Alzheimer's disease is the most fatal neurodegenerative disorder wherein the process of amyloid-beta (Abeta) amyloidogenesis appears causative. Here, we present the 3D structure of the fibrils comprising Abeta(1-42), which was obtained by using hydrogen-bonding constraints from quenched hydrogen/deuterium-exchange NMR, side-chain packing constraints from pairwise mutagenesis studies, and parallel, in-register beta-sheet arrangement from previous solid-state NMR studies. Although residues 1-17 are disordered, residues 18-42 form a beta-strand-turn-beta-strand motif that contains two intermolecular, parallel, in-register beta-sheets that are formed by residues 18-26 (beta1) and 31-42 (beta2). At least two molecules of Abeta(1-42) are required to achieve the repeating structure of a protofilament. Intermolecular side-chain contacts are formed between the odd-numbered residues of strand beta1 of the nth molecule and the even-numbered residues of strand beta2 of the (n - 1)th molecule. This interaction pattern leads to partially unpaired beta-strands at the fibrillar ends, which explains the sequence selectivity, the cooperativity, and the apparent unidirectionality of Abeta fibril growth. It also provides a structural basis for fibrillization inhibitors.

https://www.pnas.org/content/pnas/102/48/17342.full.pdf

3D structure of Alzheimer's amyloid-β(1–42) fibrils

The folding of most newly synthesized proteins in the cell requires the interaction of a variety of protein cofactors known as molecular chaperones. These molecules recognize and bind to nascent polypeptide chains and partially folded intermediates of proteins, preventing their aggregation and misfolding. There are several families of chaperones; those most involved in protein folding are the 40-kDa heat shock protein (HSP40; DnaJ), 60-kDa heat shock protein (HSP60; GroEL), and 70-kDa heat shock protein (HSP70; DnaK) families. The availability of high-resolution structures has facilitated a more detailed understanding of the complex chaperone machinery and mechanisms, including the ATP-dependent reaction cycles of the GroEL and HSP70 chaperones. For both of these chaperones, the binding of ATP triggers a critical conformational change leading to release of the bound substrate protein. Whereas the main role of the HSP70/HSP40 chaperone system is to minimize aggregation of newly synthesized proteins, the HSP60 chaperones also facilitate the actual folding process by providing a secluded environment for individual folding molecules and may also promote the unfolding and refolding of misfolded intermediates.

https://www.physiology.org/doi/pdf/10.1152/physrev.1999.79.2.425

Chaperone-Mediated Protein Folding

New coupling reagents in peptide chemistry

Rapid photoinduced electron transfer is demonstrated over a distance of greater than 40 angstroms between metallointercalators that are tethered to the 5' termini of a 15-base pair DNA duplex. An oligomeric assembly was synthesized in which the donor is Ru(phen)2dppz2+ (phen, phenanthroline, and dppz, dipyridophenazine) and the acceptor is Rh(phi)2phen3+ (phi, phenanthrenequinone diimine). These metal complexes are intercalated either one or two base steps in from the helix termini. Although the ruthenium-modified oligonucleotide hybridized to an unmodified complement luminesces intensely, the ruthenium-modified oligomer hybridized to the rhodium-modified oligomer shows no detectable luminescence. Time-resolved studies point to a lower limit of 10(9) per second for the quenching rate. No quenching was observed upon metallation of two complementary octamers by Ru(phen)3(2+) and Rh(phen)3(3+) under conditions where the phen complexes do not intercalate. The stacked aromatic heterocycles of the DNA duplex therefore serve as an efficient medium for coupling electron donors and acceptors over very long distances.

https://science.sciencemag.org/content/sci/262/5136/1025.full.pdf

Long-range photoinduced electron transfer through a DNA helix

Molecular chaperones are a functionally defined set of proteins which assist the structure formation of proteins in vivo. Without certain protective mechanisms, such as binding nascent polypeptide chains by molecular chaperones, cellular protein concentrations would lead to misfolding and aggregation. In the mammalian system, the molecular chaperones Hsp70 and Hsp90 are involved in the folding and maturation of key regulatory proteins, like steroid hormone receptors, transcription factors, and kinases, some of which are involved in cancer progression. Hsp70 and Hsp90 form a multichaperone complex, in which both are connected by a third protein called Hop. The connection of and the interplay between the two chaperone machineries is of crucial importance for cell viability. This review provides a detailed view of the Hsp70 and Hsp90 machineries, their cofactors and their mode of regulation. It summarizes the current knowledge in the field, including the ATP-dependent regulation of the Hsp70/Hsp90 multichaperone cycle and elucidates the complex interplay and their synergistic interaction.

Hsp70 and Hsp90--a relay team for protein folding.

