NMR spectroscopy combined with paramagnetic relaxation agents was used to study the positioning of the 40-residue Alzheimer Amyloid beta-peptide Abeta(1-40) in SDS micelles. 5-Doxyl stearic acid incorporated into the micelle or Mn(2+) ions in the aqueous solvent were used to determine the position of the peptide relative to the micelle geometry. In SDS solvent, the two alpha-helices induced in Abeta(1-40), comprising residues 15-24, and 29-35, respectively, are surrounded by flexible unstructured regions. NMR signals from these unstructured regions are strongly attenuated in the presence of Mn(2+) showing that these regions are positioned mostly outside the micelle. The central helix (residues 15-24) is significantly affected by 5-doxyl stearic acid however somewhat less for residues 16, 20, 22 and 23. This alpha-helix therefore resides in the SDS headgroup region with the face with residues 16, 20, 22 and 23 directed away from the hydrophobic interior of the micelle. The C-terminal helix is protected both from 5-doxyl stearic acid and Mn(2+), and should be buried in the hydrophobic interior of the micelle. The SDS micelles were characterized by diffusion and (15)N-relaxation measurements. Comparison of experimentally determined translational diffusion coefficients for SDS and Abeta(1-40) show that the size of SDS micelle is not significantly changed by interaction with Abeta(1-40).

Pro-inflammatory S100A9 protein is increasingly recognized as an important contributor to inflammation-related neurodegeneration. Here, we provide insights into S100A9 specific mechanisms of action in Alzheimer’s disease (AD). Due to its inherent amyloidogenicity S100A9 contributes to amyloid plaque formation together with Aβ. In traumatic brain injury (TBI) S100A9 itself rapidly forms amyloid plaques, which were reactive with oligomer-specific antibodies, but not with Aβ and amyloid fibrillar antibodies. They may serve as precursor-plaques for AD, implicating TBI as an AD risk factor. S100A9 was observed in some hippocampal and cortical neurons in TBI, AD and non-demented aging. In vitro S100A9 forms neurotoxic linear and annular amyloids resembling Aβ protofilaments. S100A9 amyloid cytotoxicity and native S100A9 pro-inflammatory signaling can be mitigated by its co-aggregation with Aβ, which results in a variety of micron-scale amyloid complexes. NMR and molecular docking demonstrated transient interactions between native S100A9 and Aβ. Thus, abundantly present in AD brain pro-inflammatory S100A9, possessing also intrinsic amyloidogenic properties and ability to modulate Aβ aggregation, can serve as a link between the AD amyloid and neuroinflammatory cascades and as a prospective therapeutic target.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-013-1208-4) contains supplementary material, which is available to authorized users.

The temperature‐induced structural transitions of the full length Alzheimer amyloid β‐peptide [Aβ(1–40) peptide] and fragments of it were studied using CD and 1H NMR spectroscopy. The full length peptide undergoes an overall transition from a state with a prominent population of left‐handed 31 (polyproline II; PII)‐helix at 0 °C to a random coil state at 60 °C, with an average ΔH of 6.8 ± 1.4 kJ·mol−1 per residue, obtained by fitting a Zimm–Bragg model to the CD data. The transition is noncooperative for the shortest N‐terminal fragment Aβ(1–9) and weakly cooperative for Aβ(1–40) and the longer fragments. By analysing the temperature‐dependent 3JHNHα couplings and hydrodynamic radii obtained by NMR for Aβ(1–9) and Aβ(12–28), we found that the structure transition includes more than two states. The N‐terminal hydrophilic Aβ(1–9) populates PII‐like conformations at 0 °C, then when the temperature increases, conformations with dihedral angles moving towards β‐strand at 20 °C, and approaches random coil at 60 °C. The residues in the central hydrophobic (18–28) segment show varying behaviour, but there is a significant contribution of β‐strand‐like conformations at all temperatures below 20 °C. The C‐terminal (29–40) segment was not studied by NMR, but from CD difference spectra we concluded that it is mainly in a random coil conformation at all studied temperatures. These results on structural preferences and transitions of the segments in the monomeric form of Aβ may be related to the processes leading to the aggregation and formation of fibrils in the Alzheimer plaques.

One of the grand challenges of biophysical chemistry is to understand the principles that govern protein aggregation leading to amyloid fibrils, which is a highly complex and sensitive process. This review provides a comprehensive overview of how amyloid aggregation is affected by the various in vivo constituents and conditions.

