The solvent exchange rates were reproducible within 10%, as measured at 31 and 135 min incubation time. peptide assembly are proposed. The method provides a general way to identify the core of amyloid structures and thereby pinpoint areas suitable for design of inhibitors. Amyloid diseases have in common an abnormal folding of normally soluble proteins resulting in the formation of extracellular amyloid deposits (1). Examples are Alzheimer’s Disease, Prion-transmissible Spongiform Encephalopathies, and Familial Amyloidotic Polyneuropathy (2). Through self-assembly these proteins produce regular fibrillar structures possessing a predominant -sheet conformation (3). Structural information on the core of fibrils has mainly been elucidated from fiber-diffraction studies (4), mass-spectrometry (5), and solid-state NMR spectroscopy (6), resulting in a general picture consistent with parallel or antiparallel -strands, placed perpendicularly to the fibril axis. However, a detailed structure of a fibril at the atomic level is still lacking. In this respect, more structural informatione.g., which specific amino acids make up the amyloid-forming corebecomes crucially important. In this article, we describe a NMR approach to determining the sequence-specific structural elements in the fibril stage of amyloid forming proteins. The method relies on the partial solvent protection of hydrogen-bonded amide protons connecting the -strands throughout the length of the fibril. In aqueous solutions it is expected that amide protons located on the MELK-8a hydrochloride exterior of the fibril are more accessible to solvent and therefore experience a higher hydrogen exchange rate than amide protons buried within the fibril interior. Studies using mass spectrometry, in combination with deuterium exchange, have already pointed to this possibility (7). However, mass spectrometry does not allow specific assignment of the particular amino acids involved in solvent protection. To obtain more sequence-specific information, the technique of a pulsed hydrogen/deuterium (H/D) exchange NMR experiment (8C10) was modified. Amyloid fibrils were incubated in excess of D2O during a given time. The H/D pattern, as governed by the amyloid, could then be indirectly measured using high-resolution proton NMR through rapid conversion of the fibrils into a monomeric and detectable state. By plotting the integrated peak intensity of amide protons for each residue over time, the post-trap exchange pattern could be fitted to a curve, and extrapolation to time 0 of solubilization yielded the peak intensity of amide protons present in the amyloid fibril. Internal calibration against nonexchangeable methyl groups was made and gives the relative amide-hydrogen solvent-protection value. The proposed method provides a general and quantitative tool for structural mapping of amyloid fibrils. We have used this approach to study amyloid fibrils of the highly amyloidogenic Alzheimer -peptide (A25C35; ref. 11), comprising amino acids 25C35 of A. The full-length Alzheimer A (1C43) is responsible for Alzheimer’s disease and associated dementia. Because of its similar amyloid-forming properties, the A25C35 peptide is frequently used as a convenient model system for studies of Alzheimer amyloid formation and its effects on cellular systems (12). The structural consequences of the findings and its generality are discussed. Materials and Methods Sample Preparation Amyloid Fibrils from A25C35. Thirty microliters of a concentrated (8 mg/ml) solution of the A25C35 peptide (SigmaCAldrich) was incubated for 5 days at 21C in 10 mM sodium-acetate, pH 5.4. After 5 days the initially clear solution had changed to an opaque, highly viscous gel, a characteristic feature for the formation of amyloid fibrils. Atomic Force Microscopy (AFM). The aggregated peptide solution of A25C35 was diluted to 0.05 mg/ml with distilled water and applied onto freshly cleaved ruby red mica (Goodfellow, Cambridge, U.K.). Samples were allowed to adsorb for 30 s, washed three times with distilled water, and air-dried. The bound material was imaged with a Nanoscope IIIa MELK-8a hydrochloride multimode AFM (Digital Instruments, Santa Barbara, CA), using Tapping Mode in air. A silicon probe was oscillated at 300C325 kHz, and images were collected at an optimized scan rate corresponding to 0.7C1 Hz. All images were flattened and presented in height mode by using nanoscope software (Digital Instruments). NMR Spectroscopy. Proton assignment and structural characterization of A25C35. To assign the