Cryo ultra-low-angle microtomy for XPS-depth profiling of organic coatings.

In X-ray photoelectron spectroscopy (XPS) Ar(+) ion sputtering is usually used for depth profiling. However, for such samples as organic coatings, this is not feasible because of degradation. Also, measurement of a depth profile on a conventionally prepared cross-section is not possible if, for example, sample thickness is below the smallest available measurement spot size of the XPS system. In our approach we used a rotary microtome to cut samples under a shallow tilting angle of 0.5° to obtain an extended cross-section suitable for XPS investigations. We also used liquid nitrogen cooling to ensure an exposed area of higher quality: topography measurements with a novel optical 3D microscope and by atomic force microscopy revealed the linearity of the inclined sections. With our cryo ultra-low-angle microtomy (cryo-ULAM) preparation technique we were able to determine, by XPS, elemental and chemical gradients within a 25 µm thick polyester-based organic coating deposited on steel. The gradients were related to, for example, depletion of the crosslinking agent in the sub-surface region. Complementary reflection electron energy-loss spectroscopy measurements performed on the cryo-ULAM sections also support the findings obtained from the XPS depth profiles.

Intracellular domain of nicotinic acetylcholine receptor: the importance of being unfolded.

Bioinformatics methods with subsequent verification by experimental data were applied to the structural investigation of the intracellular loop of the delta-subunit of the nicotinic acetylcholine receptor (nAChR). Three complementary methods were used: prediction of secondary structure elements, prediction of ordered/disordered protein regions and prediction of short functional binding motifs. The output of five different algorithms was used for the secondary structure construction. Most of the intracellular domain is predicted to be unfolded. The predictions correlate well with the experimental data of limited proteolysis and NMR performed on the mostly monomeric fraction of heterologously expressed Torpedo intracellular domain protein. Twelve functional binding motifs within the disordered regions of the nAChR intracellular domain are predicted. Identification of proteins that interact with the intracellular domain will provide a better understanding of protein-protein interactions involved in nAChR assembly, trafficking and clustering.

Orientational information from TEDOR spectral sidebands.

In a dipolar-coupled spin-1/2 network of the type 15N1-(13)C-15N2, an assessment of the sensitivity of the N --> C and C --> N TEDOR sideband intensities to the Euler angles defining the orientation of the two heteronuclear dipolar vectors in the 13C and 15N chemical shift (CS) tensor principal axes system has been carried out via numerical calculations. The results clearly indicate the potential of TEDOR MAS NMR spectroscopy for the characterization of the CS tensor orientation in the molecular frame. The efficacy of the method has been experimentally illustrated by TEDOR studies on a polycrystalline sample of [1, 3-(15)N2, 2-(13)C]uracil, which is one of the four bases in RNA.

2D relayed anisotropy correlation NMR: characterization of the 13C' chemical shift tensor orientation in the peptide plane of the dipeptide AibAib.

An approach to the determination of the orientation of the carbonyl chemical shift (CS) tensor in a 13C-15N-1H dipolar coupled spin network is proposed. The method involves the measurement of the Euler angles of the 13C'-15N and 15N-1H dipolar vectors in the 13C' CS tensor principal axes system, respectively, via a 13C-15N REDOR experiment and by a 2D relayed anisotropy correlation of the 13C' CSA (omega2) and 15N-1H dipolar interaction (omega1). Via numerical simulations the sensitivity of the omega1 cross sections of the 2D spectrum to the Euler angles of the 15N-1H bond vector in the 13C' CSA frame is shown. Employing the procedure outlined in this work, we have determined the orientation of the 13C' CS tensor in the peptide plane of the dipeptide AibAib-NH2 (Aib = alpha-aminoisobutyric acid). The Euler angles are found to be (chiCN, psiCN) = (34 degrees +/- 2 degrees, 88 degrees +/- 2 degrees) and (chiNH, psiNH) = (90 degrees +/- 10 degrees, 80 degrees +/- 10 degrees). From the measured Euler angles it is seen that the sigma33 and sigma22 components of the 13C' CS tensor approximately lie in the peptide plane.

Hydrogen bond directed formation of liquid-crystalline merocyanine dye assemblies.