The DnaK Chaperone System of Escherichia coli: Quaternary Structures and Interactions of the DnaK and GrpE Components

There are described novel porphyrin derivatives of the general formula ##STR1## wherein either R 1 signifies the group ##STR2## and R 2 signifies the group ##STR3## or R 1 signifies the group ##STR4## and R 2 signifies the group ##STR5## whereby X - signifies a halogen ion, an arylsulphonate ion, an alkylsulphonate ion or an alkyl sulphate ion, A signifies (C 1-8 )-alkylene and R 3 signifies (C 1-4 )-alkyl, and sulphonic acid salts of compounds of formula I in which R 1 signifies the group ##STR6## as well as a process for their manufacture and intermediates used in this process. Because of their water-solubility the novel porphyrin derivatives are suitable as label molecules for highly sensitive fluorescence immuno-assays, especially for time-resolving fluorescence immuno-assays. The coupling of the novel porphyrin derivatives to immunologically-active materials is carried out in the customary manner, for example with a water-soluble carbodiimide derivative.

Porphyrin derivatives as fluorescent markers for immunoassays

The specific attachment of bathophenanthroline-ruthenium(II) complexes as non-radioactive label molecules to synthetically 5′-NH2-modified oligonucleotides is described. After excitation by light pulses, the fluorescence of these labels can be measured by a time-resolved mode woth high sensitivity. No quenching takes place due to coupling of the Ru complexes to the DNA. Ru-complex-labelled oligonucleotides still hybridize specifically to complementary DNA sequences, and no quenching is observed in the course of the hybridization process.

Bathophenanthroline-ruthenium(II) complexes as non-radioactive labels for oligonucleotides which can be measured by time-resolved fluorescence techniques

Senile plaques, a neuropathological hallmark of Alzheimer's disease, consist primarily of insoluble aggregates of s-amyloid peptide (As). A 42-residue peptide (As1-42) appears to be the predominant form. In contrast to As1-40, As1-42 is characterized by its extreme tendency to aggregate into fibers or precipitate. A tailored biotechnological method prevents aggregation of As1-42 monomers during its production. The method is based on a protein tail fused to the amino terminus of As. 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 As. Furthermore, immobilization prevents aggregation of cleaved As. Elution from the HPLC column and all succeeding purification steps are optimized to preserve As1-42 as a monomer. Solutions of monomeric As1-42 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 As1-42 at postion 35 influence the aggregation properties. Wild-type As1-42 with methionine at position 35 has similar properties as As 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.

A Biotechnological Method Provides Access to Aggregation Competent Monomeric Alzheimer's 1–42 Residue Amyloid Peptide

The invention relates to a ruthenium complex having the formula Rusup2+ Lsub1 Lsub2 Lsub3 I wherein L 1 , L 2 and L 3 are the same or different are equal to a bi- or polycyclic ligand with at least two nitrogen-containing heterocycles, whereby at least one of these ligands is substituted with at least one group conferring water-solubility, and whereby at least one of these ligands is substituted, optionally via a spacer group, with at least one reactive group, and whereby the ligands L 1 , L 2 and L 3 are attached to the ruthenium via nitrogen atoms The invention is further related to such ruthenium complexes having coupled thereto an immunologically active material, for example, antigens, haptens or antibodies and to the use of said ruthenium complexes in fluorescence spectroscopy Specific ligands L 1 , L 2 and L 3 which are useful in the ruthenium complexes of the invention are, eg, 2,2'-bipyridine, 1,10-phenanthroline benzbathophenanthroline or bathophenanthroline groups Groups which are useful for conferring water-solubility on said ligand are, eg sulfonic acid groups which are preferably present in the form of their salts Useful spacer groups are eg an alkylene group containing 1-8 carbon atoms and which is optionally substituted with --SO 2 --NH--, --S--, --O--, --COO-- or --CO--NH-- groups Useful reactive groups to which the immunologically active material is coupled, are eg --COOH, --I, --NH 2 , --NCS or --SO 2 Hal groups The ruthenium complexes according to the present invention can be detected with great sensitivity by fluorescence spectroscopy and are thus useful in fluorescence immunoassays

Dieter Schmidt

Papers

The DnaK Chaperone System of Escherichia coli: Quaternary Structures and Interactions of the DnaK and GrpE Components

Porphyrin derivatives as fluorescent markers for immunoassays

Bathophenanthroline-ruthenium(II) complexes as non-radioactive labels for oligonucleotides which can be measured by time-resolved fluorescence techniques

A Biotechnological Method Provides Access to Aggregation Competent Monomeric Alzheimer's 1–42 Residue Amyloid Peptide

Ruthenium complexes useful as carriers for immunologically active materials