Probing dynamics within amyloid fibrils using a novel capping method.

A host of diseases involve deposition of proteinaceous amyloid fibrils, which are highly ordered, noncovalent polymers that contain a cross-b architecture. Despite great interest in these fibers, knowledge of the atomic structure of amyloid is limited owing to the difficulty of studying these large heterogeneous biomolecules, especially those formed from long polypeptide chains, with any single biophysical method. Solid-state NMR spectroscopic methods have provided information on the arrangement of the polypeptide chain within amyloid-like structures, affording constraints for secondary, tertiary, and quaternary structure. Herein we study the manner in which the polypeptide chain of b2microglobulin (b2m), a 99-residue protein that forms amyloidlike fibrils in vitro and in vivo, is accommodated within its fibril architecture. By employing a novel method that decouples the interfering contributions of dynamic exchange between fibrillar and soluble material in structural analyses by solution NMR spectroscopy, we discern which regions of b2m are structured in the core of the fibrils, which are exposed, and which are dynamic. Limited proteolysis of b2m fibrils with pepsin has shown that the N-terminal nine residues are exposed to solvent and that digestion of this sample results in a homogeneous product in which 100% of the fibrils are cleaved at a single site (Val9) (Figure 1a and Supporting Information, Figure S1). These data are consistent with NMR spectroscopy hydrogen exchange experiments that reveal limited protection in the 20 N-terminal residues of these fibrils. However, little is known about the dynamics of the polypeptide chain when it is organized into the fibril structure. Recently solidstate NMR spectroscopy methods have identified flexible regions in amyloid fibrils, 7] and previous studies have indicated that mobile regions within large macromolecules can be observed by solution NMR spectroscopy, even though the size of the systems examined would usually prohibit the use of this technique. To better understand the structural organization of the polypeptide chain in b2m amyloid-like fibrils (Figure 1 b) and to identify possible mobile regions within this system, fibril formation of b2m was monitored in real time by H–N HSQC NMR spectroscopy (Figure 2a,b). In parallel, the progression of the fibrillation reaction was monitored by fluorescence of the amyloid-specific dye thioflavin-T as well as by imaging with TEM. Typical thioflavin-T-positive longstraight and twisted amyloid-like fibrils were observed at the conclusion of the reaction (Figure 1b). The initial NMR spectrum (Figure 2a), which was acquired as soon as the protein was placed under low-pH-value conditions, is typical of acid-unfolded b2m, in which a number of intense resonances are observed with limited chemical shift dispersion, indicative of a highly unfolded polypeptide chain. As the reaction proceeds, peak intensities throughout the protein sequence are decreased as monomeric protein is recruited to the fibrillar form, leading to broadened contributions to their linewidths. At the endpoint of the reaction (after 250 h), a surprising number of peaks remains visible in the spectrum (Figure 2b). Interestingly, no chemical-shift changes are Figure 1. a) Sequence of wild-type (WT) b2m and the variant with an extended N-terminal sequence. The fragments prone to pepsinolysis are highlighted within the dashed box and positions of secondary structure and the disulfide bond in the native state are indicated. b, c) Negative-stain TEM images of fibrils formed at pH 2.5 from WT b2m (b) and N-terminally extended b2m (c).