To explore the initial stages of amyloid β peptide (Aβ42) deposition on membranes, we have studied the interaction of Aβ42 in the monomeric form with lipid monolayers and with bilayers in either the liquid-disordered or the liquid-ordered (L(o)) state, containing negatively charged phospholipids. Molecular dynamics (MD) simulations of the system have been performed, as well as experimental measurements. For bilayers in the L(o) state, in the absence of the negatively charged lipids, interaction is weak and it cannot be detected by isothermal calorimetry. However, in the presence of phosphatidic acid, or of cardiolipin, interaction is detected by different methods and in all cases interaction is strongest with lower (2.5-5 mol%) than higher (10-20 mol%) proportions of negatively charged phospholipids. Liquid-disordered bilayers consistently allowed a higher Aβ42 binding than L(o) ones. Thioflavin T assays and infrared spectroscopy confirmed a higher proportion of β-sheet formation under conditions when higher peptide binding was measured. The experimental results were supported by MD simulations. We used 100 ns MD to examine interactions between Aβ42 and three different 512 lipid bilayers consisting of palmitoylsphingomyelin, dimyristoyl phosphatidic acid, and cholesterol in three different proportions. MD pictures are different for the low- and high-charge bilayers, in the former case the peptide is bound through many contact points to the bilayer, whereas for the bilayer containing 20 mol% anionic phospholipid only a small fragment of the peptide appears to be bound. The MD results indicate that the binding and fibril formation on the membrane surface depends on the composition of the bilayer, and is the result of a subtle balance of many inter- and intramolecular interactions between the Aβ42 and membrane.

We have studied the conformation of the peptide Ac-EPKRSVAFKKTKKEVKKVATPKK (CH-1), free in solution and bound to the DNA, by Fourier-transform infrared spectroscopy. The peptide belongs to the COOH-terminal domain of histone H1 0 (residues 99 -121) and is adjacent to the central globular domain of the protein. In aqueous (D 2 O) solution the amide I is dominated by component bands at 1643 cm ؊1 and 1662 cm ؊1 , which have been assigned to random coil conformations and turns, respectively. In accordance with previous NMR results, the latter component has been interpreted as arising in turn-like conformations in rapid equilibrium with unfolded states. The peptide becomes fully structured either in 90% trifluoroethanol (TFE) solution or upon interaction with the DNA. In these conditions, the contributions of turn (1662 cm ؊1 ) and random coil components virtually disappear. In TFE, the spectrum is dominated by the ␣-helical component (1654 cm ؊1 ). The band at 1662 cm ؊1 shifts to 1670 cm ؊1 , and has been assigned to the COOH-terminal TPKK motif in a more stable turn conformation. A band at 1637 cm ؊1 , also present in TFE, has been assigned to 3 10 helical structure. The amide I band of the complexes with the DNA retains the components that were attributed to 3 10 helix and the TPKK turn. In the complexes with the DNA, the ␣-helical component observed in TFE splits into two components at 1657 cm ؊1 and 1647 cm ؊1 . Both components are inside the spectral region of ␣-helical structures. Our results support the presence of inducible helical and turn elements, both sharing the character of DNA-binding motifs.

The effects of cholesterol on the protein structure and on the ionic channel activity of purified acetylcholine receptor (AcChR) reconstituted into lipid vesicles have been studied, respectively, by Fourier-transform infrared spectroscopy and by rapid kinetics of cation influx. Reconstitution of the AcChR in asolectin phospholipid vesicles in the absence of either cholesterol or the nonpolar lipids present in crude asolectin extracts results in a considerable loss of the ability of the AcChR to support cation channel function. This functional loss is accompanied by spectral changes in the conformationally-sensitive amide I band of the protein infrared spectrum which are indicative of alteration in the protein secondary structure. Quantitative estimation of such alteration by band-fitting analysis reveals a marked decrease in ordered protein structures such as the alpha-helix and beta-pleated sheet, concomitant with an increase in less ordered structures appearing at 1644 cm-1 in the infrared spectrum. Furthermore, the addition of increasing amounts of cholesterol to the reconstituted bilayer produces a progressive, complete recovery both in the control of cation channel function and in the infrared spectrum. This restoration of AcChR structure and function by cholesterol, however, does not occur when the AcChR is reconstituted in vesicles made from purified egg phosphatidylcholine, thus suggesting that the presence in the reconstituted bilayer of phospholipids other than phosphatidylcholine may be required for cholesterol to exert its modulatory effects.(ABSTRACT TRUNCATED AT 250 WORDS)

The IR spectrum of an 16-amino acid peptide corresponding, according to NMR studies, to a 13-hairpin has been analysed. Two characteristic features distinguish its spectrum from that of an antiparallel D-sheet: the low-frequency band that in a []-sheet structure is located at =1632 cm -1 appears here at = 1620 cm -1, and the high-frequency component does not undergo the isotopic shift typical of []-sheet from 1690 to 1675 cm -1 when transferred to D20. The infrared characteristics associated with 13-hairpins have been described so far in two proteins, in one of which, whose three-dimensional structure is known from X-ray diffraction, a 13-hairpin has actually been detected.Key words: Infrared; 13-hairpin; Characterization; Conformational analysis that are known to occur in certain band constituents when transferred from one to another medium [5]. Materials and methodsPreparation of the fragment 41-56 of protein G B1 domain has been described elsewhere [6]. The peptide was analysed by IR at 2 mM concentration and pH 6.3 since it was found that under these conditions it was monomeric [2]. Solutions of the peptide were prepared in H20 and D20 media and measured in a Nicolet Magna 550 spectrometer equipped with a MCT detector. Pathlengths were 6 gna for H20 and 50 ktm for D20 measurements. Typically, 1000 scans were averaged with a nominal resolution better than 2 cm -1. Buffer contribution was subtracted and the spectra treated as described previously [5].

The effects on the protein structure produced by binding of cholinergic agomsts to purified acatylchohne r~eptor (AcChR) reconstituted into lipid vesicles, has been studsed by Fourier-transform infrared spectroscopy and dtfferentml scanning calorimetry. Spectral changes in the conformationally sensitive amlde 1 infrared band indicates that the exposme of the AcChR to the agonist ¢arbamylcholine, under conditions which drive the AcChR rote the desensitized state, produces alteratsoqs in the protein u¢¢ondary structure Quantitative estimation of these agonist-mdueed alteration5 by band-fitting analysis of the amid¢ 1 spectral band rcveal~ no apprecmble changes m the percent of ~-hclix, but a decrc~ase raft.sheet structure, concomitant with an il~erea~ m less ordered stl'ueturcs. Additionally, against binding re~ults in a concentration-dependent increase in the protein thermal stability, as indicated by the temperat are dependence of the protein infrared spectrum and by calorimetric analysis, which further suggest that AcChR desensitization indued by the chohncrglc agonist implie~ significant rearrangements in the protein structure.