Surface NMR Research Articles

Mechanism of Lys48-linked polyubiquitin chain recognition by the Mud1 UBA domain

The ubiquitin-pathway associated (UBA) domain is a 40-residue polyubiquitin-binding motif. The Schizosaccharomyces pombe protein Mud1 is an ortholog of the Saccharomyces cerevisiae DNA-damage response protein Ddi1 and binds to K48-linked polyubiquitin through its UBA domain. We have solved the crystal structure of Mud1 UBA at 1.8 angstroms resolution, revealing a canonical three-helical UBA fold. We have probed the interactions of this domain using mutagenesis, surface plasmon resonance, NMR and analytical ultracentrifugation. We show that the ubiquitin-binding surface of Mud1 UBA extends beyond previously recognized motifs and can be functionally dissected into primary and secondary ubiquitin-binding sites. Mutation of Phe330 to alanine, a residue exposed between helices 2 and 3, significantly reduces the affinity of the Mud1 UBA domain for K48-linked polyubiquitin, despite leaving the primary binding surface functionally intact. Moreover, K48-linked diubiquitin binds a single Mud1 UBA domain even in the presence of excess UBA. We therefore propose a mechanism for the recognition of K48-linked polyubiquitin chains by Mud1 in which diubiquitin units are specifically recognized by a single UBA domain.

Effect of the Addition of a Nonionic Surfactant on the Complex Poly(asparagine)−Cationic Surfactant

Poly(asparagine) (pAsn) at 0.1wt % in the presence of dodecyltrimethylammonium bromide (DTAB) and pentaoxyethylene octyl ether (C(8)E(5)) at 1:1 molar ratio leads to the formation of mixed DTAB/C(8)E(5) micelle-like aggregates onto the polypeptide as a total surfactant critical association concentration (cac) is reached, as revealed by surface tension measurements and NMR chemical shifts. Two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) capable of revealing spatial relationships among proximal protons has been performed on the pAsn-DTAB-C(8)E(5)-water system to study structural details of the surfactant-polypeptide aggregates. NOESY cross-peaks at sample temperature of 298.15 K indicate that the polypeptide interacts with the DTAB/C(8)E(5) micelle-like aggregates. The NOE intermolecular effects also show direct interactions between surfactant and polypeptide in the pAsn-DTAB-water system, whereas no interaction has been revealed in the pAsn-C(8)E(5)-water system. Furthermore, the experimental evidence suggest that the DTAB-polypeptide complex is mainly driven by the polar attraction between the two molecules.

Extrusion Monitoring of Polymer Melts Using a High‐Temperature Surface‐NMR Probe

AbstractSummary: Extrusion is one of the major ways of polymer processing, and extrusion monitoring is important to control or improve the extrusion process and the product quality. Especially methods for in‐line monitoring are desired because those enable the fastest possible response and a localization of chemical reactions in reactive processing. Methods of optical and acoustic spectroscopy have successfully been implemented so far. NMR provides a wealth of information. Surface NMR mostly gives access to molecular mobility via relaxation times. These can be correlated to temperature, composition and homogeneity which are substantial indicators for the product quality. NMR is a non‐destructive method, which does not require direct contact with the sample and it is not restricted to optical transparent materials and is therefore suitable for extrusion monitoring. The problem for the adaptation is the hostile environment in and in front of an extruder for polymer melts. Here, a NMR surface probe is presented sustaining this environment in front of an extruder. First measurements with this device are shown.High‐temperature surface‐NMR probe.magnified imageHigh‐temperature surface‐NMR probe.

