and structure of these materials. In this study, two model protein and peptide systems are presented to highlight the strengths of three surface analysis techniques for characterizing protein films: time-of-flight secondary-ion mass spectrometry (ToF-SIMS), sum-frequency generation (SFG) vibrational spectroscopy, and near-edge x-ray absorption fine structure (NEXAFS) spectroscopy. First, the orientation of Protein G B1, a rigid 6 kDa domain covalently attached to a maleimide-functionalized self-assembled monolayer, was examined using ToF-SIMS. Although the thickness of the Protein G layer was similar to the ToF-SIMS sampling depth, orientation of Protein G was successfully determined by analyzing the C{sub 2}H{sub 5}S{sup +} intensity, a secondary-ion derived from a methionine residue located at one end of the protein. Next, the secondary structure of a 13-mer leucine-lysine peptide (LK{sub 310}) adsorbed onto hydrophilic quartz and hydrophobic fluorocarbon surfaces was examined. SFG spectra indicated that the peptide's lysine side chains were ordered on the quartz surface, while the peptide's leucine side chains were ordered on the fluorocarbon surface. NEXAFS results provided complementary information about the structure of the LK{sub 310} film and the orientations of amide bonds within the LK{sub 310} peptide.

  16. Effects of adsorbed proteins, an antifouling agent and long-duration DC voltage pulses on the impedance of silicon-based neural microelectrodes.

    PubMed

    Sommakia, Salah; Rickus, Jenna L; Otto, Kevin J

    2009-01-01

    The successful use of implantable neural microelectrodes as neuroprosthetic devices depends on the mitigation of the reactive tissue response of the brain. One of the factors affecting the ultimate severity of the reactive tissue response and the in vivo electrical properties of the microelectrodes is the initial adsorption of proteins onto the surface of the implanted microelectrodes. In this study we quantify the increase in microelectrode impedance magnitude at physiological frequencies following electrode immersion in a 10% bovine serum albumin (BSA) solution. We also demonstrate the efficacy of a common antifouling molecule, poly(ethylene glycol) (PEG), in preventing a significant increase in microelectrode impedance. In addition, we show the feasibility of using long-duration DC voltage pulses to remove adsorbed proteins from the microelectrode surface. PMID:19963693

  17. Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow

    PubMed Central

    Burdick, Monica M.; Reynolds, Nathan M.; Martin, Eric W.; Hawes, Jacquelyn V.; Carlson, Grady E.; Cuckler, Chaz M.; Bates, Michael C.; Barthel, Steven R.; Dimitroff, Charles J.

    2014-01-01

    Laboratory scale to industrial scale purification of biomolecules from cell culture supernatants and lysed cell solutions can be accomplished using affinity chromatography. While affinity chromatography using porous protein A agarose beads packed in columns is arguably the most common method of laboratory scale isolation of antibodies and recombinant proteins expressing Fc fragments of IgG, it can be a time consuming and expensive process. Time and financial constraints are especially daunting in small basic science labs that must recover hundreds of micrograms to milligram quantities of protein from dilute solutions, yet lack access to high pressure liquid delivery systems and/or personnel with expertise in bioseparations. Moreover, product quantification and characterization may also excessively lengthen processing time over several workdays and inflate expenses (consumables, wages, etc.). Therefore, a fast, inexpensive, yet effective protocol is needed for laboratory scale isolation and characterization of antibodies and other proteins possessing an Fc fragment. To this end, we have devised a protocol that can be completed by limited-experience technical staff in less than 9 hr (roughly one workday) and as quickly as 4 hr, as opposed to traditional methods that demand 20+ work hours. Most required equipment is readily available in standard biomedical science, biochemistry, and (bio)chemical engineering labs, and all reagents are commercially available. To demonstrate this protocol, representative results are presented in which chimeric murine galectin-1 fused to human Fc (Gal-1hFc) from cell culture supernatant was isolated using a protein A membrane adsorber. Purified Gal-1hFc was quantified using an expedited Western blotting analysis procedure and characterized using flow cytometry. The streamlined workflow can be modified for other Fc-expressing proteins, such as antibodies, and/or altered to incorporate alternative quantification and characterization

  18. 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

  19. 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.

  20. Infrared Spectroscopy and Catalysis Research: Infrared spectra of adsorbed molecules provide important information in the study of catalysis.

    PubMed

    Eischens, R P

    1964-10-23

    The examples discussed here represent only a small part of the published work relating to infrared spectra of adsorbed molecules. The publications in this field indicate that infrared spectroscopy is being used for surface chemistry research in about 50 laboratories throughout the world. This effort is mainly devoted to problems related to catalysis, and in this field infrared spectroscopy is the most widely used physical tool for surface chemistry studies. The general acceptance of infrared spectroscopy is primarily due to the fact that it provides information which is pertinent to the understanding of surface reactions on an atomic scale. During the last decade significant progress has also been made in the classical chemical techniques of catalysis study and in utilization of physical tools which depend on phenomena of magnetism, conductivity, low-energy electron diffraction, and electron emission. Probably the most important progress has been in the field of inorganic chemistry, where dramatic advances have been made in knowledge of metal coordination compounds. Such knowledge is vital to the understanding of catalysis on metal surfaces. I believe this progress has produced an attitude of sophisticated optimism among catalysis researchers with regard to eventual understanding of heterogeneous catalysis. This attitude is closely related to the realization that there is no "secret of catalysis" which places catalytic action beyond the limits of ordinary chemical knowledge (22). This view implies that the chemical aspects of heterogeneous catalysis are not unique and that the use of solid catalysts merely provides a highly effective exposure of catalytic atoms and facilitates separation of the products from the catalyst. Many capable catalysis researchers believe that studies of homogeneous catalysis provide the most direct route for the study of heterogeneous catalysis. Obviously homogeneous reactions catalyzed by compounds containing only one or two metal atoms

  1. 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

  2. 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

  3. A novel adsorbent for protein chromatography: supermacroporous monolithic cryogel embedded with Cu2+-attached sporopollenin particles.

    PubMed

    Erzengin, Mahmut; Ünlü, Nuri; Odabaşı, Mehmet

    2011-01-21

    The aim of this study is to prepare supermacroporous cryogels embedded with Cu(2+)-attached sporopollenin particles (Cu(2+)-ASP) having large surface area for high protein adsorption capacity. Supermacroporous poly(2-hydroxyethyl methacrylate) (PHEMA)-based monolithic cryogel column embedded with Cu(2+)-ASP was prepared by radical cryo-copolymerization of 2-hydroxyethyl methacrylate (HEMA) with N,N'-methylene-bis-acrylamide (MBAAm) as cross-linker directly in a plastic syringe for affinity purification of human serum albumin (HSA). Firstly, Cu(2+) ions were attached to sporopollenin particles (SP), then the supermacroporous PHEMA cryogel with embedded Cu(2+)-ASP was produced by free radical polymerization using N,N,N',N'-tetramethylene diamine (TEMED) and ammonium persulfate (APS) as initiator/activator pair in an ice bath. Embedded particles (10 mg) in PHEMA-based cryogel column were used in the adsorption/desorption of HSA from aqueous solutions. Optimum conditions of adsorption experiments were performed at pH 8.0 phosphate buffer, with flow rate of 0.5 mL/min, and at 5°C. The maximum amount of HSA adsorption from aqueous solution was very high (677.4 mg/g SP) with initial concentration 6 mg/mL. It was observed that HSA could be repeatedly adsorbed and desorbed to the embedded Cu(2+)-ASP in PHEMA cryogel without significant loss of adsorption capacity. PMID:21176840

  4. Computational design of protein-small molecule interfaces.

    PubMed

    Allison, Brittany; Combs, Steven; DeLuca, Sam; Lemmon, Gordon; Mizoue, Laura; Meiler, Jens

    2014-02-01

    The computational design of proteins that bind small molecule ligands is one of the unsolved challenges in protein engineering. It is complicated by the relatively small size of the ligand which limits the number of intermolecular interactions. Furthermore, near-perfect geometries between interacting partners are required to achieve high binding affinities. For apolar, rigid small molecules the interactions are dominated by short-range van der Waals forces. As the number of polar groups in the ligand increases, hydrogen bonds, salt bridges, cation-π, and π-π interactions gain importance. These partial covalent interactions are longer ranged, and additionally, their strength depends on the environment (e.g. solvent exposure). To assess the current state of protein-small molecule interface design, we benchmark the popular computer algorithm Rosetta on a diverse set of 43 protein-ligand complexes. On average, we achieve sequence recoveries in the binding site of 59% when the ligand is allowed limited reorientation, and 48% when the ligand is allowed full reorientation. When simulating the redesign of a protein binding site, sequence recovery among residues that contribute most to binding was 52% when slight ligand reorientation was allowed, and 27% when full ligand reorientation was allowed. As expected, sequence recovery correlates with ligand displacement. PMID:23962892

  5. Single molecule fluorescence experiments determine protein folding transition path times

    PubMed Central

    Chung, Hoi Sung; McHale, Kevin; Louis, John M.; Eaton, William A.

    2013-01-01

    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 single-molecule property that contains all the mechanistic information on how a process occurs. As a step toward observing transition paths in protein folding we 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 Förster-resonance-energy-transfer experiments. While the folding rate coefficients differ by a factor of 10,000, the transition-path times differ by less than a factor of 5, showing that a fast-and a slow-folding protein take almost the same time to fold when folding actually happens. A very simple model based on energy landscape theory can explain this result. PMID:22363011

  6. Dynamical interaction between protein molecules and their hydration shell

    NASA Astrophysics Data System (ADS)

    Parak, Fritz G.

    2013-10-01

    The hydration shell of a protein molecule influences its functional important dynamics while the protein molecule influences the hydration shell. Neutron scattering experiments have been used to separate both effects. Neutron crystallography is used to determine the positions and the mean square displacements, , of the protons in met-myoglobin. Mean square displacements obtained by incoherent neutron scattering stem from motions occurring on a time scale faster than 100 ps. The combination of the two experimental results allows to separate three types of protein protons. The -values of lysine protons stem from motions faster than 100 ps. Half of the -values of methyl protons is caused by motions faster than 100 ps, the other halve comes from slower motions. The backbone protons move slower than 100 ps. Incoherent neutron scattering on a perdeuterated myoglobin with a 1H2O hydration shell allowed the study of the diffusion in this shell.

  7. Observation and analysis of Fano-like lineshapes in the Raman spectra of molecules adsorbed at metal interfaces

    NASA Astrophysics Data System (ADS)

    Dey, S.; Banik, M.; Hulkko, E.; Rodriguez, K.; Apkarian, V. A.; Galperin, M.; Nitzan, A.

    2016-01-01

    Surface-enhanced Raman scattering (SERS) from bipyridyl ethylene adsorbed on gold dumbbells shows Fano-like spectra at high incident light intensity. This is accompanied by an increased electronic temperature, while no vibrational anti-Stokes scattering is observed. Theory indicates that interference between vibrational and electronic Raman scattering can yield such asymmetric scattering lineshapes. The best fit to observations is however obtained by disregarding this coupling and accounting for the detailed lineshape of the continuous electronic component of the SERS.

  8. 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.

  9. Small Molecule Proteostasis Regulators for Protein Conformational Diseases

    PubMed Central

    Calamini, Barbara; Silva, Maria Catarina; Madoux, Franck; Hutt, Darren M.; Khanna, Shilpi; Chalfant, Monica A.; Saldanha, Sanjay A.; Hodder, Peter; Tait, Bradley D.; Garza, Dan; Balch, William E.; Morimoto, Richard I.

    2011-01-01

    Protein homeostasis (proteostasis) is essential for cellular and organismal health. Stress, aging, and the chronic expression of misfolded proteins, however, challenge the proteostasis machinery and the vitality of the cell. Enhanced expression of molecular chaperones, regulated by heat shock transcription factor-1 (HSF-1), has been shown to restore proteostasis in a variety of conformational disease models, suggesting a promising therapeutic approach. We describe the results of a ∼900,000 small molecule screen that identified novel classes of small molecule proteostasis regulators (PRs) that induce HSF-1-dependent chaperone expression and restore protein folding in multiple conformational disease models. The beneficial effects to proteome stability are mediated by HSF-1, DAF-16/FOXO, SKN-1/Nrf-2, and the chaperone machinery through mechanisms that are distinct from current known small molecule activators of the HSR. We suggest that modulation of the proteostasis network by PRs represents a promising therapeutic approach for the treatment of a variety of protein conformational diseases. PMID:22198733

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

    PubMed

    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

    2015-08-01

    The current predominant therapeutic 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

  11. 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

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

    PubMed Central

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

    2014-01-01

    In contrast with advances in massively parallel DNA sequencing1, high-throughput protein analyses2-4 are often limited by ensemble measurements, individual analyte purification and hence compromised quality and cost-effectiveness. Single-molecule (SM) protein detection achieved using optical methods5 is limited by the number of spectrally nonoverlapping chromophores. Here, we introduce a single molecular interaction-sequencing (SMI-Seq) technology for parallel protein interaction profiling leveraging SM advantages. DNA barcodes are attached to proteins collectively via ribosome display6 or individually via enzymatic conjugation. Barcoded proteins are assayed en masse in aqueous solution and subsequently immobilized in a polyacrylamide (PAA) thin film to construct a random SM array, where barcoding DNAs are amplified into in situ polymerase colonies (polonies)7 and analyzed by DNA sequencing. This method allows precise quantification of various proteins with a theoretical maximum array density of over one million polonies per square millimeter. Furthermore, protein interactions can be measured based on the statistics of colocalized polonies arising from barcoding DNAs of interacting proteins. Two demanding applications, G-protein coupled receptor (GPCR) and antibody binding profiling, were demonstrated. SMI-Seq enables “library vs. library” screening in a one-pot assay, simultaneously interrogating molecular binding affinity and specificity. PMID:25252978

  13. Visualizing Water Molecules in Transmembrane Proteins Using Radiolytic Labeling Methods

    SciTech Connect

    Orban, T.; Gupta, S; Palczewski, K; Chance, M

    2010-01-01

    Essential to cells and their organelles, water is both shuttled to where it is needed and trapped within cellular compartments and structures. Moreover, ordered waters within protein structures often colocalize with strategically placed polar or charged groups critical for protein function, yet it is unclear if these ordered water molecules provide structural stabilization, mediate conformational changes in signaling, neutralize charged residues, or carry out a combination of all these functions. Structures of many integral membrane proteins, including G protein-coupled receptors (GPCRs), reveal the presence of ordered water molecules that may act like prosthetic groups in a manner quite unlike bulk water. Identification of 'ordered' waters within a crystalline protein structure requires sufficient occupancy of water to enable its detection in the protein's X-ray diffraction pattern, and thus, the observed waters likely represent a subset of tightly bound functional waters. In this review, we highlight recent studies that suggest the structures of ordered waters within GPCRs are as conserved (and thus as important) as conserved side chains. In addition, methods of radiolysis, coupled to structural mass spectrometry (protein footprinting), reveal dynamic changes in water structure that mediate transmembrane signaling. The idea of water as a prosthetic group mediating chemical reaction dynamics is not new in fields such as catalysis. However, the concept of water as a mediator of conformational dynamics in signaling is just emerging, because of advances in both crystallographic structure determination and new methods of protein footprinting. Although oil and water do not mix, understanding the roles of water is essential to understanding the function of membrane proteins.

  14. Fluorous photoaffinity labeling to probe protein-small molecule interactions.

    PubMed

    Huang, Weigang; Zhang, Qisheng

    2015-01-01

    Identifying cellular targets of bioactive small molecules is essential for their applications as chemical probes or drug candidates. Of equal importance is to determine their "off-target" interactions, which usually account for unwanted properties including toxicity. Among strategies to profile small molecule-interacting proteins, photoaffinity labeling has been widely used because of its distinct advantages such as sensitivity. When combined with mass spectrometry, this approach can provide additional structural and mechanistic information, such as drug-target stoichiometry and exact interacting amino acid residues. We have described a novel fluorous photoaffinity labeling approach, in which a fluorous tag is incorporated into the photoaffinity labeling reagent to enable the enrichment of the labeled species from complex mixtures for analysis. This new feature likely makes the fluorous photoaffinity labeling approach suitable to identify transient interactions, and low-abundant, low-affinity interacting proteins in a cellular environment. PMID:25618351

  15. 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

  16. 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

  17. Single Molecule Mechanical Probing of the SNARE Protein Interactions

    PubMed Central

    Liu, W.; Montana, Vedrana; Bai, Jihong; Chapman, Edwin R.; Mohideen, U.; Parpura, Vladimir

    2006-01-01

    Exocytotic release of neurotransmitters is mediated by the ternary soluble N-ethyl maleimide-sensitive fusion protein attachment protein receptors (SNAREs) complex, comprised of syntaxin (Sx), synaptosome-associated protein of 25 kDa (SNAP25), and synaptobrevin 2 (Sb2). Since exocytosis involves the nonequilibrium process of association and dissociation of bonds between molecules of the SNARE complex, dynamic measurements at the single molecule level are necessary for a detailed understanding of these interactions. To address this issue, we used the atomic force microscope in force spectroscopy mode to show from single molecule investigations of the SNARE complex, that Sx1A and Sb2 are zippered throughout their entire SNARE domains without the involvement of SNAP25. When SNAP25B is present in the complex, it creates a local interaction at the 0 (ionic) layer by cuffing Sx1A and Sb2. Force loading rate studies indicate that the ternary complex interaction is more stable than the Sx1A-Sb2 interaction. PMID:16648158

  18. 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

  19. Conformations of polyaniline molecules adsorbed on Au(111) probed by in situ STM and ex situ XPS and NEXAFS.

    PubMed

    Lee, YiHui; Chang, ChinZen; Yau, ShuehLin; Fan, LiangJen; Yang, YawWen; Yang, LiangYueh Ou; Itaya, Kingo

    2009-05-13

    In situ scanning tunneling microscopy (STM), X-ray photoelectron spectroscopy (XPS), and near edge X-ray absorption fine structure (NEXAFS) have been used to examine the conformation of a monolayer of polyaniline (PAN) molecules produced on a Au(111) single-crystal electrode by anodization at 1.0 V [vs reversible hydrogen electrode (RHE)] in 0.10 M H(2)SO(4) containing 0.030 M aniline. The as-produced PAN molecules took on a well-defined linear conformation stretching for 500 A or more, as shown by in situ and ex situ STM. The XPS and NEXAFS results indicated that the linear PAN seen at 1.0 V assumed the form of an emeraldine salt made of PAN chains and (bi)sulfate anions. Shifting the potential from 1.0 to 0.7 V altered the shape of the PAN molecules from straight to crooked, which was ascribed to restructuring of the Au(111) electrified interface on the basis of voltammetric and XPS results. In situ STM showed that further decreasing the potential to 0.5 V transformed the crooked PAN threads into a mostly linear form again, with preferential alignment and formation of some locally ordered structures. PAN molecules could be reduced from emeraldine to leucoemeraldine as the potential was decreased to 0.2 V or less. In situ STM showed that the fully reduced PAN molecules were straight but mysteriously shortened to approximately 50 A in length. The conformation of PAN did not recuperate when the potential was shifted positively to 1.0 V. PMID:19361217

  20. 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.

  1. 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.

  2. Conductivity Measurements of Pyrrole Molecules Incorporated into Chemically Adsorbed Monolayer by Conducting Probe Technique in Atomic Force Microscope

    NASA Astrophysics Data System (ADS)

    Yamamoto, Shin‑ichi; Ogawa, Kazufumi

    2006-03-01

    A monomolecular layer containing pyrrolyl groups at the surface was prepared between two parallel Pt electrodes on a glass substrate by a chemical adsorption technique using N-[11-(trichlorosilyl)undecyl] pyrrole (PNN). Then, the pyrrolyl was polymerized with pure water by applying a DC voltage of 10 V between the two Pt electrodes. It was confirmed using an optical microscope that many electric paths were formed between the two Pt electrodes by a decoration technique using electrochemical polymerization in an aqueous medium containing pyrrole after the polymerization. Next, a conductive probe of an atomic force microscope (AFM) was used to examine an electrical polymerized path through the surface of the polypyrrolyl group in a chemically adsorbed monomolecular layer. The resistance of one electric path in the monomolecular layer was measured using an AFM with an attached Au-covered tip at room temperature. With a measurement volume of about 0.2 nm (the thickness of the electric path in the monomolecular layer) × 200 μm (the average width of the electric path) × 100 μm (the distance between the Pt electrode and the Au-covered AFM tip), the resistance at room temperature of one electric path was 4 k Ω under ambient conditions. From the results in the atmosphere, the conductivity of a super long conjugated polypyrrolyl group without any dopant in a lateral direction was ohmically estimated to be at least 6.0 × 105 S/m.

  3. 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.

  4. 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

  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. 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

  7. Orbital tomography: Molecular band maps, momentum maps and the imaging of real space orbitals of adsorbed molecules

    PubMed Central

    Offenbacher, Hannes; Lüftner, Daniel; Ules, Thomas; Reinisch, Eva Maria; Koller, Georg; Puschnig, Peter; Ramsey, Michael G.

    2015-01-01

    The frontier orbitals of molecules are the prime determinants of their chemical, optical and electronic properties. Arguably, the most direct method of addressing the (filled) frontier orbitals is ultra-violet photoemission spectroscopy (UPS). Although UPS is a mature technique from the early 1970s on, the angular distribution of the photoemitted electrons was thought to be too complex to be analysed quantitatively. Recently angle resolved UPS (ARUPS) work on conjugated molecules both, in ordered thick films and chemisorbed monolayers, has shown that the angular (momentum) distribution of the photocurrent from orbital emissions can be simply understood. The approach, based on the assumption of a plane wave final state is becoming known as orbital tomography. Here we will demonstrate, with selected examples of pentacene (5A) and sexiphenyl (6P), the potential of orbital tomography. First it will be shown how the full angular distribution of the photocurrent (momentum map) from a specific orbital is related to the real space orbital by a Fourier transform. Examples of the reconstruction of 5A orbitals will be given and the procedure for recovering the lost phase information will be outlined. We then move to examples of sexiphenyl where we interrogate the original band maps of thick sexiphenyl in the light of our understanding of orbital tomography that has developed since then. With comparison to theoretical simulations of the molecular band maps, the molecular conformation and orientation will be concluded. New results for the sexiphenyl monolayer on Al(1 1 0) will then be presented. From the band maps it will be concluded that the molecule is planarised and adopts a tilted geometry. Finally the momentum maps down to HOMO-11 will be analysed and real space orbitals reconstructed. PMID:26752804

  8. Small-molecule control of protein degradation using split adaptors.

    PubMed

    Davis, Joseph H; Baker, Tania A; Sauer, Robert T

    2011-11-18

    Targeted intracellular degradation provides a method to study the biological function of proteins and has numerous applications in biotechnology. One promising approach uses adaptor proteins to target substrates with genetically encoded degradation tags for proteolysis. Here, we describe an engineered split-adaptor system, in which adaptor assembly and delivery of substrates to the ClpXP protease depends on a small molecule (rapamycin). This degradation system does not require modification of endogenous proteases, functions robustly over a wide range of adaptor concentrations, and does not require new synthesis of adaptors or proteases to initiate degradation. We demonstrate the efficacy of this system in E. coli by degrading tagged variants of LacI repressor and FtsA, an essential cell-division protein. In the latter case, addition of rapamycin causes pronounced filamentation because daughter cells cannot divide. Strikingly, washing rapamycin away reverses this phenotype. Our system is highly modular, with clearly defined interfaces for substrate binding, protease binding, and adaptor assembly, providing a clear path to extend this system to other degradation tags, proteases, or induction systems. Together, these new reagents should be useful in controlling protein degradation in bacteria. PMID:21866931

  9. Small-molecule control of protein degradation using split adaptors

    PubMed Central

    Davis, Joseph H.; Baker, Tania A.; Sauer, Robert T.

    2011-01-01

    Targeted intracellular degradation provides a method to study the biological function of proteins and has numerous applications in biotechnology. One promising approach uses adaptor proteins to target substrates with genetically encoded degradation tags for proteolysis. Here, we describe an engineered split-adaptor system, in which adaptor assembly and delivery of substrates to the ClpXP protease depends on a small molecule (rapamycin). This degradation system does not require modification of endogenous proteases, functions robustly over a wide range of adaptor concentrations, and does not require new synthesis of adaptors or proteases to initiate degradation. We demonstrate the efficacy of this system in E. coli by degrading tagged variants of LacI repressor and FtsA, an essential cell-division protein. In the latter case, addition of rapamycin causes pronounced filamentation because daughter cells cannot divide. Strikingly, washing rapamycin away reverses this phenotype. Our system is highly modular, with clearly-defined interfaces for substrate binding, protease binding, and adaptor assembly, providing a clear path to extend this system to other degradation tags, proteases, or induction systems. Together, these new reagents should be useful in controlling protein degradation in bacteria. PMID:21866931

  10. 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.

  11. 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.).

  12. Knotting and unknotting of a protein in single molecule experiments.

    PubMed

    Ziegler, Fabian; Lim, Nicole C H; Mandal, Soumit Sankar; Pelz, Benjamin; Ng, Wei-Ping; Schlierf, Michael; Jackson, Sophie E; Rief, Matthias

    2016-07-01

    Spontaneous folding of a polypeptide chain into a knotted structure remains one of the most puzzling and fascinating features of protein folding. The folding of knotted proteins is on the timescale of minutes and thus hard to reproduce with atomistic simulations that have been able to reproduce features of ultrafast folding in great detail. Furthermore, it is generally not possible to control the topology of the unfolded state. Single-molecule force spectroscopy is an ideal tool for overcoming this problem: by variation of pulling directions, we controlled the knotting topology of the unfolded state of the 52-knotted protein ubiquitin C-terminal hydrolase isoenzyme L1 (UCH-L1) and have therefore been able to quantify the influence of knotting on its folding rate. Here, we provide direct evidence that a threading event associated with formation of either a 31 or 52 knot, or a step closely associated with it, significantly slows down the folding of UCH-L1. The results of the optical tweezers experiments highlight the complex nature of the folding pathway, many additional intermediate structures being detected that cannot be resolved by intrinsic fluorescence. Mechanical stretching of knotted proteins is also of importance for understanding the possible implications of knots in proteins for cellular degradation. Compared with a simple 31 knot, we measure a significantly larger size for the 52 knot in the unfolded state that can be further tightened with higher forces. Our results highlight the potential difficulties in degrading a 52 knot compared with a 31 knot. PMID:27339135

  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. 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

  15. 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

  16. Selectivity by Small-Molecule Inhibitors of Protein Interactions Can Be Driven by Protein Surface Fluctuations

    PubMed Central

    Johnson, David K.; Karanicolas, John

    2015-01-01

    Small-molecules that inhibit interactions between specific pairs of proteins have long represented a promising avenue for therapeutic intervention in a variety of settings. Structural studies have shown that in many cases, the inhibitor-bound protein adopts a conformation that is distinct from its unbound and its protein-bound conformations. This plasticity of the protein surface presents a major challenge in predicting which members of a protein family will be inhibited by a given ligand. Here, we use biased simulations of Bcl-2-family proteins to generate ensembles of low-energy conformations that contain surface pockets suitable for small molecule binding. We find that the resulting conformational ensembles include surface pockets that mimic those observed in inhibitor-bound crystal structures. Next, we find that the ensembles generated using different members of this protein family are overlapping but distinct, and that the activity of a given compound against a particular family member (ligand selectivity) can be predicted from whether the corresponding ensemble samples a complementary surface pocket. Finally, we find that each ensemble includes certain surface pockets that are not shared by any other family member: while no inhibitors have yet been identified to take advantage of these pockets, we expect that chemical scaffolds complementing these “distinct” pockets will prove highly selective for their targets. The opportunity to achieve target selectivity within a protein family by exploiting differences in surface fluctuations represents a new paradigm that may facilitate design of family-selective small-molecule inhibitors of protein-protein interactions. PMID:25706586

  17. Scrutinizing individual CoTPP molecule adsorbed on coinage metal surfaces from the interplay of STM experiment and theory

    NASA Astrophysics Data System (ADS)

    Houwaart, Torsten; Le Bahers, Tangui; Sautet, Philippe; Auwärter, Willi; Seufert, Knud; Barth, Johannes V.; Bocquet, Marie-Laure

    2015-05-01

    The cobalt tetraphenyl porphyrin (CoTPP) molecule and its adsorption on clean Cu and Ag surfaces are comparatively analyzed within the Density Functional Theory (DFT) framework. Different sets of exchange-correlation functionals - the Local Density Approximation (LDA) and the Gradient Generalized Approximation (along with the PBE functional and the semi-empirical Grimme's corrections of dispersion) - are compared. Two prominent structural adsorption properties are disclosed in all sets of calculations: an asymmetric saddle deformation of CoTPP with an enhanced tilting of the upwards bent pyrroles and a single adsorption site where the Co center occupies a bridge position and one molecular axis (along the direction of the lowered pair of opposite pyrroles) is aligned with the dense-packed < 1 1 bar 0 > substrate direction. The similarities between Cu(111) and Ag(111) surfaces extend to the interfacial electronic structure with similar electronic redistribution and molecular charging. However subtle differences between the two substrates are revealed with bias-dependent STM simulations, especially in the low-bias imaging range. The STM calculations underline the difficulty for the commonly used GGA + D2 DFT framework to quantitatively predict the energy positions of the frontier molecular orbitals (MOs).

