Elastin is the principal protein component of the elastic fiber in vertebrate tissue. The waters of hydration in the elastic fiber are believed to play a critical role in the structure and function of this largely hydrophobic, amorphous protein. (13)C CPMAS NMR spectra are acquired for elastin samples with different hydration levels. The spectral intensities in the aliphatic region undergo significant changes as 70% of the water in hydrated elastin is removed. In addition, dramatic differences in the CPMAS spectra of hydrated, lyophilized, and partially dehydrated elastin samples over a relatively small temperature range (-20 degrees C to 37 degrees C) are observed. Results from other experiments, including (13)C T(1) and (1)H T(1 rho) measurements, direct polarization with magic-angle spinning, and static CP of the hydrated and lyophilized elastin preparations, also support the model that there is significant mobility in fully hydrated elastin. Our results support models in which water plays an integral role in the structure and proper function of elastin in vertebrate tissue.

We present a new solid-state NMR approach, based on 1H spin diffusion with X-nucleus (15N, 13C, 31P) detection, for investigating the structure of membrane proteins. For any segment with a resolvable signal in the X-nucleus spectrum, the depth of insertion into the lipid bilayer can be determined. The technique represents the adaptation of the Goldman−Shen 1H spin-diffusion experiment with X-nucleus detection to proteins in hydrated lipid bilayers (>25% water by weight) in the gel state at 240 K. The experiments are demonstrated on the 21-kDa channel-forming domain of the toxin-like colicin E1 molecule incorporated into lipid vesicles. More than 32% of the protons in our sample are in mobile H2O molecules, which can be selected efficiently by the 1H T 2 filter in the Goldman−Shen sequence. The transfer of 1H magnetization from mobile H2O to the colicin E1 channel domain is 80% complete within only 5 ms. This transfer to the protein, probed by the amide 15N signals, is faster than the transfer to the rigid protons on average, proving that most of the protein is preferentially located between the water and the lipid bilayer. From the spin-diffusion and dipolar-dephasing data, 60% of the 24 lysine side groups are shown to be highly mobile. Quantitative depth profiling is demonstrated using the 31P in the lipid phosphate head groups and the 13C nuclei in the lipid acyl chains as distance markers for the spin diffusion.

The first experimental determination of the chemical shielding tensor of a singlet carbene center, 1,3,4,5tetramethylimidazol-2-ylidene (1), is reported. Detailed ab initio theoretical calculations of the chemical shielding tensors are reported for local density functional theory (LDFT) and the Hartree-Fock levels (HF) in the LORG and IGLO frameworks. The chemical shielding anisotropy is quite large, which is characteristic of the lowest-energy-singlet carbene centers. The anisotropy at the carbene center in 1 is ~240 ppm. This is one of the largest anisotropies observed for carbon in a strictly organic framework. The calculations indicate that the orientation of the chemical shielding tensor of the carbene is such that the most shielded component is perpendicular to the molecular plane, with the intermediate component oriented approximately along the direction of the lone pair, and the least shielded component perpendicular to the other two. This orientation, as well as the relative size of the anisotropy, agrees with calculations on the singlet carbenes 1, imidazol-2-ylidene (2), and :CF2. The size of the anisotropy is predicted to be enhanced for the parent carbene :CH2-

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The structure of an elastin-mimetic model peptide, (VPGVG) 6, was proposed by combining data obtained from quantitative use of the conformation-dependent 13 C NMR chemical shifts, twodimensional off magic angle spinning spin-diffusion solid-state NMR with 13 C double labeling of the peptides, rotational-echo double-resonance of 13 C, 15 N double labeling peptides, and statistical distribution of the backbone torsion angles of Val-Pro, Pro-Gly, and Gly-Val-Gly sequences from PDB. In essence, this approach drew upon several sets of data to formulate the resulting model, namely, that there is a distribution of conformations in this polypeptide. The Val-16 residue adopts torsion angles (φ, ψ) ) (-90 ( 15°, 120 ( 15°). In contrast, bimodal distributions for the central residues of the (VPGVG) subunit were detected. For Pro-12, (φ, ψ) ) (-60 ( 15°, 120 ( 15°), 80%, and (φ, ψ) ) (-60 ( 15°, -30 ( 15°), 20%; for Gly-13, (φ, ψ) ) (90 ( 30°, -15 ( 30°) and (φ, ψ) ) (-90 ( 15°, 0 ( 15°), 60%, and (φ, ψ) ) (-105 ( 75°, 150 ( 30°), (φ, ψ) ) (120 ( 60°, 150 ( 30°), (φ, ψ) ) (-120 ( 60°, -150 ( 30°) and (φ, ψ) ) (120 ( 60°, -150 ( 30°), 40%. For Val-14, (φ, ψ) ) (-110 ( 20°, 130 ( 20°), 70%, and (φ, ψ) ) (-75 ( 15°, -15 ( 15°) 30%. To reconcile the torsion angles corresponding to the main component in the conformational distributions, a type-II β-turn structure was assigned to about 40% of the Pro-Gly pair.