Dynamic nuclear polarization of amyloidogenic peptide nanocrystals: GNNQQNY, a core segment of the yeast prion protein Sup35p

Dynamic nuclear polarization (DNP) permits a approximately 10(2)-10(3) enhancement of the nuclear spin polarization and therefore increases sensitivity in nuclear magnetic resonance (NMR) experiments. Here, we demonstrate the efficient transfer of DNP-enhanced (1)H polarization from an aqueous, radical-containing solvent matrix into peptide crystals via (1)H-(1)H spin diffusion across the matrix-crystal interface. The samples consist of nanocrystals of the amyloid-forming peptide GNNQQNY(7-13), derived from the yeast prion protein Sup35p, dispersed in a glycerol-water matrix containing a biradical polarizing agent, TOTAPOL. These crystals have an average width of 100-200 nm, and their known crystal structure suggests that the size of the biradical precludes its penetration into the crystal lattice; therefore, intimate contact of the molecules in the nanocrystal core with the polarizing agent is unlikely. This is supported by the observed differences between the time-dependent growth of the enhanced polarization in the solvent versus the nanocrystals. Nevertheless, DNP-enhanced magic-angle spinning (MAS) spectra recorded at 5 T and 90 K exhibit an average signal enhancement epsilon approximately 120. This is slightly lower than the DNP enhancement of the solvent mixture surrounding the crystals (epsilon approximately 160), and we show that it is consistent with spin diffusion across the solvent-matrix interface. In particular, we correlate the expected DNP enhancement to several properties of the sample, such as crystal size, the nuclear T(1), and the average (1)H-(1)H spin diffusion constant. The enhanced (1)H polarization was subsequently transferred to (13)C and (15)N via cross-polarization, and allowed rapid acquisition of two-dimensional (13)C-(13)C correlation data.     

First real-time measurement of the evolving 2H/1H ratio during water evaporation from plant leaves

We have studied the temporal behaviour of the deuterium isotope ratio of water vapour emerging from a freshly cut plant leaf placed in a dry nitrogen atmosphere. The leaf material was placed directly inside the sample gas cell of the stable isotope ratio infrared spectrometer. At the reduced pressure (～40 mbar) inside the cell, the appearance of water evaporating from the leaf is easily probed by the spectrometer, as well as the evolving isotope ratios, with a precision of about 1 ‰. The demonstration experiment we describe measures the ²H/¹H isotope ratio only, but the experiment can be easily extended to include the ¹⁸O/¹⁶O and ¹⁷O/¹⁶O isotope ratios. Plant leaf water isotope ratios provide important information towards quantification of the different components in the ecosystem water and carbon dioxide exchange.     

High-resolution solid-state NMR structure of Alanyl-Prolyl-Glycine

We present a de novo high-resolution structure of the peptide Alanyl-Prolyl-Glycine using a combination of sensitive solid-state NMR techniques that each yield precise structural constraints. High-quality ¹³C–¹³C distance constraints are extracted by fitting rotational resonance width (R²W) experiments using Multimode Multipole Floquet Theory and experimental chemical shift anisotropy (CSA) orientations. In this strategy, a structure is first calculated using DANTE-REDOR and torsion angle measurements and the resulting relative CSA orientations are used as an input parameter in the ¹³C–¹³C distance calculations. Finally, a refined structure is calculated using all the constraints. We investigate the effect of different structural constraints on structure quality, as determined by comparison to the crystal structure and also self-consistency of the calculated structures. Inclusion of all or subsets of these constraints into CNS calculations resulted in high-quality structures (0.02 Å backbone RMSD using all 11 constraints).     