  18. 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

  19. Pluripotency transcription factor Sox2 is strongly adsorbed by heparin but requires a protein transduction domain for cell internalization

    SciTech Connect

    Albayrak, Cem; Yang, William C.; Swartz, James R.

    2013-02-15

    Highlights: ► Both R9Sox2 and Sox2 bind heparin with comparable affinity. ► Both R9Sox2 and Sox2 bind to fibroblasts, but only R9Sox2 is internalized. ► Internalization efficiency of R9Sox2 is 0.3% of the administered protein. ► Heparan sulfate adsorption may be part of a mechanism for managing cell death. -- Abstract: The binding of protein transduction domain (PTD)-conjugated proteins to heparan sulfate is an important step in cellular internalization of macromolecules. Here, we studied the pluripotency transcription factor Sox2, with or without the nonaarginine (R9) PTD. Unexpectedly, we observed that Sox2 is strongly adsorbed by heparin and by the fibroblasts without the R9 PTD. However, only the R9Sox2 fusion protein is internalized by the cells. These results collectively show that binding to heparan sulfate is not sufficient for cellular uptake, thereby supporting a recent hypothesis that other proteins play a role in cell internalization of PTD-conjugated proteins.

  20. 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.

  1. The importance of individual protein molecule dynamics in developing and assessing solid state protein preparations.

    PubMed

    Hill, John J; Shalaev, Evgenyi Y; Zografi, George

    2014-09-01

    Processing protein solutions into the solid state is a common approach for generating stable amorphous protein mixtures that are suitable for long-term storage. Great care is typically given to protecting the protein native structure during the various drying steps that render it into the amorphous solid state. However, many studies illustrate that chemical and physical degradations still occur in spite of this amorphous material having good glassy properties and it being stored at temperatures below its glass transition temperature (Tg). Because of these persistent issues and recent biophysical studies that have refined the debate ascribing meaning to the molecular dynamical transition temperature and Tg of protein molecules, we provide an updated discussion on the impact of assessing and managing localized, individual protein molecule nondiffusive motions in the context of proteins being prepared into bulk amorphous mixtures. Our aim is to bridge the pharmaceutical studies addressing bulk amorphous preparations and their glassy behavior, with the biophysical studies historically focused on the nondiffusive internal protein dynamics and a protein's activity, along with their combined efforts in assessing the impact of solvent hydrogen-bonding networks on local stability. We also provide recommendations for future research efforts in solid-state formulation approaches. PMID:24867196

  2. Electrostatic Similarities between Protein and Small Molecule Ligands Facilitate the Design of Protein-Protein Interaction Inhibitors

    PubMed Central

    Zhang, Kam Y. J.

    2013-01-01

    One of the underlying principles in drug discovery is that a biologically active compound is complimentary in shape and molecular recognition features to its receptor. This principle infers that molecules binding to the same receptor may share some common features. Here, we have investigated whether the electrostatic similarity can be used for the discovery of small molecule protein-protein interaction inhibitors (SMPPIIs). We have developed a method that can be used to evaluate the similarity of electrostatic potentials between small molecules and known protein ligands. This method was implemented in a software called EleKit. Analyses of all available (at the time of research) SMPPII structures indicate that SMPPIIs bear some similarities of electrostatic potential with the ligand proteins of the same receptor. This is especially true for the more polar SMPPIIs. Retrospective analysis of several successful SMPPIIs has shown the applicability of EleKit in the design of new SMPPIIs. PMID:24130741

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

    PubMed

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

    2016-07-01

    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

  4. Theory and applications of refractive index-based optical microscopy to measure protein mass transfer in spherical adsorbent particles.

    PubMed

    Bankston, Theresa E; Stone, Melani C; Carta, Giorgio

    2008-04-25

    This work provides the theoretical foundation and a range of practical application examples of a recently developed method to measure protein mass transfer in adsorbent particles using refractive index-based optical microscopy. A ray-theoretic approach is first used to predict the behavior of light traveling through a particle during transient protein adsorption. When the protein concentration gradient in the particle is sharp, resulting in a steep refractive index gradient, the rays bend and intersect, thereby concentrating light in a sharp ring that marks the position of the adsorption front. This behavior is observed when mass transfer is dominated by pore diffusion and the adsorption isotherm is highly favorable. Applications to protein cation-exchange, hydrophobic interaction, and affinity adsorption are then considered using, as examples, the three commercial, agarose-based stationary phases SP-Sepharose-FF, Butyl Sepharose 4FF, and MabSelect. In all three cases, the method provides results that are consistent with measurements based on batch adsorption and previously published data confirming its utility for the determination of protein mass transfer kinetics under a broad range of practically relevant conditions. PMID:18353343

  5. Nanoporous Gyroid-Structured Epoxy from Block Copolymer Templates for High Protein Adsorbability.

    PubMed

    Wang, Xin-Bo; Lin, Tze-Chung; Hsueh, Han-Yu; Lin, Shih-Chieh; He, Xiao-Dong; Ho, Rong-Ming

    2016-06-28

    Nanoporous epoxy with gyroid texture is fabricated by using a nanoporous polymer with gyroid-forming nanochannels as a template for polymerization of epoxy. The nanoporous polymer template is obtained from the self-assembly of degradable block copolymer, polystyrene-b-poly(l-lactide) (PS-PLLA), followed by hydrolysis of PLLA blocks. Templated polymerization can be conducted under ambient conditions to create well-defined, bicontinuous epoxy networks in a PS matrix. By taking advantage of multistep curing of epoxy, well-ordered robust nanoporous epoxy can be obtained after removal of PS template, giving robust porous materials. The through-hole nanoporous epoxy in the film state can be used as a coated layer to enhance the adsorbability for both lysozyme and bovine serum albumin. PMID:27245380

  6. Small Molecule Probes That Perturb A Protein-protein Interface In Antithrombin

    PubMed Central

    Xin, Dongyue; Holzenburg, Andreas

    2014-01-01

    Small molecule probes for perturbing protein-protein interactions (PPIs) in vitro can be useful if they cause the target proteins to undergo biomedically relevant changes to their tertiary and quaternary structures. Application of the Exploring Key Orientations (EKO) strategy (J. Am. Chem. Soc., 2013, 135, 167 – 173) to a piperidinone-piperidine chemotype 1 indicated specific derivatives were candidates to perturb a protein-protein interface in the α-antithrombin dimer; those particular derivatives of 1 were prepared and tested. In the event, most of them significantly accelerated oligomerization of monomeric α-antithrombin, which is metastable in its oligomeric state. This assertion is supported by data from gel electrophoresis (non-denaturing PAGE; throughout) and probe-induced loss of α-antithrombin’s inhibitor activity in a reaction catalyzed by thrombin. Kinetics of α-antithrombin oligomerization induced by the target compounds were examined. It was found that probes with O-benzyl-protected serine side-chains are the most active catalysts in the series, and reasons for this, based on modeling experiments, are proposed. Overall, this study reveals one of the first examples of small molecules designed to act at a protein-protein interface relevant to oligomerization of a serpin (ie α-antithrombin). The relevance of this to formation of oligomeric serpin fibrils, associated with the disease states known as “serpinopathies”, is discussed. PMID:25396040

  7. Small Molecule Probes That Perturb A Protein-protein Interface In Antithrombin.

    PubMed

    Xin, Dongyue; Holzenburg, Andreas; Burgess, Kevin

    2014-12-01

    Small molecule probes for perturbing protein-protein interactions (PPIs) in vitro can be useful if they cause the target proteins to undergo biomedically relevant changes to their tertiary and quaternary structures. Application of the Exploring Key Orientations (EKO) strategy (J. Am. Chem. Soc., 2013, 135, 167 - 173) to a piperidinone-piperidine chemotype 1 indicated specific derivatives were candidates to perturb a protein-protein interface in the α-antithrombin dimer; those particular derivatives of 1 were prepared and tested. In the event, most of them significantly accelerated oligomerization of monomeric α-antithrombin, which is metastable in its oligomeric state. This assertion is supported by data from gel electrophoresis (non-denaturing PAGE; throughout) and probe-induced loss of α-antithrombin's inhibitor activity in a reaction catalyzed by thrombin. Kinetics of α-antithrombin oligomerization induced by the target compounds were examined. It was found that probes with O-benzyl-protected serine side-chains are the most active catalysts in the series, and reasons for this, based on modeling experiments, are proposed. Overall, this study reveals one of the first examples of small molecules designed to act at a protein-protein interface relevant to oligomerization of a serpin (ie α-antithrombin). The relevance of this to formation of oligomeric serpin fibrils, associated with the disease states known as "serpinopathies", is discussed. PMID:25396040

  8. 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.

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

    NASA Astrophysics Data System (ADS)

    Meng, Qingyong; Meyer, Hans-Dieter

    2015-10-01

    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.

  10. 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.

  11. Unintended consequences? Water molecules at biological and crystallographic protein-protein interfaces.

    PubMed

    Ahmed, Mostafa H; Habtemariam, Mesay; Safo, Martin K; Scarsdale, J Neel; Spyrakis, Francesca; Cozzini, Pietro; Mozzarelli, Andrea; Kellogg, Glen E

    2013-12-01

    The importance of protein-protein interactions (PPIs) is becoming increasingly appreciated, as these interactions lie at the core of virtually every biological process. Small molecule modulators that target PPIs are under exploration as new therapies. One of the greatest obstacles faced in crystallographically determining the 3D structures of proteins is coaxing the proteins to form "artificial" PPIs that lead to uniform crystals suitable for X-ray diffraction. This work compares interactions formed naturally, i.e., "biological", with those artificially formed under crystallization conditions or "non-biological". In particular, a detailed analysis of water molecules at the interfaces of high-resolution (≤2.30 Å) X-ray crystal structures of protein-protein complexes, where 140 are biological protein-protein complex structures and 112 include non-biological protein-protein interfaces, was carried out using modeling tools based on the HINT forcefield. Surprisingly few and relatively subtle differences were observed between the two types of interfaces: (i) non-biological interfaces are more polar than biological interfaces, yet there is better organized hydrogen bonding at the latter; (ii) biological associations rely more on water-mediated interactions with backbone atoms compared to non-biological associations; (iii) aromatic/planar residues play a larger role in biological associations with respect to water, and (iv) Lys has a particularly large role at non-biological interfaces. A support vector machines (SVMs) classifier using descriptors from this study was devised that was able to correctly classify 84% of the two interface types. PMID:24076743

  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. PMID:22226554

  14. 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.

  15. 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

  16. Adsorption and trace detection of pharmacologically significant 5-methylthio-1, 3, 4-thiadiazole-2-thiol molecule adsorbed on silver nanocolloids and understanding the role of Albrecht's “A” and Herzberg-Teller contributions in the SERS spectra

    NASA Astrophysics Data System (ADS)

    Chowdhury, Joydeep; Chandra, Subhendu; Ghosh, Manash

    2015-01-01

    The surface enhanced Raman scattering (SERS) spectra of biologically and industrially significant 5-methylthio-1, 3, 4-thiadiazole 2-thiol molecule have been investigated. The SERS spectra at various concentrations of the adsorbate are compared with the Fourier transform Infrared (FTIR) and normal Raman spectra (NRS) of the probe molecule recorded in different environmental conditions. The optimized molecular structures of the most probable thione and the thiol forms of the molecule have been estimated from the density functional theory (DFT) calculations. The vibrational signatures of the molecule have been assigned from the potential energy distributions (PEDs). The detail vibrational analyses reveal that ∼54% of the thione form of the molecule is prevalent in the solid state and its population increases to ∼65% in ACN solvent medium. Concentration dependent SERS, together with the 2-dimensional correlation spectra (2D-COS), corroborate the presence of both the thione and the thiol forms of the molecule even in the surface adsorbed state. The orientations of the thione and the thiol forms of the molecule on the nanocolloidal silver surface have been predicted from the surface selection rule. The selective enhancement of Raman bands in the SERS spectra have been explored from the view of the Albrecht's "A" and Herzberg-Teller (HT) charge transfer (CT) contribution.

  17. Adsorption and trace detection of pharmacologically significant 5-methylthio-1, 3, 4-thiadiazole-2-thiol molecule adsorbed on silver nanocolloids and understanding the role of Albrecht's "A" and Herzberg-Teller contributions in the SERS spectra.

    PubMed

    Chowdhury, Joydeep; Chandra, Subhendu; Ghosh, Manash

    2015-01-25

    The surface enhanced Raman scattering (SERS) spectra of biologically and industrially significant 5-methylthio-1, 3, 4-thiadiazole 2-thiol molecule have been investigated. The SERS spectra at various concentrations of the adsorbate are compared with the Fourier transform Infrared (FTIR) and normal Raman spectra (NRS) of the probe molecule recorded in different environmental conditions. The optimized molecular structures of the most probable thione and the thiol forms of the molecule have been estimated from the density functional theory (DFT) calculations. The vibrational signatures of the molecule have been assigned from the potential energy distributions (PEDs). The detail vibrational analyses reveal that ∼54% of the thione form of the molecule is prevalent in the solid state and its population increases to ∼65% in ACN solvent medium. Concentration dependent SERS, together with the 2-dimensional correlation spectra (2D-COS), corroborate the presence of both the thione and the thiol forms of the molecule even in the surface adsorbed state. The orientations of the thione and the thiol forms of the molecule on the nanocolloidal silver surface have been predicted from the surface selection rule. The selective enhancement of Raman bands in the SERS spectra have been explored from the view of the Albrecht's "A" and Herzberg-Teller (HT) charge transfer (CT) contribution. PMID:25168230

  18. 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.

  19. Capillary electrophoresis-mass spectrometry of basic proteins using a new physically adsorbed polymer coating. Some applications in food analysis.

    PubMed

    Simó, Carolina; Elvira, Carlos; González, Nieves; San Román, J; Barbas, Coral; Cifuentes, Alejandro

    2004-07-01

    A new physically adsorbed capillary coating for capillary electrophoresis-mass spectrometry (CE-MS) of basic proteins is presented, which is easily obtained by flushing the capillary with a polymer aqueous solution for two min. This coating significantly reduces the electrostatic adsorption of a group of basic proteins (i.e., cytochrome c, lysozyme, and ribonuclease A) onto the capillary wall allowing their analysis by CE-MS. The coating protocol is compatible with electrospray inonization (ESI)-MS via the reproducible separation of the standard basic proteins (%RSD values (n = 5) < 1% for analysis time reproducibility and < 5% for peak heights, measured from the total ion electropherograms (TIEs) within the same day). The LODs determined using cytochrome c with total ion current and extracted ion current defection were 24.5 and 2.9 fmol, respectively. Using this new coating lysozymes from chicken and turkey egg white could be easily distinguished by CE-MS, demonstrating the usefulness of this method to differentiate animal species. Even after sterilization at 120 degrees C for 30 min, lysozyme could be detected, as well as in wines at concentrations much lower than the limit marked by the EC Commission Regulation. Adulteration of minced meat with 5% of egg-white could also be analysed by our CE-MS protocol. PMID:15237406

  20. Paramagnetic Nanoparticles Leave Their Mark on Nuclear Spins of Transiently Adsorbed Proteins.

    PubMed

    Zanzoni, Serena; Pedroni, Marco; D'Onofrio, Mariapina; Speghini, Adolfo; Assfalg, Michael

    2016-01-13

    The successful application of nanomaterials in biosciences necessitates an in-depth understanding of how they interface with biomolecules. Transient associations of proteins with nanoparticles (NPs) are accessible by solution NMR spectroscopy, albeit with some limitations. The incorporation of paramagnetic centers into NPs offers new opportunities to explore bio-nano interfaces. We propose NMR paramagnetic relaxation enhancement as a new tool to detect NP-binding surfaces on proteins with increased sensitivity, also extending the applicability of NMR investigations to heterogeneous biomolecular mixtures. The adsorption of ubiquitin on gadolinium-doped fluoride-based NPs produced residue-specific NMR line-broadening effects mapping to a contiguous area on the surface of the protein. Importantly, an identical paramagnetic fingerprint was observed in the presence of a competing protein-protein association equilibrium, exemplifying possible interactions taking place in crowded biological media. The interaction was further characterized using isothermal titration calorimetry and upconversion emission measurements. The data indicate that the used fluoride-based NPs are not biologically inert but rather are capable of biomolecular recognition. PMID:26683352

  1. Effect of acidification and heating on the rheological properties of oil-water interfaces with adsorbed milk proteins.

    PubMed

    Mellema, M; Isenbart, J G

    2004-09-01

    The behavior of casein and whey proteins at the oil-water interface was studied using a dynamic drop tensiometer (DDT). The dilational modulus of the interface was measured for aqueous solutions of skim milk powder (SMP) and whey protein concentrate (WPC) with various additions (salt, calcium, lactose) and (order of) various processing steps. Acidification or heating was performed before or after creation of the interface. The elastic properties of oil-water interfaces with adsorbed milk proteins could partly determine the rate of partial coalescence and resulting product instability. For WPC, preacidification slows down the adsorption, but the modulus is not affected. This is probably because, although the whey proteins change conformation more slowly at the interface, still a homogeneous film is formed. If postacidification is applied, coarsening of the protein film leads to loss of interfacial rigidity. Preheating of the aqueous phase with WPC leads to denaturation and aggregation, but the aggregates formed are still surface active and give high moduli. If preheating of a WPC solution is followed by postacidification, the resulting modulus is high (approximately 60 mN/m). The oil-water interfacial properties of SMP are only minimally affected by preheating or by choice of powder (low, medium, or high heat). At low pH, however, aggregates are formed that are less surface active, and interfacial moduli are lower. If measurements are performed at high temperature (i.e., if postheating is applied), for both SMP and WPC systems, moduli became much lower (approximately 10 mN/m). This is probably because of accelerated rearrangements, leading to the formation of inhomogeneous film structures. PMID:15375034

  2. Water-mediated influence of a crowded environment on internal vibrations of a protein molecule.

    PubMed

    Kuffel, Anna; Zielkiewicz, Jan

    2016-02-01

    The influence of crowding on the protein inner dynamics is examined by putting a single protein molecule close to one or two neighboring protein molecules. The presence of additional molecules influences the amplitudes of protein fluctuations. Also, a weak dynamical coupling of collective velocities of surface atoms of proteins separated by a layer of water is detected. The possible mechanisms of these phenomena are described. The cross-correlation function of the collective velocities of surface atoms of two proteins was decomposed into the Fourier series. The amplitude spectrum displays a peak at low frequencies. Also, the results of principal component analysis suggest that the close presence of an additional protein molecule influences the high-amplitude, low-frequency modes in the most prominent way. This part of the spectrum covers biologically important protein motions. The neighbor-induced changes in the inner dynamics of the protein may be connected with the changes in the velocity power spectrum of interfacial water. The additional protein molecule changes the properties of solvation water and in this way it can influence the dynamics of the second protein. It is suggested that this phenomenon may be described, at first approximation, by a damped oscillator driven by an external random force. This model was successfully applied to conformationally rigid Choristoneura fumiferana antifreeze protein molecules. PMID:26805932

  3. 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.

  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. 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

  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. 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.

  8. 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

  9. Single-Molecule Microscopy Reveals Plasma Membrane Microdomains Created by Protein-Protein Networks that Exclude or Trap Signaling Molecules in T Cells

    PubMed Central

    Douglass, Adam D.; Vale, Ronald D.

    2010-01-01

    Summary Membrane subdomains have been implicated in T cell signaling, although their properties and mechanisms of formation remain controversial. Here, we have used single-molecule and scanning confocal imaging to characterize the behavior of GFP-tagged signaling proteins in Jurkat T cells. We show that the coreceptor CD2, the adaptor protein LAT, and tyrosine kinase Lck cocluster in discrete microdomains in the plasma membrane of signaling T cells. These microdomains require protein-protein interactions mediated through phosphorylation of LAT and are not maintained by interactions with actin or lipid rafts. Using a two color imaging approach that allows tracking of single molecules relative to the CD2/LAT/Lck clusters, we demonstrate that these microdomains exclude and limit the free diffusion of molecules in the membrane but also can trap and immobilize specific proteins. Our data suggest that diffusional trapping through protein-protein interactions creates microdomains that concentrate or exclude cell surface proteins to facilitate T cell signaling. PMID:15960980

  10. Small molecule inhibitors of PSD95-nNOS protein-protein interactions as novel analgesics.

    PubMed

    Lee, Wan-Hung; Xu, Zhili; Ashpole, Nicole M; Hudmon, Andy; Kulkarni, Pushkar M; Thakur, Ganesh A; Lai, Yvonne Y; Hohmann, Andrea G

    2015-10-01

    Aberrant increases in NMDA receptor (NMDAR) signaling contributes to central nervous system sensitization and chronic pain by activating neuronal nitric oxide synthase (nNOS) and generating nitric oxide (NO). Because the scaffolding protein postsynaptic density 95kDA (PSD95) tethers nNOS to NMDARs, the PSD95-nNOS complex represents a therapeutic target. Small molecule inhibitors IC87201 (EC5O: 23.94 μM) and ZL006 (EC50: 12.88 μM) directly inhibited binding of purified PSD95 and nNOS proteins in AlphaScreen without altering binding of PSD95 to ErbB4. Both PSD95-nNOS inhibitors suppressed glutamate-induced cell death with efficacy comparable to MK-801. IC87201 and ZL006 preferentially suppressed phase 2A pain behavior in the formalin test and suppressed allodynia induced by intraplantar complete Freund's adjuvant administration. IC87201 and ZL006 suppressed mechanical and cold allodynia induced by the chemotherapeutic agent paclitaxel (ED50s: 2.47 and 0.93 mg/kg i.p. for IC87201 and ZL006, respectively). Efficacy of PSD95-nNOS disruptors was similar to MK-801. Motor ataxic effects were induced by MK-801 but not by ZL006 or IC87201. Finally, MK-801 produced hyperalgesia in the tail-flick test whereas IC87201 and ZL006 did not alter basal nociceptive thresholds. Our studies establish the utility of using AlphaScreen and purified protein pairs to establish and quantify disruption of protein-protein interactions. Our results demonstrate previously unrecognized antinociceptive efficacy of ZL006 and establish, using two small molecules, a broad application for PSD95-nNOS inhibitors in treating neuropathic and inflammatory pain. Collectively, our results demonstrate that disrupting PSD95-nNOS protein-protein interactions is effective in attenuating pathological pain without producing unwanted side effects (i.e. motor ataxia) associated with NMDAR antagonists. PMID:26071110

  11. 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

  12. 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.

  13. Conformational changes of α-lactalbumin adsorbed at oil-water interfaces: interplay between protein structure and emulsion stability.

    PubMed

    Zhai, Jiali; Hoffmann, Søren V; Day, Li; Lee, Tzong-Hsien; Augustin, Mary Ann; Aguilar, Marie-Isabel; Wooster, Tim J

    2012-02-01

    The conformation and structural dimensions of α-lactalbumin (α-La) both in solution and adsorbed at oil-water interfaces of emulsions were investigated using synchrotron radiation circular dichroism (SRCD) spectroscopy, front-face tryptophan fluorescence (FFTF) spectroscopy, and dual polarization interferometry (DPI). The near-UV SRCD and the FFTF results demonstrated that the hydrophobic environment of the aromatic residues located in the hydrophobic core of native α-La was significantly altered upon adsorption, indicating the unfolding of the hydrophobic core of α-La upon adsorption. The far-UV SRCD results showed that adsorption of α-La at oil-water interfaces created a new non-native secondary structure that was more stable to thermally induced conformational changes. Specifically, the α-helical conformation increased from 29.9% in solution to 45.8% at the tricaprylin-water interface and to 58.5% at the hexadecane-water interface. However, the β-sheet structure decreased from 18.0% in solution to less than 10% at both oil-water interfaces. The DPI study showed that adsorption of α-La to a hydrophobic C18-water surface caused a change in the dimensions of α-La from the native globule-like shape (2.5-3.7 nm) to a compact/dense layer approximately 1.1 nm thick. Analysis of the colloidal stability of α-La stabilized emulsions showed that these emulsions were physically stable against droplet flocculation at elevated temperatures both in the absence and in the presence of 120 mM NaCl. In the absence of salt, the thermal stability of emulsions was due to the strong electrostatic repulsion provided by the adsorbed α-La layer, which was formed after the adsorption and structural rearrangement. In the presence of salt, although the electrostatic repulsion was reduced via electrostatic screening, heating did not induce strong and permanent droplet flocculation. The thermal stability of α-La stabilized emulsions in the presence of salt is a combined effect of

  14. Momentum resolved electron stimulated desorption ion angular distribution, a new technique, probing the low frequency motion of adsorbed molecules on single crystal surfaces

    SciTech Connect

    Ahner, J.; Mocuta, D.; Yates, J.T. Jr.

    1999-07-01

    A new technique, momentum resolved electron stimulated desorption ion angular distribution (ESDIAD), provides a method for taking snapshots of the zero-point position and lateral momentum of particles adsorbed on crystalline surfaces. By employing state-of-the-art electronics and computer technology it is possible to record for each desorbing particle the desorption direction together with the flight time. High momentum and directional resolved images are obtained, with time-of-flight resolution in the picosecond range and data acquisition rates up to 100 kHz. This enables us to deconvolute spatial and momentum contributions to the ESDIAD pattern and to map the low frequency motion of the adsorbed particles. These maps reflect the adsorbate interactions with the substrate and with neighboring species on the substrate. For selected examples it is demonstrated that by measuring the three dimensional momentum vector for each desorbing particle it is possible to probe the lowest energy states of adsorbed species, as well as to measure the momentum distribution when the adsorbed species gains thermal energy. Such information can be used as a basis for thinking about anisotropies in lateral motion of particles on surfaces. One major opportunity involves the study of dissimilar chemisorbed species which, when imaged together in momentum and real space, give new insights into the first stages of interaction between the species, leading ultimately to a chemical reaction. {copyright} {ital 1999 American Vacuum Society.}

  15. 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

  16. 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

  17. 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).

