Neurotoxic assemblies of the amyloid b-protein (Ab) have been linked strongly to the pathogenesis of Alzheimer's disease (AD). Here, we sought to monitor the earliest step in Ab assembly, the creation of a folding nucleus, from which oligomeric and fibrillar assemblies emanate. To do so, limited proteolysis/mass spectrometry was used to identify protease-resistant segments within monomeric Ab(1-40) and Ab(1-42). The results revealed a 10-residue, protease-resistant segment, Ala21-Ala30, in both peptides. Remarkably, the homologous decapeptide, Ab(21-30), displayed identical protease resistance, making it amenable to detailed structural study using solution-state NMR. Structure calculations revealed a turn formed by residues Val24-Lys28. Three factors contribute to the stability of the turn, the intrinsic propensities of the Val-Gly-Ser-Asn and Gly-Ser-Asn-Lys sequences to form a b-turn, long-range Coulombic interactions between Lys28 and either Glu22 or Asp23, and hydrophobic interaction between the isopropyl and butyl side chains of Val24 and Lys28, respectively. We postulate that turn formation within the Val24-Lys28 region of Ab nucleates the intramolecular folding of Ab monomer, and from this step, subsequent assembly proceeds. This model provides a mechanistic basis for the pathologic effects of amino acid substitutions at Glu22 and Asp23 that are linked to familial forms of AD or cerebral amyloid angiopathy. Our studies also revealed that common C-terminal peptide segments within Ab(1-40) and Ab(1-42) have distinct structures, an observation of relevance for understanding the strong disease association of increased Ab(1-42) production. Our results suggest that therapeutic approaches targeting the Val24-Lys28 turn or the Ab(1-42)-specific C-terminal fold may hold promise.

In a calcium-dependent interaction critical for blood coagulation, vitamin K-dependent blood coagulation proteins bind cell membranes containing phosphatidylserine via gamma-carboxyglutamic acid-rich (Gla) domains. Gla domain-mediated protein-membrane interaction is required for generation of thrombin, the terminal enzyme in the coagulation cascade, on a physiologic time scale. We determined by X-ray crystallography and NMR spectroscopy the lysophosphatidylserine-binding site in the bovine prothrombin Gla domain. The serine head group binds Gla domain-bound calcium ions and Gla residues 17 and 21, fixed elements of the Gla domain fold, predicting the structural basis for phosphatidylserine specificity among Gla domains. Gla domains provide a unique mechanism for protein-phospholipid membrane interaction. Increasingly Gla domains are being identified in proteins unrelated to blood coagulation. Thus, this membrane-binding mechanism may be important in other physiologic processes.

Amyloid ␤-protein (A␤) oligomers may be the proximate neurotoxins in Alzheimer's disease (AD). Recently, to elucidate the oligomerization pathway, we studied A␤ monomer folding and identified a decapeptide segment of A␤, 21 Ala-22 Glu-23 Asp-24 Val-25 Gly-26 Ser-27 Asn-28 Lys-29 Gly-30 Ala, within which turn formation appears to nucleate monomer folding. The turn is stabilized by hydrophobic interactions between Val-24 and Lys-28 and by longrange electrostatic interactions between Lys-28 and either Glu-22 or Asp-23. We hypothesized that turn destabilization might explain the effects of amino acid substitutions at Glu-22 and Asp-23 that cause familial forms of AD and cerebral amyloid angiopathy. To test this hypothesis, limited proteolysis, mass spectrometry, and solution-state NMR spectroscopy were used here to determine and compare the structure and stability of the A␤(21-30) turn within wild-type A␤ and seven clinically relevant homologues. In addition, we determined the relative differences in folding free energies (⌬⌬G f) among the mutant peptides. We observed that all of the disease-associated amino acid substitutions at Glu-22 or Asp-23 destabilized the turn and that the magnitude of the destabilization correlated with oligomerization propensity. The Ala21Gly (Flemish) substitution, outside the turn proper (Glu-22-Lys-28), displayed a stability similar to that of the wild-type peptide. The implications of these findings for understanding A␤ monomer folding and disease causation are discussed.A bundant evidence links the amyloid ␤-protein (A␤) with the neuropathogenesis of Alzheimer's disease (AD) (for recent reviews, see refs. 1 and 2). A␤ is a normal metabolite of the A␤ precursor (A␤PP), from which A␤ is produced by endoproteolysis (3). Two predominant forms of A␤ exist in vivo, A␤40 and A␤42, which are 40-and 42-aa in length, respectively (2, 4, 5). Recent experimental and clinical evidence suggests that the primary neurotoxins in AD are A␤ oligomers or protofibrils (1, 6-10). Understanding the folding and oligomerization of nascent A␤ monomers thus has become an especially important aspect of current strategies for understanding AD etiology and developing therapeutic agents.We have applied a multidisciplinary approach to the A␤ assembly problem. Initial studies used limited proteolysis coupled with mass spectrometry to determine whether monomeric A␤ possessed any stable or quasistable structure that could protect the peptide from proteolysis. Surprisingly, a 10-residue segment within both A␤40 and A␤42, Ala-21-Ala-30, was identified (11). The homologous decapeptide, A␤(21-30), displayed protease resistance identical to that of full-length A␤, suggesting that this region could organize monomer folding and thus be a folding nucleus. This suggestion was consistent with the observation that many folding nuclei studied in isolation are structurally stable (12-16). In fact, NMR studies of the A␤(21-30) peptide revealed a turn in the Val-24-Lys-28 region that was stabilized by hydrophobic interactions between...