Combined interaction of triple hydrogen bonding, dipolar pi-pi aggregation and micro-segregation in a melamine-barbituric acid dye assembly leads to a columnar mesophase which could be characterized by optical polarising microscopy, differential scanning calorimetry and X-ray diffraction.

Redor in IS1S2 systems.

An approach to the determination of the 2-(13)C' chemical shift (CS) tensor orientation in pyrimidine bases via heteronuclear MAS NMR spectroscopy is presented. Considering a dipolar coupled spin 1/2 network of the type S1-I-S2 consisting of directly bonded heteronuclear spins, we have carried out numerical simulations to assess the sensitivity of I-S REDOR spinning sidebands to the Euler angles defining the orientation of the I-S1 and I-S2 dipolar vectors in the I spin CS tensor principal axes system. Our investigations clearly demonstrate the potential of I-S REDOR studies in IS1S2 systems for obtaining with high reliability and accuracy the I spin chemical shift tensor orientation in the molecular frame spanned by the two internuclear vectors I-S1 and I-S2. The significant contribution to the observed REDOR sideband intensities from anti-phase operator terms which are present at the start of the data acquisition is illustrated. The procedure for the recording and analysis of the I-S REDOR spectra in IS1S2 systems is presented and the measurement of the 2-(13)C' CS tensor orientation in a polycrystalline sample of [1,3-(15)N2, 2-(13)C] uracil, which is one of the four bases in RNA, is experimentally demonstrated.

REDOR with adiabatic dephasing pulses

The response of a spin (1/2) ensemble, at thermal equilibrium and experiencing chemical shift anisotropy (CSA), to the application of adiabatic inversion pulses has been studied under magic-angle spinning (MAS). Numerical simulations and experimental studies on such systems, carried out under slow spinning conditions, show that the response to adiabatic inversion pulses has much more favorable characteristics than the response to conventional rectangular pulses. We have also explored the possibilities of employing adiabatic 180 degrees pulses as dephasing pulses in rotational-echo double-resonance (REDOR) experiments. Our results show that it is indeed possible to employ such adiabatic inversion pulses conveniently in REDOR experiments to eliminate resonance offset and H(1) inhomogeneity effects which may arise from the usage of conventional rectangular 180 degrees pulses. Copyright 2000 Academic Press.

15N chemical shift tensor magnitude and orientation in the molecular frame of uracil determined via MAS NMR.

The potential of heteronuclear MAS NMR spectroscopy for the characterization of (15)N chemical shift (CS) tensors in multiply labeled systems has been illustrated, in one of the first studies of this type, by a measurement of the chemical shift tensor magnitude and orientation in the molecular frame for the two (15)N sites of uracil. Employing polycrystalline samples of (15)N(2) and 2-(13)C, (15)N(2)-labeled uracil, we have measured, via (15)N-(13)C REDOR and (15)N-(1)H dipolar-shift experiments, the polar and azimuthal angles (θ, psi) of orientation of the (15)N-(13)C and (15)N-(1)H dipolar vectors in the (15)N CS tensor frame. The (θ(NC), psi(NC)) angles are determined to be (92 +/- 10 degrees, 100 +/- 5 degrees ) and (132 +/- 3 degrees, 88 +/- 10 degrees ) for the N1 and N3 sites, respectively. Similarly, (θ(NH), psi(NH)) are found to be (15 +/- 5 degrees, -80 +/- 10 degrees ) and (15 +/- 5 degrees, 90 +/- 10 degrees ) for the N1 and N3 sites, respectively. These results obtained based only on MAS NMR measurements have been compared with the data reported in the literature.

Characterization of 15N chemical shift tensors via 15N-13C REDOR and 1N-1H dipolar-shift CPMAS NMR spectroscopy.

As part of our studies on the characterization of 15N chemical shift anisotropy (CSA) via magic angle spinning (MAS) NMR spectroscopy, we have investigated via numerical simulations the sensitivity of two different REDOR experimental protocols to the angles defining the orientation of the 15N-13C' bond vector in the principal axis system of the 15N CSA tensor of the amide nitrogen in a peptide bond. Additionally, employing polycrystalline samples of 15N and 13C', 15N-labeled acetanilide, we have obtained, in a first study of this type, the orientation of the 15N CSA tensor in the molecular frame by orienting the tensor with respect to the 15N-3C' and 15N-1H dipolar vectors via 15N-13C' REDOR and 15N-1H dipolar-shift MAS experiments, respectively.