  18. Single-molecule DNA detection using a novel SP1 protein nanopore.

    PubMed

    Wang, Hai-Yan; Li, Yang; Qin, Li-Xia; Heyman, Arnon; Shoseyov, Oded; Willner, Itamar; Long, Yi-Tao; Tian, He

    2013-02-28

    SP1 protein as a new type of biological nanopore is described and is utilized to distinguish single-stranded DNA at the single-molecule level. Using the SP1 nanopore to investigate single molecule detection broadens the existing research areas of pore-forming biomaterials from unsymmetrical biological nanopores to symmetrical biological nanopores. This novel nanopore could provide a good candidate for single-molecule detection and characterization of biomaterial applications. PMID:23340583

  19. 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

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

    PubMed Central

    Bidlingmaier, Scott; Liu, Bin

    2016-01-01

    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

  1. 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

  2. High-resolution mass spectrometry of small molecules bound to membrane proteins.

    PubMed

    Gault, Joseph; Donlan, Joseph A C; Liko, Idlir; Hopper, Jonathan T S; Gupta, Kallol; Housden, Nicholas G; Struwe, Weston B; Marty, Michael T; Mize, Todd; Bechara, Cherine; Zhu, Ya; Wu, Beili; Kleanthous, Colin; Belov, Mikhail; Damoc, Eugen; Makarov, Alexander; Robinson, Carol V

    2016-04-01

    Small molecules are known to stabilize membrane proteins and to modulate their function and oligomeric state, but such interactions are often hard to precisely define. Here we develop and apply a high-resolution, Orbitrap mass spectrometry-based method for analyzing intact membrane protein-ligand complexes. Using this platform, we resolve the complexity of multiple binding events, quantify small molecule binding and reveal selectivity for endogenous lipids that differ only in acyl chain length. PMID:26901650

  3. 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.

  4. 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

  5. [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

  6. 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.

  7. 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

  8. Real-time single-molecule observations of proteins at the solid-liquid interface

    NASA Astrophysics Data System (ADS)

    Langdon, Blake Brianna

    Non-specific protein adsorption to solid surfaces is pervasive and observed across a broad spectrum of applications including biomaterials, separations, pharmaceuticals, and biosensing. Despite great interest in and considerable literature dedicated to the phenomena, a mechanistic understanding of this complex phenomena is lacking and remains controversial, partially due to the limits of ensemble-averaging techniques used to study it. Single-molecule tracking (SMT) methods allow us to study distinct protein dynamics (e.g. adsorption, desorption, diffusion, and intermolecular associations) on a molecule-by-molecule basis revealing the protein population and spatial heterogeneity inherent in protein interfacial behavior. By employing single-molecule total internal reflection fluorescence microscopy (SM-TIRFM), we have developed SMT methods to directly observe protein interfacial dynamics at the solid-liquid interface to build a better mechanistic understanding of protein adsorption. First, we examined the effects of surface chemistry (e.g. hydrophobicity, hydrogen-bonding capacity), temperature, and electrostatics on isolated protein desorption and interfacial diffusion for fibrinogen (Fg) and bovine serum albumin (BSA). Next, we directly and indirectly probed the effects of protein-protein interactions on interfacial desorption, diffusion, aggregation, and surface spatial heterogeneity on model and polymeric thin films. These studies provided many useful insights into interfacial protein dynamics including the following observations. First, protein adsorption was reversible, with the majority of proteins desorbing from all surface chemistries within seconds. Isolated protein-surface interactions were relatively weak on both hydrophobic and hydrophilic surfaces (apparent desorption activation energies of only a few kBT). However, proteins could dynamically and reversibly associate at the interface, and these interfacial associations led to proteins remaining on the

  9. 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

  10. 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. PMID:24001944

  11. 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.

  12. 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

  13. Development of bispecific molecules for the in situ detection of protein-protein interactions and protein phosphorylation.

    PubMed

    van Dieck, Jan; Schmid, Volker; Heindl, Dieter; Dziadek, Sebastian; Schraeml, Michael; Gerg, Michael; Massoner, Petra; Engel, Alfred M; Tiefenthaler, Georg; Vural, Serhat; Stritt, Simon; Tetzlaff, Fabian; Soukupova, Monika; Kopetzki, Erhard; Bossenmaier, Birgit; Thomas, Marlene; Klein, Christian; Mertens, Alfred; Heller, Astrid; Tacke, Michael

    2014-03-20

    Investigation of protein-protein interactions (PPIs) and protein phosphorylation in clinical tissue samples can offer valuable information about the activation status and function of proteins involved in disease progression. However, existing antibody-based methods for phosphorylation detection have been found to lack specificity, and methods developed for examining PPIs in vitro cannot be easily adapted for tissues samples. In this study, we eliminated some of these limitations by developing a specific immunohistochemical staining method that uses "dual binders" (DBs), which are bispecific detection agents consisting of two Fab fragment molecules joined by a flexible linker, to detect PPIs and protein phosphorylation. We engineered DBs by selecting Fab fragments with fast off-rate kinetics, which allowed us to demonstrate that stable target binding was achieved only upon simultaneous, cooperative binding to both epitopes. We show that DBs specifically detect the activated HER2/HER3 complex in formalin-fixed, paraffin-embedded cancer cells and exhibit superior detection specificity for phospho-HER3 compared to the corresponding monoclonal antibody. Overall, the performance of DBs makes them attractive tools for future development for clinical applications. PMID:24529991

  14. 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.

  15. 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.

  16. Terminal Supraparticle Assemblies from Similarly Charged Protein Molecules and Nanoparticles

    PubMed Central

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

    2015-01-01

    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 as demonstrated by enzymatic nitrate reduction initiated by light absorption in the nanoparticle. PMID:24845400

  17. 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.

  18. 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

  19. 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

  20. Effective Long-Range Attraction between Protein Molecules in Solutions Studied by Small Angle Neutron Scattering

    SciTech Connect

    Liu Yun; Chen, W.-R.; Chen, S.-H.; Fratini, Emiliano; Baglioni, Piero

    2005-09-09

    Small angle neutron scattering intensity distributions taken from cytochrome C and lysozyme protein solutions show a rising intensity at a very small wave vector Q, which can be interpreted in terms of the presence of a weak long-range attraction between protein molecules. This interaction has a range several times that of the diameter of the protein molecule, much greater than the range of the screened electrostatic repulsion. We show evidence that this long-range attraction is closely related to the type of anion present and ion concentration in the solution.

  1. 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. PMID:26147918

  2. 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

  3. Probing small-molecule microarrays with tagged proteins in cell lysates.

    PubMed

    Pop, Marius S; Wassaf, Dina; Koehler, Angela N

    2014-01-01

    The technique of small-molecule microarray (SMM) screening is based on the ability of small molecules to bind to various soluble proteins. This type of interaction is easily detected by the presence of a fluorescence signal produced by labeled antibodies that specifically recognize a unique sequence (tag) present on the target protein. The fluorescent signal intensity values are determined based on signal-to-noise ratios (SNRs). SMM screening is a high-throughput, unbiased method that can rapidly identify novel direct ligands for various protein targets. This binding-based assay format is generally applicable to most proteins, but it is especially useful for protein targets that do not possess an enzymatic activity. SMMs enable screening a protein in a purified form or in the context of a cellular lysate, likely providing a more physiologically relevant screening environment. PMID:25445177

  4. Role of Solvation Effects in Protein Denaturation: From Thermodynamics to Single Molecules and Back

    PubMed Central

    England, Jeremy L.; Haran, Gilad

    2011-01-01

    Protein stability often is studied in vitro through the use of urea and guanidinium chloride, chemical cosolvents that disrupt protein native structure. Much controversy still surrounds the underlying mechanism by which these molecules denature proteins. Here we review current thinking on various aspects of chemical denaturation. We begin by discussing classic models of protein folding and how the effects of denaturants may fit into this picture through their modulation of the collapse, or coil-globule transition, which typically precedes folding. Subsequently, we examine recent molecular dynamics simulations that have shed new light on the possible microscopic origins of the solvation effects brought on by denaturants. It seems likely that both denaturants operate by facilitating solvation of hydrophobic regions of proteins. Finally, we present recent single-molecule fluorescence studies of denatured proteins, the analysis of which corroborates the role of denaturants in shifting the equilibrium of the coil-globule transition. PMID:21219136

  5. Nanomolar-Potency Small Molecule Inhibitor of STAT5 Protein

    PubMed Central

    2014-01-01

    We herein report the design and synthesis of the first nanomolar binding inhibitor of STAT5 protein. Lead compound 13a, possessing a phosphotyrosyl-mimicking salicylic acid group, potently and selectively binds to STAT5 over STAT3, inhibits STAT5–SH2 domain complexation events in vitro, silences activated STAT5 in leukemic cells, as well as STAT5′s downstream transcriptional targets, including MYC and MCL1, and, as a result, leads to apoptosis. We believe 13a represents a useful probe for interrogating STAT5 function in cells as well as being a potential candidate for advanced preclinical trials. PMID:25419444

  6. A Materiomics Approach to Spider Silk: Protein Molecules to Webs

    NASA Astrophysics Data System (ADS)

    Tarakanova, Anna; Buehler, Markus J.

    2012-02-01

    The exceptional mechanical properties of hierarchical self-assembling silk biopolymers have been extensively studied experimentally and in computational investigations. A series of recent studies has been conducted to examine structure-function relationships across different length scales in silk, ranging from atomistic models of protein constituents to the spider web architecture. Silk is an exemplary natural material because its superior properties stem intrinsically from the synergistic cooperativity of hierarchically organized components, rather than from the superior properties of the building blocks themselves. It is composed of beta-sheet nanocrystals interspersed within less orderly amorphous domains, where the underlying molecular structure is dominated by weak hydrogen bonding. Protein chains are organized into fibrils, which pack together to form threads of a spider web. In this article we survey multiscale studies spanning length scales from angstroms to centimeters, from the amino acid sequence defining silk components to an atomistically derived spider web model, with the aim to bridge varying levels of hierarchy to elucidate the mechanisms by which structure at each composite level contributes to organization and material phenomena at subsequent levels. The work demonstrates that the web is a highly adapted system where both material and hierarchical structure across all length scales is critical for its functional properties.

  7. Cholesterol ester transfer protein: a molecule with three faces?

    PubMed

    Stevenson, C G

    1998-12-01

    The pathogenesis of atherosclerosis continues to be a focus of intensive study. One of the more recent players in the atherosclerosis drama is cholesterol ester transfer protein (CETP). CETP is primarily involved in lipid transfer between lipoproteins, for example, from high-density lipoproteins (HDL) to apo B-containing lipoproteins, but CETP has also been found to take up cholesterol directly from cells without the co-participation of lipoproteins, and it is still not clear whether CETP should be classified as a beneficial or as a harmful protein. Some of the important evidence for these conflicting theories is examined here, with special reference to situations where CETP appears to be proatherogenic, instances where CETP seems to assume an antiatherogenic role, and situations where CETP seems to be both proatherogenic and antiatherogenic. In addition, the metabolic context of CETP and the modification of CETP substrates play crucial roles that are not always recognized when judgements about the role of CETP in atherosclerosis are recorded. PMID:9885774

  8. Scanning a DNA molecule for bound proteins using hybrid magnetic and optical tweezers.

    PubMed

    van Loenhout, Marijn T J; De Vlaminck, Iwijn; Flebus, Benedetta; den Blanken, Johan F; Zweifel, Ludovit P; Hooning, Koen M; Kerssemakers, Jacob W J; Dekker, Cees

    2013-01-01

    The functional state of the genome is determined by its interactions with proteins that bind, modify, and move along the DNA. To determine the positions and binding strength of proteins localized on DNA we have developed a combined magnetic and optical tweezers apparatus that allows for both sensitive and label-free detection. A DNA loop, that acts as a scanning probe, is created by looping an optically trapped DNA tether around a DNA molecule that is held with magnetic tweezers. Upon scanning the loop along the λ-DNA molecule, EcoRI proteins were detected with ~17 nm spatial resolution. An offset of 33 ± 5 nm for the detected protein positions was found between back and forwards scans, corresponding to the size of the DNA loop and in agreement with theoretical estimates. At higher applied stretching forces, the scanning loop was able to remove bound proteins from the DNA, showing that the method is in principle also capable of measuring the binding strength of proteins to DNA with a force resolution of 0.1 pN/[Formula: see text]. The use of magnetic tweezers in this assay allows the facile preparation of many single-molecule tethers, which can be scanned one after the other, while it also allows for direct control of the supercoiling state of the DNA molecule, making it uniquely suitable to address the effects of torque on protein-DNA interactions. PMID:23755219

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

    PubMed Central

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

    2011-01-01

    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. PMID:21931220

  10. Dynamics of Protein Folding and Cofactor Binding Monitored by Single-Molecule Force Spectroscopy

    PubMed Central

    Cao, Yi; Li, Hongbin

    2011-01-01

    Many proteins in living cells require cofactors to carry out their biological functions. To reach their functional states, these proteins need to fold into their unique three-dimensional structures in the presence of their cofactors. Two processes, folding of the protein and binding of cofactors, intermingle with each other, making the direct elucidation of the folding mechanism of proteins in the presence of cofactors challenging. Here we use single-molecule atomic force microscopy to directly monitor the folding and cofactor binding dynamics of an engineered metal-binding protein G6-53 at the single-molecule level. Using the mechanical stability of different conformers of G6-53 as sensitive probes, we directly identified different G6-53 conformers (unfolded, apo- and Ni2+-bound) populated along the folding pathway of G6-53 in the presence of its cofactor Ni2+. By carrying out single-molecule atomic force microscopy refolding experiments, we monitored kinetic evolution processes of these different conformers. Our results suggested that the majority of G6-53 folds through a binding-after-folding mechanism, whereas a small fraction follows a binding-before-folding pathway. Our study opens an avenue to utilizing force spectroscopy techniques to probe the folding dynamics of proteins in the presence of cofactors at the single-molecule level, and we anticipated that this method can be used to study a wide variety of proteins requiring cofactors for their function. PMID:22004755

  11. Single-molecule fluorescence imaging to quantify membrane protein dynamics and oligomerization in living plant cells.

    PubMed

    Wang, Xiaohua; Li, Xiaojuan; Deng, Xin; Luu, Doan-Trung; Maurel, Christophe; Lin, Jinxing

    2015-12-01

    Measuring the mobility and interactions of proteins is key to understanding cellular signaling mechanisms; however, quantitative analysis of protein dynamics in living plant cells remains a major challenge. Here we describe an automated, single-molecule protocol based on total internal reflection fluorescence microscopy (TIRFM) imaging that allows protein tracking and subunit counting in living plant cells. This protocol uses TIRFM to image transgenic plant tissues expressing fluorescently tagged proteins that are localized to the plasma membrane. Next, a tracking algorithm quantifies dynamic changes in fluorescent protein motion types, temporary particle displacement and protein photobleaching steps. This protocol allows researchers to study the kinetic characteristics of heterogeneously distributed proteins. The approach has potential applications for studies of protein dynamics and subunit stoichiometry for a wide variety of plasma membrane and intracellular proteins in living plant cells and other biological specimens visualized by TIRFM or other fluorescence imaging techniques. The whole protocol can be completed in 5-6 h. PMID:26584445

  12. Small-Molecule Hydrophobic Tagging Induced Degradation of HaloTag Fusion Proteins

    PubMed Central

    Neklesa, Taavi K.; Tae, Hyun Seop; Schneekloth, Ashley R.; Stulberg, Michael J.; Corson, Timothy W.; Sundberg, Thomas B.; Raina, Kanak; Holley, Scott A.; Crews, Craig M.

    2011-01-01

    The ability to regulate any protein of interest in living systems with small molecules remains a challenge. We hypothesized that appending a hydrophobic moiety to the surface of a protein would mimic the partially denatured state of the protein, thus engaging the cellular quality control machinery to induce its proteasomal degradation. We designed and synthesized bifunctional small molecules that bind a bacterial dehalogenase (HaloTag protein) and present a hydrophobic group on its surface. Remarkably, hydrophobic tagging of the HaloTag protein with an adamantyl moiety induced the degradation of cytosolic, isoprenylated, and transmembrane fusion proteins in cell culture. We demonstrated the in vivo utility of hydrophobic tagging by degrading proteins expressed in zebrafish embryos and by inhibiting RasG12V-driven tumor progression in mice. Therefore, hydrophobic tagging of HaloTag fusion proteins affords small molecule control over any protein of interest, making it an ideal system for validating potential drug targets in disease models. PMID:21725302

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

    DOE PAGESBeta

    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

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

    PubMed Central

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

    2015-01-01

    Disordered proteins 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). 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). PMID:26507530

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

    PubMed

    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

  16. 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).

  17. 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

  18. 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

  19. Conformational Switching in a Light-Harvesting Protein as Followed by Single-Molecule Spectroscopy

    PubMed Central

    Gall, Andrew; Ilioaia, Cristian; Krüger, Tjaart P.J.; Novoderezhkin, Vladimir I.; Robert, Bruno; van Grondelle, Rienk

    2015-01-01

    Among the ultimate goals of protein physics, the complete, experimental description of the energy paths leading to protein conformational changes remains a challenge. Single protein fluorescence spectroscopy constitutes an approach of choice for addressing protein dynamics, and, among naturally fluorescing proteins, light-harvesting (LH) proteins from purple bacteria constitute an ideal object for such a study. LHs bind bacteriochlorophyll a molecules, which confer on them a high intrinsic fluorescence yield. Moreover, the electronic properties of these pigment-proteins result from the strong excitonic coupling between their bound bacteriochlorophyll a molecules in combination with the large energetic disorder due to slow fluctuations in their structure. As a result, the position and probability of their fluorescence transition delicately depends on the precise realization of the disorder of the set of bound pigments, which is governed by the LH protein dynamics. Analysis of these parameters using time-resolved single-molecule fluorescence spectroscopy thus yields direct access to the protein dynamics. Applying this technique to the LH2 protein from Rhodovulum (Rdv.) sulfidophilum, the structure—and consequently the fluorescence properties—of which depends on pH, allowed us to follow a single protein, pH-induced, reversible, conformational transition. Hence, for the first time, to our knowledge, a protein transition can be visualized through changes in the electronic structure of the intrinsic cofactors, at a level of a single LH protein, which opens a new, to our knowledge, route for understanding the changes in energy landscape that underlie protein function and adaptation to the needs of living organisms. PMID:26039172

  20. 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

  1. 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

  2. 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

  3. A rapid identification of hit molecules for target proteins via physico-chemical descriptors.

    PubMed

    Mukherjee, Goutam; Jayaram, B

    2013-06-21

    We report here a novel computationally fast protocol (RASPD) for identifying good candidates for any target protein from any molecule/million molecule database. A QSAR-type equation sets up the extent of complementarity of the physico-chemical properties of the target protein and the candidate molecule and an estimate of the binding energy is generated. A correlation coefficient of 0.84 and an average error ±1.45 kcal mol(-1) are obtained for the calculated protein-ligand binding energies against experiment for more than 380 protein-ligand complexes. RASPD is seen to perform better than other popular scoring functions in predicting binding energies. The most interesting feature of this methodology is that it takes only a fraction of a second for calculating the binding energy of any ligand without docking in the active site of the target protein as opposed to several minutes for regular docking and scoring methods, while the accuracy in sorting good candidates remains comparable to that of conventional techniques. An entire million compound library, a (~10(5) compound) natural product library and a (~10(5) compound) NCI database can be scanned against a specified target protein within a few minutes for identifying hit molecules. The RASPD methodology is freely accessible at . PMID:23646352

  4. 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

  5. Design of small molecules targeting I-BAR proteins.

    PubMed

    Cao, Meng; Chang, Weiwei; Zheng, Ming; Xie, Li; Zhang, Yu; Cai, Jin; Chen, Junqing; Zhan, Xi; Ji, Min; Gu, Ning

    2015-01-01

    Missing in metastasis (MIM, also MTSS1) is a member of the inverse Bin-Amphiphysin-Rvs (I-BAR) family that senses and stabilizes negative membrane protrusions. Abnormal expression of MIM has been frequently associated with a subset of human cancers and may play different roles in different stages of tumor progression. Overexpression of MIM-I-BAR in 293A cells potentiated the cell growth and increased the toxic response to paclitaxel. To modulate the function of MIM within cells, we designed several short peptide derivatives to target I-BAR dimerization. One of these derivatives had a cyclic configuration with a potency to disrupt the dimerization of MIM or ABBA proteins in vitro, and to be readily internalized into cells. Exposure of cells expressing MIM-I-BAR to this compound abolished increased susceptibility to paclitaxel and partially inhibited the IBAR- mediated endocytosis. Our data suggests that this cyclic peptide can be used as a tool to study the function of intracellular MIM and as a lead to develop a therapy targeting human diseases involving abnormal MIM expressions. PMID:25410498

  6. 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

  7. 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

  8. Protein kinase small molecule inhibitors for rheumatoid arthritis: Medicinal chemistry/clinical perspectives

    PubMed Central

    Malemud, Charles J; Blumenthal, David E

    2014-01-01

    Medicinal chemistry strategies have contributed to the development, experimental study of and clinical trials assessment of the first type of protein kinase small molecule inhibitor to target the Janus kinase/Signal Transducers and Activators of Transcription (JAK/STAT) signaling pathway. The orally administered small molecule inhibitor, tofacitinib, is the first drug to target the JAK/STAT pathway for entry into the armamentarium of the medical therapy of rheumatoid arthritis. The introduction of tofacitinib into general rheumatologic practice coupled with increasing understanding that additional cellular signal transduction pathways including the mitogen-activated protein kinase and phosphatidylinositide-3-kinase/Akt/mammalian target of rapamycin pathways as well as spleen tyrosine kinase also contribute to immune-mediated inflammatory in rheumatoid arthritis makes it likely that further development of orally administered protein kinase small molecule inhibitors for rheumatoid arthritis will occur in the near future. PMID:25232525

  9. Direct visual evidence for chemical mechanism of SERRS of the S-complex of pyrimidine molecule adsorbed on silver nanoparticle via charge transfer.

    PubMed

    Zhang, Xin; Wang, Peijie; Sheng, Shaoxiang; Zhang, Lisheng; Fang, Yan

    2014-01-01

    In this paper, the S-complex of pyrimidine molecule absorbed on silver clusters was employed as a model molecule to study the enhancement mechanism in surface-enhanced resonance Raman scattering (SERRS). We described the chemical enhancement of SERRS through charge transfer (CT) from Ag20 to pyrimidine on resonance excitation, and electromagnetic enhancement through intracluster charge redistribution (CR) on the electronic intracluster collective oscillation excitation. It is shown that SERRS process of the pyrimidine molecule absorbed on silver clusters with different incident wavelength are dominated by different enhancement mechanisms. Both experimental and theoretical works have been performed to understand the CT process in SERRS. PMID:24287052

  10. Enhancement of spin polarization in transport through protein-like single-helical molecules

    NASA Astrophysics Data System (ADS)

    Wu, Hai-Na; Wang, Xiao; Zhang, Ya-Jing; Yi, Guang-Yu; Gong, Wei-Jiang

    2016-06-01

    We investigate the spin-polarized electron transport through the single-helical molecules connected with two normal metallic leads. On the basis of an effective model Hamiltonian, influences of the structural parameters on the conductance and the spin polarization are calculated by using the Landauer-Büttiker formula. The optimal structural parameters for the maximal spin polarization are analyzed. Our results show that the dephasing term is an important factor to enhance the spin polarization, in addition to the intrinsic parameters of the single-helical molecule. This work can be helpful in optimizing the spin polarization in the protein-like single-helical molecules.

  11. 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.

  12. DEPTH: a web server to compute depth and predict small-molecule binding cavities in proteins.

    PubMed

    Tan, Kuan Pern; Varadarajan, Raghavan; Madhusudhan, M S

    2011-07-01

    Depth measures the extent of atom/residue burial within a protein. It correlates with properties such as protein stability, hydrogen exchange rate, protein-protein interaction hot spots, post-translational modification sites and sequence variability. Our server, DEPTH, accurately computes depth and solvent-accessible surface area (SASA) values. We show that depth can be used to predict small molecule ligand binding cavities in proteins. Often, some of the residues lining a ligand binding cavity are both deep and solvent exposed. Using the depth-SASA pair values for a residue, its likelihood to form part of a small molecule binding cavity is estimated. The parameters of the method were calibrated over a training set of 900 high-resolution X-ray crystal structures of single-domain proteins bound to small molecules (molecular weight <1.5  KDa). The prediction accuracy of DEPTH is comparable to that of other geometry-based prediction methods including LIGSITE, SURFNET and Pocket-Finder (all with Matthew's correlation coefficient of ∼0.4) over a testing set of 225 single and multi-chain protein structures. Users have the option of tuning several parameters to detect cavities of different sizes, for example, geometrically flat binding sites. The input to the server is a protein 3D structure in PDB format. The users have the option of tuning the values of four parameters associated with the computation of residue depth and the prediction of binding cavities. The computed depths, SASA and binding cavity predictions are displayed in 2D plots and mapped onto 3D representations of the protein structure using Jmol. Links are provided to download the outputs. Our server is useful for all structural analysis based on residue depth and SASA, such as guiding site-directed mutagenesis experiments and small molecule docking exercises, in the context of protein functional annotation and drug discovery. PMID:21576233

  13. 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. PMID:25042595

  14. 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

  15. 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

  16. Hot spots and transient pockets: predicting the determinants of small-molecule binding to a protein-protein interface.

    PubMed

    Metz, Alexander; Pfleger, Christopher; Kopitz, Hannes; Pfeiffer-Marek, Stefania; Baringhaus, Karl-Heinz; Gohlke, Holger

    2012-01-23

    Protein-protein interfaces are considered difficult targets for small-molecule protein-protein interaction modulators (PPIMs ). Here, we present for the first time a computational strategy that simultaneously considers aspects of energetics and plasticity in the context of PPIM binding to a protein interface. The strategy aims at identifying the determinants of small-molecule binding, hot spots, and transient pockets, in a protein-protein interface in order to make use of this knowledge for predicting binding modes of and ranking PPIMs with respect to their affinity. When applied to interleukin-2 (IL-2), the computationally inexpensive constrained geometric simulation method FRODA outperforms molecular dynamics simulations in sampling hydrophobic transient pockets. We introduce the PPIAnalyzer approach for identifying transient pockets on the basis of geometrical criteria only. A sequence of docking to identified transient pockets, starting structure selection based on hot spot information, RMSD clustering and intermolecular docking energies, and MM-PBSA calculations allows one to enrich IL-2 PPIMs from a set of decoys and to discriminate between subgroups of IL-2 PPIMs with low and high affinity. Our strategy will be applicable in a prospective manner where nothing else than a protein-protein complex structure is known; hence, it can well be the first step in a structure-based endeavor to identify PPIMs. PMID:22087639

  17. Probing redox proteins on a gold surface by single molecule fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Elmalk, Abdalmohsen T.; Salverda, Jante M.; Tabares, Leandro C.; Canters, Gerard W.; Aartsma, Thijs J.

