Novel Observation of a Circular Dichroism Band Originating from Amyloid Fibril

Abstract

IntroductionCircular dichroism (CD) is one of the most useful physical techniques for determining the structures and monitoring structural changes of peptides, proteins, nucleic acids, and so on. The technique operates by the differential absorption of the left-and right-handed components of circularly polarized light by chiral molecules in solution. 1 In addition to the chirality of a monomolecule in solution, the chirality of a macromolecule, such as liquid crystals, 2-4 and homoassociates of porphyrin 5 were also detected by CD spectra.The far-ultraviolet (far-UV) CD spectra (below 250 nm) of peptides and proteins are extremely sensitive to the secondary structure, and the near-UV spectra reflect the contributions of aromatic side chains, disulfide bonds, and induced CD bands of prosthetic groups.In recent years, several kinds of peptides and proteins, which polymerize and form amyloid fibrils, have been focused on due to their neurotoxicity. 6 A non-amyloid β-component (NAC) is one of the amyloidogenic peptides which have been identified as the second major constituent in the plaques of a brain with Alzheimer's disease. 7 The amyloid formation by peptides and proteins was mainly detected by the far-UV CD spectrum, especially near 218 nm, which indicates the existence of a β-conformation. Until now, near-UV CD studies of amyloid-forming peptides have been confined to the analysis of aromatic residues. 8,9 It has recently been demonstrated that a peptide fragment of NAC (NAC(1-13); EQVTNVGGAVVTG) forms amyloid fibrils. 10With an extension of far-UV CD measurements of NAC(1-13), we studied in detail the near-UV CD spectra and found a novel band. In this paper, the results of CD analyses of NAC(1-13) in the far-UV and near-UV regions are presented and discussed concerning the formation of amyloid fibrils. ExperimentalPeptide synthesis NAC(1-13) was synthesized by the Fmoc strategy using a simultaneous multiple-peptide synthesizer, Model PSSM-8 (Shimadzu Corp., Kyoto, Japan). Each residue was coupled for 30 min with PyBOP/HOBt (Watanabe Chemical Industries, Hiroshima, Japan). The peptide was cleavaged from the Alkoresin (Watanabe Chemical Industries, Hiroshima, Japan) using 5% anisole and 1% 1,2-ethanedithiol in trifluoroacetic acid for 1.5 h at room temperature. The cleaved peptide was washed with diethyl ether and solubilized with 25% acetonitrile in 0.01 N HCl.Crude NAC(1-13) was purified by reverse-phase HPLC using an ODS column (20 mm × 150 mm) heated at 40˚C with a linear gradient of 20 to 25% acetonitrile in an aqueous solution of 0.01N HCl over 10 min at a flow rate of 9.9 ml/min. The molecular weight of the peptide was determined by matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The (M+H) + value of NAC(1-13) was calculated to be 1231.4 and was found to be 1231.7 experimentally. The peptide purity was found to be greater than 95% by analytical HPLC. Purified peptides were lyophilized and stored at -20˚C until used. Preparation of peptide samplesA sample soluti...

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“…25,32,34,35 Circular dichroism of the peptide backbone absorption band is also sensitive to secondary structure and has given similar results. 36,37 Fluorescence spectroscopy has been used to detect conformational changes either by noncovalent labeling with dyes such as 1-anilino-8-naphthalenesulfonate (ANS) that are specific for exposed hydrophobic patches [38][39][40][41] or by covalent attachment of fluorescent dyes. 42,43 Bovine β-lactoglobulin variant A…”

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