Unambiguous Assignment of Short‐ and Long‐Range Structural Restraints by Solid‐State NMR Spectroscopy with Segmental Isotope Labeling

We present an efficient method for the reduction of spectral complexity in the solid‐state NMR spectra of insoluble protein assemblies, without loss of signal intensity. The approach is based on segmental isotope labeling by using the split intein DnaE from Nostoc punctiforme. We show that the segmentally ¹³C,¹⁵N‐labeled prion domain of HET‐s exhibits significantly reduced spectral overlap while retaining the wild‐type structure and spectral quality. A large number of unambiguous distance restraints were thus collected from a single two‐dimensional ¹³C,¹³C cross‐correlation spectrum. The observed resonances could be unambiguously identified as intramolecular without the need for preparing a dilute, less sensitive sample.     

Structural changes of bulk polyethylene by stress impact as studied by solid‐state NMR

Solid‐state ¹³C‐ and ¹H‐NMR spectra of bulk high‐density polyethylene samples, cylindrical in form, to which stress impacts were applied with a home‐made stress‐impact apparatus, were measured. The fraction of the noncrystalline component increases with an increase in the stress‐impact strength. In the crystalline region, the monoclinic crystalline component appear with the stress impact, in addition to the major orthorhombic crystalline component. Furthermore, dynamic characterization was carried out on the basis of the observed values of the relaxation parameters ¹H T₂ and T_CH of the ¹H and ¹³C nuclei. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 82: 2268–2272, 2001     

The curing of UF resins studied by low‐resolution 1H‐NMR

A series of UF resins and one MUF resin were studied by low‐resolution ¹H‐NMR. The mobility of the resin during curing could be followed by measuring the spin‐spin relaxation time (T₂) with curing time. The relative curing behavior was similar to that found by traditional gel time measurements. In addition, extra features in the T₂ plots with curing time showed at what point the bulk of the condensation reactions took place. The speed of cure was also related to the chemical groups in the liquid resin, and it was found that the linear methylol groups were mainly responsible for the curing speed of the resins. By studying the curing with different hardener levels and glue concentrations it was found that a UF resin is more sensitive to the glue mix concentration than an MUF resin. A cured resin was also studied after curing to investigate postcuring effects. Water seemed to play the biggest role in the postcure, with substantial amounts present immediately after cure, which decreased with curing time and aging. For the low mol ratio resins studied here further curing reactions did not seem to play a major role in the post curing phenomenon. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 75: 754–765, 2000     

Characterization of a Cyclic Nucleotide‐Activated K+ Channel and its Lipid Environment by Using Solid‐State NMR Spectroscopy

Voltage‐gated ion channels are large tetrameric multidomain membrane proteins that play crucial roles in various cellular transduction pathways. Because of their large size and domain‐related mobility, structural characterization has proved challenging. We analyzed high‐resolution solid‐state NMR data on different isotope‐labeled protein constructs of a bacterial cyclic nucleotide‐activated K⁺ channel (MlCNG) in lipid bilayers. We could identify the different subdomains of the 4×355 residue protein, such as the voltage‐sensing domain and the cyclic nucleotide binding domain. Comparison to ssNMR data obtained on isotope‐labeled cell membranes suggests a tight association of negatively charged lipids to the channel. We detected spectroscopic polymorphism that extends beyond the ligand binding site, and the corresponding protein segments have been associated with mutant channel types in eukaryotic systems. These findings illustrate the potential of ssNMR for structural investigations on large membrane‐embedded proteins, even in the presence of local disorder.     

Protocols for the Sequential Solid‐State NMR Spectroscopic Assignment of a Uniformly Labeled 25 kDa Protein: HET‐s(1‐227)

The sequence‐specific resonance assignment of a protein forms the basis for studies of molecular structure and dynamics, as well as to functional assay studies by NMR spectroscopy. Here we present a protocol for the sequential ¹³C and ¹⁵N resonance assignment of uniformly [¹⁵N,¹³C]‐labeled proteins, based on a suite of complementary three‐dimensional solid‐state NMR spectroscopy experiments. It is directed towards the application to proteins with more than about 100 amino acid residues. The assignments rely on a walk along the backbone by using a combination of three experiments that correlate nitrogen and carbon spins, including the well‐dispersed C^β resonances. Supplementary spectra that correlate further side‐chain resonances can be important for identifying the amino acid type, and greatly assist the assignment process. We demonstrate the application of this assignment protocol for a crystalline preparation of the N‐terminal globular domain of the HET‐s prion, a 227‐residue protein.     