    2012-06-01

    The interaction between the fluorescently labeled redox protein, azurin, and a thin gold film is characterized using single-molecule fluorescence intensity and lifetime measurements. Fluorescence quenching starts at distances below 2.3 nm from the gold surface. At shorter distances the quantum yield may decrease down to fourfold for direct attachment of the protein to bare gold. Outside of the quenching range, up to fivefold enhancement of the fluorescence is observed on average with increasing roughness of the gold layer. Fluorescence-detected redox activity of individual azurin molecules, with a lifetime switching ratio of 0.4, is demonstrated for the first time close to a gold surface.

  18. A Bidirectional System for the Dynamic Small Molecule Control of Intracellular Fusion Proteins

    PubMed Central

    Kuzin, Alexander P.; Lew, Scott; Seetharaman, Jayaraman; Acton, Thomas B.; Kornhaber, Gregory J.; Xiao, Rong; Montelione, Gaetano Thomas; Tong, Liang; Crews, Craig M.

    2014-01-01

    Small molecule control of intracellular protein levels allows temporal and dose-dependent regulation of protein function. Recently, we developed a method to degrade proteins fused to a mutant dehalogenase (HaloTag2) using small molecule hydrophobic tags (HyTs). Here, we introduce a complementary method to stabilize the same HaloTag2 fusion proteins, resulting in a unified system allowing bidirectional control of cellular protein levels in a temporal and dose-dependent manner. From a small molecule screen, we identified N-(3,5-dichloro-2-ethoxybenzyl)-2H-tetrazol-5-amine as a nanomolar HALoTag2 Stabilizer (HALTS1) that reduces the Hsp70:HaloTag2 interaction, thereby preventing HaloTag2 ubiquitination. Finally, we demonstrate the utility of the HyT/HALTS system in probing the physiological role of therapeutic targets by modulating HaloTag2-fused oncogenic H-Ras, which resulted in either the cessation (HyT) or acceleration (HALTS) of cellular transformation. In sum, we present a general platform to study protein function, whereby any protein of interest fused to HaloTag2 can be either degraded 10-fold or stabilized 5-fold using two corresponding compounds. PMID:23978068

  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. 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

  1. 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

  2. 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

  3. Tuneable surface shear forces to physically displace nonspecific molecules in protein biomarker detection.

    PubMed

    Vaidyanathan, Ramanathan; Rauf, Sakandar; Shiddiky, Muhammad J A; Trau, Matt

    2014-11-15

    We report a simple method to remove nonspecifically adsorbed species from sensor surface and also improve the detection sensitivity of the sensor using tuneable alternating current (ac) electrohydrodynamics (ac-EHD) forces. These forces generated within few nanometers of an electrode surface (i.e., double layer) engender fluid flow within a serpentine channel containing a long array of the asymmetric electrode pairs, and can easily be tuned externally by changing the frequency and amplitude of the ac-EHD field. Under the optimized experimental conditions, we achieved a 3.5-fold reduction in nonspecific adsorption of non-target proteins with a 1000-fold enhancement in detection sensitivity of the device for the analysis of human epidermal growth factor receptor 2 (HER2) protein spiked in serum. This approach can be applicable in diverse fields including biosensors, cellular and molecular separation systems and biomedical applications to remove/reduce nonspecific adsorption of molecular and cellular species. PMID:24880656

  4. Protein-Observed Fluorine NMR: A Bioorthogonal Approach for Small Molecule Discovery.

    PubMed

    Arntson, Keith E; Pomerantz, William C K

    2016-06-01

    The (19)F isotope is 100% naturally abundant and is the second most sensitive and stable NMR-active nucleus. Unlike the ubiquitous hydrogen atom, fluorine is nearly absent in biological systems, making it a unique bioorthogonal atom for probing molecular interactions in biology. Over 73 fluorinated proteins have been studied by (19)F NMR since the seminal studies of Hull and Sykes in 1974. With advances in cryoprobe production and fluorinated amino acid incorporation strategies, protein-based (19)F NMR offers opportunities to the medicinal chemist for characterizing and ultimately discovering new small molecule protein ligands. This review will highlight new advances using (19)F NMR for characterizing small molecule interactions with both small and large proteins as well as detailing NMR resonance assignment challenges and amino acid incorporation approaches. PMID:26599421

  5. A Small Molecule Inhibitor of Inducible Heat Shock Protein 70 (HSP70)

    PubMed Central

    Leu, J. I-Ju; Pimkina, Julia; Frank, Amanda; Murphy, Maureen E.; George, Donna L.

    2009-01-01

    SUMMARY The multifunctional, stress-inducible, molecular chaperone HSP70 has important roles in aiding protein folding and maintaining protein homeostasis. HSP70 expression is elevated in many cancers, contributing to tumor cell survival and resistance to therapy. We have determined that a small molecule called 2-Phenylethynesulfonamide (PES) interacts selectively with HSP70, and leads to a disruption of the association between HSP70 and several of its co-chaperones and substrate proteins. Treatment of cultured tumor cells with PES promotes cell death that is associated with protein aggregation, impaired autophagy, and inhibition of lysosomal function. Moreover, this small molecule is able to suppress tumor development and enhance survival in a mouse model of Myc-induced lymphomagenesis. The data demonstrate that PES disrupts actions of HSP70 in multiple cell signaling pathways offering an opportunity to better understand the diverse functions of this molecular chaperone, and also to aid in the development of new cancer therapies. PMID:19818706

  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. Improving protein-ligand docking with flexible interfacial water molecules using SWRosettaLigand.

    PubMed

    Li, Linqing; Xu, Weiwei; Lü, Qiang

    2015-11-01

    Computational protein-ligand docking is of great importance in drug discovery and design. Conformational changes greatly affect the results of protein-ligand docking, especially when water molecules take part in mediating protein ligand interactions or when large conformational changes are observed in the receptor backbone interface. We have developed an improved protocol, SWRosettaLigand, based on the RosettaLigand protocol. This approach incorporates the flexibility of interfacial water molecules and modeling of the interface of the receptor into the original RosettaLigand. In a coarse sampling step, SWRosettaLigand pre-optimizes the initial position of the water molecules, docks the ligand to the receptor with explicit water molecules, and minimizes the predicted structure with water molecules. The receptor backbone interface is treated as a loop and perturbed and refined by kinematic closure, or cyclic coordinate descent algorithm, with the presence of the ligand. In two cross-docking test sets, it was identified that for 8 out of 14, and 16 out of 22, test instances, the top-ranked structures by SWRosettaLigand achieved better accuracy than other protocols. PMID:26515196

  10. Detection of protein-small molecule binding using a self-referencing external cavity laser biosensor.

    PubMed

    Zhang, Meng; Peh, Jessie; Hergenrother, Paul J; Cunningham, Brian T

    2014-04-23

    High-throughput screening has enabled the identification of small molecule modulators of important drug targets via well-established colorimetric or fluorimetric activity assays. However, existing methods to identify small molecule binders of nonenzymatic protein targets lack either the simplicity (e.g., require labeling one of the binding partners with a reporter) or throughput inherent in enzymatic assays widely used for HTS. Thus, there is intense interest in the development of high-throughput technologies for label-free detection of protein-small molecule interactions. Here we describe a novel self-referencing external cavity laser (ECL) biosensor approach that achieves high resolution and high sensitivity, while eliminating thermal noise with subpicometer wavelength accuracy. Using the self-referencing ECL biosensor, we demonstrate detection of binding between small molecules and a variety of immobilized protein targets, pairs that have binding affinities or inhibition constants ranging from subnanomolar to low micromolar. Finally, a "needle-in-the-haystack" screen for inhibitors against carbonic anhydrase isozyme II is performed, in which known inhibitors are clearly differentiated from inactive molecules within a compound library. PMID:24720510

  11. 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.

  12. Visualizing and quantifying protein polySUMOylation at the single-molecule level.

    PubMed

    Yang, Yong; Zhang, Chun-yang

    2014-01-21

    Protein polySUMOylation, the attachment of small ubiquitin-like modifier (SUMO) chains to the target protein, is associated with a variety of physiological processes. However, the analysis of protein polySUMOylation is often complicated by the heterogeneity of SUMO-target conjugates. Here, we develop a new strategy to visualize and quantify polySUMOylation at the single-molecule level by integrating the tetracysteine (TC) tag labeling technology and total internal reflection fluorescence (TIRF)-based single-molecule imaging. As a proof-of-concept, we employ the human SUMO-2 as the model. The addition of TC tag to SUMO-2 can specifically translate the SUMO-mediated modification into visible fluorescence signal without disturbing the function of SUMO-2. The SUMO monomers display homogeneous fluorescence spots at the single-molecule level, whereas the mixed SUMO chains exhibit nonuniform fluorescence spots with a wide range of intensities. Analysis of the number and the brightness of fluorescence spots enable quantitative measurement of the polySUMOylation degree inside the cells under different physiological conditions. Due to the frequent occurrence of posttranslational modification by polymeric chains in cells, this single-molecule strategy has the potential to be broadly applied for studying protein posttranslational modification in normal cellular physiology and disease etiology. PMID:24383460

  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. Mechanical Biochemistry of Proteins One Molecule at a Time*S

    PubMed Central

    Oberhauser, Andres F.; Carrión-Vázquez, Mariano

    2008-01-01

    The activity of proteins and their complexes often involves the conversion of chemical energy (stored or supplied) into mechanical work through conformational changes. Mechanical forces are also crucial for the regulation of the structure and function of cells and tissues. Thus, the shape of eukaryotic cells (and by extension, that of the multicellular organisms they form) is the result of cycles of mechanosensing, mechanotransduction, and mechanoresponse. Recently developed single-molecule atomic force microscopy techniques can be used to manipulate single molecules, both in real time and under physiological conditions, and are ideally suited to directly quantify the forces involved in both intra- and intermolecular protein interactions. In combination with molecular biology and computer simulations, these techniques have been applied to characterize the unfolding and refolding reactions in a variety of proteins. Single-molecule mechanical techniques are providing fundamental information on the structure and function of proteins and are becoming an indispensable tool to understand how these molecules fold and work. PMID:18195002

  15. A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening.

    PubMed

    Yu, Yong; New, Siu Yee; Lin, Jiaxian; Su, Xiaodi; Tan, Yen Nee

    2015-01-01

    We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold nanoclusters (Au NCs) within the drug-loaded protein, based on the differential fluorescence signal emitted by the Au NCs. Albumin proteins such as human serum albumin (HSA) and bovine serum albumin (BSA) are selected as the model proteins. Four small molecular drugs (e.g., ibuprofen, warfarin, phenytoin, and sulfanilamide) of different binding affinities to the albumin proteins are tested. It was found that the formation rate of fluorescent Au NCs inside the drug loaded albumin protein under denaturing conditions (i.e., 60 °C or in the presence of urea) is slower than that formed in the pristine protein (without drugs). Moreover, the fluorescent intensity of the as-formed NCs is found to be inversely correlated to the binding affinities of these drugs to the albumin proteins. Particularly, the higher the drug-protein binding affinity, the slower the rate of Au NCs formation, and thus a lower fluorescence intensity of the resultant Au NCs is observed. The fluorescence intensity of the resultant Au NCs therefore provides a simple measure of the relative binding strength of different drugs tested. This method is also extendable to measure the specific drug-protein binding constant (KD) by simply varying the drug content preloaded in the protein at a fixed protein concentration. The measured results match well with the values obtained using other prestige but more complicated methods. PMID:26555855

  16. Small-molecule-mediated rescue of protein function by an inducible proteolytic shunt

    PubMed Central

    Pratt, Matthew R.; Schwartz, Edmund C.; Muir, Tom W.

    2007-01-01

    Controlling protein function through posttranslational manipulations has emerged as an attractive complementary technology to existing genetic systems. Often these methods involve developing pharmacological agents to probe protein function without the need to generate a unique compound for each protein family. One common strategy uses small molecules that act as chemical inducers of dimerization by mediating the interaction of two proteins. Herein we report the use of a chemical inducer of dimerization for the development of a posttranslational technology for the manipulation of protein function. This system, split ubiquitin for the rescue of function (SURF), places the complementation of genetically split ubiquitin under the control of rapamycin-induced dimerization of FK506-binding protein and FKBP12-rapamycin-binding protein. Before complementation a “degron” dooms a protein of interest for destruction by the proteasome. Addition of rapamycin results in a proteolytic shunt away from degradation by inducing ubiquitin complementation and cleavage of the protein of interest from the degron. Importantly, the native protein is rescued. We characterized this system with firefly luciferase and went on to apply it to members of three important classes of proteins: proteases (caspase-3), kinases (v-Src), and transcription factors (Smad3). This general strategy should allow for inducible rescue of a variety of proteins in such a way that their native structure and function are maintained. PMID:17563385

  17. Robust assessment of protein complex formation in vivo via single-molecule intensity distributions of autofluorescent proteins

    NASA Astrophysics Data System (ADS)

    Meckel, Tobias; Semrau, Stefan; Schaaf, Marcel J. M.; Schmidt, Thomas

    2011-07-01

    The formation of protein complexes or clusters in the plasma membrane is essential for many biological processes, such as signaling. We develop a tool, based on single-molecule microscopy, for following cluster formation in vivo. Detection and tracing of single autofluorescent proteins have become standard biophysical techniques. The determination of the number of proteins in a cluster, however, remains challenging. The reasons are (i) the poor photophysical stability and complex photophysics of fluorescent proteins and (ii) noise and autofluorescent background in live cell recordings. We show that, despite those obstacles, the accurate fraction of signals in which a certain (or set) number of labeled proteins reside, can be determined in an accurate an robust way in vivo. We define experimental conditions under which fluorescent proteins exhibit predictable distributions of intensity and quantify the influence of noise. Finally, we confirm our theoretical predictions by measurements of the intensities of individual enhanced yellow fluorescent protein (EYFP) molecules in living cells. Quantification of the average number of EYFP-C10HRAS chimeras in diffraction-limited spots finally confirm that the membrane anchor of human Harvey rat sarcoma (HRAS) heterogeneously distributes in the plasma membrane of living Chinese hamster ovary cells.

  18. Degradation of HaloTag-fused nuclear proteins using bestatin-HaloTag ligand hybrid molecules.

    PubMed

    Tomoshige, Shusuke; Naito, Mikihiko; Hashimoto, Yuichi; Ishikawa, Minoru

    2015-10-14

    We have developed a protein knockdown technology using hybrid small molecules designed as conjugates of a ligand for the target protein and a ligand for ubiquitin ligase cellular inhibitor of apoptosis protein 1 (cIAP1). However, this technology has several limitations. Here, we report the development of a novel protein knockdown system to address these limitations. In this system, target proteins are fused with HaloTag to provide a common binding site for a degradation inducer. We designed and synthesized small molecules consisting of alkyl chloride as the HaloTag-binding degradation inducer, which binds to HaloTag, linked to BE04 (2), which binds to cIAP1. Using this system, we successfully knocked down HaloTag-fused cAMP responsive element binding protein 1 (HaloTag-CREB1) and HaloTag-fused c-jun (HaloTag-c-jun), which are ligand-unknown nuclear proteins, in living cells. HaloTag-binding degradation inducers can be synthesized easily, and are expected to be useful as biological tools for pan-degradation of HaloTag-fused proteins. PMID:26338696

  19. Single-molecule resolution of protein structure and interfacial dynamics on biomaterial surfaces

    PubMed Central

    McLoughlin, Sean Yu; Kastantin, Mark; Schwartz, Daniel K.; Kaar, Joel L.

    2013-01-01

    A method was developed to monitor dynamic changes in protein structure and interfacial behavior on surfaces by single-molecule Förster resonance energy transfer. This method entails the incorporation of unnatural amino acids to site-specifically label proteins with single-molecule Förster resonance energy transfer probes for high-throughput dynamic fluorescence tracking microscopy on surfaces. Structural changes in the enzyme organophosphorus hydrolase (OPH) were monitored upon adsorption to fused silica (FS) surfaces in the presence of BSA on a molecule-by-molecule basis. Analysis of >30,000 individual trajectories enabled the observation of heterogeneities in the kinetics of surface-induced OPH unfolding with unprecedented resolution. In particular, two distinct pathways were observed: a majority population (∼ 85%) unfolded with a characteristic time scale of 0.10 s, and the remainder unfolded more slowly with a time scale of 0.7 s. Importantly, even after unfolding, OPH readily desorbed from FS surfaces, challenging the common notion that surface-induced unfolding leads to irreversible protein binding. This suggests that protein fouling of surfaces is a highly dynamic process because of subtle differences in the adsorption/desorption rates of folded and unfolded species. Moreover, such observations imply that surfaces may act as a source of unfolded (i.e., aggregation-prone) protein back into solution. Continuing study of other proteins and surfaces will examine whether these conclusions are general or specific to OPH in contact with FS. Ultimately, this method, which is widely applicable to virtually any protein, provides the framework to develop surfaces and surface modifications with improved biocompatibility. PMID:24235137

  20. 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.

  1. 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.

  2. CSM-lig: a web server for assessing and comparing protein-small molecule affinities.

    PubMed

    Pires, Douglas E V; Ascher, David B

    2016-07-01

    Determining the affinity of a ligand for a given protein is a crucial component of drug development and understanding their biological effects. Predicting binding affinities is a challenging and difficult task, and despite being regarded as poorly predictive, scoring functions play an important role in the analysis of molecular docking results. Here, we present CSM-Lig (http://structure.bioc.cam.ac.uk/csm_lig), a web server tailored to predict the binding affinity of a protein-small molecule complex, encompassing both protein and small-molecule complementarity in terms of shape and chemistry via graph-based structural signatures. CSM-Lig was trained and evaluated on different releases of the PDBbind databases, achieving a correlation of up to 0.86 on 10-fold cross validation and 0.80 in blind tests, performing as well as or better than other widely used methods. The web server allows users to rapidly and automatically predict binding affinities of collections of structures and assess the interactions made. We believe CSM-lig would be an invaluable tool for helping assess docking poses, the effects of multiple mutations, including insertions, deletions and alternative splicing events, in protein-small molecule affinity, unraveling important aspects that drive protein-compound recognition. PMID:27151202

  3. 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

  4. Enhanced Raman spectroscopy of molecules adsorbed on carbon-doped TiO₂ obtained from titanium carbide: a visible-light-assisted renewable substrate.

    PubMed

    Kiran, Vankayala; Sampath, Srinivasan

    2012-08-01

    Titanium carbide (TiC) is an electrically conducting material with favorable electrochemical properties. In the present studies, carbon-doped TiO(2) (C-TiO(2)) has been synthesized from TiC particles, as well as TiC films coated on stainless steel substrate via thermal annealing under various conditions. Several C-TiO(2) substrates are synthesized by varying experimental conditions and characterized by UV-visible spectroscopy, photoluminescence, X-ray diffraction, and X-ray photoelectron spectroscopic techniques. C-TiO(2) in the dry state (in powder form as well as in film form) is subsequently used as a substrate for enhancing Raman signals corresponding to 4-mercaptobenzoic acid and 4-nitrothiophenol by utilizing chemical enhancement based on charge-transfer interactions. Carbon, a nonmetal dopant in TiO(2), improves the intensities of Raman signals, compared to undoped TiO(2). Significant dependence of Raman intensity on carbon doping is observed. Ameliorated performance obtained using C-TiO(2) is attributed to the presence of surface defects that originate due to carbon as a dopant, which, in turn, triggers charge transfer between TiO(2) and analyte. The C-TiO(2) substrates are subsequently regenerated for repetitive use by illuminating an analyte-adsorbed substrate with visible light for a period of 5 h. PMID:22738214

  5. Small-Molecule Stabilization of the 14-3-3/Gab2 Protein-Protein Interaction (PPI) Interface.

    PubMed

    Bier, David; Bartel, Maria; Sies, Katharina; Halbach, Sebastian; Higuchi, Yusuke; Haranosono, Yu; Brummer, Tilman; Kato, Nobuo; Ottmann, Christian

    2016-04-19

    Small-molecule modulation of protein-protein interactions (PPIs) is one of the most promising new areas in drug discovery. In the vast majority of cases only inhibition or disruption of PPIs is realized, whereas the complementary strategy of targeted stabilization of PPIs is clearly under-represented. Here, we report the example of a semi-synthetic natural product derivative-ISIR-005-that stabilizes the cancer-relevant interaction of the adaptor protein 14-3-3 and Gab2. The crystal structure of ISIR-005 in complex with 14-3-3 and the binding motif of Gab2 comprising two phosphorylation sites (Gab2pS210pT391) showed how the stabilizing molecule binds to the rim-of-the-interface of the protein complex. Only in the direct vicinity of 14-3-3/Gab2pT391 site is a pre-formed pocket occupied by ISIR-005; binding of the Gab2pS210 motif to 14-3-3 does not create an interface pocket suitable for the molecule. Accordingly, ISIR-005 only stabilizes the binding of the Gab2pT391 but not the Gab2pS210 site. This study represents structural and biochemical proof of the druggability of the 14-3-3/Gab2 PPI interface with important implications for the development of PPI stabilizers. PMID:26644359

  6. 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. PMID:19933333

  7. Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

    PubMed Central

    Monico, Carina; Belcastro, Gionata; Vanzi, Francesco; Pavone, Francesco S.; Capitanio, Marco

    2014-01-01

    The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable. PMID:25226304

  8. Free energy barriers for escape of water molecules from protein hydration layer.

    PubMed

    Roy, Susmita; Bagchi, Biman

    2012-03-01

    Free energy barriers separating interfacial water molecules from the hydration layer at the surface of a protein to the bulk are obtained by using the umbrella sampling method of free energy calculation. We consider hydration layer of chicken villin head piece (HP-36) which has been studied extensively by molecular dynamics simulations. The free energy calculations reveal a strong sensitivity to the secondary structure. In particular, we find a region near the junction of first and second helix that contains a cluster of water molecules which are slow in motion, characterized by long residence times (of the order of 100 ps or more) and separated by a large free energy barrier from the bulk water. However, these "slow" water molecules constitute only about 5-10% of the total number of hydration layer water molecules. Nevertheless, they play an important role in stabilizing the protein conformation. Water molecules near the third helix (which is the important helix for biological function) are enthalpically least stable and exhibit the fastest dynamics. Interestingly, barrier height distributions of interfacial water are quite broad for water surrounding all the three helices (and the three coils), with the smallest barriers found for those near the helix-3. For the quasi-bound water molecules near the first and second helices, we use well-known Kramers' theory to estimate the residence time from the free energy surface, by estimating the friction along the reaction coordinate from the diffusion coefficient by using Einstein relation. The agreement found is satisfactory. We discuss the possible biological function of these slow, quasi-bound (but transient) water molecules on the surface. PMID:22288939

  9. 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

  10. Small-Molecule Inhibitor Leads of Ribosome-Inactivating Proteins Developed Using the Doorstop Approach

    PubMed Central

    Pang, Yuan-Ping; Park, Jewn Giew; Wang, Shaohua; Vummenthala, Anuradha; Mishra, Rajesh K.; McLaughlin, John E.; Di, Rong; Kahn, Jennifer Nielsen; Tumer, Nilgun E.; Janosi, Laszlo; Davis, Jon; Millard, Charles B.

    2011-01-01

    Ribosome-inactivating proteins (RIPs) are toxic because they bind to 28S rRNA and depurinate a specific adenine residue from the α-sarcin/ricin loop (SRL), thereby inhibiting protein synthesis. Shiga-like toxins (Stx1 and Stx2), produced by Escherichia coli, are RIPs that cause outbreaks of foodborne diseases with significant morbidity and mortality. Ricin, produced by the castor bean plant, is another RIP lethal to mammals. Currently, no US Food and Drug Administration-approved vaccines nor therapeutics exist to protect against ricin, Shiga-like toxins, or other RIPs. Development of effective small-molecule RIP inhibitors as therapeutics is challenging because strong electrostatic interactions at the RIP•SRL interface make drug-like molecules ineffective in competing with the rRNA for binding to RIPs. Herein, we report small molecules that show up to 20% cell protection against ricin or Stx2 at a drug concentration of 300 nM. These molecules were discovered using the doorstop approach, a new approach to protein•polynucleotide inhibitors that identifies small molecules as doorstops to prevent an active-site residue of an RIP (e.g., Tyr80 of ricin or Tyr77 of Stx2) from adopting an active conformation thereby blocking the function of the protein rather than contenders in the competition for binding to the RIP. This work offers promising leads for developing RIP therapeutics. The results suggest that the doorstop approach might also be applicable in the development of other protein•polynucleotide inhibitors as antiviral agents such as inhibitors of the Z-DNA binding proteins in poxviruses. This work also calls for careful chemical and biological characterization of drug leads obtained from chemical screens to avoid the identification of irrelevant chemical structures and to avoid the interference caused by direct interactions between the chemicals being screened and the luciferase reporter used in screening assays. PMID:21455295

  11. 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).

  12. A novel single-molecule study to determine protein--protein association constants.

    PubMed

    Ratcliff, G C; Erie, D A

    2001-06-20

    Atomic force microscopy (AFM) is traditionally used as an imaging technique to gain qualitative information for a biological system. We have successfully used the imaging capabilities of the AFM to determine protein-protein association constants. We have developed a method to measure the molecular weight of a protein based on its volume determined from AFM images. Our volume determination method allows for rapid, accurate analysis of large protein populations. On the basis of the measured volume, the fraction of monomers as dimers was determined for the DNA helicase UvrD, and the dissociation constant (K(d)) for the helicase was calculated. We determined a K(d) for UvrD of 1.4 microM, which is in good agreement with published K(d) data obtained from analytical ultracentrifugation (AUC) studies. Our method provides a rapid method for determining protein-protein association constants. PMID:11403593

  13. 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.

  14. The translocation time of DNA and protein molecules in solid-state nanopores

    NASA Astrophysics Data System (ADS)

    Ledden, Bradley; Rollings, Ryan; Talaga, David; Li, Jiali

    2011-03-01

    The time that a biopolymer takes to translocate through a nanopore contains the properties of the polymer including its size, conformation, electrical charge and charge distribution. We measured the dependence of the translocation times on the size, charge and charge distribution, voltage, and conformation states of DNA and protein molecules. To quantitatively fit the time distributions measured, 1-D Langevin and 1-D Fokker-Planck equations were used for DNA and native state proteins. Kramers reaction rate theory was used to fit the time distribution of unfolded proteins. It was observed that native-state protein and DNA translocation approximately follows simple one-dimensional biased diffusion of charged particles. Due to the heterogeneous charge sequence of polypeptides, unfolded proteins obey a coupled electrophoretic and thermally activated process that is sequence specific. Deviations between models and experimental results as well as future challenges for single molecule DNA and protein characterization using solid-state nanopores will be discussed. Funding support provided by NHGRI/NIH R21HG003290, NHGRI /NIH R21HG00477, and NIH R01GM071684 to DST.

