NMR studies of amyloid b-peptides (Ab) in aqueous solution provide a novel way in which to characterize the apparent Alzheimer's disease-related conformational polymorphism of Ab. In the aqueous medium, neither of the polypeptides Ab ox or Ab(1-42) ox (both of which contain a methionine sulfoxide at position 35) is folded into a globular structure, but they both deviate from random coil behavior by local conformational preferences of several short segments along the amino-acid sequence. Differences between the solution structures of Ab(1-40) ox and Ab ox are indicated only by decreased flexibility of the region from about residue 32 to the C-terminus in Ab (1 -42) ox when compared to Ab(1 -40) ox . The lack of the observation of more extensive conformational differences between the two molecules is intriguing, considering that Ab (1 -42) ox in aqueous solution has much higher plaquecompetence than Ab(1 -40) ox .Keywords: Alzheimer's disease; aqueous solution; NMR; polypeptide; aggregation.Alzheimer's disease (AD) is associated with the progressive accumulation of amyloid deposits in the brain and is identified by extracellular neuritic plaques and neurofibrillary tangles [1]. The major components of the plaques are polypeptides with 39 -43 amino-acid residues, which are commonly referred to as 'amyloid b-peptides' (Ab). Once it is cleaved from the much larger amyloid precursor protein (APP), which is a glycoprotein containing a single hydrophobic transmembrane region, Ab has a high propensity for the formation of b-sheet-containing aggregates [2]. Production of Ab and its aggregation and deposition appear to play a causative role in the onset of AD, and genetic factors or brain injuries may accelerate this process [3,4].Ab occurs as a heterogeneous ensemble of variant polypeptides in body fluids as well as in supernatants of cell cultures and brain amyloid deposits [5 -7]. Heterogeneity occurs at both chain ends as well as in nonterminal sequence positions. For example, a substantial proportion of the peptides in brain deposits contain methionine in position 35 as sulfoxide (Ab ox ) [6]. The heterogeneity at the C-terminus is of particular importance. About 90% of the molecules end at position 40 [Ab(x240)] and 10% at position 42 [Ab(x242)]. The extension by the dipeptide segment Ile41-Ala42 results in dramatically increased aggregation propensity when compared with the corresponding Ab(x240) [8,9]. The importance of this effect lies in the fact that some individuals with inherited early onset AD bear a mutation which increases the proportion of Ab(x242) [10][11][12].In this paper we investigate whether the well documented, widely different competence for fibril formation of Ab ox and Ab(1-42) ox [13] might be related to different conformational properties of the monomeric forms of the two polypeptides. NMR spectroscopy in aqueous solution was used to study the two polypeptides Ab ox and Ab(1 -42) ox , which were both generated in a dedicated procedure to yield monomeric species that could be dissolved i...

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We have constructed a two-dimensional database of the proteome of Haemophilus influenzae, a bacterium of medical interest of which the complete genome, comprising about 1742 open reading frames, has been sequenced. The soluble protein fraction of the microorganism was analyzed by two-dimensional electrophoresis, using immobilized pH gradient strips of various pH regions, gels with different acrylamide concentrations and buffers with different trailing ions. In order to visualize low-copy-number gene products, we employed a series of protein extraction and sample application approaches and several chromatographic steps, including heparin chromatography, chromatofocusing and hydrophobic interaction chromatography. We have also analyzed the cell envelope-bound protein fraction using either immobilized pH gradient strips or a two-detergent system with a cationic detergent in the first and an anionic detergent in the second-dimensional separation. Different proteins (502) were identified by matrix-assisted laser desorption/ionization mass spectrometry and amino acid composition analysis. This is at present one of the largest two-dimensional proteome databases.

Senile plaques, a neuropathological hallmark of Alzheimer's disease, consist primarily of insoluble aggregates of beta-amyloid peptide (A beta). A 42-residue peptide (A beta 1-42) appears to be the predominant form. In contrast to A beta 1-40, A beta 1-42 is characterized by its extreme tendency to aggregate into fibers or precipitate. A tailored biotechnological method prevents aggregation of A beta 1-42 monomers during its production. The method is based on a protein tail fused to the amino terminus of A beta. This tail leads to a high expression in E. coli, and a histidine affinity tag facilitates purification. Selective cleavage of the fusion tail is performed with cyanogen bromide by immobilizing the fusion protein on a reversed phase chromatography column. Cleavage then occurs only at the methionine positioned at the designed site but not at the methionine contained in the membrane anchor sequence of A beta. Furthermore, immobilization prevents aggregation of cleaved A beta. Elution from the HPLC column and all succeeding purification steps are optimized to preserve A beta 1-42 as a monomer. Solutions of monomeric A beta 1-42 spontaneously aggregate into fibers within hours. This permits the investigation of the transition of monomers into fibers and the correlation of physico-chemical properties with biological activities. Mutations of A beta 1-42 at position 35 influence the aggregation properties. Wild-type A beta 1-42 with methionine at position 35 has similar properties as A beta with a methionine sulfoxide residue. The fiber formation tendency, however, is reduced when position 35 is occupied by a glutamine, serine, leucine, or a glutamic acid residue.