Molecular Basis for the Specificity of p27 Toward Cyclin-dependent Kinases that Regulate Cell Division

The cyclin-dependent kinase inhibitors (CKIs) bind to and directly regulate the catalytic activity of cyclin-dependent kinase (Cdk)/cyclin complexes involved in cell cycle control and do not regulate other, closely related Cdks. We showed previously that the CKI, p27, binds to Cdk2/cyclin A though a sequential mechanism that involves folding-on-binding. The first step in the kinetic mechanism is interaction of a small, highly dynamic domain of p27 (domain 1) with the cyclin subunit of the Cdk2/cyclin A complex, followed by much slower binding of a more lengthy and less flexible domain (domain 2) to Cdk2. The second step requires folding of domain 2 into the kinase inhibitory conformation. Rapid binding of p27 domain 1 to cyclin A tethers the inhibitor to the binary Cdk2/cyclin A complex, which reduces the entropic barrier associated with slow binding of domain 2 to the catalytic subunit. We show here that p27/cyclin interactions are an important determinant of p27 specificity towards cell cycle Cdks. We used surface plasmon resonance, limited proteolysis, mass spectrometry, and NMR spectroscopy to study the interaction of p27 with Cdk2/cyclin A, and with another Cdk complex, Cdk5/p25, that is involved in neurodegeneration. Importantly, Cdk5/p35 (the parent complex of Cdk5/p25) is not regulated by p27 in neurons. Our results show that p27 binds to Cdk5 and Cdk2 with similar, slow kinetics. However, p27 fails to interact with p25 within the Cdk5/p25 complex, which we believe prevents formation of a kinetically trapped, inhibited p27/Cdk5/p25 complex in vivo. The helical topology of p25 is very similar to that of cyclin A. However, p25 lacks the MRAIL sequence in one helix that, in the cell cycle cyclins, mediates specific interactions with domain 1 of p21 and p27. Our results strongly suggest that p21 and p27, related Cdk inhibitors, select their cell cycle regulatory Cdk targets by binding specifically to the cyclin subunit of these Cdk/cyclin complexes as a first step in a sequential, folding-on-binding mechanism.

A continuously recirculating optical pumping apparatus for high xenon polarization and surface NMR studies

A continuously recirculating xenon optical pumping apparatus was constructed and coupled to a 200 MHz NMR spectrometer. Steady-state 129Xe polarizations as high as 69% were achieved approximately 5 min after circulation was begun, and this polarization is >3.5 times larger than observed for single pass operation. Using this apparatus, spin polarization induced nuclear Overhauser effect (SPINOE) experiments were performed on porous silicon samples and on 13CD 3OD adsorbed on TiO 2-coated optical fibers to evaluate the capabilities of the system for studying low surface areas samples. The 13C enhancement achieved was approximately 14. The recirculating optical pumping system provides high xenon polarization at moderate laser powers for a variety of NMR studies.

Biodegradable comb polyesters containing polyelectrolyte backbones facilitate the preparation of nanoparticles with defined surface structure and bioadhesive properties

AbstractA major challenge in oral peptide and protein delivery remains the search for suitable carrier systems. Therefore, a new concept was investigated combining a modified three‐dimensional architecture, increased hydrophilicity of poly(lactic‐co‐glycolic acid) (PLGA) and charged groups in a single polymer. Biodegradable comb PLGA were synthesized by grafting short PLGA chains onto different poly(vinyl alcohol) (PVA) based backbone polyols, poly(2‐sulfobutyl‐vinyl alcohol) and poly(diethylaminoethyl‐vinyl alcohol). The polyelectrolyte backbones were obtained by etherification of PVA with charge‐containing pendent groups. The comb polymer structure could be confirmed by nuclear magnetic resonance, infrared spectroscopy, differential scanning calorimetry, elemental analysis and measurement of intrinsic viscosity. Nanoparticles (NP), as potential mucosal carriers systems, were prepared by controlled precipitation and investigated as a function of polymer composition. The amphiphilic character and the three‐dimensional architecture of the novel polyesters allowed the preparation of small nanoparticles even without the use of surfactants. Surface NMR, surface charge and hydrophobicity determination indicate a core–corona‐like NP structure, especially in the case of negatively charged polyesters. A structural model is proposed for the NP with an inner polyester core and an outer charged‐groups‐containing surface, depending on polymer composition and backbone charge density. The higher the polymer backbone charge density, the more pronounced its influence on the nanoparticle surface properties. The possibility of preparing NP without the use of a surfactant, as well as of designing the NP surface characteristics by polymer backbone charge density and polymer hydrophilic–hydrophobic balance, will be a major advantage in protein adsorption, bioadhesion and organ distribution. This makes these biodegradable polymers promising candidates for colloidal protein and peptide delivery. Copyright © 2003 John Wiley & Sons, Ltd.

