The 42-residue, beta-(1-42) peptide is the predominant protein component of amyloid plaque core deposits in Alzheimer's disease (AD). The beta-(1-42) nucleates amyloid formation. In aqueous solution at physiological pH, the synthetic beta-(1-42) peptide undergoes the following conformational transition: random coil or alpha-helix (soluble, monomeric)--> beta-sheet (soluble, aggregated)--> beta-sheet (insoluble, aggregated, amyloid-like). Because these conformational transformations are important in enhancing the neurotoxicity of the beta-peptide, and are presumably involved in amyloidosis in AD, a major goal of research is now focused on identifying substances that prevent the beta-sheet formation and the accompanying precipitation of beta-peptide into amyloid. The development of such inhibitors would be greatly enhanced by knowledge of the intermediate structures formed during the course of amyloidosis. Unfortunately, the beta-(1-42) peptide has not been amenable to traditional three-dimensional protein structural analysis by X-ray or NMR. The beta-(1-42) peptide does not produce crystals suitable for X-ray diffraction and likewise does not adopt a single, well defined structure in aqueous solution over several days which is required for long-term multi-dimensional NMR data acquisitions. The applicant shows the feasibility of characterizing the synthetic beta-(1-42) peptide in aqueous solution by NMR. The success results from two procedures: (1) pre-treatment of the synthetic peptide with trifluoroacetic acid to dissociate pre-aggregated material and (2) supplementing the aqueous solution with sodium dodecyl sulfate (SDS). The SDS micelle is negatively charged and mimics a membrane-like surface. Preliminary circular dichroism (CD) and NMR data establish that in water-SDS solution the beta-(1-42) peptide folds into a predominantly monomeric beta-sheet structure, analogous to its native and biologically relevant structure formed during aggregation. Most significantly, this structure probably represents a very early intermediate that forms during amyloidosis, and is thus a target for the design of therapeutic inhibitors. The ability of SDS to prevent aggregation is analogous to other recent work, where the micelle, hexadecyl-N-methylpiperidinium (HMP) bromide, inhibited aggregation of the beta-(1-40) peptide. There are three specific aims. First, the three-dimensional structure will be determined using a combination of well-established techniques: multi-dimensional NMR spectroscopy, distance geometry, and molecular dynamics. Assignment of the proton NMR spectra, and the determination of the secondary structures will be obtained from interpretation of the NMR data, which include NOE, vicinal coupling constants, and temperature coefficients of the amide-NH chemical shifts. Second, the dynamics of the interaction of the peptide in aqueous SDS solution will be explored using 15N and 13C NMR relaxation studies. Third, putative inhibitor binding sites or surfaces of the beta-(1-42) peptide will be identified. These NMR binding studies will be performed with known inhibitors of amyloidosis such as HMP. Together, the three-dimensional solution structure, plus the NMR binding studies and related molecular modeling procedures will facilitate the design of therapeutic procedures to curtail the beta-(1-42) (soluble)--> beta-(1-42) (insoluble) conversion that occurs in AD.