eleven amide protons of the peptide A25C35 nuclear Overhauser effect spectroscopy (NOESY) spectra were recorded in both deuterated trifluoroethanol (TFEvalues (weighing function 1/and Table ?Table22 show the final protection factors of amide.The freeze-dried A25C35 amyloid deposits readily dissolved in DMSO em -d /em 6 with a measured rate of 0.04C0.05 min?1 at 31C, sufficiently fast to allow the analysis of H/D exchange patterns by using one-dimensional 1H NMR spectroscopy (Fig. being the most protected. In addition, quantitative values for the solvent accessibility for each involved residue could be obtained. Based on our data, two models of peptide assembly are proposed. The method provides a general way to identify the core of amyloid structures and thereby pinpoint areas suitable for design of inhibitors. Amyloid diseases have in common an abnormal folding of normally soluble proteins resulting in the formation of extracellular amyloid deposits (1). Examples are Alzheimer’s Disease, Prion-transmissible Spongiform Encephalopathies, and Familial Amyloidotic Polyneuropathy (2). Through self-assembly these proteins produce regular fibrillar structures possessing a predominant -sheet conformation (3). Structural information on the core of fibrils has mainly been elucidated from fiber-diffraction studies (4), mass-spectrometry (5), and solid-state NMR spectroscopy (6), resulting in a general picture consistent with parallel or antiparallel -strands, placed perpendicularly to the fibril axis. However, a detailed structure of a fibril at the atomic level is still lacking. In this respect, more structural informatione.g., which specific amino acids make up the amyloid-forming corebecomes crucially important. In this article, we describe a NMR approach to determining the sequence-specific structural elements in the fibril stage of amyloid forming proteins. The method relies on the partial solvent protection of hydrogen-bonded amide protons connecting the -strands throughout the length of the fibril. In aqueous solutions it is expected that amide protons located on the exterior of the fibril are more accessible to solvent and therefore experience a higher hydrogen exchange rate MELK-8a hydrochloride than amide protons buried within the fibril interior. Studies using mass spectrometry, in combination with deuterium exchange, have already pointed to this possibility (7). However, mass spectrometry does not allow specific assignment of the particular amino acids involved in solvent protection. To obtain more sequence-specific information, the technique of a pulsed hydrogen/deuterium (H/D) exchange NMR experiment (8C10) was modified. Amyloid fibrils were incubated in excess of D2O during a given time. The H/D pattern, as governed by the amyloid, could then be indirectly measured using high-resolution proton NMR through rapid conversion of the fibrils into a monomeric and detectable state. By plotting the integrated peak intensity of amide protons for each residue over time, the post-trap exchange pattern could be fitted to a curve, and extrapolation to time 0 of solubilization yielded the peak intensity of amide protons present in the amyloid fibril. Internal calibration against nonexchangeable methyl groups was made and gives the relative amide-hydrogen solvent-protection value. The proposed method provides a general and quantitative tool for structural mapping of amyloid fibrils. We have used this approach to study amyloid fibrils of the highly amyloidogenic Alzheimer -peptide (A25C35; ref. 11), comprising amino acids 25C35 of A. The full-length Alzheimer A (1C43) is responsible for Alzheimer’s disease and associated dementia. Because of its similar amyloid-forming properties, the A25C35 peptide is frequently used as a convenient model system for studies of Alzheimer amyloid formation and its effects on cellular systems (12). The structural consequences of the findings and its generality are discussed. Materials and Methods Sample Preparation Amyloid Fibrils from A25C35. Thirty microliters of a concentrated (8 mg/ml) solution of the A25C35 peptide (SigmaCAldrich) was incubated for 5 days at 21C in 10 mM sodium-acetate, pH 5.4. After 5 days the initially clear solution had changed to an opaque, highly viscous gel, a characteristic feature for the formation of amyloid fibrils. Atomic Force Microscopy (AFM). The Pax6 aggregated peptide solution of A25C35 was diluted to 0.05 mg/ml with distilled water and applied onto freshly cleaved ruby red mica (Goodfellow, Cambridge, U.K.). Samples were allowed to adsorb.