Conformational transitions of the cross-linking domains of elastin during self-assembly.

Elastin is the intrinsically disordered polymeric protein imparting the exceptional properties of extension and elastic recoil to the extracellular matrix of most vertebrates. The monomeric precursor of elastin, tropoelastin, as well as polypeptides containing smaller subsets of the tropoelastin sequence, can self-assemble through a colloidal phase separation process called coacervation. Present understanding suggests that self-assembly is promoted by association of hydrophobic domains contained within the tropoelastin sequence, whereas polymerization is achieved by covalent joining of lysine side chains within distinct alanine-rich, α-helical cross-linking domains. In this study, model elastin polypeptides were used to determine the structure of cross-linking domains during the assembly process and the effect of sequence alterations in these domains on assembly and structure. CD temperature melts indicated that partial α-helical structure in cross-linking domains at lower temperatures was absent at physiological temperature. Solid-state NMR demonstrated that ß-strand structure of the cross-linking domains dominated in the coacervate state, although α-helix was predominant after subsequent cross-linking of lysine side chains with genipin. Mutation of lysine residues to hydrophobic amino acids, tyrosine or alanine, leads to increased propensity for ß-structure and the formation of amyloid-like fibrils, characterized by thioflavin-T binding and transmission electron microscopy. These findings indicate that cross-linking domains are structurally labile during assembly, adapting to changes in their environment and aggregated state. Furthermore, the sequence of cross-linking domains has a dramatic effect on self-assembly properties of elastin-like polypeptides, and the presence of lysine residues in these domains may serve to prevent inappropriate ordered aggregation.

Dynamic equilibria between monomeric and oligomeric misfolded states of the mammalian prion protein measured by 19F NMR.

The assembly of misfolded proteins is a critical step in the pathogenesis of amyloid and prion diseases, although the molecular mechanisms underlying this phenomenon are not completely understood. Here, we use (19)F NMR spectroscopy to examine the thermodynamic driving forces surrounding formation of ß-sheet-rich oligomers early in the misfolding and aggregation pathway of the mammalian prion protein. We show that initial assembly of a small octameric intermediate is entropically driven, while further assembly to putative prefibrillar aggregates is driven by a favorable change in enthalpy. Kinetic data suggest that formation of the ß-octamer represents a rate-limiting step in the assembly of prion aggregates. A disease-related mutation (F198S) known to destabilize the native state of PrP was also found to stabilize the ß-octamer, suggesting that it can influence susceptibility to prion disease through two distinct mechanisms. This study provides new insight into the misfolding pathway leading to critical oligomers of the prion protein and suggests a physical basis for increased assembly of the F198S mutant.

Dimerization of the transmembrane domain of human tetherin in membrane mimetic environments.

Tetherin/Bst-2 is a cell surface protein that can act as a restriction factor against a number of enveloped viruses, including HIV-1. It acts by tethering new virus particles to the host cell membrane, promoting their internalization and degradation. Tetherin is a type II membrane protein, with an N-terminal transmembrane domain, an extracellular coiled-coil domain, and a C-terminal GPI anchor. This double membrane anchor is important for anti-HIV activity, as is dimerization of the coiled-coil domain, but despite recent crystal structures of the coiled-coil ectodomains of human and mouse tetherin, the topology of tetherin with respect to host and viral membranes has yet to be determined. The tetherin transmembrane domain is also thought to mediate interactions with the HIV-1 encoded integral membrane protein Vpu, which is an antagonist of tetherin, through direct binding to the transmembrane region of Vpu. Using a combination of SDS-PAGE, size exclusion chromatography, and pyrene excimer fluorescence, we show that in the absence of the coiled-coil domain the transmembrane domain of human tetherin forms parallel homodimers in membrane mimetic environments. Transmembrane domain dimerization does not require disulfide bond formation and is favored in TFE, SDS micelles, and POPC liposomes. This observation has implications for functional models of tetherin, suggesting that both transmembrane domains in the dimeric molecule are inserted into the same lipid bilayer, rather than into opposing membranes.

Solid-State NMR characterization of autofluorescent fibrils formed by the elastin-derived peptide GVGVAGVG.

The characterization of the molecular structure and physical properties of self-assembling peptides is an important aspect of optimizing their utility as scaffolds for biomaterials and other applications. Here we report the formation of autofluorescent fibrils by an octapeptide (GVGVAGVG) derived via a single amino acid substitution in one of the hydrophobic repeat elements of human elastin. This is the shortest and most well-defined peptide so far reported to exhibit intrinsic fluorescence in the absence of a discrete fluorophore. Structural characterization by FTIR and solid-state NMR reveals a predominantly ß-sheet conformation for the peptide in the fibrils, which are likely assembled in an amyloid-like cross-ß structure. Investigation of dynamics and the effects of hydration on the peptide are consistent with a rigid, water excluded structure, which has implications for the likely mechanism of intrinsic fibril fluorescence.

Core structure of amyloid fibrils formed by residues 106-126 of the human prion protein.

Peptides comprising residues 106-126 of the human prion protein (PrP) exhibit many features of the full-length protein. PrP(106-126) induces apoptosis in neurons, forms fibrillar aggregates, and can mediate the conversion of native cellular PrP (PrP(C)) to the scrapie form (PrP(Sc)). Despite a wide range of biochemical and biophysical studies on this peptide, including investigation of its propensity for aggregation, interactions with cell membranes, and PrP-like toxicity, the structure of amyloid fibrils formed by PrP(106-126) remains poorly defined. In this study we use solid-state nuclear magnetic resonance to define the secondary and quaternary structure of PrP(106-126) fibrils. Our results reveal that PrP(106-126) forms in-register parallel beta sheets, stacked in an antiparallel fashion within the mature fibril. The close intermolecular contacts observed in the fibril core provide a rational for the sequence-dependent behavior of PrP(106-126), and provide a basis for further investigation of its biological properties.

Morphology and secondary structure of stable beta-oligomers formed by amyloid peptide PrP(106-126).

The formation of nonfibrillar oligomers has been proposed to be a common element of the aggregation pathway of amyloid peptides. Here we describe the first detailed investigation of the morphology and secondary structure of stable oligomers formed by a peptide comprising residues 106-126 of the human prion protein (PrP). These oligomers have an apparent hydrodynamic radius of approximately 30 nm and are more membrane-active than monomeric or fibrillar PrP(106-126). Circular dichroism and solid state NMR data support formation of an extended beta-strand by the hydrophobic core of PrP(106-126), while negative thioflavin-T binding implies an absence of cross-beta structure in nonfibrillar oligomers.

The gelsolin family of actin regulatory proteins: modular structures, versatile functions.

This issue of FEBS Letters includes two manuscripts describing structural studies of gelsolin, the best-characterized member of a superfamily of actin binding proteins that sever, cap, and in some cases nucleate and bundle actin filaments. The manuscripts by Narayan et al. and Irobi et al. provide snapshots of gelsolin domains activated by calcium and in complex with the actin monomer, revealing new insights into the remarkable actin regulatory activities of this versatile protein. These studies build upon nearly a quarter of a century of research on gelsolin's effects on actin dynamics and its role in normal and diseased cells. In the following minireview, we summarize the structural studies that have provided insights into gelsolin's severing and capping activities and look to the future of work on this remarkable molecule.