Kristin K. Kumashiro

Solid-State 13C NMR Reveals Effects of Temperature and Hydrationon Elastin

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.

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 α-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.

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.

Resolving Nitrogen-15 and Proton Chemical Shifts for Mobile Segments of Elastin with Two-dimensional NMR Spectroscopy

Background: Little is known about the structure of elastin, the abundant elastomeric protein in vertebrate tissue. Results: Observed chemical shifts do not match predictions for random coil, helix, or sheet. Conclusion: The structural makeup of elastin is heterogeneous and possibly unique in nature. Significance: The chemical shift index needs refinement for structural studies of Gly-rich proteins. In this study, one- and two-dimensional NMR experiments are applied to uniformly 15N-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.

Backbone motion in elastin's hydrophobic domains as detected by 2H 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.

CATALYTICALLY ENCHANCED SYSTEMS FOR HYDROGEN STORAGE

Values for the ∆H ‡ of dehydrogenation NaAlH4 to Na3AlH6/Al and Na3AlH6 to NaH/Al and the reverse hydrogenation reactions have been determined through kinetic studies of 2 mol % Ti and Zr doped hydride. We find that the experimentally determined values of ∆H ‡ closely match those predicted by the reaction profile diagrams. This demonstrates that the kinetics of both hydrogenation reactions are limited by the micro-reverse, of the fundamental, Al-H bond breaking process. Solid state 1 H NMR studies have revealed that there are two distinguishable populations of hydrogen in both doped and undoped NaAlH4. The extremely narrow signal (1-2 kHz) that is observed for one of the hydrogen species establishes that it has unusually high solid state mobility. The results of our studies also demonstrate that doping the hydride results in a bulk effect on Al-H bonding and changes structure of the hydride. On the basis of these and X-ray diffraction studies, we developed a new “Na + substitution” model of the doped hydride, which is strongly supported by ESR studies. The arsino pincer complex, IrH2{C6H32,6-(CH2AsBu t 2)2} has been found to be an active catalyst for the dehydrogenation of a variety of cycloalkanes to arenes as well as the reverse hydrogenation reaction. However, development of a hydrogen storage system based on this technology seems impractical as only a maximum, 20 % conversion to the arene can be achieved.

Tropoelastin Implants That Accelerate Wound Repair

A novel, pure, synthetic material is presented that promotes the repair of full-thickness skin wounds. The active component is tropoelastin and leverages its ability to promote new blood vessel formation and its cell recruiting properties to accelerate wound repair. Key to the technology is the use of a novel heat-based, stabilized form of human tropoelastin which allows for tunable resorption. This implantable material contributes a tailored insert that can be shaped to the wound bed, where it hydrates to form a conformable protein hydrogel. Significant benefits in the extent of wound healing, dermal repair, and regeneration of mature epithelium in healthy pigs are demonstrated. The implant is compatible with initial co-treatment with full- and split-thickness skin grafts. The implants superiority to sterile bandaging, commercial hydrogel and dermal regeneration template products is shown. On this basis, a new concept for a prefabricated tissue repair material for point-of-care treatment of open wounds is provided.