Organic surface analysis with time-of-flight SIMS

In the last few years static secondary ion mass spectrometry (SSIMS) has proved to be a versatile and indispensable method for determining the composition and the structure of the outermost molecular layers of a surface. In particular when using a high-resolution time-of-flight (TOF) mass analyser a high sensitivity can be obtained with SSIMS. In this review it will be shown that the analysis of surfaces with a well-defined chemical structure by means of SSIMS has given detailed insight into the relation between the structure of the fragment ions formed by ion bombardment of the surface and the original surface structure. These studies have also improved the possibilities for quantifying the SSIMS results. In addition, the better knowledge about the ionformation process can be used for the analysis of surfaces of unknown composition and structure. Finally, some recent applications of SSIMS will be presented.Dedicated to Professor Günther Tölg on the occasion of his 60th birthday     

Importance of tensor asymmetry for the analysis of 2H NMR spectra from deuterated aromatic rings

We have used ab initio calculations to compute all of the tensor elements of the electric field gradient for each carbon-deuterium bond in the ring of deuterated 3-methyl-indole. Previous analyses have ignored the smaller tensor elements perpendicular to principal component Vzz which is aligned with the C-2H bond (local bond z-axis). At each ring position, the smallest element Vxx is in the molecular plane and Vyy is normal to the plane of the ring. The asymmetry parameter = (Vyy - Vxx)/Vzz ranges from 0.07 at C4 to 0.11 at C2. We used the perpendicular (off-bond) tensor elements, in concert with an improved understanding of the indole ring geometry, to analyze prototype 2H NMR spectra from well-oriented, hydrated peptide/lipid samples. For each of the four tryptophans of membrane-spanning gramicidin A (gA) channels, the inclusion of the perpendicular elements changes the deduced ring tilt by nearly 10 and increases the ring principal order parameter Szz for overall "wobble" with respect to the membrane normal (molecular z-axis). With the improved analysis, the magnitude of Szz for the outermost indole rings of Trp13 and Trp15 is indistinguishable from that observed previously for backbone atoms (0.93 +/- 0.03). For the Trp9 and Trp11 rings, which are slightly more buried within the membrane, Szz is slightly lower (0.86 +/- 0.03). The results show that the perpendicular elements are important for the detailed analysis of 2H NMR spectra from aromatic ring systems.     

Cryogenic sample exchange NMR probe for magic angle spinning dynamic nuclear polarization

We describe a cryogenic sample exchange system that dramatically improves the efficiency of magic angle spinning (MAS) dynamic nuclear polarization (DNP) experiments by reducing the time required to change samples and by improving long-term instrument stability. Changing samples in conventional cryogenic MAS DNP/NMR experiments involves warming the probe to room temperature, detaching all cryogenic, RF, and microwave connections, removing the probe from the magnet, replacing the sample, and reversing all the previous steps, with the entire cycle requiring a few hours. The sample exchange system described here—which relies on an eject pipe attached to the front of the MAS stator and a vacuum jacketed dewar with a bellowed hole—circumvents these procedures. To demonstrate the excellent sensitivity, resolution, and stability achieved with this quadruple resonance sample exchange probe, we have performed high precision distance measurements on the active site of the membrane protein bacteriorhodopsin. We also include a spectrum of the tripeptide N-f-MLF-OH at 100 K which shows 30 Hz linewidths.     

Direct Spectroscopic Evidence for 1ΔgO2 Production from the Photolysis of Vanadium(V)-Peroxo Complexes in Aqueous Solution

The chemiluminescence of ¹DL_gO₂ expelled from the UV photolysis of vanadium(V)-peroxo (VP) complexes in aqueous solution was observed for the first time. Using phenalenone photosensitization as a standard reference, the ¹O₂ quantum yields from the photolysis of 12 VP complexes were measured. No good correlation was found between the relative DNA-photocleavage activities and the ¹O₂ quantum yields of these complexes. This lack of correlation underscores the significant contribution of the complex-DNA binding interaction in determining the observed DNA-photocleavage activity of the complex. The small ¹O₂ quantum yields observed in these complexes were interpreted in terms of the minor ¹O₂ pho-toelimination channel relative to other more efficient relaxation processes.