The Alzheimer peptide fragment Aβ(12-28) was studied at millimolar concentration by parallel experiments with high-resolution nuclear magnetic resonance (NMR) and circular dichroism (CD) in solution at a pH close to the isoelectric point of the peptide. A preparation procedure using low temperature and low ionic strength buffer gave a sample with stable and reproducible properties. Reversible changes in secondary structure and state of aggregation were studied by variation of temperature. High-temperature promotes aggregation and β-sheet induction, whereas low-temperature shifts the equilibrium toward low molecular weight fractions and less β-sheet like structure. NMR diffusion experiments show that the dominating, most low molecular weight fraction is monomeric. With increasing temperature, residues F 20 A 21 E 22 , overlapping with the so-called central hydrophobic segment of the Aβ peptide, exhibit the most pronounced R-proton NMR secondary chemical shift changes from random coil toward more β-sheet like structure. High ionic strength also promotes aggregation and β-sheet induction. The combined spectroscopic results, including also molecular weight estimations by cutoff filters, are summarized in a scheme in which monomeric mostly random coil and heterogeneous aggregated partly β-sheet forms of the peptide are in a temperature-dependent equilibrium, a situation which corresponds to an early stage of the fibrillogenesis.

The dynamics of monomeric Alzheimer Abeta1-40 in aqueous solution was studied using heteronuclear NMR experiments. 15N NMR relaxation rates of amide groups report on the dynamics in the peptide chain and make it possible to estimate structural propensities from temperature-dependent relaxation data and chemical shifts change analysis. The persistence length of the polypeptide chain was determined using a model in which the influence of neighboring residue relaxation is assumed to decay exponentially as a function of distance. The persistence length of the Abeta1-40 monomer was found to decrease from eight to three residues when temperature was increased from 3 to 18 degrees C. At 3 degrees C the peptide shows structural propensities that correlate well with the suggested secondary structure regions of the peptide to be present in the fibrils, and with the alpha-helical structure in membrane-mimicking systems. Our data leads to a structural model for the monomeric soluble beta-peptide with six different regions of secondary structure propensities. The peptide has two regions with beta-strand propensity (residues 16-24 and 31-40), two regions with high PII-helix propensity (residues 1-4 and 11-15) and two unstructured regions with higher mobility (residues 5-10 and 25-30) connecting the structural elements.

PFG-NMR methods were used to measure the translational diffusion coefficients for the Ab peptide involved in Alzheimer's disease and also for a series of fragments of this peptide. The peptides ranged from a pentamer to the full length Ab(1-40). They were studied at 25• C and physiological pH in aqueous solution. The measured diffusion coefficients, including those of known monomeric peptides, were fitted without systematic deviations to a scaling law function of the molecular mass. We concluded that under these conditions Ab(1-40) is in monomeric form. From the diffusion coefficient data, hydrodynamic radii r H were evaluated for the peptides. When combining our results on non-or weakly structured peptides with previously reported results on denatured proteins, we found that the hydrodynamic radii for the combined dataset could be well described by the same scaling law relating them to the molecular weight. The same law would even encompass data on single amino acids and di-and tripeptides measured by classical methods. From the above-mentioned experimental data, scaling law parameters were determined. The relation between the measured hydrodynamic radius .r H / and the molecular weight of the polypeptide chain .M r / for amino acids, peptides and denatured proteins is r H = 0.27M 0.50 rÅ . There is a remarkably good fit to this function for the measured hydrodynamic radii in a large range, almost three orders of magnitude, of molecular weights. The numerical value of the exponent, 0.5, is an indication that these polymers behave as Gaussian chains.

Transportan is a 27-residue peptide (GWTLN SAGYL LGKIN LKALA ALAKK IL-amide) which has the ability to penetrate into living cells carrying a hydrophilic load. Transportan is a chimeric peptide constructed from the 12 N-terminal residues of galanin in the N-terminus with the 14-residue sequence of mastoparan in the C-terminus and a connecting lysine. Circular dichroism studies of transportan and mastoparan show that both peptides have close to random coil secondary structure in water. Sodium dodecyl sulfate (SDS) micelles induce 60% helix in transportan and 75% helix in mastoparan. The 600 MHz (1)H NMR studies of secondary structure in SDS micelles confirm the helix in mastoparan and show that in transportan the helix is localized to the mastoparan part. The less structured N-terminus of transportan has a secondary structure similar to that of the same sequence in galanin [Ohman, A., et al. (1998) Biochemistry 37, 9169-9178]. The position of mastoparan and transportan relative to the SDS micelle surface was studied by adding spin-labeled 5-doxyl- or 12-doxyl-stearic acid or Mn2+ to the peptide/micelle system. The combined results show that the peptides are for the most part buried in the SDS micelles. Only the C-terminal parts of both peptides and the central segment connecting the two parts of transportan are clearly surface exposed. For mastoparan, the secondary chemical shifts of the amide protons were found to vary periodically and display a pattern almost identical to those reported for mastoparan in phospholipid bicelles [Vold, R., et al. (1997) J. Biomol. NMR 9, 329-335], indicating similar structures and interactions in the two membrane-mimicking environments.