Backbone motion in elastin's hydrophobic domains as detected by (2) H NMR spectroscopy.

The elasticity of vertebrate tissue originates from the insoluble, cross-linked protein elastin. Here, the results of variable-temperature (2) H NMR spectra are reported for hydrated elastin that has been enriched at the Hα position in its abundant glycines. Typical powder patterns reflecting averaged quadrupolar parameters are observed for the frozen protein, as opposed to the two, inequivalent deuterons that are detected in a powder sample of enriched glycine. The spectra of the hydrated elastin at warmer temperatures are dominated by a strong central peak with features close to the baseline, reflective of both isotropic and very weakly anisotropic motions.

Resolving nitrogen-15 and proton chemical shifts for mobile segments of elastin with two-dimensional NMR spectroscopy.

In this study, one- and two-dimensional NMR experiments are applied to uniformly (15)N-enriched synthetic elastin, a recombinant human tropoelastin that has been cross-linked to form an elastic hydrogel. Hydrated elastin is characterized by large segments that undergo "liquid-like" motions that limit the efficiency of cross-polarization. The refocused insensitive nuclei enhanced by polarization transfer experiment is used to target these extensive, mobile regions of this protein. Numerous peaks are detected in the backbone amide region of the protein, and their chemical shifts indicate the completely unstructured, "random coil" model for elastin is unlikely. Instead, more evidence is gathered that supports a characteristic ensemble of conformations in this rubber-like protein.

De-Pake-ing transform analysis of fully deuterated malonic acid.

The analysis of deuterium wideline NMR spectra has been an essential step in characterizing the dynamics of molecules in the solid-state. Although clearly important, the identification of quadrupolar coupling constants (QCCs) from the powder patterns is often complicated by poor sensitivity and/or spectral overlap. Previously, others have demonstrated the utility of "de-Pake-ing", a mathematical transform that yields the QCCs in a straightforward manner for symmetric (eta=0) sites. In this short paper, we describe our analysis of a powder sample of perdeutero-malonic acid, a molecule with two distinct deuteron environments and asymmetries. The methylene sites are immediately amenable to the standard de-Pake-ing transform analysis due to their low asymmetry. However, the de-Pake-ing methodology for the acid deuterons, for which the asymmetry deviates significantly from zero, requires more analysis to extract their QCCs. The impact of this work on the future application of de-Pake-ing to a wider range of samples is also discussed.

Structural insights into the elastin mimetic (LGGVG)6 using solid-state 13C NMR experiments and statistical analysis of the PDB.

Elastin is a crosslinked hydrophobic protein found in abundance in vertebrate tissue and is the source of elasticity in connective tissues and blood vessels. The repeating polypeptide sequences found in the hydrophobic domains of elastin have been the focus of many studies that attempt to understand the function of the native protein on a molecular scale. In this study, the central residues of the (LGGVG)(6) elastin mimetic are targeted. Using a combination of a statistical analysis based on structures in the Brookhaven Protein Data Bank (PDB), 1D cross-polarization magic-angle-spinning (CPMAS) NMR spectroscopy, and 2D off-magic-angle-spinning (OMAS) spin-diffusion experiments, it is determined that none of the residues are found in a singular regular, highly ordered structure. Instead, like the poly(VPGVG) elastin mimetics, there are multiple conformations and significant disorder. Furthermore, the conformational ensembles are not reflective of proteins generally, as in the PDB, suggesting that the structure distributions in elastin mimetics are unique to these peptides and are a salient feature of the functional model of the native protein.

Heterogeneity in the conformation of valine in the elastin mimetic (LGGVG)6 as shown by solid-state 13C NMR SPEctroscopy.

Elastin is an abundant protein found in vertebrates and is the source of elasticity in connective tissues and blood vessels. The repeating polypeptide sequences found in the hydrophobic domains of elastin have been the focus of many studies that attempt to understand the function of the native protein on a molecular scale. In this communication, the (LGGVG)6 elastin mimetic is characterized by solid-state 13C NMR spectroscopy. Through the use of a combination of a statistical analysis based on the Protein Data Bank, one-dimensional cross-polarization magic-angle-spinning NMR spectroscopy, and two-dimensional off-magic-angle-spinning spin-diffusion experiments, it is determined that this tandem repeat does not form a regular, highly ordered structure. Instead, like the poly(VPGVG) elastin mimetics, the valine has a twofold heterogeneity, although the conformations of these two populations differ from one peptide to the other.

Cooperativity between the hydrophobic and cross-linking domains of elastin.

The principal protein component of the elastic fiber found in elastic tissues is elastin, an amorphous, cross-linked biopolymer that is assembled from a high molecular weight monomer. The hydrophobic and cross-linking domains of elastin have been considered separate and independent, such that changes to one region are not thought to affect the other. However, results from these solid-state 13C NMR experiments demonstrate that cooperativity in protein folding exists between the two domain types. The sequence of the EP20-24-24 polypeptide has three hydrophobic sequences from exons 20 and 24 of the soluble monomer tropoelastin, interspersed with cross-linking domains constructed from exons 21 and 23. In the middle of each cross-linking domain is a "hinge" sequence. When this pentapeptide is replaced with alanines, as in EP20-24-24[23U], its properties are changed. In addition to the expected increase in alpha-helical content and the resulting increase in rigidity of the cross-linking domains, changes to the organization of the hydrophobic regions are also observed. Using one-dimensional CPMAS (cross-polarization with magic angle spinning) techniques, including spectral editing and relaxation measurements, evidence for a change in dynamics to both domain types is observed. Furthermore, it is likely that the methyl groups of the leucines of the hydrophobic domains are also affected by the substitution to the hinge region of the cross-linking sequences. This cooperativity between the two domain types brings new questions to the phenomenon of coacervation in elastin polypeptides and strongly suggests that functional models for the protein must include a role for the cross-linking regions.

13C CPMAS NMR studies of the elastin-like polypeptide (LGGVG)n.

The elucidation of structure-function relationships in insoluble elastin is often approached using elastin-like polypeptides. In this manner, the characterization of the different regions in this extensive biopolymer may be facilitated in a "piece-wise" manner. Our solid-state NMR experiments indicate that (LGGVG)n has structural similarities to elastin and some elastin peptides, providing support for the utility of the mimetic peptides. Furthermore, previous NMR and CD studies indicated that the structure of the elastin-like polypeptide (LGGVG)n in solution is best described as a "conformational ensemble" with a mixture of type I and II beta-turns, in addition to unfolded regions. Our data indicate that the peptide does not adopt a single conformation in the solid state, lending further support to models for elastin that involve significant conformational heterogeneity.