Alzheimer disease is characterized by abnormal protein deposits in the brain, such as extracellular amyloid plaques and intracellular neurofibrillary tangles. The tangles are made of a protein called tau comprising 441 residues in its longest isoform. Tau belongs to the class of natively unfolded proteins, binds to and stabilizes microtubules, and partially folds into an ordered β-structure during aggregation to Alzheimer paired helical filaments (PHFs). Here we show that it is possible to overcome the size limitations that have traditionally hampered detailed nuclear magnetic resonance (NMR) spectroscopy studies of such large nonglobular proteins. This is achieved using optimal NMR pulse sequences and matching of chemical shifts from smaller segments in a divide and conquer strategy. The methodology reveals that 441-residue tau is highly dynamic in solution with a distinct domain character and an intricate network of transient long-range contacts important for pathogenic aggregation. Moreover, the single-residue view provided by the NMR analysis reveals unique insights into the interaction of tau with microtubules. Our results establish that NMR spectroscopy can provide detailed insight into the structural polymorphism of very large nonglobular proteins.

Conkunitzin-S1 (Conk-S1) is a 60-residue neurotoxin from the venom of the cone snail Conus striatus that interacts with voltage-gated potassium channels. Conk-S1 shares sequence homology with Kunitz-type proteins but contains only two out of the three highly conserved cysteine bridges, which are typically found in these small, basic protein modules. In this study the three-dimensional structure of Conk-S1 has been solved by multidimensional NMR spectroscopy. The solution structure of recombinant Conk-S1 shows that a Kunitz fold is present, even though one of the highly conserved disulfide cross-links is missing. Introduction of a third, homologous disulfide bond into Conk-S1 results in a functional toxin with similar affinity for Shaker potassium channels. The affinity of Conk-S1 can be enhanced by a pore mutation within the Shaker channel pore indicating an interaction of Conk-S1 with the vestibule of potassium channels.Kunitz domain proteins, like the bovine pancreatic trypsin inhibitor (BPTI) 1 (1) or the dendrotoxins (2) are small, basic proteins that contain three highly conserved disulfide bonds. The three disulfide cross-links make these extracellular proteins extremely stable. Two different general functions are known for the different Kunitz proteins. One group, including BPTI, consists of potent protease inhibitors. The complex of BPTI and trypsin is exceptionally stable, with an association constant Ͼ10 MϪ1 (3). The dendrotoxins belong to another group of Kunitz peptides found in the venom of the black mamba, which block different potassium channels with a high degree of specificity and selectivity (4). In snake and scorpion venoms a diverse set of different potassium channel blockers have been characterized (2).Despite the great variety of toxins from the venoms of the predatory cone snails, relatively few have been identified so far that interact with potassium channels (5). Most conotoxins are small, peptidic toxins that typically contain 10 -30 amino acids and bind with high affinity and specificity to various ligand-or voltage-gated ion channels. One striking feature of these peptides is that they usually contain a diverse complement of posttranslational modifications, like C-terminal amidation, hydroxyprolines, or glycosylation of serine or threonine (6). Conotoxins can be broadly divided into two groups, the non-disulfide-rich peptides and the disulfide-rich conotoxins. The latter conotoxins are further separated into several families based on cysteine bridge pattern and biological activities (5).The potassium channel-targeted toxin conkunitzin-S1 (Conk-S1) from the venom of Conus striatus is the first member of a new family of polypeptides. Recently it has been shown that Conk-S1 blocks Shaker potassium channels with an IC 50 of less than 100 nM.2 Compared with most toxins from Conus venoms, Conk-S1 is unusually long (60 amino acids). The only post-translational modification of this peptide is the amidation of the C-terminal carboxylic acid. Conk-S1 shares 33% sequence identity with BPTI and 3...

High-resolution solid-state NMR spectroscopy can provide structural information of proteins that cannot be studied by X-ray crystallography or solution NMR spectroscopy. Here we demonstrate that it is possible to determine a protein structure by solid-state NMR to a resolution comparable to that by solution NMR. Using an iterative assignment and structure calculation protocol, a large number of distance restraints was extracted from 1H/1H mixing experiments recorded on a single uniformly labeled sample under magic angle spinning conditions. The calculated structure has a coordinate precision of 0.6 Å and 1.3 Å for the backbone and side chain heavy atoms, respectively, and deviates from the structure observed in solution. The approach is expected to be applicable to larger systems enabling the determination of high-resolution structures of amyloid or membrane proteins.

The major bottleneck of rapid NMR‐based structure determination is data analysis. The solution to this problem is a method that enables automatic high‐resolution protein‐structure determination from unassigned, experimental NMR data without manual intervention in less than 24 h.

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