Solid-State NMR Studies of Elastin and Elastin Peptides

The skin and blood vessels of a vertebrate have a uniquely resilient and elastic quality to them. These properties are traced to elastin, the principal protein component of the fibers that comprise a large portion of these elastic tissues. Numerous reviews have been written on the biology and chemistry of this unique protein [1–3]. Briefly, the elastin gene encodes tropoelastin, which is crosslinked in post-translational modification to form insoluble elastin or, simply, elastin. The molecular weight of tropoelastin is large, with molecular weights ranging from 70 to 80 kDa, and its high-resolution structure is not yet solved. Generally, tropoelastin and insoluble elastin are considered to have two “domains,” namely, hydrophobic and crosslinking. Much attention has been focused on the hydrophobic regions that are dominated with the small nonpolar amino acids, glycine, alanine, proline, and valine. A number of repeating polypeptide sequences are found in this domain. Among them are (VPGVG)n and (PGVGVA)n . The crosslinking domain is rich with alanines, with a typical repeat sequence of (KAAK)n or (KAAAK)n . In portions of tropoelastin, the hydrophobic and crosslinking domains alternate. Figure 1 illustrates several domains of rat tropoelastin, as reported by Pierce et al. [4]. To date, there is limited information on the threedimensional structure of insoluble elastin. If one were to consider the size and nature of tropoelastin, then it would be easy to see why this problem is so difficult. That is, the predominance of the small hydrophobic residues and the presence of the crosslinks are the root causes for the insolubility of amorphous, or “mature,” elastin in all but the harshest conditions. Hence, solution nuclear magnetic resonance (NMR) and X-ray crystallography are virtually useless for high-resolution structure determination. Indeed, elastin has more in common with synthetic organic polymers than with many proteins characterized thus far with NMR spectroscopy. In the past, numerous models have emerged to explain the elasticity of elastin [5–8]. They range from the most disordered and globular to ones with significant degrees of order. As examples of the former, Hoeve and Flory used thermodynamic measurements to suggest that elastin was much like rubber, with long hydrophobic chains interspersed randomly with crosslinks [5]. In contrast, the “oiled coils” from predictive methods [6] and the “β-spiral” from structural studies of elastin-based peptides [7–10] suggest that this polymer has a much greater degree of order. More recent computational studies on the elastin peptides have provided some new insights [11,12]. It is generally accepted, though, that the Alarich crosslinking domains are mostly α-helical, whereas the hydrophobic domain’s organization is much less clear. Again, the lack of site-, residue-, and sequence-specific data, such as those obtained by solution and solid-state NMR spectroscopy, has greatly hampered the understanding of the native protein’s structure–function relationships. Two basic approaches have emerged as viable ways to characterize this intriguing protein by solid-state NMR spectroscopy. One focuses on the native (or nativelike) elastin, while the other uses smaller model peptides. Studies of the native protein would be most physiologically relevant, when drawing conclusions regarding structure–function relationships. The preparation of elastin from connective tissue is straightforward [13–15], and large quantities are easily obtained. With purified elastin samples, various groups [13,16–22] have characterized the natural-abundance 13C populations present in the native protein, complete, in most cases, with the waters of hydration. To complement this approach, methods for isotopic enrichment of a given residue type have also emerged [23–25]. These labeling schemes are essential for NMR studies targeting key amino acid types in elastin, and the power of solid-state NMR as a high-resolution structural tool is becoming more evident as these findings are reported. Alternatively, now-classic approaches in elastin biochemistry have focused on mimetics, most notably, the repeating polypenta and hexapeptides, as studied by Urry [7–10,26] and Tamburro [27–29]. The use of these smaller peptides circumvents the problems associated with the polymeric nature and insolubility of elastin. Typically, the rationale for using these peptides is based on the fact that the hydrophobic regions of elastin have an abundance of these somewhat unusual repeating motifs, and elasticity has been assumed to originate from this domain. In addition, the repeating polypeptides possess properties similar to the native tropoelastin, such as coacervation, and can