SNMR test measurements in Austria

In 1997, surface NMR measurements were conducted for the first time in Austria to test the possible application of this new technique for groundwater research in typical sites of Austria. The measurements were done in areas where other geophysical, hydrological and logging data were also available. From the test measurements, it could be concluded that in most areas, where this method was tested, no reliable data could be obtained due to the high background noise. At locations where a low noise level allowed proper measurements, the results gave valuable additional information for groundwater exploration. These results were confirmed by drillings.

Smooth and block inversion of surface NMR amplitudes and decay times using simulated annealing

This paper introduces a new 1-D inversion scheme for surface nuclear magnetic resonance (SNMR) amplitudes and decay times, using the optimized random search algorithm simulated annealing (SA). As an alternative to the smooth inversion used in other SNMR inversion techniques, the new scheme can also use block inversion, similar to 1-D geoelectrics. In smooth inversion, the number and thickness of inversion layers as well as the degree and type of the smoothness constraint, for example, the parameter of regularization, is most decisive on the results of the inversion. Therefore, the results are highly ambiguous, depending on the type and degree of regularization. To improve the mapping of aquifers with sharp boundaries and to overcome the ambiguity of the smooth inversion, a different approach has therefore been chosen, using block inversion. Using both smooth and block inversions, respectively, the results for two synthetic sets of SNMR data and for field data from a site in northern Germany with well-known hydrogeological properties are discussed. The results of block inversion and smooth inversion using a regularization that favors more block-like structures correspond to the sharp aquifer boundaries of the synthetic models and the site. The conventional smooth inversion gives only a rough estimate of the aquifer location.

Minimal Heparin/Heparan Sulfate Sequences for Binding to Fibroblast Growth Factor-1

The glycosaminoglycans heparin and heparan sulfate (HS) bind to fibroblast growth factor FGF1 and promote its dimerization, a proposed prerequisite for binding to a cellular receptor and triggering mitogenic signals. The problem of minimal structural requirements for heparin/HS sequences to bind FGF1 was approached by surface plasmon resonance (SPR), NMR spectroscopy, and MALDI mass spectrometry studies using the three synthetic tetrasaccharides GlcNSO36OR-IdoA2SO3-GlcNSO36OR′-IdoA2SO3OPr (AA, R = R′ = SO3; BA, R = H, R′ = SO3; BB, R = R′ = H; Pr, propyl). AA and BA significantly interact with the protein, whereas BB is practically inactive. The NMR spectra show that, whereas the interaction of AA primarily involves the GlcNSO36SO3IdoA2SO3 disaccharide moiety at its nonreducing end, residues at both the nonreducing (NR) and reducing side (R) appear to be involved in the weaker complex of BA. Furthermore, MALDI experiments show that, in addition to 1:1 protein:tetrasaccharide complexes, AA and BA are able to form 2:1 complexes, indicating that heparin/HS-induced dimerization of FGF1 requires only one 6-OSO3 group per tetrasaccharide.

Physicochemical properties of resorcinol-type calix[4]arenes phosphoric esters having four alkyl side chains in aqueous solution

The physicochemical properties of phosphate esters of resorcinol-type of calix[4]arenes([4]Ar5P-R n ; side chain length n=6, 8, and 12) in aqueous solutions have been characterized by several techniques including surface tension and NMR. It is found that the critical micelle concentrations (cmcs) of [4]Ar5P-R n are insensitive against the alkyl side chain length, whereas the polarity of aggregates remarkably depends on the alkyl side chain length. From static light scattering and NMR measurements it is suggested that the shapes of aggregates of [4]Ar5P-R n are not globular and the alkyl side chains of [4]Ar5P-R n gather loosely in the aggregates. The mixed properties of [4[Ar5P-R 12 and hexaethyleneglycol dodecylether have also been investigated. The mixed cmcs are considerably greater than those of the respective surfactant cmcs and the interaction parameter calculated using a modified regular solution theory is +2.7. These results indicate that the repulsive interactions occur in the mixed micelles.