The NMR solution structure of the ion channel peptaibol chrysospermin C bound to dodecylphosphocholine micelles.

Chrysospermin C is a 19-residue peptaibol capable of forming transmembrane ion channels in phospholipid bilayers. The conformation of chrysospermin C bound to dodecylphosphocholine micelles has been solved using heteronuclear NMR spectroscopy. Selective 15N-labeling and 13C-labeling of specific alpha-aminoisobutyric acid residues was used to obtain complete stereospecific assignments for all eight alpha-aminoisobutyric acid residues. Structures were calculated using 339 distance constraints and 40 angle constraints obtained from NMR data. The NMR structures superimpose with mean global rmsd values to the mean structure of 0. 27 A (backbone heavy atoms) and 0.42 A (all heavy atoms). Chrysospermin C bound to decylphosphocholine micelles displays two well-defined helices at the N-terminus (residues Phe1-Aib9) and C-terminus (Aib13-Trp-ol19). A slight bend preceding Pro14, i.e. encompassing residues 10-12, results in an angle of approximately 38 degrees between the mean axes of the two helical regions. The bend structure observed for chrysospermin C is compatible with the sequences of all 18 long peptaibols and may represent a common 'active' conformation. The structure of chrysospermin C shows clear hydrophobic and hydrophilic surfaces which would be appropriate for the formation of oligomeric ion channels.

Magic angle spinning NMR spectroscopy with composite RF pulses.

Using numerical optimization procedures it is shown that it is possible to design composite 180 degrees RF pulses for MAS NMR spectroscopy by explicitly taking into account the variation of the resonance offset of each crystallite during the application of the RF pulses. When using composite RF pulses in experiments such as TOSS, where the delays between the RF pulses have to be critically adjusted, an optimization of these delays can lead to the desired performance characteristics.

Brain potentials related to voluntary hand tracking, motivation and attention.

While there are many behavioural studies available investigating human hand tracking performance, there are none that include recording of cerebral potentials. Sixteen subjects tracked a visual or a tactile target by moving a stylus with their right hand. They voluntarily started the stimulus, which moved for 1 s in a first random direction, then for 1's in another direction. The stimulus was given to the left field of vision or tactually to the left palm. Tracking was compared to no-tracking controls. The voluntary initiation was preceded by a Bereitschaftspotential (BP), the change in direction by a contingent negative variation (CNV). Both BP and CNV showed a characteristic right hemispheric parietooccipital (visual task) or centro-parietal asymmetry (tactile task) due to the attention paid to the expected stimulus event ("directed attention potential", DAP). While the DAP outlasted stimulus onset by 0.2 s, fronto-midline areas switched already to positivity 100 ms prior to stimulus onset and more than 300 ms prior to the change in direction. Large P300-like components were elicited by stimulus onset and change in direction.

Frontal hemispheric differences in the Bereitschaftspotential associated with writing and drawing.

Twenty right-handed subjects participated in a study investigating the cerebral potentials related to three complex actions: (1) writing one's own signature, (2) drawing a pentagram, and (3) fast meaningless scribbling. The Bereitschaftspotential (BP, readiness potential) started as early as 3 s prior to writing, 2.5 s prior to drawing, but only 1.5 s prior to scribbling. In all three tasks, the BP had its earliest onset over the supplementary motor area (SMA). BP topography was shifted towards the frontal lobes when compared to encephalographic activity reflecting simple finger movements, and was very weak in retro-rolandic leads. The side of the performing hand, as assessed from scribbling, was reflected in a contralateral preponderance of the precentral BP. The maximum BP (about 6 microV) was, in all three tasks, located in FCz (mid fronto-central) overlying the SMA. This location is different from that for simple finger movements, when the maximum is at the vertex. Hemispheric differences were found over the frontal cortex and were characteristic for the verbal and spatial tasks involved: for writing, the BP was significantly larger left frontally than right (even after considering the effect of the performing hand from scribbling), and the difference was largest prior to the onset of movement; for drawing, the BP was larger over the right than over the left frontal lobe, and the difference was largest during the movement.