Structural characterization of the caveolin scaffolding domain in association with cholesterol-rich membranes.

Members of the caveolin protein family are implicated in the formation of caveolae and play important roles in a number of signaling pathways and in the regulation of various proteins. We employ complementary spectroscopic methods to study the structure of the caveolin scaffolding domain (CSD) in caveolin-1 fragments, while bound to cholesterol-rich membranes. This key domain is thought to be involved in multiple critical functions that include protein recognition, oligomerization, and cholesterol binding. In our membrane-bound peptides, residues within the flanking intramembrane domain (IMD) are found to adopt an α-helical structure, consistent with its commonly believed helical hairpin conformation. Intriguingly, in these same peptides, we observe a ß-stranded conformation for residues in the CSD, contrasting with earlier reports, which commonly do not reflect ß-structure. Our experimental data based on solid-state NMR, CD, and FTIR are found to be consistent with computational analyses of the secondary structure preference of the primary sequence. We discuss how our structural data of membrane binding Cav fragments may match certain general features of cholesterol-binding domains and could be consistent with the role for CSD in protein recognition and homo-oligomerization.

Amyloid-like fibrils from a domain-swapping protein feature a parallel, in-register conformation without native-like interactions.

The formation of amyloid-like fibrils is characteristic of various diseases, but the underlying mechanism and the factors that determine whether, when, and how proteins form amyloid, remain uncertain. Certain mechanisms have been proposed based on the three-dimensional or runaway domain swapping, inspired by the fact that some proteins show an apparent correlation between the ability to form domain-swapped dimers and a tendency to form fibrillar aggregates. Intramolecular ß-sheet contacts present in the monomeric state could constitute intermolecular ß-sheets in the dimeric and fibrillar states. One example is an amyloid-forming mutant of the immunoglobulin binding domain B1 of streptococcal protein G, which in its native conformation consists of a four-stranded ß-sheet and one α-helix. Under native conditions this mutant adopts a domain-swapped dimer, and it also forms amyloid-like fibrils, seemingly in correlation to its domain-swapping ability. We employ magic angle spinning solid-state NMR and other methods to examine key structural features of these fibrils. Our results reveal a highly rigid fibril structure that lacks mobile domains and indicate a parallel in-register ß-sheet structure and a general loss of native conformation within the mature fibrils. This observation contrasts with predictions that native structure, and in particular intermolecular ß-strand interactions seen in the dimeric state, may be preserved in "domain-swapping" fibrils. We discuss these observations in light of recent work on related amyloid-forming proteins that have been argued to follow similar mechanisms and how this may have implications for the role of domain-swapping propensities for amyloid formation.

The aggregation-enhancing huntingtin N-terminus is helical in amyloid fibrils.

The 17-residue N-terminus (htt(NT)) directly flanking the polyQ sequence in huntingtin (htt) N-terminal fragments plays a crucial role in initiating and accelerating the aggregation process that is associated with Huntington's disease pathogenesis. Here we report on magic-angle-spinning solid-state NMR studies of the amyloid-like aggregates of an htt N-terminal fragment. We find that the polyQ portion of this peptide exists in a rigid, dehydrated amyloid core that is structurally similar to simpler polyQ fibrils and may contain antiparallel ß-sheets. In contrast, the htt(NT) sequence in the aggregates is composed in part of a well-defined helix, which likely also exists in early oligomeric aggregates. Further NMR experiments demonstrate that the N-terminal helical segment displays increased dynamics and water exposure. Given its specific contribution to the initiation, rate, and mechanism of fibril formation, the helical nature of htt(NT) and its apparent lack of effect on the polyQ fibril core structure seem surprising. The results provide new details about these disease-associated aggregates and also provide a clear example of an amino acid sequence that greatly enhances the rate of amyloid formation while itself not taking part in the amyloid structure. There is an interesting mechanistic analogy to recent reports pointing out the early-stage contributions of transient intermolecular helix-helix interactions in the aggregation behavior of various other amyloid fibrils.

Phosphatidylcholine "wobble" in vesicles assessed by high-resolution 13C field cycling NMR spectroscopy.

High resolution (13)C NMR field cycling (covering 11.7 down to 0.002 T) relaxation studies of the sn-2 carbonyl of phosphatidylcholines in vesicles provide a detailed look at the dynamics of this position of the phospholipid in vesicles. The spin-lattice relaxation rate, R(1), observed down to 0.05 T is the result of dipolar and CSA relaxation components characterized by a single correlation time tau(c), with a small contribution from a faster motion contributing to CSA relaxation. At lower fields, R(1) increases further with a correlation time consistent with vesicle tumbling. The tau(c) is particularly interesting since it is 2-3 times slower than what is observed for (31)P of the same phospholipid. However, cholesterol increases the tau(c) for both (31)P and (13)C sites to the same value, approximately 25 ns. These observations suggest faster local motion dominates the dipolar relaxation of the (31)P, while a slower rotation or wobble dominates the relaxation of the carbonyl carbon by the alpha-CH(2) group. The faster motion must be damped with the sterol present. As a general methodology, high resolution (13)C field cycling may be useful for quantifying dynamics in other complex systems as long as a (13)C label (without attached protons) can be introduced.