Two-dimensional structure of beta-amyloid(10-35) fibrils.

Beta-amyloid (Abeta) peptides are the main protein component of the pathognomonic plaques found in the brains of patients with Alzheimer's disease. These heterogeneous peptides adopt a highly organized fibril structure both in vivo and in vitro. Here we use solid-state NMR on stable, homogeneous fibrils of Abeta(10-35). Specific interpeptide distance constraints are determined with dipolar recoupling NMR on fibrils prepared from a series of singly labeled peptides containing (13)C-carbonyl-enriched amino acids, and skipping no more that three residues in the sequence. From these studies, we demonstrate that the peptide adopts the structure of an extended parallel beta-sheet in-register at pH 7.4. Analysis of DRAWS data indicates interstrand distances of 5.3 +/- 0.3 A (mean +/- standard deviation) throughout the entire length of the peptide, which is compatible only with a parallel beta-strand in-register. Intrastrand NMR constraints, obtained from peptides containing labels at two adjacent amino acids, confirm the secondary structural findings obtained using DRAWS. Using peptides with (13)C incorporated at the carbonyl position of adjacent amino acids, structural transitions from alpha-helix to beta-sheet were observed at residues 19 and 20, but using similar techniques, no evidence for a turn could be found in the putative turn region comprising residues 25-29. Implications of this extended parallel organization for Abeta(10-35) for overall fibril formation, stability, and morphology based upon specific amino acid contacts are discussed.

Propagating structure of Alzheimer's beta-amyloid(10-35) is parallel beta-sheet with residues in exact register.

The pathognomonic plaques of Alzheimer's disease are composed primarily of the 39- to 43-aa beta-amyloid (Abeta) peptide. Crosslinking of Abeta peptides by tissue transglutaminase (tTg) indicates that Gln15 of one peptide is proximate to Lys16 of another in aggregated Abeta. Here we report how the fibril structure is resolved by mapping interstrand distances in this core region of the Abeta peptide chain with solid-state NMR. Isotopic substitution provides the source points for measuring distances in aggregated Abeta. Peptides containing a single carbonyl 13C label at Gln15, Lys16, Leu17, or Val18 were synthesized and evaluated by NMR dipolar recoupling methods for the measurement of interpeptide distances to a resolution of 0.2 A. Analysis of these data establish that this central core of Abeta consists of a parallel beta-sheet structure in which identical residues on adjacent chains are aligned directly, i. e., in register. Our data, in conjunction with existing structural data, establish that the Abeta fibril is a hydrogen-bonded, parallel beta-sheet defining the long axis of the Abeta fibril propagation.

Structure-function relationships in side chain lactam cross-linked peptide models of a conserved N-terminal domain of apolipoprotein E.

Bioactive peptides have multiple conformations in solution but adopt well-defined conformations at lipid surfaces and in interactions with receptors. We have used side chain lactam cross-links to stabilize secondary structures in the following peptide models of a conserved N-terminal domain of apolipoprotein E (cross-link periodicity in parentheses): I, H2N-GQTLSEQVQEELLSSQVTQELRAG-COOH (none); III, [sequence; see text] (i to i + 3); IV,[sequence; see text] (i to i + 4); IVa, [sequence, see text] (i to i + 4) (lactams above the sequence, potential salt bridges below the sequence). We previously demonstrated [Luo et al. (1994) Biochemistry 33, 12367-12377; Braddock et al. (1996) Biochemistry 35, 13975-13984] that peptide III, containing lactam cross-links between the i and i + 3 side chains, enhances specific binding of LDL via a receptor other than the LDL-receptor. Peptide III in solution consists of two short alpha helices connected by a non alpha helical segment. Here we examine the hypothesis that the domain modeled by peptide III is one antipode of a conformational switch. To model another antipode of the switch, we introduced two strategic modifications into peptide III to examine structure-function relationships in this domain: (1) the spacing of the lactam cross-links was changed (i to i + 4 in peptides IV and IVa) and (2) peptides IV and IVa contain the two alternative sequences at a site of a possible end-capping interaction in peptide III. The structure of peptide IV, determined by 2D-NMR, is alpha helical across its entire length. Despite the remarkable degree of structural order, peptide IV is biologically inactive. In contrast, peptides III and possibly IVa contain a central interruption of the alpha helix, which appears necessary for biological activity. These and other studies support the hypothesis that this domain is a conformational switch which, to the extent that it models apolipoprotein E itself, may modulate interactions between apo E and its various receptors.

Dipolar recoupling NMR of biomolecular self-assemblies: determining inter- and intrastrand distances in fibrilized Alzheimer's beta-amyloid peptide.

We demonstrate a new method for investigating the structure of self-associating biopolymers using dipolar recoupling NMR techniques. This approach was applied to the study of fibrillar beta-amyloid (Abeta) peptides (the primary component of the plaques of Alzheimer's disease) containing only a single isotopic spin label (13C), by employing the DRAWS (dipolar recoupling with a windowless sequence) technique to measure 13C-13C distances. The 'single-label' approach simplified analysis of DRAWS data, since only interstrand contacts are present, without the possibility of any intrastrand contacts. As previously reported [T.L.S. Benzinger, D.M. Gregory, T.S. Burkoth, H. Miller-Auer, D.G. Lynn, R.E. Botto, S.C. Meredith, Proc. Natl. Acad. Sci. 95 (1998) 13407.], contacts of approximately 5 A were observed at all residues studied, consistent with an extended parallel beta-sheet structure with each amino acid in exact register. Here, we propose that our strategy is completely generalizable, and provides a new approach for characterizing any iterative, self-associating biopolymer. Towards the end of generalizing and refining our approach, in this paper we evaluate several issues raised by our previous analyses. First, we consider the effects of double-quantum (DQ) transverse relaxation processes. Next, we discuss the effects of various multiple-spin geometries on modeling of DRAWS data. Several practical issues are also discussed: these include (1) the use of DQ filtering experiments, either to corroborate DRAWS data, or as a rapid screening assessment of the proper placement of isotopic spin labels; and (2) the comparison of solid samples prepared by either lyophilization or freezing. Finally, data obtained from the use of single labels is compared with that obtained in doubly 13C-labeled model compounds of known crystal structure. It is shown that such data are obtainable in far more complex peptide molecules. These data,taken together, refine the DRAWS method, and demonstrate its precision and utility in obtaining high resolution structural data in complex biomolecular aggregates such as Abeta.