Microstructure and morphology of polyphenylacetylene prepared in compressed carbon dioxide

Polyphenylacetylene was prepared by transition metal catalysis in compressed (liquid and supercritical) CO2. Surface morphology, IR and NMR spectroscopic properties of the resulting materials are discussed in terms of the microstructure of polymers. Copyright © 2001 John Wiley & Sons, Ltd.

Optimal Design Features of Camelized Human Single-domain Antibody Libraries

We have constructed a human V(H) library based on a camelized V(H) sequence. The library was constructed with complete randomization of 19 of the 23 CDR3 residues and was panned against two monoclonal antibody targets to generate V(H) sequences for determination of the antigen contact residue positions. Furthermore, the feasibility and desirability of introducing a disulfide bridge between CDR1 and CDR3 was investigated. Sequences derived from the library showed a bias toward the use of C-terminal CDR3 residues as antigen contact residues. Mass spectrometric analyses indicated that CDR1-CDR3 disulfide formation was universal. However, surface plasmon resonance and NMR data showed that the CDR3 constraint imposed by the disulfide bridge was not always desirable. Very high yields of soluble protein products and lack of protein aggregation, as demonstrated by the quality of the (1)H-(15)N HSQC spectra, indicated that the V(H) sequence for library construction was a good choice. These results should be useful in the design of V(H) libraries with optimal features.

The Conversion of Methanol: A Probe Reaction for Hydroxyapatite

Stoichiometric and nonstoichiometric hydroxyapatites are characterized by surface area, ICP, XPS and NMR MAS. The dehydration of methanol is shown to be unimolecular but poisoned by both alcohol and water. Rate constants and the ratio of the adsorption equilibrium constants of alcohol and water are obtained with the former appearing to be related to the surface Ca/P values. Infrared spectroscopy and temperature-programmed desorption and reaction show that hydroxyapatite, after exposure to methanol, contains surface methoxy groups and related experiments with deuterated methanol show that the dehydration process involves the interaction of methanol with surface protium.

Characterization of Interaction between Butylbenzene Sulfonates and Cetyl Pyridinium Chloride in a Mixed Aggregate System

Surface active short chain alkyl benzene sulfonates, which are known as hydrotropes, interact electrostatically as well as hydrophobically with cationic surfactants. The mixtures of butyl benzene sulfonate (BBS-) with cetyl pyridinium chloride (CPyCl), as shown by surface tensiometry, rheological techniques, and IR and NMR spectroscopy form rodlike micelles or vesicles. These microstructures give rise to highly viscous solutions even at very low concentrations in the millimolar region. The CPy+/BBS- complexes also show similar behavior in the absence of electrolytes. The steric hindrance due to the butyl group to the interaction with CPyCl is at a maximum with tert-butyl benzene sulfonate (TBBS-) as compared to n-butyl benzene sulfonate (NBBS-) and iso-butyl benzene sulfonate (IBBS-). The molecular interactions between surfactant/hydrotrope pairs are simulated using the MM2 force field in the presence and absence of water. The presence of water reduces the electrostatic interaction by solvation and reinfo...

Counterion effect on the phase behavior of perfluoropolyether carboxylates: micelles and liquid crystals in water

Perfluoropolyether carboxylates having sodium, potassium or ammonium counterions, and hydrophobic chains, mostly ClC 3F 6O terminated, have been investigated in aqueous binary systems, with the focus laid upon the counterion effect on the major self-assembly features. Micellization and liquid crystal formation have been detected via surface tension, conductivity, optical microscopy and NMR.