  15. 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

  16. Enhancements to the Rosetta Energy Function Enable Improved Identification of Small Molecules that Inhibit Protein-Protein Interactions

    PubMed Central

    Karanicolas, John

    2015-01-01

    Protein-protein interactions are among today’s most exciting and promising targets for therapeutic intervention. To date, identifying small-molecules that selectively disrupt these interactions has proven particularly challenging for virtual screening tools, since these have typically been optimized to perform well on more “traditional” drug discovery targets. Here, we test the performance of the Rosetta energy function for identifying compounds that inhibit protein interactions, when these active compounds have been hidden amongst pools of “decoys.” Through this virtual screening benchmark, we gauge the effect of two recent enhancements to the functional form of the Rosetta energy function: the new “Talaris” update and the “pwSHO” solvation model. Finally, we conclude by developing and validating a new weight set that maximizes Rosetta’s ability to pick out the active compounds in this test set. Looking collectively over the course of these enhancements, we find a marked improvement in Rosetta’s ability to identify small-molecule inhibitors of protein-protein interactions. PMID:26484863

  17. Single-molecule study of protein-DNA target search mechanisms for dimer-active protein complexes

    NASA Astrophysics Data System (ADS)

    Landry, Markita; Huang, Wai Mun; Chemla, Yann

    2012-02-01

    Protein-DNA interactions are essential to cellular processes, many of which require proteins to recognize a specific DNA target-site. This search process is well-documented for monomeric proteins, but not as well understood for systems that require dimerization at the target site for activity. We present a single-molecule study of the target-search mechanism of Protelomerase TelK, a recombinase-like protein that is only active as a dimer. We observe that TelK undergoes 1D diffusion on non-target DNA as a monomer, as expected, but becomes immobile on DNA as a dimer or oligomer despite the absence of its target site. We further show that TelK condenses non-target DNA upon dimerization, forming a tightly bound nucleo-protein complex. Together with simulations, our results suggest a search model whereby monomers diffuse along DNA, and subsequently dimerize to form an active complex on target DNA. These results show that target-finding occurs faster than nonspecific dimerization at biologically relevant protein concentrations. This model may provide insights into the search mechanisms of proteins that are active as multimeric complexes for a more accurate and comprehensive model for the target-search process by sequence specific proteins.

  18. MALDI- or ESI? Pros and cons for protein and small molecules

    SciTech Connect

    Olivares, J. A.

    2004-01-01

    Mass spectrometry has become a very popular technique in the analytical characterization of elements and molecules that range from inorganic, organic, and biological species. This popularity has soared in the past 15 years primarily through the development of ionization sources that can easily ionize large organic and biological molecules, intact and/or with controlled fragmentation. The two primary ionization mechanisms responsible for this capability are Matrix Assisted Laser Desorption Ionization (MALDI) and Electrospray Ionization (ESI). The development of the latter resulted in the 2002 Nobel Prize in Chemistry Engineering for John Fenn. This capability has presented a new paradigm allowing the field of proteomics to break through, with the characterization of major fractions of the proteins in a biological cell. The sensitivity, specificity, and structural characterization of available today using these techniques will be discussed with some examples in the characterization of both large and small molecules and relative merits of each technology.

  19. Dissecting single-molecule signal transduction in carbon nanotube circuits with protein engineering

    PubMed Central

    Choi, Yongki; Olsen, Tivoli J.; Sims, Patrick C.; Moody, Issa S.; Corso, Brad L.; Dang, Mytrang N.; Weiss, Gregory A.; Collins, Philip G.

    2013-01-01

    Single molecule experimental methods have provided new insights into biomolecular function, dynamic disorder, and transient states that are all invisible to conventional measurements. A novel, non-fluorescent single molecule technique involves attaching single molecules to single-walled carbon nanotube field-effective transistors (SWNT FETs). These ultrasensitive electronic devices provide long-duration, label-free monitoring of biomolecules and their dynamic motions. However, generalization of the SWNT FET technique first requires design rules that can predict the success and applicability of these devices. Here, we report on the transduction mechanism linking enzymatic processivity to electrical signal generation by a SWNT FET. The interaction between SWNT FETs and the enzyme lysozyme was systematically dissected using eight different lysozyme variants synthesized by protein engineering. The data prove that effective signal generation can be accomplished using a single charged amino acid, when appropriately located, providing a foundation to widely apply SWNT FET sensitivity to other biomolecular systems. PMID:23323846

  20. 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.

  1. 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

  2. Terminal protection of small-molecule-linked DNA for sensitive electrochemical detection of protein binding via selective carbon nanotube assembly.

    PubMed

    Wu, Zhan; Zhen, Zhen; Jiang, Jian-Hui; Shen, Guo-Li; Yu, Ru-Qin

    2009-09-01

    Small-molecule-linked DNA has emerged as a versatile tool for the interaction assay between small organic molecules and their protein receptors. We report herein the proof-of-principle of a terminal protection assay of small-molecule-linked DNA. This assay is based on our new finding that single-stranded DNA (ssDNA) terminally tethered to a small molecule is protected from the degradation by exonuclease I (Exo I) when the small molecule moiety is bound to its protein target. This finding translates the binding of small molecules to proteins into the presence of a specific DNA sequence, which enables us to probe the interaction between small organic molecules and their protein targets using various DNA sequence amplification and detection technologies. On the basis of selective assembly of single-walled carbon nanotubes (SWNTs) with surface-tethered small-molecule-linked ssDNA not protected by protein binding, a novel electrochemical strategy for terminal protection assay has been developed. Through detecting the redox signal mediated by SWNT assembly on a 16-mercaptohexadecanoic acid-blocked electrode, this strategy is able to ensure substantial signal amplification and a low background current. This strategy is demonstrated for quantitative analysis of the interaction of folate with a tumor biomarker of folate receptor (FR), and a detection limit of 3 pM FR is readily achieved with desirable specificity and sensitivity, indicating that the terminal protection assay can offer a promising platform for small molecule-protein interaction studies. PMID:19655753

  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. 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.

  5. 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

  6. 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

  7. Counting numbers of synaptic proteins: absolute quantification and single molecule imaging techniques.

    PubMed

    Patrizio, Angela; Specht, Christian G

    2016-10-01

    The ability to count molecules is essential to elucidating cellular mechanisms, as these often depend on the absolute numbers and concentrations of molecules within specific compartments. Such is the case at chemical synapses, where the transmission of information from presynaptic to postsynaptic terminals requires complex interactions between small sets of molecules. Be it the subunit stoichiometry specifying neurotransmitter receptor properties, the copy numbers of scaffold proteins setting the limit of receptor accumulation at synapses, or protein packing densities shaping the molecular organization and plasticity of the postsynaptic density, all of these depend on exact quantities of components. A variety of proteomic, electrophysiological, and quantitative imaging techniques have yielded insights into the molecular composition of synaptic complexes. In this review, we compare the different quantitative approaches and consider the potential of single molecule imaging techniques for the quantification of synaptic components. We also discuss specific neurobiological data to contextualize the obtained numbers and to explain how they aid our understanding of synaptic structure and function. PMID:27335891

  8. 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. PMID:26757808

  9. 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

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

    PubMed Central

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

    2011-01-01

    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 coopted 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. PMID:22053884

  11. 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

  12. 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.

  13. Mapping the protein-binding sites for iridium(iii)-based CO-releasing molecules.

    PubMed

    Caterino, Marco; Petruk, Ariel A; Vergara, Alessandro; Ferraro, Giarita; Marasco, Daniela; Doctorovich, Fabio; Estrin, Dario A; Merlino, Antonello

    2016-07-26

    A combination of mass spectrometry, Raman microspectroscopy, circular dichroism and X-ray crystallography has been used to obtain detailed information on the reaction of an iridium-based CO-releasing molecule (Ir-CORM), Cs2IrCl5CO, with a model protein, bovine pancreatic ribonuclease. The results show that Ir-compound fragments bind to the N-terminal amine and close to histidine and methionine side chains, and the CO ligand is retained for a long time. The data provide helpful information for identifying protein targets for Ir-CORMs and for studying the mechanism that allows them to exhibit their interesting biological properties. PMID:27411388

  14. 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.

  15. 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

  16. 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.

  17. Single-molecule protein unfolding and translocation by an ATP-fueled proteolytic machine

    PubMed Central

    Aubin-Tam, Marie-Eve; Olivares, Adrian O.; Sauer, Robert T.; Baker, Tania A.; Lang, Matthew J.

    2011-01-01

    All cells employ ATP-powered proteases for protein-quality control and regulation. In the ClpXP protease, ClpX is a AAA+ machine that recognizes specific protein substrates, unfolds these molecules, and then translocates the denatured polypeptide through a central pore and into ClpP for degradation. Here, we use optical-trapping nanometry to probe the mechanics of enzymatic unfolding and translocation of single molecules of a multidomain substrate. Our experiments demonstrate the capacity of ClpXP and ClpX to perform mechanical work under load, reveal very fast and highly cooperative unfolding of individual substrate domains, suggest a translocation step size of 5–8 amino acids, and support a power-stroke model of denaturation in which successful enzyme-mediated unfolding of stable domains requires coincidence between mechanical pulling by the enzyme and a transient stochastic reduction in protein stability. We anticipate that single-molecule studies of the mechanical properties of other AAA+ proteolytic machines will reveal many shared features with ClpXP. PMID:21496645

  18. 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

  19. Single Molecule Photobleaching (SMPB) Technology for Counting of RNA, DNA, Protein and Other Molecules in Nanoparticles and Biological Complexes by TIRF Instrumentation

    PubMed Central

    Zhang, Hui; Guo, Peixuan

    2014-01-01

    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, EMBO J, 2007, 26:527; Zhang et al, RNA, 2007, 13:1793) 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. PMID:24440482

  20. 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

  1. 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

  2. Probing the Small-Molecule Inhibition of an Anticancer Therapeutic Protein-Protein Interaction Using a Solid-State Nanopore.

    PubMed

    Kwak, Dong-Kyu; Chae, Hongsik; Lee, Mi-Kyung; Ha, Ji-Hyang; Goyal, Gaurav; Kim, Min Jun; Kim, Ki-Bum; Chi, Seung-Wook

    2016-05-01

    Nanopore sensing is an emerging technology for the single-molecule-based detection of various biomolecules. In this study, we probed the anticancer therapeutic p53 transactivation domain (p53TAD)/MDM2 interaction and its inhibition with a small-molecule MDM2 antagonist, Nutlin-3, using low-noise solid-state nanopores. Although the translocation of positively charged MDM2 through a nanopore was detected at the applied negative voltage, this MDM2 translocation was almost completely blocked upon formation of the MDM2/GST-p53TAD complex owing to charge conversion. In combination with NMR data, the nanopore measurements showed that the addition of Nutlin-3 rescued MDM2 translocation, indicating that Nutlin-3 disrupted the MDM2/GST-p53TAD complex, thereby releasing MDM2. Taken together, our results reveal that solid-state nanopores can be a valuable platform for the ultrasensitive, picomole-scale screening of small-molecule drugs against protein-protein interaction (PPI) targets. PMID:27038437

  3. Hemoglobin-mimetic oxygen adsorbent prepared via self-assembly of cysteinyl bolaamphiphiles.

    PubMed

    Lee, Chaemyeong; Kim, Min-Chul; Lee, Sang-Yup

    2016-06-01

    In this study, a novel cysteinyl bolaamphiphilic molecule was synthesized and its self-assembled planar suprastructure was applied as a biomimetic matrix to create a hemoglobin-mimetic oxygen adsorbent that exploits the ability of cysteine thiols to bind hemin. Self-assembly of the cysteinyl bolaamphiphilic molecule exposed cysteine thiols on its surface in the presence of β-mercaptoethanol, known to reduce disulfide bonds, without which, helically coiled structures were generated. The self-assembled planar structure was used as a soft matrix to create a hemoglobin-mimetic oxygen adsorbent. The surface-exposed cysteine thiols were used to attach hemin, producing a hemin-bound, planar structure mimicking hemoglobin. This hemoglobin mimic strongly adsorbed oxygen and remained stable up to 50°C. The cysteinyl bolaamphiphile self-assembled structure provided a biomimetic platform that allowed for the association of biological substances in a manner similar to natural proteins. PMID:26970824

  4. Polymer scaling laws of unfolded and intrinsically disordered proteins quantified with single-molecule spectroscopy

    PubMed Central

    Hofmann, Hagen; Soranno, Andrea; Borgia, Alessandro; Gast, Klaus; Nettels, Daniel; Schuler, Benjamin

    2012-01-01

    The dimensions of unfolded and intrinsically disordered proteins are highly dependent on their amino acid composition and solution conditions, especially salt and denaturant concentration. However, the quantitative implications of this behavior have remained unclear, largely because the effective theta-state, the central reference point for the underlying polymer collapse transition, has eluded experimental determination. Here, we used single-molecule fluorescence spectroscopy and two-focus correlation spectroscopy to determine the theta points for six different proteins. While the scaling exponents of all proteins converge to 0.62 ± 0.03 at high denaturant concentrations, as expected for a polymer in good solvent, the scaling regime in water strongly depends on sequence composition. The resulting average scaling exponent of 0.46 ± 0.05 for the four foldable protein sequences in our study suggests that the aqueous cellular milieu is close to effective theta conditions for unfolded proteins. In contrast, two intrinsically disordered proteins do not reach the Θ-point under any of our solvent conditions, which may reflect the optimization of their expanded state for the interactions with cellular partners. Sequence analyses based on our results imply that foldable sequences with more compact unfolded states are a more recent result of protein evolution. PMID:22984159

  5. Heat Shock Protein-70 Inhibition by the Small-Molecule 2-phenylethynesulfonamide Impairs Protein Clearance Pathways in Tumor Cells

    PubMed Central

    Leu, J. I-Ju; Pimkina, Julia; Pandey, Pooja; Murphy, Maureen E.; George, Donna L.

    2011-01-01

    The evolutionarily conserved stress-inducible HSP70 molecular chaperone plays a central role in maintaining protein quality control in response to various forms of stress. Constitutively elevated HSP70 expression is a characteristic of many tumor cells and contributes to their survival. We recently identified the small-molecule 2-phenylethyenesulfonamide (PES) as a novel HSP70 inhibitor. Here we present evidence that PES-mediated inhibition of HSP70-family proteins in tumor cells results in an impairment of the two major protein degradation systems, namely the autophagy-lysosome system as well as the proteasome pathway. HSP70-family proteins work closely with the HSP90 molecular chaperone to maintain the stability and activities of their many client proteins and PES causes a disruption in the HSP70/HSP90 chaperone system. As a consequence, many cellular proteins, including known HSP70/HSP90 substrates, accumulate in detergent-insoluble cell fractions, indicative of aggregation and functional inactivation. Overall, PES simultaneously disrupts several cancer-critical survival pathways, supporting the idea of targeting HSP70 as a potential approach for cancer therapeutics. PMID:21636681

  6. Single-molecule denaturation and degradation of proteins by the AAA+ ClpXP protease

    PubMed Central

    Shin, Yongdae; Davis, Joseph H.; Brau, Ricardo R.; Martin, Andreas; Kenniston, Jon A.; Baker, Tania A.; Sauer, Robert T.; Lang, Matthew J.

    2009-01-01

    ClpXP is an ATP-fueled molecular machine that unfolds and degrades target proteins. ClpX, an AAA+ enzyme, recognizes specific proteins, and then uses cycles of ATP hydrolysis to denature any native structure and to translocate the unfolded polypeptide into ClpP for degradation. Here, we develop and apply single-molecule fluorescence assays to probe the kinetics of protein denaturation and degradation by ClpXP. These assays employ a single-chain variant of the ClpX hexamer, linked via a single biotin to a streptavidin-coated surface, and fusion substrates with an N-terminal fluorophore and a C-terminal GFP-titin-ssrA module. In the presence of adenosine 5′-[γ-thio]triphosphate (ATPγS), ClpXP degrades the titin-ssrA portion of these substrates but stalls when it encounters GFP. Exchange into ATP then allows synchronous resumption of denaturation and degradation of GFP and any downstream domains. GFP unfolding can be monitored directly, because intrinsic fluorescence is quenched by denaturation. The time required for complete degradation coincides with loss of the substrate fluorophore from the protease complex. Fitting single-molecule data for a set of related substrates provides time constants for ClpX unfolding, translocation, and a terminal step that may involve product release. Comparison of these single-molecule results with kinetics measured in bulk solution indicates similar levels of microscopic and macroscopic ClpXP activity. These results support a stochastic engagement/unfolding mechanism that ultimately results in highly processive degradation and set the stage for more detailed single-molecule studies of machine function. PMID:19892734

  7. 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.

  8. Chemoselective small molecules that covalently modify one lysine in a non-enzyme protein in plasma

    SciTech Connect

    Choi, Sungwook; Connelly, Stephen; Reixach, Natàlia; Wilson, Ian A.; Kelly, Jeffery W.

    2010-02-19

    A small molecule that could bind selectively to and then react chemoselectively with a non-enzyme protein in a complex biological fluid, such as blood, could have numerous practical applications. Herein, we report a family of designed stilbenes that selectively and covalently modify the prominent plasma protein transthyretin in preference to more than 4,000 other human plasma proteins. They react chemoselectively with only one of eight lysine {epsilon}-amino groups within transthyretin. The crystal structure confirms the expected binding orientation of the stilbene substructure and the anticipated conjugating amide bond. These covalent transthyretin kinetic stabilizers exhibit superior amyloid inhibition potency compared to their noncovalent counterparts, and they prevent cytotoxicity associated with amyloidogenesis. Though there are a few prodrugs that, upon metabolic activation, react with a cysteine residue inactivating a specific non-enzyme, we are unaware of designed small molecules that react with one lysine {epsilon}-amine within a specific non-enzyme protein in a complex biological fluid.

  9. A hybrid multi-loop genetic-algorithm/simplex/spatial-grid method for locating the optimum orientation of an adsorbed protein on a solid surface

    NASA Astrophysics Data System (ADS)

    Wei, Tao; Mu, Shengjing; Nakano, Aiichiro; Shing, Katherine

    2009-05-01

    Atomistic simulation of protein adsorption on a solid surface in aqueous environment is computationally demanding, therefore the determination of preferred protein orientations on the solid surface usually serves as an initial step in simulation studies. We have developed a hybrid multi-loop genetic-algorithm/simplex/spatial-grid method to search for low adsorption-energy orientations of a protein molecule on a solid surface. In this method, the surface and the protein molecule are treated as rigid bodies, whereas the bulk fluid is represented by spatial grids. For each grid point, an effective interaction region in the surface is defined by a cutoff distance, and the possible interaction energy between an atom at the grid point and the surface is calculated and recorded in a database. In searching for the optimum position and orientation, the protein molecule is translated and rotated as a rigid body with the configuration obtained from a previous Molecular Dynamic simulation. The orientation-dependent protein-surface interaction energy is obtained using the generated database of grid energies. The hybrid search procedure consists of two interlinked loops. In the first loop A, a genetic algorithm (GA) is applied to identify promising regions for the global energy minimum and a local optimizer with the derivative-free Nelder-Mead simplex method is used to search for the lowest-energy orientation within the identified regions. In the second loop B, a new population for GA is generated and competitive solution from loop A is improved. Switching between the two loops is adaptively controlled by the use of similarity analysis. We test the method for lysozyme adsorption on a hydrophobic hydrogen-terminated silicon (110) surface in implicit water (i.e., a continuum distance-dependent dielectric constant). The results show that the hybrid search method has faster convergence and better solution accuracy compared with the conventional genetic algorithm.

  10. 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.

  11. Small-molecule inhibitors that target protein-protein interactions in the RAD51 family of recombinases.

    PubMed

    Scott, Duncan E; Coyne, Anthony G; Venkitaraman, Ashok; Blundell, Tom L; Abell, Chris; Hyvönen, Marko

    2015-02-01

    The development of small molecules that inhibit protein-protein interactions continues to be a challenge in chemical biology and drug discovery. Herein we report the development of indole-based fragments that bind in a shallow surface pocket of a humanised surrogate of RAD51. RAD51 is an ATP-dependent recombinase that plays a key role in the repair of double-strand DNA breaks. It both self-associates, forming filament structures with DNA, and interacts with the BRCA2 protein through a common "FxxA" tetrapeptide motif. We elaborated previously identified fragment hits that target the FxxA motif site and developed small-molecule inhibitors that are approximately 500-fold more potent than the initial fragments. The lead compounds were shown to compete with the BRCA2-derived Ac-FHTA-NH2 peptide and the self-association peptide of RAD51, but they had no effect on ATP binding. This study is the first reported elaboration of small-molecular-weight fragments against this challenging target. PMID:25470112

  12. 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

  13. 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

  14. 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

  15. Spin-dependent electron transport in protein-like single-helical molecules

    PubMed Central

    Guo, Ai-Min; Sun, Qing-Feng

    2014-01-01

    We report on a theoretical study of spin-dependent electron transport through single-helical molecules connected by two nonmagnetic electrodes, and explain the experiment of significant spin-selective phenomenon observed in α-helical protein and the contradictory results between the protein and single-stranded DNA. Our results reveal that the α-helical protein is an efficient spin filter and the spin polarization is robust against the disorder. These results are in excellent agreement with recent experiments [Mishra D, et al. (2013) Proc Natl Acad Sci USA 110(37):14872–14876; Göhler B, et al. (2011) Science 331(6019):894–897] and may facilitate engineering of chiral-based spintronic devices. PMID:25071198

  16. 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.

  17. 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

  18. Single-molecule Förster resonance energy transfer study of protein dynamics under denaturing conditions

    PubMed Central

    Kuzmenkina, Elza V.; Heyes, Colin D.; Nienhaus, G. Ulrich

    2005-01-01

    Proteins are highly complex systems, exhibiting a substantial degree of structural variability in their folded state. In the presence of denaturants, the heterogeneity is greatly enhanced, and fluctuations among vast numbers of folded and unfolded conformations occur via many different pathways. Here, we have studied the structure and dynamics of the small enzyme ribonuclease HI (RNase H) in the presence of the chemical denaturant guanidinium chloride (GdmCl) using single-molecule fluorescence microscopy, with a particular focus on the characterization of the unfolded-state ensemble. A dye pair was specifically attached to the enzyme to measure structural changes through Förster resonance energy transfer (FRET). Enzyme immobilization on star-polymer surfaces that were specially developed for negligible interaction with folded and unfolded proteins enabled us to monitor conformational changes of individual proteins for several hundred seconds. FRET efficiency histograms were calculated from confocal scan images. They showed an expansion of the unfolded proteins with increasing GdmCl concentration. Cross-correlation analysis of donor and acceptor fluorescence intensity time traces from single molecules revealed reconfiguration of the polypeptide chain on a timescale of ≈20 μs at 1.7 M GdmCl. Slow conformational dynamics gave rise to characteristic, stepwise FRET efficiency changes. Transitions between folded and unfolded enzyme molecules occurred on the 100-s timescale, in excellent agreement with bulk denaturation experiments. Transitions between unfolded conformations were more frequent, with characteristic times of ≈2 s. These data were analyzed to obtain information on the free energy landscape of RNase H in the presence of chemical denaturants. PMID:16221762

  19. Probing Protein Multidimensional Conformational Fluctuations by Single-Molecule Multiparameter Photon Stamping Spectroscopy

    PubMed Central

    2015-01-01

    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

  20. 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.

  1. 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

  2. Chemical Dimerizers in Three-Hybrid Systems for Small Molecule-Target Protein Profiling.

    PubMed

    De Clercq, Dries J H; Tavernier, Jan; Lievens, Sam; Van Calenbergh, Serge

    2016-08-19

    The identification of the molecular targets and mechanisms underpinning the beneficial or detrimental effects of small-molecule leads and drugs constitutes a crucial aspect of current drug discovery. Over the last two decades, three-hybrid (3H) systems have progressively taken an important position in the armamentarium of small molecule-target protein profiling technologies. Yet, a prerequisite for successful 3H analysis is the availability of appropriate chemical inducers of dimerization. Herein, we present a comprehensive and critical overview of the chemical dimerizers specifically applied in both yeast and mammalian three-hybrid systems for small molecule-target protein profiling within the broader scope of target deconvolution and drug discovery. Furthermore, examples and alternative suggestions for typical components of chemical dimerizers for 3H systems are discussed. As illustrated, more tools have become available that increase the sensitivity and efficiency of 3H-based screening platforms. Hence, it is anticipated that the great potential of 3H systems will further materialize in important contributions to drug discovery. PMID:27267544

  3. Improving the representation of peptide-like inhibitor and antibiotic molecules in the Protein Data Bank.

    PubMed

    Dutta, Shuchismita; Dimitropoulos, Dimitris; Feng, Zukang; Persikova, Irina; Sen, Sanchayita; Shao, Chenghua; Westbrook, John; Young, Jasmine; Zhuravleva, Marina A; Kleywegt, Gerard J; Berman, Helen M

    2014-06-01

    With the accumulation of a large number and variety of molecules in the Protein Data Bank (PDB) comes the need on occasion to review and improve their representation. The Worldwide PDB (wwPDB) partners have periodically updated various aspects of structural data representation to improve the integrity and consistency of the archive. The remediation effort described here was focused on improving the representation of peptide-like inhibitor and antibiotic molecules so that they can be easily identified and analyzed. Peptide-like inhibitors or antibiotics were identified in over 1000 PDB entries, systematically reviewed and represented either as peptides with polymer sequence or as single components. For the majority of the single-component molecules, their peptide-like composition was captured in a new representation, called the subcomponent sequence. A novel concept called "group" was developed for representing complex peptide-like antibiotics and inhibitors that are composed of multiple polymer and nonpolymer components. In addition, a reference dictionary was developed with detailed information about these peptide-like molecules to aid in their annotation, identification and analysis. Based on the experience gained in this remediation, guidelines, procedures, and tools were developed to annotate new depositions containing peptide-like inhibitors and antibiotics accurately and consistently. PMID:24173824

  4. 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.

  5. One Protein to Rule them All: Modulation of Cell Surface Receptors and Molecules by HIV Nef

    PubMed Central

    Landi, Alessia; Iannucci, Veronica; Nuffel, Anouk Van; Meuwissen, Pieter; Verhasselt, Bruno

    2011-01-01

    The HIV-1, HIV-2 and SIV Nef protein are known to modulate the expression of several cell surface receptors and molecules to escape the immune system, to alter T cell activation, to enhance viral replication, infectivity and transmission and overall to ensure the optimal environment for infection outcome. Consistent and continuous efforts have been made over the years to characterize the modulation of expression of each of these molecules, in the hope that a better understanding of these processes essential for HIV infection and/or pathogenesis will eventually highlight new therapeutic targets. In this article we provide an extensive review of the knowledge gained so far on this important and evolving topic. PMID:22103833

  6. 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

  7. 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

  8. 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.

  9. 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. PMID:15373829

  10. Single Molecule FRET of Protein-Nucleic Acid and Protein-Protein complexes: Surface Passivation and Immobilization

    PubMed Central

    Lamichhane, Rajan; Solem, Amanda; Black, Will; Rueda, David

    2010-01-01

    Single-molecule fluorescence spectroscopy reveals the real time dynamics that occur during biomolecular interactions that would otherwise be hidden by the ensemble average. It also removes the requirement to synchronize reactions, thus providing a very intuitive approach to study kinetics of biological systems. Surface immobilization is commonly used to increase observation times to the minute time scale, but it can be detrimental if the sample interacts non-specifically with the surface. Here, we review detailed protocols to prevent such interactions by passivating the surface or by trapping the molecules inside surface immobilized lipid vesicles. Finally, we discuss recent examples where these methods were applied to study the dynamics of important cellular processes at the single molecule level. PMID:20554047

  11. 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

  12. Integration and oligomerization of Bax protein in lipid bilayers characterized by single molecule fluorescence study.