Solid‐state 13C‐NMR and study of the subproducts obtained from corn industry

The two types of commercial corn flour subproducts from the Brazilian corn industry containing starch with low fat and with fat were investigated by solid‐state NMR techniques to improve their uses. From the NMR techniques used, it was characterized that after a treatment the quantity of fibers decreased. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 84: 1680–1685, 2002; DOI 10.1002/app.10547     

Blends of natural rubber and polyurethane latices studied by 1H wideline and 13C MAS solid‐state NMR

¹H wideline and ¹³C magic‐angle spinning NMR have been used to study the morphology and dynamics of latex‐cast and solution‐cast film blends of natural rubber (NR) and a polyurethane (PU) based on poly(?‐caprolactone) diol and isophorone di‐isocyanate. ¹H T₁ and T_1ρ relaxation times have been measured, and the extent of interpenetration of the NR and PU constituents has been monitored using the Goldman–Shen technique. The NMR spectra and relaxation properties indicated that the NR and PI constituents largely occupy separated domains on a distance scale of >10 nm. The Goldman–Shen experiments indicated that there was slightly greater contact between NR and PU in the solution‐cast samples than in the latex‐cast. The tensile properties of the films have been measured. The tensile strength and initial Young's modulus pass through a maximum at a PU content of about 50 wt%. © 2002 Society of Chemical Industry     

Kinetics of the solid‐state polymerization of nylon‐6

The kinetics of the thermally induced solid‐state polymerization (SSP) of nylon‐6 were examined in both a fixed‐bed reactor and a rotary reactor. Factors such as the regulator content, the reaction temperature and time, the particle size, the type and geometry of the nylon‐6 prepolymer, the nitrogen gas flow rate, the water content of the nitrogen gas flow, and the polymerization process were studied. The results showed that the regulator content, the reaction temperature and time, and the particle size were the primary factors, and that the others were negligible. Moreover, the SSP rate and number‐average molecular weight (M_n) increased with increasing reaction temperature and time and decreasing particle size. The SSP rate and M_n had maximum values with increasing regulator content in an experimental range of 0.03–0.07 wt %. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 84: 616–621, 2002; DOI 10.1002/app.10341     

In-cell solid-state NMR as a tool to study proteins in large complexes

A major limitation of solution NMR is molecular tumbling, which is often too slow for detection. Here we demonstrate that solid-state NMR spectroscopy in combination with flash freezing of cells can be used to detect proteins in the cellular environment and provides information on backbone chemical shifts.     

Surface Binding of TOTAPOL Assists Structural Investigations of Amyloid Fibrils by Dynamic Nuclear Polarization NMR Spectroscopy

Dynamic nuclear polarization (DNP) NMR can enhance sensitivity but often comes at the price of a substantial loss of resolution. Two major factors affect spectral quality: low‐temperature heterogeneous line broadening and paramagnetic relaxation enhancement (PRE) effects. Investigations by NMR spectroscopy, isothermal titration calorimetry (ITC), and EPR revealed a new substantial affinity of TOTAPOL to amyloid surfaces, very similar to that shown by the fluorescent dye thioflavin‐T (ThT). As a consequence, DNP spectra with remarkably good resolution and still reasonable enhancement could be obtained at very low TOTAPOL concentrations, typically 400 times lower than commonly employed. These spectra yielded several long‐range constraints that were difficult to obtain without DNP. Our findings open up new strategies for structural studies with DNP NMR spectroscopy on amyloids that can bind the biradical with affinity similar to that shown towards ThT.     

Chemical Structures Formed in Electron Beam Irradiated Poly(tetrafluoroethylene‐co‐hexafluoropropylene) (FEP)

Summary: The perfluorinated copolymer poly(tetrafluoroethylene‐co‐hexafluoropropylene) (FEP) was electron beam irradiated under vacuum at various temperatures ranging from room temperature to a temperature above the melting temperature of FEP. Changes of the chemical structure were analyzed by ¹⁹F solid‐state NMR and IR spectroscopy. Trifluoromethyl end groups were generated as a result of main chain scission at all irradiation temperatures studied. In addition, trifluoromethyl side groups in various environments and double bond structures were formed. Quantitative analysis showed that long‐chain branches were formed at irradiation temperatures above 200 °C. Furthermore, the hexafluoropropylene (HFP) units in FEP were found to be less sensitive to radiation than the perfluoropropyl vinyl ether (PPVE) units in poly[tetrafluoroethylene‐co‐(perfluoropropyl vinyl ether)] (PFA).

¹⁹F solid‐state NMR spectra of FEP.     

Solid‐state NMR and morphological studies of poly(ethylene‐co‐vinyl acetate)/poly(vinyl acetate) blends

To obtain a correlation among structure–morphology–mobility–compatibility properties of poly(ethylene‐co‐vinyl acetate) (EVA)/poly(vinyl acetate) (PVAc) blends, we have used scanning electron microscopy and solid‐state nuclear magnetic resonance in our investigations. The results are discussed in terms of blends, component dispersion, plasticization effect, and domain mobilities to acquire a response of the correlation between structural properties. © 1999 John Wiley & Sons, Inc. J Appl Polym Sci 74: 2990–2996, 1999     

A Combined Solid‐State NMR and MD Characterization of the Stability and Dynamics of the HET‐s(218‐289) Prion in its Amyloid Conformation

Dynamic and rigid : The prion HET‐s(218–289) consists, in its amyloid form as shown here, of highly ordered and rigid parts and a very dynamic loop, which could be of great importance for fibril formation. Indeed, MD simulations explain the experimental NMR results and describe the dynamics of the salt‐bridge network that stabilizes the amyloid fibril, a feature not easily accessible by experiment.