Surface NMR within a geophysical study of an aquifer at Haldensleben (Germany)

The surface nuclear magnetic resonance (SNMR) method has been tested at a site in Haldensleben, northern Germany, to assess the suitability of this new method for groundwater exploration and environmental investigations. More information is obtained by SNMR, particularly with respect to aquifer parameters, than with other geophysical techniques. SNMR measurements were carried out at three borehole locations, together with 2D and 1D direct current geoelectrics, as well as ground‐penetrating radar, and well logging (induction log, gamma‐ray log and pulsed neutron‐gamma log). Permeabilities were calculated from the grain‐size distributions of core material determined in the laboratory. It is demonstrated that the SNMR method is able to detect groundwater and the results are in good agreement with other geophysical and hydrogeological data. Using the SNMR method, the water content of the unsaturated and saturated zones (i.e. porosity of an aquifer) can be reliably determined. This information and resistivity data permit in situ determination of other aquifer parameters. Comparison of the SNMR results with borehole data clearly shows that the water content determined by SNMR is the free or mobile water in the pores. The permeabilities estimated from the SNMR decay times are similar to those derived from sieve analysis of core material. Thus, the combination of SNMR with geoelectric methods promises to be a powerful tool for studying aquifer properties.

Surface Nuclear Magnetic Resonance Imaging of Large Systems

The general theory of surface NMR imaging of large electromagnetically active systems is considered, motivated by geophysical applications. A general imaging equation is derived for the NMR voltage response, valid for arbitrary transmitter and receiver loop geometry and arbitrary conductivity structure of the sample. When the conductivity grows to the point where the electromagnetic skin depth becomes comparable to the sample size, significant diffusive retardation effects occur that strongly affect the signal. Accounting for these now allows more accurate imaging than previously possible. It is shown that the time constant ${T}_{1}$ may in principle be inferred directly from the diffusive tail of the signal.

Surface and colloid properties of cyclic amides III. Surface activity and micellization of N-butyl-2-pyrrolidone in water

The aggregation and surface activity of N-butyl-2-pyrrolidone (BP) in water is investigated using surface tension, solubilization, fluorescence and NMR methods. The micelle point is high (˜1.0 M) as expected for the butyl chain length, and the aggregation number is approximately 5, with pre-association below the c.m.c. Despite the low aggregation number, the fluorescence data indicate that the interior of the aggregate is similar to pure liquid BP. The adsorption of BP at the solution/air interface goes to a maxmum near the c.m.c, corresponding to an area of 45 ± 1 Å2/molecule.

Analysis of Polymer Materials by Surface NMR via the MOUSE

Applications are discussed of a novel NMR device, the NMR MOUSE (mobileuniversalsurfaceexplorer), for characterization of polymers. Different properties of elastomers can be related to an effective transverse relaxation parameterT2eff. Effects of multiecho sequences influence the decay curve and can be described in terms ofB0inhomogeneity and spin-lock effects. Furthermore, the signal-to-noise ratio (S/N) can be improved by use of steady-state free precession (SSFP) pulse sequences modified for use in inhomogeneous magnetic fields.

Mixed Micellar Properties of Nonionic Saccharide and Anionic Fluorocarbon Surfactants in Aqueous Solution

Mixed micellar properties of nonionic saccharides (n-decyllactobionamide, C10Glu2;n-dodecyllactobionamide, C12Glu2) and anionic fluorocarbon surfactants (lithium perfluorooctanesulfonate, LiFOS) in aqueous solutions have been studied by means of surface tensiometry, NMR, and light scattering. The interaction parameters estimated from the modified regular solution theory are −5.0 for the C10Glu2/LiFOS system and −0.2 for the C12Glu2/LiFOS system. This difference could be due to the interaction of the surfactant tails. The average aggregation number is almost identical over a wide mole fraction of C12Glu2for the C12Glu2/LiFOS system, while it shows a minimum at a C10Glu2mole fraction of 0.4 for the C10Glu2/LiFOS system. It is also found from NMR measurements that the segmental motions of surfactants in the mixed micelles of C12Glu2and LiFOS are not restricted, whereas they are restricted at a C10Glu2mole fraction of 0.4 for the C10Glu2/LiFOS system. These motional changes as well as micellar composition reflect the solubilization of decafluorobiphenyl in the mixed micelles.