    PubMed

    Luo, Lu; Yang, Jun; Liu, Dongxiang

    2014-11-14

    Bax is a pro-apoptotic Bcl-2 family protein. The activated Bax translocates to mitochondria, where it forms pore and permeabilizes the mitochondrial outer membrane. This process requires the BH3-only activator protein (i.e. tBid) and can be inhibited by anti-apoptotic Bcl-2 family proteins such as Bcl-xL. Here by using single molecule fluorescence techniques, we studied the integration and oligomerization of Bax in lipid bilayers. Our study revealed that Bax can bind to lipid membrane spontaneously in the absence of tBid. The Bax pore formation undergoes at least two steps: pre-pore formation and membrane insertion. The activated Bax triggered by tBid or BH3 domain peptide integrates on bilayers and tends to form tetramers, which are termed as pre-pore. Subsequent insertion of the pre-pore into membrane is highly dependent on the composition of cardiolipin in lipid bilayers. Bcl-xL can translocate Bax from membrane to solution and inhibit the pore formation. The study of Bax integration and oligomerization at the single molecule level provides new evidences that may help elucidate the pore formation of Bax and its regulatory mechanism in apoptosis. PMID:25288797

  13. 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

  14. 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

  15. Peptide nanofibers modified with a protein by using designed anchor molecules bearing hydrophobic and functional moieties.

    PubMed

    Miyachi, Ayaka; Takahashi, Tsuyoshi; Matsumura, Sachiko; Mihara, Hisakazu

    2010-06-11

    Self-assembly of peptides and proteins is a key feature of biological functions. Short amphiphilic peptides designed with a beta-sheet structure can form sophisticated nanofiber structures, and the fibers are available as nanomaterials for arranging biomolecules. Peptide FI (H-PKFKIIEFEP-OH) self-assembles into nanofibers with a coiled fine structure, as reported in our previous work. We have constructed anchor molecules that have both a binding moiety for the fiber structure and a functional unit capable of capturing target molecules, with the purpose of arranging proteins on the designed peptide nanofibers. Designed anchors containing an alkyl chain as a binding unit and biotin as a functional moiety were found to bind to peptide fibers FI and F2i (H-ALEAKFAAFEAKLA-NH(2)). The surface-exposed biotin moiety on the fibers could capture an anti-biotin antibody. Moreover, hydrophobic dipeptide anchor units composed of iminodiacetate connected to Phe-Phe or Ile-Ile and a peptide composed of six histidine residues connected to biotin could also connect FI peptide fibers to the anti-biotin antibody through the chelation of Ni(2+) ions. This strategy of using designed anchors opens a novel approach to constructing nanoscale protein arrays on peptide nanomaterials. PMID:20419712

  16. 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

  17. 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

  18. 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.

  19. Analysis of multiple compound-protein interactions reveals novel bioactive molecules.

    PubMed

    Yabuuchi, Hiroaki; Niijima, Satoshi; Takematsu, Hiromu; Ida, Tomomi; Hirokawa, Takatsugu; Hara, Takafumi; Ogawa, Teppei; Minowa, Yohsuke; Tsujimoto, Gozoh; Okuno, Yasushi

    2011-03-01

    The discovery of novel bioactive molecules advances our systems-level understanding of biological processes and is crucial for innovation in drug development. For this purpose, the emerging field of chemical genomics is currently focused on accumulating large assay data sets describing compound-protein interactions (CPIs). Although new target proteins for known drugs have recently been identified through mining of CPI databases, using these resources to identify novel ligands remains unexplored. Herein, we demonstrate that machine learning of multiple CPIs can not only assess drug polypharmacology but can also efficiently identify novel bioactive scaffold-hopping compounds. Through a machine-learning technique that uses multiple CPIs, we have successfully identified novel lead compounds for two pharmaceutically important protein families, G-protein-coupled receptors and protein kinases. These novel compounds were not identified by existing computational ligand-screening methods in comparative studies. The results of this study indicate that data derived from chemical genomics can be highly useful for exploring chemical space, and this systems biology perspective could accelerate drug discovery processes. PMID:21364574

  20. 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.

  1. 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.

  2. Targeting GLI proteins in human cancer by small molecules (WO2014116651 A1): a patent evaluation.

    PubMed

    Hadden, M Kyle

    2015-05-01

    The invention reviewed in this patent evaluation is the synthesis and application of small molecule inhibitors of Gli transcriptional activity as potential anticancer agents. The oncogenic nature of Gli proteins has been traditionally associated with the hedgehog (Hh) signaling pathway; however, the recent identification of aberrant Gli activation unrelated to Hh signaling has prompted drug discovery efforts directly targeting Gli proteins. The central core of the compounds described in this patent (WO2014116651 A1) is structurally analogous to the pyrazoline scaffold previously disclosed by these inventors. Data describing the inhibitory activity of these compounds against the Hh pathway in vitro and in Hh-dependent in vivo models of human cancer are not provided. For this patent disclosure, the inventors primarily focus on the anticancer properties of their compounds in lung and lung-related malignancies. The compounds are moderately active in these models, but they do not exhibit the overall preclinical profile generally required for advancement into clinical trials. PMID:25772316

  3. Benzofuran Small Molecules as Potential Inhibitors of Human Protein Kinases. A Review.

    PubMed

    Kwiecień, Halina; Goszczyńska, Agata; Rokosz, Paulina

    2016-01-01

    Kinases are known to regulate the majority of human cellular processes such as communication, division, metabolism, survival and apoptosis therefore they can be promising targets in cancer diseases, viral infection and in other disorders. Small molecules acting as selective human protein kinase inhibitors are very attractive pharmacological targets. This review presents a number of examples of biologically active natural and synthetic benzo[b]furans and their derivatives, such as benzo[b]furan-2- and 3-ones, benzo[b]furan-2- and 3-carboxylic acids, as well as benzo[c]furans as potential inhibitors of various human protein kinases. The pathways of function and implication of the inhibitors in cancer and other diseases are discussed. PMID:26648467

  4. Activation of the Proapoptotic Bcl-2 Protein Bax by a Small Molecule Induces Tumor Cell Apoptosis

    PubMed Central

    Zhao, Guoping; Zhu, Yanglong; Eno, Colins O.; Liu, Yanlong; DeLeeuw, Lynn; Burlison, Joseph A.; Chaires, Jonathan B.; Trent, John O.

    2014-01-01

    The proapoptotic Bcl-2 protein Bax by itself is sufficient to initiate apoptosis in almost all apoptotic paradigms. Thus, compounds that can facilitate disruptive Bax insertion into mitochondrial membranes have potential as cancer therapeutics. In our study, we have identified small-molecule compounds predicted to associate with the Bax hydrophobic groove by a virtual-screen approach. Among these, one lead compound (compound 106) promotes Bax-dependent but not Bak-dependent apoptosis. Importantly, this compound alters Bax protein stability in vitro and promotes the insertion of Bax into mitochondria, leading to Bax-dependent permeabilization of the mitochondrial outer membrane. Furthermore, as a single agent, compound 106 inhibits the growth of transplanted tumors, probably by inducing apoptosis in tumors. Our study has revealed a compound that activates Bax and induces Bax-dependent apoptosis, which may lead to the development of new therapeutic agents for cancer. PMID:24421393

  5. Partial restoration of protein synthesis rates by the small molecule ISRIB prevents neurodegeneration without pancreatic toxicity

    PubMed Central

    Halliday, M; Radford, H; Sekine, Y; Moreno, J; Verity, N; le Quesne, J; Ortori, C A; Barrett, D A; Fromont, C; Fischer, P M; Harding, H P; Ron, D; Mallucci, G R

    2015-01-01

    Activation of the PERK branch of the unfolded protein response (UPR) in response to protein misfolding within the endoplasmic reticulum (ER) results in the transient repression of protein synthesis, mediated by the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2α). This is part of a wider integrated physiological response to maintain proteostasis in the face of ER stress, the dysregulation of which is increasingly associated with a wide range of diseases, particularly neurodegenerative disorders. In prion-diseased mice, persistently high levels of eIF2α cause sustained translational repression leading to catastrophic reduction of critical proteins, resulting in synaptic failure and neuronal loss. We previously showed that restoration of global protein synthesis using the PERK inhibitor GSK2606414 was profoundly neuroprotective, preventing clinical disease in prion-infected mice. However, this occured at the cost of toxicity to secretory tissue, where UPR activation is essential to healthy functioning. Here we show that pharmacological modulation of eIF2α-P-mediated translational inhibition can be achieved to produce neuroprotection without pancreatic toxicity. We found that treatment with the small molecule ISRIB, which restores translation downstream of eIF2α, conferred neuroprotection in prion-diseased mice without adverse effects on the pancreas. Critically, ISRIB treatment resulted in only partial restoration of global translation rates, as compared with the complete restoration of protein synthesis seen with GSK2606414. ISRIB likely provides sufficient rates of protein synthesis for neuronal survival, while allowing some residual protective UPR function in secretory tissue. Thus, fine-tuning the extent of UPR inhibition and subsequent translational de-repression uncouples neuroprotective effects from pancreatic toxicity. The data support the pursuit of this approach to develop new treatments for a range of neurodegenerative

  6. Partial restoration of protein synthesis rates by the small molecule ISRIB prevents neurodegeneration without pancreatic toxicity.

    PubMed

    Halliday, M; Radford, H; Sekine, Y; Moreno, J; Verity, N; le Quesne, J; Ortori, C A; Barrett, D A; Fromont, C; Fischer, P M; Harding, H P; Ron, D; Mallucci, G R

    2015-01-01

    Activation of the PERK branch of the unfolded protein response (UPR) in response to protein misfolding within the endoplasmic reticulum (ER) results in the transient repression of protein synthesis, mediated by the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2α). This is part of a wider integrated physiological response to maintain proteostasis in the face of ER stress, the dysregulation of which is increasingly associated with a wide range of diseases, particularly neurodegenerative disorders. In prion-diseased mice, persistently high levels of eIF2α cause sustained translational repression leading to catastrophic reduction of critical proteins, resulting in synaptic failure and neuronal loss. We previously showed that restoration of global protein synthesis using the PERK inhibitor GSK2606414 was profoundly neuroprotective, preventing clinical disease in prion-infected mice. However, this occured at the cost of toxicity to secretory tissue, where UPR activation is essential to healthy functioning. Here we show that pharmacological modulation of eIF2α-P-mediated translational inhibition can be achieved to produce neuroprotection without pancreatic toxicity. We found that treatment with the small molecule ISRIB, which restores translation downstream of eIF2α, conferred neuroprotection in prion-diseased mice without adverse effects on the pancreas. Critically, ISRIB treatment resulted in only partial restoration of global translation rates, as compared with the complete restoration of protein synthesis seen with GSK2606414. ISRIB likely provides sufficient rates of protein synthesis for neuronal survival, while allowing some residual protective UPR function in secretory tissue. Thus, fine-tuning the extent of UPR inhibition and subsequent translational de-repression uncouples neuroprotective effects from pancreatic toxicity. The data support the pursuit of this approach to develop new treatments for a range of neurodegenerative

  7. 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. PMID:24567151

  8. Adsorbed Water Illustration

    NASA Technical Reports Server (NTRS)

    2008-01-01

    The Thermal and Electrical Conductivity Probe on NASA's Phoenix Mars Lander detected small and variable amounts of water in the Martian soil.

    In this schematic illustration, water molecules are represented in red and white; soil minerals are represented in green and blue. The water, neither liquid, vapor, nor solid, adheres in very thin films of molecules to the surfaces of soil minerals. The left half illustrates an interpretation of less water being adsorbed onto the soil-particle surface during a period when the tilt, or obliquity, of Mars' rotation axis is small, as it is in the present. The right half illustrates a thicker film of water during a time when the obliquity is greater, as it is during cycles on time scales of hundreds of thousands of years. As the humidity of the atmosphere increases, more water accumulates on mineral surfaces. Thicker films behave increasingly like liquid water.

    The Phoenix Mission is led by the University of Arizona, Tucson, on behalf of NASA. Project management of the mission is by NASA's Jet Propulsion Laboratory, Pasadena, Calif. Spacecraft development is by Lockheed Martin Space Systems, Denver.

  9. Syntenin-1 and Ezrin Proteins Link Activated Leukocyte Cell Adhesion Molecule to the Actin Cytoskeleton*

    PubMed Central

    Tudor, Cicerone; te Riet, Joost; Eich, Christina; Harkes, Rolf; Smisdom, Nick; Bouhuijzen Wenger, Jessica; Ameloot, Marcel; Holt, Matthew; Kanger, Johannes S.; Figdor, Carl G.; Cambi, Alessandra; Subramaniam, Vinod

    2014-01-01

    Activated leukocyte cell adhesion molecule (ALCAM) is a type I transmembrane protein member of the immunoglobulin superfamily of cell adhesion molecules. Involved in important pathophysiological processes such as the immune response, cancer metastasis, and neuronal development, ALCAM undergoes both homotypic interactions with other ALCAM molecules and heterotypic interactions with the surface receptor CD6 expressed at the T cell surface. Despite biochemical and biophysical evidence of a dynamic association between ALCAM and the actin cytoskeleton, no detailed information is available about how this association occurs at the molecular level. Here, we exploit a combination of complementary microscopy techniques, including FRET detected by fluorescence lifetime imaging microscopy and single-cell force spectroscopy, and we demonstrate the existence of a preformed ligand-independent supramolecular complex where ALCAM stably interacts with actin by binding to syntenin-1 and ezrin. Interaction with the ligand CD6 further enhances these multiple interactions. Altogether, our results propose a novel biophysical framework to understand the stabilizing role of the ALCAM supramolecular complex engaged to CD6 during dendritic cell-T cell interactions and provide novel information on the molecular players involved in the formation and signaling of the immunological synapse at the dendritic cell side. PMID:24662291

  10. Nanovalved Adsorbents for CH4 Storage.

    PubMed

    Song, Zhuonan; Nambo, Apolo; Tate, Kirby L; Bao, Ainan; Zhu, Minqi; Jasinski, Jacek B; Zhou, Shaojun J; Meyer, Howard S; Carreon, Moises A; Li, Shiguang; Yu, Miao

    2016-05-11

    A novel concept of utilizing nanoporous coatings as effective nanovalves on microporous adsorbents was developed for high capacity natural gas storage at low storage pressure. The work reported here for the first time presents the concept of nanovalved adsorbents capable of sealing high pressure CH4 inside the adsorbents and storing it at low pressure. Traditional natural gas storage tanks are thick and heavy, which makes them expensive to manufacture and highly energy-consuming to carry around. Our design uses unique adsorbent pellets with nanoscale pores surrounded by a coating that functions as a valve to help manage the pressure of the gas and facilitate more efficient storage and transportation. We expect this new concept will result in a lighter, more affordable product with increased storage capacity. The nanovalved adsorbent concept demonstrated here can be potentially extended for the storage of other important gas molecules targeted for diverse relevant functional applications. PMID:27124722

  11. 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.

  12. 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

  13. 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.

  14. High Mobility Group Box Protein 1 (HMGB1): The Prototypical Endogenous Danger Molecule

    PubMed Central

    Yang, Huan; Wang, Haichao; Chavan, Sangeeta S; Andersson, Ulf

    2015-01-01

    High mobility group box protein 1 (HMGB1) is an evolutionary ancient nuclear protein that exerts divergent biological tasks inside and outside of cells. The functions of HMGB1 depend on location, binding partners and redox states of the molecule. In the nucleus, HMGB1 organizes DNA and nucleosomes and regulates gene transcription. Upon cell activation or injury, nuclear HMGB1 can translocate to the cytoplasm, where it is involved in inflammasome activation and pyroptosis, as well as regulation of the autophagy/apoptosis balance. When actively secreted or passively released into the extracellular milieu, HMGB1 has cytokine, chemokine, neuroimmune and metabolic activities. Thus, HMGB1 plays multiple roles in the pathogenesis of inflammatory diseases and mediates immune responses that range from inflammation and bacterial killing to tissue repair. HMGB1 has been associated with divergent clinical conditions such as sepsis, rheumatoid arthritis and atherosclerosis. HMGB1 initiates and perpetuates immune responses during infectious and sterile inflammation, as the archetypical alarmin and damage-associated molecular pattern (DAMP) molecule. We here describe advances in the understanding of HMGB1 biology with focus on recent findings of its mission as a DAMP in danger sensing and as a therapeutic target in inflammatory diseases. PMID:26605648

  15. Nucleic Acid Chaperone Activity of HIV-1 NC Proteins Investigated by Single Molecule DNA Stretching

    NASA Astrophysics Data System (ADS)

    Williams, Mark C.; Gorelick, Robert J.; Musier-Forsyth, Karin; Bloomfield, Victor A.

    2002-03-01

    HIV-1 Nucleocapsid Protein (NC) is a nucleic acid chaperone protein that is responsible for facilitating numerous nucleic acid rearrangements throughout the reverse transcription cycle of HIV-1. To understand the mechanism of NC’s chaperone function, we carried out single molecule DNA stretching studies in the presence of NC and mutant forms of NC. Using an optical tweezers instrument, we stretch single DNA molecules from the double-stranded helical state to the single-stranded (coil) state. Based on the observed cooperativity of DNA force-induced melting, we find that the fraction of melted base pairs at room temperature is increased dramatically in the presence of NC. Thus, upon NC binding, increased thermal fluctuations cause continuous melting and reannealing of base pairs so that DNA strands are able to rapidly sample configurations in order to find the lowest energy state. While NC destabilizes the double-stranded form of DNA, a mutant form of NC that lacks the zinc finger structures does not. DNA stretching experiments carried out in the presence of NC variants containing more subtle changes in the zinc finger structures were conducted to elucidate the contribution of each individual finger to NC’s chaperone activity, and these results will be reported.

  16. Mechanism of Inhibition of Cholesteryl Ester Transfer Protein by Small Molecule Inhibitors.

    PubMed

    Chirasani, Venkat R; Sankar, Revathi; Senapati, Sanjib

    2016-08-25

    Cholesteryl ester transfer protein (CETP) facilitates the bidirectional exchange of cholesteryl esters and triglycerides between high-density lipoproteins and low- or very low-density lipoproteins. Recent studies have shown that the impairment of lipid exchange processes of CETP can be an effective strategy for the treatment of cardiovascular diseases (CVDs). Understanding the molecular mechanism of CETP inhibition has, therefore, attracted tremendous attention in recent past. In this study, we explored the detailed mechanism of CETP inhibition by a series of recently reported small molecule inhibitors that are currently under preclinical testing. Our results from molecular dynamics simulations and protein-ligand docking studies suggest that the hydrophobic interactions between the CETP core tunnel residues and inhibitor moieties play a pivotal role, and physical occlusion of the CETP tunnel by these small molecules is the primary mechanism of CETP inhibition. Interestingly, bound inhibitors were found to increase the plasticity of CETP, which was explained by principal component analysis that showed a larger space of sampling of CETP C-domain due to inhibitor binding. The atomic-level details presented here could help accelerate the structure-based drug-discovery processes targeting CETP for CVD therapeutics. PMID:27111423

  17. Small Molecule Antagonizes Autoinhibition and Activates AMP-activated Protein Kinase in Cells*

    PubMed Central

    Pang, Tao; Zhang, Zhen-Shan; Gu, Min; Qiu, Bei-Ying; Yu, Li-Fang; Cao, Peng-Rong; Shao, Wei; Su, Ming-Bo; Li, Jing-Ya; Nan, Fa-Jun; Li, Jia

    2008-01-01

    AMP-activated protein kinase (AMPK) serves as an energy sensor and is considered a promising drug target for treatment of type II diabetes and obesity. A previous report has shown that mammalian AMPK α1 catalytic subunit including autoinhibitory domain was inactive. To test the hypothesis that small molecules can activate AMPK through antagonizing the autoinhibition in α subunits, we screened a chemical library with inactive human α1394 (α1, residues 1-394) and found a novel small-molecule activator, PT1, which dose-dependently activated AMPK α1394, α1335, α2398, and even heterotrimer α1β1γ1. Based on PT1-docked AMPK α1 subunit structure model and different mutations, we found PT1 might interact with Glu-96 and Lys-156 residues near the autoinhibitory domain and directly relieve autoinhibition. Further studies using L6 myotubes showed that the phosphorylation of AMPK and its downstream substrate, acetyl-CoA carboxylase, were dose-dependently and time-dependently increased by PT1 with-out an increase in cellular AMP:ATP ratio. Moreover, in HeLa cells deficient in LKB1, PT1 enhanced AMPK phosphorylation, which can be inhibited by the calcium/calmodulin-dependent protein kinase kinases inhibitor STO-609 and AMPK inhibitor compound C. PT1 also lowered hepatic lipid content in a dose-dependent manner through AMPK activation in HepG2 cells, and this effect was diminished by compound C. Taken together, these data indicate that this small-molecule activator may directly activate AMPK via antagonizing the autoinhibition in vitro and in cells. This compound highlights the effort to discover novel AMPK activators and can be a useful tool for elucidating the mechanism responsible for conformational change and autoinhibitory regulation of AMPK. PMID:18321858

  18. 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

  19. 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

  20. Single-molecule Observation of Protein Folding in Symmetric GroEL-(GroES)2 Complexes*

    PubMed Central

    Takei, Yodai; Iizuka, Ryo; Ueno, Taro; Funatsu, Takashi

    2012-01-01

    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

  1. 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.

  2. Identification of Small-Molecule Inhibitors of the Antiapoptotic Protein Myeloid Cell Leukaemia-1 (Mcl-1).

    PubMed

    Beekman, Andrew M; O'Connell, Maria A; Howell, Lesley A

    2016-04-19

    Protein-protein interactions (PPIs) control many cellular processes in cancer and tumour growth. Of significant interest is the role PPIs play in regulating apoptosis. The overexpression of the antiapoptosis regulating Bcl-2 family of proteins is commonly observed in several cancers, leading to resistance towards both radiation and chemotherapies. From this family, myeloid cell leukemia-1 (Mcl-1) has proven the most difficult to target, and one of the leading causes of treatment resistance. Exploiting the selective PPI between the apoptosis-regulating protein Noxa and Mcl-1, utilising a fluorescence polarization assay, we have identified four small molecules with the ability to modulate Mcl-1. The identified compounds were computationally modelled and docked against the Mcl-1 binding interface to obtain structural information about their binding sites allowing for future analogue design. When examined for their activity towards pancreatic cell lines that overexpress Mcl-1 (MiaPaCa-2 and BxPC-3), the identified compounds demonstrated growth inhibition, suggesting effective Mcl-1 modulation. PMID:26616140

  3. Predicting the order in which contacts are broken during single molecule protein stretching experiments

    PubMed Central

    Sułkowska, Joanna I.; Kloczkowski, Andrzej; Sen, Taner Z.; Cieplak, Marek; Jernigan, Robert L.

    2008-01-01

    We combine two methods to enable the prediction of the order in which contacts are broken under external stretching forces in single molecule experiments. These two methods are Gô-like models and elastic network models. The Gô-like models have shown remarkable success in representing many aspects of protein behavior, including the reproduction of experimental data obtained from atomic force microscopy. The simple elastic network models are often used successfully to predict the fluctuations of residues around their mean positions, comparing favorably with the experimentally measured crystallographic B-factors. The behavior of biomolecules under external forces has been demonstrated to depend principally on their elastic properties and the overall shape of their structure. We have studied in detail the muscle protein titin and green fluorescent protein and tested for ten other proteins. First, we stretch the proteins computationally by performing stochastic dynamics simulations with the Gô-like model. We obtain the force–displacement curves and unfolding scenarios of possible mechanical unfolding. We then use the elastic network model to calculate temperature factors (B-factors) and compare the slowest modes of motion for the stretched proteins and compare them with the predicted order of breaking contacts between residues in the Gô-like model. Our results show that a simple Gaussian network model is able to predict contacts that break in the next time stage of stretching. Additionally, we have found that the contact disruption is strictly correlated with the highest force exerted by the backbone on these residues. Our prediction of bond-breaking agrees well with the unfolding scenario obtained with the Gô-like model. We anticipate that this method will be a useful new tool for interpreting stretching experiments. PMID:17932935

  4. Nectin-like molecule 1 is a protein 4.1N associated protein and recruits protein 4.1N from cytoplasm to the plasma membrane.

    PubMed

    Zhou, Yan; Du, Guangwei; Hu, Xiaoyan; Yu, Shun; Liu, Yaobo; Xu, Yaqin; Huang, Xiaowei; Liu, Jin; Yin, Bin; Fan, Ming; Peng, Xiaozhong; Qiang, Boqin; Yuan, Jiangang

    2005-05-20

    Nectins are immunoglobulin superfamily adhesion molecules that participate in the organization of epithelial and endothelial junctions. Sharing high homology with the poliovirus receptor (PVR/CD155), nectins were also named poliovirus receptor-related proteins (PRRs). Four nectins and five nectin-like molecules have been identified. Here we describe the cloning and characterization of human and mouse nectin-like molecular 1 (NECL1). Human and mouse NECL1 share 87.3% identity at the amino acid level. NECL1 contains an ectodomain made of three immunoglobulin-like domains, and a cytoplasmic region homologous to those of glycophorin C and contactin-associated protein. RNA blot and in situ hybridization analysis showed that NECL1 predominantly expressed in the central nervous system, mainly in neuronal cell bodies in a variety of brain regions including the cerebellum, cerebral cortex and hippocampus. In vitro binding assay proved the association of NECL1 with protein 4.1N. NECL1 localizes to the cell-cell junctions and recruits protein 4.1N to the plasma membranes through its C-terminus, thus may regulate the function of the cell-cell junction. We propose that the NECL1 and protein 4.1N complex is involved in the morphological development, stability, and dynamic plasticity of the nervous system. PMID:15893517

  5. 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

  6. Discovery and structural characterization of a small molecule 14-3-3 protein-protein interaction inhibitor

    SciTech Connect

    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

    2013-02-14

    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 kD{sub a} 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 14-3-3{zeta} 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.

  7. Discovery and structural characterization of a small molecule 14-3-3 protein-protein interaction inhibitor.

    PubMed

    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

    2011-09-27

    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 kD(a) 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

  8. 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

  9. IP-FCM Measures Physiologic Protein-Protein Interactions Modulated by Signal Transduction and Small-Molecule Drug Inhibition

    PubMed Central

    Smith, Stephen E. P.; Bida, Anya T.; Davis, Tessa R.; Sicotte, Hugues; Patterson, Steven E.; Gil, Diana; Schrum, Adam G.

    2012-01-01

    Protein-protein interactions (PPI) mediate the formation of intermolecular networks that control biological signaling. For this reason, PPIs are of outstanding interest in pharmacology, as they display high specificity and may represent a vast pool of potentially druggable targets. However, the study of physiologic PPIs can be limited by conventional assays that often have large sample requirements and relatively low sensitivity. Here, we build on a novel method, immunoprecipitation detected by flow cytometry (IP-FCM), to assess PPI modulation during either signal transduction or pharmacologic inhibition by two different classes of small-molecule compounds. First, we showed that IP-FCM can detect statistically significant differences in samples possessing a defined PPI change as low as 10%. This sensitivity allowed IP-FCM to detect a PPI that increases transiently during T cell signaling, the antigen-inducible interaction between ZAP70 and the T cell antigen receptor (TCR)/CD3 complex. In contrast, IP-FCM detected no ZAP70 recruitment when T cells were stimulated with antigen in the presence of the src-family kinase inhibitor, PP2. Further, we tested whether IP-FCM possessed sufficient sensitivity to detect the effect of a second, rare class of compounds called SMIPPI (small-molecule inhibitor of PPI). We found that the first-generation non-optimized SMIPPI, Ro-26-4550, inhibited the IL-2:CD25 interaction detected by IP-FCM. This inhibition was detectable using either a recombinant CD25-Fc chimera or physiologic full-length CD25 captured from T cell lysates. Thus, we demonstrate that IP-FCM is a sensitive tool for measuring physiologic PPIs that are modulated by signal transduction and pharmacologic inhibition. PMID:23029201

  10. 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

  11. 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

  12. The Catalytic Activity of Protein-Disulfide Isomerase Requires a Conformationally Flexible Molecule

    SciTech Connect

    Tian, G.; Kober, F; Lewandrowski, U; Sickmann, A; Lennarz, W; Schindelin, H

    2008-01-01

    Protein-disulfide isomerase (PDI) catalyzes the formation of the correct pattern of disulfide bonds in secretory proteins. A low resolution crystal structure of yeast PDI described here reveals large scale conformational changes compared with the initially reported structure, indicating that PDI is a highly flexible molecule with its catalytic domains, a and a?, representing two mobile arms connected to a more rigid core composed of the b and b? domains. Limited proteolysis revealed that the linker between the a domain and the core is more susceptible to degradation than that connecting the a? domain to the core. By restricting the two arms with inter-domain disulfide bonds, the molecular flexibility of PDI, especially that of its a domain, was demonstrated to be essential for the enzymatic activity in vitro and in vivo. The crystal structure also featured a PDI dimer, and a propensity to dimerize in solution and in the ER was confirmed by cross-linking experiments and the split green fluorescent protein system. Although sedimentation studies suggested that the self-association of PDI is weak, we hypothesize that PDI exists as an interconvertible mixture of monomers and dimers in the endoplasmic reticulum due to its high abundance in this compartment.

  13. High-throughput single-molecule force spectroscopy for membrane proteins

    NASA Astrophysics Data System (ADS)

    Bosshart, Patrick D.; Casagrande, Fabio; Frederix, Patrick L. T. M.; Ratera, Merce; Bippes, Christian A.; Müller, Daniel J.; Palacin, Manuel; Engel, Andreas; Fotiadis, Dimitrios

    2008-09-01

    Atomic force microscopy-based single-molecule force spectroscopy (SMFS) is a powerful tool for studying the mechanical properties, intermolecular and intramolecular interactions, unfolding pathways, and energy landscapes of membrane proteins. One limiting factor for the large-scale applicability of SMFS on membrane proteins is its low efficiency in data acquisition. We have developed a semi-automated high-throughput SMFS (HT-SMFS) procedure for efficient data acquisition. In addition, we present a coarse filter to efficiently extract protein unfolding events from large data sets. The HT-SMFS procedure and the coarse filter were validated using the proton pump bacteriorhodopsin (BR) from Halobacterium salinarum and the L-arginine/agmatine antiporter AdiC from the bacterium Escherichia coli. To screen for molecular interactions between AdiC and its substrates, we recorded data sets in the absence and in the presence of L-arginine, D-arginine, and agmatine. Altogether ~400 000 force-distance curves were recorded. Application of coarse filtering to this wealth of data yielded six data sets with ~200 (AdiC) and ~400 (BR) force-distance spectra in each. Importantly, the raw data for most of these data sets were acquired in one to two days, opening new perspectives for HT-SMFS applications.

  14. 4 °C preparation of ferrite nanoparticles having protein molecules immobilized on their surfaces

    NASA Astrophysics Data System (ADS)

    Nishimura, K.; Hasegawa, M.; Ogura, Y.; Nishi, T.; Kataoka, K.; Handa, H.; Abe, M.

    2002-05-01

    Trypsin, a proteolytic enzyme or a protein, was immobilized onto the surfaces of ferrite (a Fe3O4-γFe2O3 mixed solution) fine particles, ˜8 nm in size, during the process in which the particles were synthesized from an aqueous solution. The process was performed in the open air at a temperature as low as 4 °C and on near-neutral condition of pH⩽9, which is compatible with most of the bioactive molecules as well as trypsin. Therefore this technique is advantageous for preparing magnetite particles having biomolecules immobilized on their surfaces, which will be used for biomedical applications utilizing magnetic separation technique.

  15. 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

  16. Structural Elucidation of a Small Molecule Inhibitor of Protein Disulfide Isomerase

    PubMed Central

    2015-01-01

    Compound libraries provide a starting point for multiple biological investigations, but the structural integrity of compounds is rarely assessed experimentally until a late stage in the research process. Here, we describe the discovery of a neuroprotective small molecule that was originally incorrectly annotated with a chemical structure. We elucidated the correct structure of the active compound using analytical chemistry, revealing it to be the natural product securinine. We show that securinine is protective in a cell model of Huntington disease and identify the binding site of securinine to its target, protein disulfide isomerase using NMR chemical shift perturbation studies. We show that securinine displays favorable pharmaceutical properties, making it a promising compound for in vivo studies in neurodegenerative disease models. In addition to finding this unexpected activity of securinine, this study provides a systematic roadmap to those who encounter compounds with incorrect structural annotation in the course of screening campaigns. PMID:26500720

  17. Small Molecule Substrate Phosphorylation Site Inhibitors of Protein Kinases: Approaches and Challenges

    PubMed Central

    2015-01-01

    Protein kinases are important mediators of cellular communication and attractive drug targets for many diseases. Although success has been achieved with developing ATP-competitive kinase inhibitors, the disadvantages of ATP-competitive inhibitors have led to increased interest in targeting sites outside of the ATP binding pocket. Kinase inhibitors with substrate-competitive, ATP-noncompetitive binding modes are promising due to the possibility of increased selectivity and better agreement between biochemical and in vitro potency. However, the difficulty of identifying these types of inhibitors has resulted in significantly fewer small molecule substrate phosphorylation site inhibitors being reported compared to ATP-competitive inhibitors. This review surveys reported substrate phosphorylation site inhibitors and methods that can be applied to the discovery of such inhibitors, including a discussion of the challenges inherent to these screening methods. PMID:25494294

  18. 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

  19. Discovery and mechanistic study of a small molecule inhibitor for motor protein KIFC1.

    PubMed

    Wu, Jiaquan; Mikule, Keith; Wang, Wenxian; Su, Nancy; Petteruti, Philip; Gharahdaghi, Farzin; Code, Erin; Zhu, Xiahui; Jacques, Kelly; Lai, Zhongwu; Yang, Bin; Lamb, Michelle L; Chuaqui, Claudio; Keen, Nicholas; Chen, Huawei

    2013-10-18

    Centrosome amplification is observed in many human cancers and has been proposed to be a driver of both genetic instability and tumorigenesis. Cancer cells have evolved mechanisms to bundle multiple centrosomes into two spindle poles to avoid multipolar mitosis that can lead to chromosomal segregation defects and eventually cell death. KIFC1, a kinesin-14 family protein, plays an essential role in centrosomal bundling in cancer cells, but its function is not required for normal diploid cell division, suggesting that KIFC1 is an attractive therapeutic target for human cancers. To this end, we have identified the first reported small molecule inhibitor AZ82 for KIFC1. AZ82 bound specifically to the KIFC1/microtubule (MT) binary complex and inhibited the MT-stimulated KIFC1 enzymatic activity in an ATP-competitive and MT-noncompetitive manner with a Ki of 0.043 μM. AZ82 effectively engaged with the minus end-directed KIFC1 motor inside cells to reverse the monopolar spindle phenotype induced by the inhibition of the plus end-directed kinesin Eg5. Treatment with AZ82 caused centrosome declustering in BT-549 breast cancer cells with amplified centrosomes. Consistent with genetic studies, our data confirmed that KIFC1 inhibition by a small molecule holds promise for targeting cancer cells with amplified centrosomes and provided evidence that functional suppression of KIFC1 by inhibiting its enzymatic activity could be an effective means for developing cancer therapeutics. PMID:23895133

  20. Suppressing RNA silencing with small molecules and the viral suppressor of RNA silencing protein p19.

    PubMed

    Danielson, Dana C; Filip, Roxana; Powdrill, Megan H; O'Hara, Shifawn; Pezacki, John P

    2015-08-01

    RNA silencing is a gene regulatory and host defense mechanism whereby small RNA molecules are engaged by Argonaute (AGO) proteins, which facilitate gene knockdown of complementary mRNA targets. Small molecule inhibitors of AGO represent a convenient method for reversing this effect and have applications in human therapy and biotechnology. Viral suppressors of RNA silencing, such as p19, can also be used to suppress the pathway. Here we assess the compatibility of these two approaches, by examining whether synthetic inhibitors of AGO would inhibit p19-siRNA interactions. We observe that aurintricarboxylic acid (ATA) is a potent inhibitor of p19's ability to bind siRNA (IC50 = 0.43 μM), oxidopamine does not inhibit p19:siRNA interactions, and suramin is a mild inhibitor of p19:siRNA interactions (IC50 = 430 μM). We observe that p19 and suramin are compatible inhibitors of RNA silencing in human hepatoma cells. Our data suggests that at least some inhibitors of AGO may be used in combination with p19 to inhibit RNA silencing at different points in the pathway. PMID:26079891

  1. 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.

  2. 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.

  3. 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.

  4. Single-Molecule Studies of Unlabeled Full-Length p53 Protein Binding to DNA.

    PubMed

    Nuttall, Philippa; Lee, Kidan; Ciccarella, Pietro; Carminati, Marco; Ferrari, Giorgio; Kim, Ki-Bum; Albrecht, Tim

    2016-03-10

    p53 is an antitumor protein that plays an important role in apoptosis, preserving genomic stability and preventing angiogenesis, and it has been implicated in a large number of human cancers. For this reason it is an interesting target for both fundamental studies, such as the mechanism of interaction with DNA, and applications in biosensing. Here, we report a comprehensive study of label-free, full length p53 (flp53) and its interaction with engineered double-stranded DNA in vitro, at the single-molecule level, using atomic force microscopy (AFM) imaging and solid-state nanopore sensing. AFM data show that dimeric and tetrameric p53 bind to the DNA in a sequence-specific manner, confirming previously reported relative binding affinities. The statistical significance is tested using both the Grubbs test and stochastic simulations. For the first time, ultralow noise solid-state nanopore sensors are employed for the successful differentiation between bare DNA and p53/DNA complexes. Furthermore, translocation statistics reflect the binding affinities of different DNA sequences, in accordance with AFM data. Our results thus highlight the potential of solid-state nanopore sensors for single-molecule biosensing, especially when labeling is either not possible or at least not a viable option. PMID:26855037

  5. 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

  6. 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.

  7. 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.

  8. STAT1:DNA sequence-dependent binding modulation by phosphorylation, protein:protein interactions and small-molecule inhibition

    PubMed Central

    Bonham, Andrew J.; Wenta, Nikola; Osslund, Leah M.; Prussin, Aaron J.; Vinkemeier, Uwe; Reich, Norbert O.

    2013-01-01

    The DNA-binding specificity and affinity of the dimeric human transcription factor (TF) STAT1, were assessed by total internal reflectance fluorescence protein-binding microarrays (TIRF-PBM) to evaluate the effects of protein phosphorylation, higher-order polymerization and small-molecule inhibition. Active, phosphorylated STAT1 showed binding preferences consistent with prior characterization, whereas unphosphorylated STAT1 showed a weak-binding preference for one-half of the GAS consensus site, consistent with recent models of STAT1 structure and function in response to phosphorylation. This altered-binding preference was further tested by use of the inhibitor LLL3, which we show to disrupt STAT1 binding in a sequence-dependent fashion. To determine if this sequence-dependence is specific to STAT1 and not a general feature of human TF biology, the TF Myc/Max was analysed and tested with the inhibitor Mycro3. Myc/Max inhibition by Mycro3 is sequence independent, suggesting that the sequence-dependent inhibition of STAT1 may be specific to this system and a useful target for future inhibitor design. PMID:23180800

  9. The Mycobacterium tuberculosis Outer Membrane Channel Protein CpnT Confers Susceptibility to Toxic Molecules

    PubMed Central

    Danilchanka, Olga; Pires, David

    2015-01-01

    Mycobacterium tuberculosis, the causative agent of tuberculosis, is protected from toxic solutes by an effective outer membrane permeability barrier. Recently, we showed that the outer membrane channel protein CpnT is required for efficient nutrient uptake by M. tuberculosis and Mycobacterium bovis BCG. In this study, we found that the cpnT mutant of M. bovis BCG is more resistant than the wild type to a large number of drugs and antibiotics, including rifampin, ethambutol, clarithromycin, tetracycline, and ampicillin, by 8- to 32-fold. Furthermore, the cpnT mutant of M. bovis BCG was 100-fold more resistant to nitric oxide, a major bactericidal agent required to control M. tuberculosis infections in mice. Thus, CpnT constitutes the first outer membrane susceptibility factor in slow-growing mycobacteria. The dual functions of CpnT in uptake of nutrients and mediating susceptibility to toxic molecules are reflected in macrophage infection experiments: while loss of CpnT was detrimental for M. bovis BCG in macrophages that enable bacterial replication, presumably due to inadequate nutrient uptake, it conferred a survival advantage in macrophages that mount a strong bactericidal response. Importantly, the cpnT gene showed a significantly higher density of nonsynonymous mutations in drug-resistant clinical M. tuberculosis strains, indicating that CpnT is under selective pressure in human tuberculosis and/or during chemotherapy. Our results indicate that the CpnT channel constitutes an outer membrane gateway controlling the influx of nutrients and toxic molecules into slow-growing mycobacteria. This study revealed that reducing protein-mediated outer membrane permeability might constitute a new drug resistance mechanism in slow-growing mycobacteria. PMID:25645841

  10. Construction of a functional S-layer fusion protein comprising an immunoglobulin G-binding domain for development of specific adsorbents for extracorporeal blood purification.

    PubMed

    Völlenkle, Christine; Weigert, Stefan; Ilk, Nicola; Egelseer, Eva; Weber, Viktoria; Loth, Fritz; Falkenhagen, Dieter; Sleytr, Uwe B; Sára, Margit

    2004-03-01

    The chimeric gene encoding a C-terminally-truncated form of the S-layer protein SbpA from Bacillus sphaericus CCM 2177 and two copies of the Fc-binding Z-domain was constructed, cloned, and heterologously expressed in Escherichia coli HMS174(DE3). The Z-domain is a synthetic analogue of the B-domain of protein A, capable of binding the Fc part of immunoglobulin G (IgG). The S-layer fusion protein rSbpA(31-1068)/ZZ retained the specific properties of the S-layer protein moiety to self-assemble in suspension and to recrystallize on supports precoated with secondary cell wall polymer (SCWP), which is the natural anchoring molecule for the S-layer protein in the bacterial cell wall. Due to the construction principle of the S-layer fusion protein, the ZZ-domains remained exposed on the outermost surface of the protein lattice. The binding capacity of the native or cross-linked monolayer for human IgG was determined by surface plasmon resonance measurements. For batch adsorption experiments, 3-microm-diameter, biocompatible cellulose-based, SCWP-coated microbeads were used for recrystallization of the S-layer fusion protein. In the case of the native monolayer, the binding capacity for human IgG was 5.1 ng/mm(2), whereas after cross-linking with dimethyl pimelimidate, 4.4 ng of IgG/mm(2) was bound. This corresponded to 78 and 65% of the theoretical saturation capacity of a planar surface for IgGs aligned in the upright position, respectively. Compared to commercial particles used as immunoadsorbents to remove autoantibodies from sera of patients suffering from an autoimmune disease, the IgG binding capacity of the S-layer fusion protein-coated microbeads was at least 20 times higher. For that reason, this novel type of microbeads should find application in the microsphere-based detoxification system. PMID:15006773

  11. 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

  12. 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

  13. Substrate Pathways in the Nitrogenase MoFe Protein by Experimental Identification of Small Molecule Binding Sites

    PubMed Central

    2016-01-01

    In the nitrogenase molybdenum-iron (MoFe) protein, we have identified five potential substrate access pathways from the protein surface to the FeMo-cofactor (the active site) or the P-cluster using experimental structures of Xe pressurized into MoFe protein crystals from Azotobacter vinelandii and Clostridium pasteurianum. Additionally, all published structures of the MoFe protein, including those from Klebsiella pneumoniae, were analyzed for the presence of nonwater, small molecules bound to the protein interior. Each pathway is based on identification of plausible routes from buried small molecule binding sites to both the protein surface and a metallocluster. Of these five pathways, two have been previously suggested as substrate access pathways. While the small molecule binding sites are not conserved among the three species of MoFe protein, residues lining the pathways are generally conserved, indicating that the proposed pathways may be accessible in all three species. These observations imply that there is unlikely a unique pathway utilized for substrate access from the protein surface to the active site; however, there may be preferred pathways such as those described here. PMID:25710326

  14. Separation of the attractive and repulsive contributions to the adsorbate-adsorbate interactions of polar adsorbates on Si(100)

    NASA Astrophysics Data System (ADS)

    Lin, Ying-Hsiu; Jeng, Horng-Tay; Lin, Deng-Sung

    2015-11-01

    Dissociative adsorption of H2O, NH3, CH3OH and CH3NH2 polar molecules on the Si(100) surface results in a 1:1 mixture of two adsorbates (H and multi-atomic fragment A = OH, NH2, CH3O, CH3NH, respectively) on the surface. By using density functional theory (DFT) calculations, the adsorption geometry, the total energies and the charge densities for various possible ordered structures of the mixed adsorbate layer have been found. Analyzing the systematic trends in the total energies unveils concurrently the nearest-neighbor interactions ENN and the next nearest-neighbor interactions ENNN between two polar adsorbates A. In going from small to large polar adsorbates, ENN's exhibit an attractive-to-repulsive crossover behavior, indicating that they include competing attractive and repulsive contributions. Exploration of the charge density distributions allows the estimation of the degree of charge overlapping between immediately neighboring A's, the resulting contribution of the steric repulsions, and that of the attractive interactions to the corresponding ENN's. The attractive contributions to nearest neighboring adsorbate-adsorbate interactions between the polar adsorbates under study are shown to result from hydrogen bonds or dipole-dipole interactions.

  15. Mechanism of amyloid β-protein dimerization determined using single-molecule AFM force spectroscopy

    NASA Astrophysics Data System (ADS)

    Lv, Zhengjian; Roychaudhuri, Robin; Condron, Margaret M.; Teplow, David B.; Lyubchenko, Yuri L.

    2013-10-01

    Aβ42 and Aβ40 are the two primary alloforms of human amyloid β-protein (Aβ). The two additional C-terminal residues of Aβ42 result in elevated neurotoxicity compared with Aβ40, but the molecular mechanism underlying this effect remains unclear. Here, we used single-molecule force microscopy to characterize interpeptide interactions for Aβ42 and Aβ40 and corresponding mutants. We discovered a dramatic difference in the interaction patterns of Aβ42 and Aβ40 monomers within dimers. Although the sequence difference between the two peptides is at the C-termini, the N-terminal segment plays a key role in the peptide interaction in the dimers. This is an unexpected finding as N-terminal was considered as disordered segment with no effect on the Aβ peptide aggregation. These novel properties of Aβ proteins suggests that the stabilization of N-terminal interactions is a switch in redirecting of amyloids form the neurotoxic aggregation pathway, opening a novel avenue for the disease preventions and treatments.

  16. Single molecule compression reveals intra-protein forces drive cytotoxin pore formation

    PubMed Central

    Czajkowsky, Daniel M; Sun, Jielin; Shen, Yi; Shao, Zhifeng

    2015-01-01

    Perfringolysin O (PFO) is a prototypical member of a large family of pore-forming proteins that undergo a significant reduction in height during the transition from the membrane-assembled prepore to the membrane-inserted pore. Here, we show that targeted application of compressive forces can catalyze this conformational change in individual PFO complexes trapped at the prepore stage, recapitulating this critical step of the spontaneous process. The free energy landscape determined from these measurements is in good agreement with that obtained from molecular dynamics simulations showing that an equivalent internal force is generated by the interaction of the exposed hydrophobic residues with the membrane. This hydrophobic force is transmitted across the entire structure to produce a compressive stress across a distant, otherwise stable domain, catalyzing its transition from an extended to compact conformation. Single molecule compression is likely to become an important tool to investigate conformational transitions in membrane proteins. DOI: http://dx.doi.org/10.7554/eLife.08421.001 PMID:26652734

  17. Protein-DNA interactions in high speed AFM: single molecule diffusion analysis of human RAD54.

    PubMed

    Sanchez, Humberto; Suzuki, Yuki; Yokokawa, Masatoshi; Takeyasu, Kunio; Wyman, Claire

    2011-11-01

    High-speed AFM (atomic force microscopy also called scanning force microscopy) provides nanometre spatial resolution and sub-second temporal resolution images of individual molecules. We exploit these features to study diffusion and motor activity of the RAD54 DNA repair factor. Human RAD54 functions at critical steps in recombinational-DNA repair. It is a member of the Swi2/Snf2 family of chromatin remodelers that translocate on DNA using ATP hydrolysis. A detailed single molecular description of DNA-protein interactions shows intermediate states and distribution of variable states, usually hidden by ensemble averaging. We measured the motion of individual proteins using single-particle tracking and observed that random walks were affected by imaging-buffer composition. Non-Brownian diffusion events were characterized in the presence and in the absence of nucleotide cofactors. Double-stranded DNA immobilized on the surface functioned as a trap reducing Brownian motion. Distinct short range slides and hops on DNA were visualized by high-speed AFM. These short-range interactions were usually inaccessible by other methods based on optical resolution. RAD54 monomers displayed a diffusive behavior unrelated to the motor activity. PMID:21986699

  18. 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

  19. 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. PMID:24906121

  20. 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

  1. Single DNA molecule stretching measures the activity of chemicals that target the HIV-1 nucleocapsid protein

    PubMed Central

    Cruceanu, Margareta; Stephen, Andrew G.; Beuning, Penny J.; Gorelick, Robert J.; Fisher, Robert J.; Williams, Mark C.

    2006-01-01

    We develop a biophysical method for investigating chemical compounds that target the nucleic acid chaperone activity of HIV-1 nucleocapsid protein (NCp7). We used an optical tweezers instrument to stretch single λ-DNA molecules through the helix-to-coil transition in the presence of NCp7 and various chemical compounds. The change in the helix-coil transition width induced by wild-type NCp7 and its zinc finger variants correlates with in vitro nucleic acid chaperone activity measurements and in vivo assays. The compound-NC interaction measured here reduces NCp7’s capability to alter the transition width. Purified compounds from the NCI Diversity set, 119889, 119911, and 119913 reduce the chaperone activity of 5 nM NC in aqueous solution at 10 nM, 25 nM, and 100 nM concentration, respectively. Similarly, gallein reduced the activity of 4 nM NC at 100 nM concentration. Further analysis allows us to dissect the impact of each compound on both sequence-specific and non-sequence-specific DNA binding of NC, two of the main components of NC’s nucleic acid chaperone activity. These results suggest that DNA stretching experiments can be used to screen chemical compounds targeting NC proteins, and to further explore the mechanisms by which these compounds interact with NC and alter its nucleic acid chaperone activity. PMID:17034752

  2. Early Growth Response Protein 1 Promotes Restenosis by Upregulating Intercellular Adhesion Molecule-1 in Vein Graft

    PubMed Central

    Zhang, Kui; Cao, Jian; Dong, Ran; Du, Jie

    2013-01-01

    Objectives. To verify the relationship between Egr-1 and vein graft restenosis and investigate the related mechanisms. Methods. Mouse vein graft models were established in Egr-1 knockout (KO) and wild-type (WT) mice. The vein grafts in the mice were taken for pathological examination and immunohistochemical analysis. The endothelial cells (ECs) were stimulated by using a computer-controlled cyclic stress unit. BrdU staining and PCR were used to detect ECs proliferation activity and Egr-1 and ICAM-1 mRNA expression, respectively. Western-blot analysis was also used to detect expression of Egr-1 and intercellular adhesion molecule-1 (ICAM-1) proteins. Results. The lumens of vein grafts in Egr-1 KO mice were wider than in WT mice. ECs proliferation after mechanical stretch stimulation was suppressed by Egr-1 knockout (P < 0.05). Both in vein grafts and ECs from WT mice after mechanical stretch stimulation, mRNA expression and protein of Egr-1 and ICAM-1 showed increases (P < 0.05). However, ICAM-1 expression was significantly suppressed in ECs from Egr-1 knockout mice (P < 0.05). Conclusions. Egr-1 may promote ECs proliferation and result in vein graft restenosis by upregulating the expression of ICAM-1. As a key factor of vein graft restenosis, it could be a target for the prevention of restenosis after CABG surgery. PMID:24386503

  3. Continuous Reduction of Protein-Bound Uraemic Toxins with Improved Oxidative Stress by Using the Oral Charcoal Adsorbent AST-120 in Haemodialysis Patients

    PubMed Central

    Yamamoto, Suguru; Kazama, Junichiro J.; Omori, Kentaro; Matsuo, Koji; Takahashi, Yoshimitsu; Kawamura, Kazuko; Matsuto, Takayuki; Watanabe, Hiroshi; Maruyama, Toru; Narita, Ichiei

    2015-01-01

    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. PMID:26395517

  4. 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.

  5. Addressing the Requirements of High-Sensitivity Single-Molecule Imaging of Low-Copy-Number Proteins in Bacteria.

    PubMed

    Tuson, Hannah H; Aliaj, Alisa; Brandes, Eileen R; Simmons, Lyle A; Biteen, Julie S

    2016-05-18

    Single-molecule fluorescence super-resolution imaging and tracking provide nanometer-scale information about subcellular protein positions and dynamics. These single-molecule imaging experiments can be very powerful, but they are best suited to high-copy number proteins where many measurements can be made sequentially in each cell. We describe artifacts associated with the challenge of imaging a protein expressed in only a few copies per cell. We image live Bacillus subtilis in a fluorescence microscope, and demonstrate that under standard single-molecule imaging conditions, unlabeled B. subtilis cells display punctate red fluorescent spots indistinguishable from the few PAmCherry fluorescent protein single molecules under investigation. All Bacillus species investigated were strongly affected by this artifact, whereas we did not find a significant number of these background sources in two other species we investigated, Enterococcus faecalis and Escherichia coli. With single-molecule resolution, we characterize the number, spatial distribution, and intensities of these impurity spots. PMID:26888309

  6. Examining Adsorbed Polymer Conformations with Fluorescence Imaging

    NASA Astrophysics Data System (ADS)

    Parkes, Maria; Chennaoui, Mourad; Wong, Janet; Tribology Group, Dept. of Mechanical Engineering Team

    2011-03-01

    The conformation of adsorbed polymers can have significant impact on their properties such as dynamics and elasticity as well as their ability to take part in reactions with other molecules. Experimental research to determine adsorbed polymer conformation has relied mainly on atomic force microscopy (AFM) studies. During an AFM scan, the contact between the scanning probe and the polymer could affect the polymer conformation, particularly where parts of the polymer might have formed projected loops and tails. In this work, conformations of model polymers are examined with total internal reflection fluorescence microscopy (TIRFM). The advantage of TIRFM over AFM is that TIRFM is a non contact technique. Lambda DNA labelled along its length with fluorescent probes was adsorbed in a projected 2D -- 3D state. With TIRFM, the relationship between intensity and depth was used as a basis to determine how the conformation of the adsorbed polymers evolved with time using our custom algorithm.

  7. Quantitative real-time kinetics of optogenetic proteins CRY2 and CIB1/N using single-molecule tools

    PubMed Central

    Cui, Yi; Choudhury, Samrat Roy; Irudayaraj, Joseph

    2015-01-01

    In this work we evaluate the interaction of two optogenetic protein variants (CIB1, CIBN) with their complementary protein CRY2 by single-molecule tools in cell-free extracts. After validating the blue light induced co-localization of CRY2 and CIB1/N by Förster resonance energy transfer (FRET) in live cells, a fluorescence correlation spectroscopy (FCS) based method was developed to quantitatively determine the in vitro association of the extracted proteins. Our experiments suggest that CIB1, in comparison with CIBN, possesses a better coupling efficiency with CRY2 due to its intact protein structure and lower diffusion rate within 300 s detection window. PMID:24780222

  8. Two-dimensional convex-molecule fluid model for surface adsorption of proteins: Effect of soft interaction on adsorption equilibria

    NASA Astrophysics Data System (ADS)

    Mahata, Paritosh; Das, Sovan Lal

    2014-12-01

    Adsorption of proteins on membrane surfaces plays an important role in cell biological processes. In this work, we develop a two-dimensional fluid model for proteins. The protein molecules have been modeled as two-dimensional convex and soft particles. The Lennard-Jones potential for circular particles and Kihara (12,6) potential for elliptical particles with hard core have been used to model pairwise intermolecular interactions. The equation of state of the fluid model has been derived using Weeks-Chandler-Andersen decomposition and it involves three parameters, an attraction, a repulsion, and a size parameter, which depend on the shape and core size of the molecules. For validation of the model, a two-dimensional molecular dynamics simulation has been performed. Finally, the model has been applied to study the adsorption of proteins on a flat membrane. In comparison with the existing model of hard and convex particles for protein adsorption, our model predicts a higher packing fraction for the adsorption equilibria. Although the present work is based on Lennard-Jones-type interaction, it can be extended for other specific soft interactions between convex molecules. Thus the model has general applicability for any other two-dimensional adsorption systems of molecules with soft interaction.

  9. Single molecule spectroscopic characterization of a far-red fluorescent protein (HcRed) from the Anthozoa coral Heteractis crispa

    NASA Astrophysics Data System (ADS)

    Cotlet, Mircea; Habuchi, Satoshi; Whitier, Jennifer E.; Werner, James H.; De Schryver, Frans C.; Hofkens, Johan; Goodwin, Peter M.

    2006-02-01

    We report on the photophysical properties of a far-red intrinsic fluorescent protein by means of single molecule and ensemble spectroscopic methods. The green fluorescent protein (GFP) from Aequorea victoria is a popular fluorescent marker with genetically encoded fluorescence and which can be fused to any biological structure without affecting its function. GFP and its variants provide emission colors from blue to yellowish green. Red intrinsic fluorescent proteins from Anthozoa species represent a recent addition to the emission color palette provided by GFPs. Red intrinsic fluorescent markers are on high demand in protein-protein interaction studies based on fluorescence-resonance energy transfer or in multicolor tracking studies or in cellular investigations where autofluorescence possesses a problem. Here we address the photophysical properties of a far-red fluorescent protein (HcRed), a mutant engineered from a chromoprotein cloned from the sea anemone Heteractis crispa, by using a combination of ensemble and single molecule spectroscopic methods. We show evidence for the presence of HcRed protein as an oligomer and for incomplete maturation of its chromophore. Incomplete maturation results in the presence of an immature (yellow) species absorbing/fluorescing at 490/530-nm. This yellow chromophore is involved in a fast resonance-energy transfer with the mature (purple) chromophore. The mature chromophore of HcRed is found to adopt two conformations, a Transoriented form absorbing and 565-nm and non-fluorescent in solution and a Cis-oriented form absorbing at 590-nm and emitting at 645-nm. These two forms co-exist in solution in thermal equilibrium. Excitation-power dependence fluorescence correlation spectroscopy of HcRed shows evidence for singlet-triplet transitions in the microseconds time scale and for cis-trans isomerization occurring in a time scale of tens of microseconds. Single molecule fluorescence data recorded from immobilized HcRed proteins, all

  10. Chemical synthesis of the precursor molecule of the Aequorea green fluorescent protein, subsequent folding, and development of fluorescence

    PubMed Central

    Nishiuchi, Yuji; Inui, Tatsuya; Nishio, Hideki; Bódi, József; Kimura, Terutoshi; Tsuji, Frederick I.; Sakakibara, Shumpei

    1998-01-01

    The present paper describes the total chemical synthesis of the precursor molecule of the Aequorea green fluorescent protein (GFP). The molecule is made up of 238 amino acid residues in a single polypeptide chain and is nonfluorescent. To carry out the synthesis, a procedure, first described in 1981 for the synthesis of complex peptides, was used. The procedure is based on performing segment condensation reactions in solution while providing maximum protection to the segment. The effectiveness of the procedure has been demonstrated by the synthesis of various biologically active peptides and small proteins, such as human angiogenin, a 123-residue protein analogue of ribonuclease A, human midkine, a 121-residue protein, and pleiotrophin, a 136-residue protein analogue of midkine. The GFP precursor molecule was synthesized from 26 fully protected segments in solution, and the final 238-residue peptide was treated with anhydrous hydrogen fluoride to obtain the precursor molecule of GFP containing two Cys(acetamidomethyl) residues. After removal of the acetamidomethyl groups, the product was dissolved in 0.1 M Tris⋅HCl buffer (pH 8.0) in the presence of DTT. After several hours at room temperature, the solution began to emit a green fluorescence (λmax = 509 nm) under near-UV light. Both fluorescence excitation and fluorescence emission spectra were measured and were found to have the same shape and maxima as those reported for native GFP. The present results demonstrate the utility of the segment condensation procedure in synthesizing large protein molecules such as GFP. The result also provides evidence that the formation of the chromophore in GFP is not dependent on any external cofactor. PMID:9811837

  11. 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. PMID:26158404

  12. Single Molecule Series A New View of Protein Synthesis: Mapping the Free Energy Landscape of the Ribosome Using Single-Molecule FRET

    PubMed Central

    Munro, James B.; Vaiana, Andrea; Sanbonmatsu, Kevin Y.; Blanchard, Scott C.

    2010-01-01

    This article reviews the application of single-molecule fluorescence resonance energy transfer (smFRET) methods to the study of protein synthesis catalyzed by the ribosome. smFRET is a powerful new technique that can be used to investigate dynamic processes within enzymes spanning many orders of magnitude. The application of wide-field smFRET imaging methods to the study of dynamic processes in the ribosome offers a new perspective on the mechanism of protein synthesis. Using this technique, the structural and kinetic parameters of tRNA motions within wild-type and specifically mutated ribosome complexes have been obtained that provide valuable new insights into the mechanism and regulation of translation elongation. The results of these studies are discussed in the context of current knowledge of the ribosome mechanism from both structural and biophysical perspectives. PMID:18286627

  13. 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

  14. 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.

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. 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

  20. Single Molecule Analysis of Protein Free U2/U6 snRNAs

    PubMed Central

    Guo, Zhuojun; Karunatilaka, Krishanthi S.; Rueda, David

    2009-01-01

    Spliceosomes catalyze the maturation of precursor mRNAs from yeast to humans. Their catalytic core comprises three small nuclear RNAs (U2, U5 and U6) involved in substrate positioning and catalysis. It has been postulated, but never shown experimentally, that the U2/U6 complex adopts at least two conformations that reflect different activation states. We have used single-molecule fluorescence to probe the structural dynamics of a protein-free RNA complex modeling U2/U6 from yeast and mutants of highly conserved regions. Our data show the presence of at least three distinct conformations in equilibrium. The minimal folding pathway consists of a two-step process with an obligatory intermediate. The first step is strongly magnesium dependent and we provide evidence suggesting the second corresponds to the formation of the genetically conserved helix IB. Site-specific mutations in the highly conserved AGC triad and the U80 base in U6 suggest that the observed conformational dynamics correlate with residues that play an important role in splicing. PMID:19881500

  1. 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.

  2. Synergic interaction between amyloid precursor protein and neural cell adhesion molecule promotes neurite outgrowth

    PubMed Central

    Chen, Keping; Lu, Huixia; Gao, Tianli; Xue, Xiulei; Wang, Chunling; Miao, Fengqin

    2016-01-01

    Alzheimer's disease (AD) is one of the most common neurodegenerative diseases worldwide. The main features of AD are the pathological changes of density and distribution of intracellular neurofibrillary tangles (NFT) and extracellular amyloid plaques. The processing of amyloid beta precursor protein (APP) to β-amyloid peptide (Aβ) is one of the critical events in the pathogenesis of AD. In this study, we evaluated the role of the interaction of neural cell adhesion molecule (NCAM) and APP in neurite outgrowth using two different experimental systems: PC12E2 cells and hippocampal neurons that were isolated from wild type, APP knock-in and APP knock-out mice. PC12E2 cells or hippocampal neurons were co-cultured with NCAM-negative or NCAM-positive fibroblasts L929 cells. We found that APP promoted neurite outgrowth of PC12E2 cells and hippocampal neurons in either the presence or absence of NCAM. Secreted APP can rescue the neurite outgrowth in hippocampal neurons from APP knock-out mice. The interaction of APP and NCAM had synergic effect in promoting neurite outgrowth in both PC12E2 cells and hippocampal neurons. Our results suggested that the interaction of APP with NCAM played an important role in AD development and therefore could be a potential therapeutic target for AD treatment. PMID:26883101

  3. 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

  4. 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

  5. Podocalyxin-like protein 1 functions as an immunomodulatory molecule in breast cancer cells.

    PubMed

    Amo, Laura; Tamayo-Orbegozo, Estíbaliz; Maruri, Natalia; Buqué, Aitziber; Solaun, Miren; Riñón, Marta; Arrieta, Arantza; Larrucea, Susana

    2015-11-01

    Podocalyxin-like protein 1 (PCLP1), a CD34-related sialomucin involved in the regulation of cellular morphology and adhesion, is expressed by a number of normal cells and various tumor cells. In breast malignancies PCLP1 overexpression has been associated with the most aggressive, metastatic cancers and poor prognosis. These observations suggest that PCLP1 expression could provide a mechanism to evade the immune response, thereby promoting metastatic progression of cancer. In the present work, we aimed to determine the effect of PCLP1 overexpressed in MCF7 breast cancer cells on natural killer (NK) cell cytotoxicity, dendritic cell maturation, and agonist-induced T cell proliferation. The results showed that PCLP1 expressed in MCF7 breast cancer cells confers resistance to NK cell-mediated cytolysis and impairs T cell proliferation. Furthermore, PCLP1 decreased the levels of NK cell activating receptors NKG2D, NKp30, NKp44, NKp46, DNAM-1, and CD16 on cell surface in a contact-dependent manner. Moreover, NK cells acquired PCLP1 from MCF7 cells by a process known as trogocytosis. These data reveal a new function of PCLP1 expressed on tumor cells as an immunomodulatory molecule, which may represent a mechanism to evade the immune response. PMID:26276714

  6. High Mobility Group Box Protein 1 (HMGB1)-Partner Molecule Complexes Enhance Cytokine Production by Signaling Through the Partner Molecule Receptor

    PubMed Central

    Hreggvidsdóttir, Hulda Sigridur; Lundberg, Anna M; Aveberger, Ann-Charlotte; Klevenvall, Lena; Andersson, Ulf; Harris, Helena Erlandsson

    2012-01-01

    The nuclear protein high mobility group box protein 1 (HMGB1) promotes inflammation upon extracellular release. HMGB1 induces proinflammatory cytokine production in macrophages via Toll-like receptor (TLR)-4 signaling in a redox-dependent fashion. Independent of its redox state and endogenous cytokine-inducing ability, HMGB1 can form highly immunostimulatory complexes by interaction with certain proinflammatory mediators. Such complexes have the ability to enhance the induced immune response up to 100-fold, compared with induction by the ligand alone. To clarify the mechanisms for these strong synergistic effects, we studied receptor requirements. Interleukin (IL)-6 production was assessed in supernatants from cultured peritoneal macrophages from mice each deficient in one of the HMGB1 receptors (receptor for advanced glycation end products [RAGE], TLR2 or TLR4) or from wild-type controls. The cultures were stimulated with the TLR4 ligand lipopolysaccaride (LPS), the TLR2 ligand Pam3CysSerLys4 (Pam3CSK4), noninflammatory HMGB1 or each TLR ligand in complex with noninflammatory HMGB1. The activity of the HMGB1-TLR ligand complexes relied on engagement of the same receptor as for the noncomplexed TLR ligand, since HMGB1-LPS complexes used TLR4 and HMGB1-Pam3CSK4 complexes used TLR2. Deletion of any of the intracellular adaptor molecules used by TLR2 (myeloid differentiation factor-88 [MyD88], TIR domain–containing adaptor protein [TIRAP]) or TLR4 (MyD88, TIRAP, TIR domain–containing adaptor-inducing interferon-β [TRIF], TRIF-related adaptor molecule [TRAM]) had similar effects on HMGB1 complex activation compared with noncomplexed LPS or Pam3CSK4. This result implies that the enhancing effects of HMGB1-partner molecule complexes are not regulated by the induction of additional signaling cascades. Elucidating HMGB1 receptor usage in processes where HMGB1 acts alone or in complex with other molecules is essential for the understanding of basic HMGB1 biology and

  7. A novel lumazine synthase molecule from Brucella significantly promotes the immune-stimulation effects of antigenic protein.

    PubMed

    Du, Z Q; Wang, J Y

    2015-01-01

    Brucella, an intracellular parasite that infects some livestock and humans, can damage or destroy the reproductive system of livestock. The syndrome is referred to as brucellosis and often occurs in pastoral areas; it is contagious from livestock to humans. In this study, the intact Brucella suis outer membrane protein 31 (omp31) gene was cloned, recombinantly expressed, and examined as a subunit vaccine candidate. The intact Brucella lumazine synthase (bls) gene was cloned and recombinantly expressed to study polymerization function in vitro. Non-reducing gel electrophoresis showed that rBs-BLS existed in different forms in vitro, including as a dimer and a pentamer. An enzyme-linked immunosorbent assay result showed that rOmp31 protein could induce production of an antibody in rabbits. However, the rOmp31-BLS fusion protein could elicit a much higher antibody titer in rabbits; this construct involved fusion of the Omp31 molecule with the BLS molecule. Our results indicate that Omp31 is involved in immune stimulation, while BLS has a polymerizing function based on rOmp31-BLS fusion protein immunogenicity. These data suggest that Omp31 is an ideal subunit vaccine candidate and that the BLS molecule is a favorable transport vector for antigenic proteins. PMID:26535621

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. Preparation of core-shell structure Fe3 O4 @SiO2 superparamagnetic microspheres immoblized with iminodiacetic acid as immobilized metal ion affinity adsorbents for His-tag protein purification.

    PubMed

    Ni, Qian; Chen, Bing; Dong, Shaohua; Tian, Lei; Bai, Quan

    2016-04-01

    The core-shell structure Fe3 O4 /SiO2 magnetic microspheres were prepared by a sol-gel method, and immobiled with iminodiacetic acid (IDA) as metal ion affinity ligands for protein adsorption. The size, morphology, magnetic properties and surface modification of magnetic silica nanospheres were characterized by various modern analytical instruments. It was shown that the magnetic silica nanospheres exhibited superparamagnetism with saturation magnetization values of up to 58.1 emu/g. Three divalent metal ions, Cu(2+) , Ni(2+) and Zn(2+) , were chelated on the Fe3 O4 @SiO2 -IDA magnetic microspheres to adsorb lysozyme. The results indicated that Ni(2+) -chelating magnetic microspheres had the maximum adsorption capacity for lysozyme of 51.0 mg/g, adsorption equilibrium could be achieved within 60 min and the adsorbed protein could be easily eluted. Furthermore, the synthesized Fe3 O4 @SiO2 -IDA-Ni(2+) magnetic microspheres were successfully applied for selective enrichment lysozyme from egg white and His-tag recombinant Homer 1a from the inclusion extraction expressed in Escherichia coli. The result indicated that the magnetic microspheres showed unique characteristics of high selective separation behavior of protein mixture, low nonspecific adsorption, and easy handling. This demonstrates that the magnetic silica microspheres can be used efficiently in protein separation or purification and show great potential in the pretreatment of the biological sample. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26268650

  14. Single Molecule Measurements of Interaction Free Energies Between the Proteins Within Binary and Ternary SNARE Complexes

    PubMed Central

    Liu, W.; Montana, Vedrana; Parpura, Vladimir; Mohideen, U.

    2010-01-01

    We use an Atomic Force Microscope based single molecule measurements to evaluate the activation free energy in the interaction of SNARE proteins syntaxin 1A, SNAP25B and synaptobrevin 2 which regulate intracellular fusion of vesicles with target membranes. The dissociation rate of the binary syntaxin-synaptobrevin and the ternary syntaxin-SNAP25B-synaptobrevin complex was measured from the rupture force distribution as a function of the rate of applied force. The temperature dependence of the spontaneous dissociation rate was used to obtain the activation energy to the transition state of 19.8 ± 3.5 kcal/mol = 33 ± 6 kBT and 25.7 ± 3.0 kcal/mol = 43 ± 5 kBT for the binary and ternary complex, respectively. They are consistent with those measured previously for the ternary complex in lipid membranes and are of order expected for bilayer fusion and pore formation. The ΔG was 12.4–16.6 kcal/mol = 21–28 kBT and 13.8–18.0 kcal/mol = 23–30 kBT for the binary and ternary complex, respectively. The ternary complex was more stable by 1.4 kcal/mol = 2.3 kBT, consistent with the spontaneous dissociation rates. The higher adhesion energies and smaller molecular extensions measured with SNAP25B point to its possible unique and important physiological role in tethering/docking the vesicle in closer proximity to the plasma membrane and increasing the probability for fusion completion. PMID:20107522

  15. A novel small molecule inhibitor of the DNA repair protein Ku70/80.

    PubMed

    Weterings, Eric; Gallegos, Alfred C; Dominick, Lauren N; Cooke, Laurence S; Bartels, Trace N; Vagner, Josef; Matsunaga, Terry O; Mahadevan, Daruka

    2016-07-01

    Non-Homologous End-Joining (NHEJ) is the predominant pathway for the repair of DNA double strand breaks (DSBs) in human cells. The NHEJ pathway is frequently upregulated in several solid cancers as a compensatory mechanism for a separate DSB repair defect or for innate genomic instability, making this pathway a powerful target for synthetic lethality approaches. In addition, NHEJ reduces the efficacy of cancer treatment modalities which rely on the introduction of DSBs, like radiation therapy or genotoxic chemotherapy. Consequently, inhibition of the NHEJ pathway can modulate a radiation- or chemo-refractory disease presentation. The Ku70/80 heterodimer protein plays a pivotal role in the NHEJ process. It possesses a ring-shaped structure with high affinity for DSBs and serves as the first responder and central scaffold around which the rest of the repair complex is assembled. Because of this central position, the Ku70/80 dimer is a logical target for the disruption of the entire NHEJ pathway. Surprisingly, specific inhibitors of the Ku70/80 heterodimer are currently not available. We here describe an in silico, pocket-based drug discovery methodology utilizing the crystal structure of the Ku70/80 heterodimer. We identified a novel putative small molecule binding pocket and selected several potential inhibitors by computational screening. Subsequent biological screening resulted in the first identification of a compound with confirmed Ku-inhibitory activity in the low micro-molar range, capable of disrupting the binding of Ku70/80 to DNA substrates and impairing Ku-dependent activation of another NHEJ factor, the DNA-PKCS kinase. Importantly, this compound synergistically sensitized human cell lines to radiation treatment, indicating a clear potential to diminish DSB repair. The chemical scaffold we here describe can be utilized as a lead-generating platform for the design and development of a novel class of anti-cancer agents. PMID:27130816

  16. 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

  17. 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

  18. Differential expression of cruzipain- and gp63-like molecules in the phytoflagellate trypanosomatid Phytomonas serpens induced by exogenous proteins.

    PubMed

    Elias, Camila G R; Chagas, Michel G; Souza-Gonçalves, Ana Luiza; Pascarelli, Bernardo M O; d'Avila-Levy, Claudia M; Branquinha, Marta H; Santos, André L S

    2012-01-01

    Phytomonas serpens synthesizes metallo- and cysteine-proteases that are related to gp63 and cruzipain, respectively, two virulence factors produced by pathogenic trypanosomatids. Here, we described the cellular distribution of gp63- and cruzipain-like molecules in P. serpens through immunocytochemistry and confocal fluorescence microscopy. Both proteases were detected in distinct cellular compartments, presenting co-localization in membrane domains and intracellular regions. Subsequently, we showed that exogenous proteins modulated the production of both protease classes, but in different ways. Regarding the metalloprotease, only fetal bovine serum (FBS) influenced the gp63 expression, reducing its surface exposition (≈30%). Conversely, the cruzipain-like molecule was differentially modulated according to the proteins: human and bovine albumins reduced its expression around 50% and 35%, respectively; mucin and FBS did not alter its production, while IgG and hemoglobin drastically enhanced its surface exposition around 7- and 11-fold, respectively. Additionally, hemoglobin induced an augmentation in the cell-associated cruzipain-like activity in a dose-dependent manner. A twofold increase of the secreted cruzipain-like protein was detected after parasite incubation with 1% hemoglobin compared to the parasites incubated in PBS-glucose. The results showed the ability of P. serpens in modulating the expression and the activity of proteolytic enzymes after exposition to exogenous proteins, with emphasis in its cruzipain-like molecules. PMID:22033075

  19. 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.

  20. Structure and properties of water film adsorbed on mica surfaces

    NASA Astrophysics Data System (ADS)

    Zhao, Gutian; Tan, Qiyan; Xiang, Li; Cai, Di; Zeng, Hongbo; Yi, Hong; Ni, Zhonghua; Chen, Yunfei

    2015-09-01

    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.

  1. 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.

  2. In situ ATR-IR spectroscopy study of adsorbed protein: Visible light denaturation of bovine serum albumin on TiO2

    NASA Astrophysics Data System (ADS)

    Bouhekka, A.; Bürgi, T.

    2012-11-01

    In this work in situ Fourier transform infrared-attenuated total reflection (FTIR-ATR) spectroscopy in a flow-through cell was used to study the effect of visible light irradiation on bovine serum albumin (BSA) adsorbed on porous TiO2 films. The experiments were performed in water at concentrations of 10-6 mol/l at room temperature. The curve fitting method of the second derivative spectra allowed us to explore details of the secondary structure of pure BSA in water and conformation changes upon adsorption as well as during and after illumination by visible light. The results clearly show that visible light influences the conformation of adsorbed BSA. The appearance of a shift of the amide I band, in the original spectra, from 1653 cm-1 to 1648 cm-1, is interpreted by the creation of random coil in the secondary structure of adsorbed BSA. The second derivative analysis of infrared spectra permits direct quantitative analysis of the secondary structural components of BSA, which show that the percentage of α-helix decreases during visible light illumination whereas the percentage of random coil increases.

  3. Efficient protein knockdown of HaloTag-fused proteins using hybrid molecules consisting of IAP antagonist and HaloTag ligand.

    PubMed

    Tomoshige, Shusuke; Hashimoto, Yuichi; Ishikawa, Minoru

    2016-07-15

    We previously reported a protein knockdown system for HaloTag-fused proteins using hybrid small molecules consisting of alkyl chloride, which binds covalently to HaloTag, linked to BE04 (2), a bestatin (3) derivative with an affinity for cellular inhibitor of apoptosis protein 1 (cIAP1, a kind of ubiquitin ligase). This system addressed several limitations of prior protein knockdown technology, and was applied to degrade two HaloTag-fused proteins. However, the degradation activity of these hybrid small molecules was not potent. Therefore, we set out to improve this system. We report here the design, synthesis and biological evaluation of novel hybrid compounds 4a and 4b consisting of alkyl chloride linked to IAP antagonist MV1 (5). Compounds 4a and 4b were confirmed to reduce the levels of HaloTag-fused tumor necrosis factor α (HaloTag-TNFα), HaloTag-fused cell division control protein 42 (HaloTag-Cdc42), and unfused HaloTag protein in living cells more potently than did BE04-linked compound 1b. Analysis of the mode of action revealed that the reduction of HaloTag-TNFα is proteasome-dependent, and is also dependent on the linker structure between MV1 (5) and alkyl chloride. These compounds appear to induce ubiquitination at the HaloTag moiety of HaloTag-fused proteins. Our results indicate that these newly synthesized MV1-type hybrid compounds, 4a and 4b, are efficient tools for protein knockdown for HaloTag-fused proteins. PMID:27236416

  4. A single-molecule view of DNA replication: the dynamic nature of multi-protein complexes revealed.

    PubMed

    Geertsema, Hylkje J; van Oijen, Antoine M

    2013-10-01

    Recent advances in the development of single-molecule approaches have made it possible to study the dynamics of biomolecular systems in great detail. More recently, such tools have been applied to study the dynamic nature of large multi-protein complexes that support multiple enzymatic activities. In this review, we will discuss single-molecule studies of the replisome, the protein complex responsible for the coordinated replication of double-stranded DNA. In particular, we will focus on new insights obtained into the dynamic nature of the composition of the DNA-replication machinery and how the dynamic replacement of components plays a role in the regulation of the DNA-replication process. PMID:23890728

  5. A highly purified, fluorescently labeled in vitro translation system for single-molecule studies of protein synthesis.

    PubMed

    Fei, Jingyi; Wang, Jiangning; Sternberg, Samuel H; MacDougall, Daniel D; Elvekrog, Margaret M; Pulukkunat, Dileep K; Englander, Michael T; Gonzalez, Ruben L

    2010-01-01

    Single-molecule fluorescence resonance energy transfer (smFRET) has emerged as a powerful tool for mechanistic investigations of increasingly complex biochemical systems. Recently, we and others have successfully used smFRET to directly investigate the role of structural dynamics in the function and regulation of the cellular protein synthesis machinery. A significant challenge to these experiments, and to analogous experiments in similarly complex cellular machineries, is the need for specific and efficient fluorescent labeling of the biochemical system at locations that are both mechanistically informative and minimally perturbative to the biological activity. Here, we describe the development of a highly purified, fluorescently labeled in vitro translation system that we have successfully designed for smFRET studies of protein synthesis. The general approaches we outline should be amenable to single-molecule fluorescence studies of other complex biochemical systems. PMID:20580967

  6. A fluorogenic probe for SNAP-tagged plasma membrane proteins based on the solvatochromic molecule Nile Red.

    PubMed

    Prifti, Efthymia; Reymond, Luc; Umebayashi, Miwa; Hovius, Ruud; Riezman, Howard; Johnsson, Kai

    2014-03-21

    A fluorogenic probe for plasma membrane proteins based on the dye Nile Red and SNAP-tag is introduced. It takes advantage of Nile Red, a solvatochromic molecule highly fluorescent in an apolar environment, such as cellular membranes, but almost dark in a polar aqueous environment. The probe possesses a tuned affinity for membranes allowing its Nile Red moiety to insert into the lipid bilayer of the plasma membrane, becoming fluorescent, only after its conjugation to a SNAP-tagged plasma membrane protein. The fluorogenic character of the probe was demonstrated for different SNAP-tag fusion proteins, including the human insulin receptor. This work introduces a new approach for generating a powerful turn-on probe for "no-wash" labeling of plasma membrane proteins with numerous applications in bioimaging. PMID:24471525

  7. 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.

  8. 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