Margaret Sunde

The structure of amyloid fibrils by electron microscopy and X-ray diffraction.

Publisher Summary The chapter discusses the structural analysis of amyloid fibrils by electron microscopy and X-ray diffraction. A method to isolate amyloid fibrils from tissue was developed, and such preparations of purified fibrils exhibited the classic staining and birefringent characteristics of crude amyloid samples. The further use of high-resolution electron microscopy, examining ultrathin sections of tissue and isolated material, confirmed the fibrillar form as the characteristic appearance of amyloid in the electron microscope. With the ability to prepare samples of isolated amyloid fibrils it began to be possible to probe the molecular structure of the fibrils using X-ray diffraction. The initial use of this technique produced diffraction patterns that showed that the fibrils were composed of polypeptide chains, extended in the so-called cross-β-conformation. Subsequent analyses by X-ray diffraction on a variety of amyloids and also by NMR analysis have confirmed that the protein chains in all amyloid fibrils are predominantly in the cross-β-conformation. The structures of the soluble, globular forms of several amyloidogenic proteins have been determined by the single crystal X-ray crystallography. The origins of the different amyloidoses appear quite diverse, with some arising from a genetic mutation and others apparently from polypeptide misprocessing or from an unusual accumulation of full-length wild-type protein.

Cryo‐electron microscopy structure of an SH3 amyloid fibril and model of the molecular packing

Amyloid fibrils are assemblies of misfolded proteins and are associated with pathological conditions such as Alzheimers disease and the spongiform encephalopathies. In the amyloid diseases, a diverse group of normally soluble proteins self‐assemble to form insoluble fibrils. X‐ray fibre diffraction studies have shown that the protofilament cores of fibrils formed from the various proteins all contain a cross‐β‐scaffold, with β‐strands perpendicular and β‐sheets parallel to the fibre axis. We have determined the threedimensional structure of an amyloid fibril, formed by the SH3 domain of phosphatidylinositol‐3′‐kinase, using cryo‐electron microscopy and image processing at 25 Å resolution. The structure is a double helix of two protofilament pairs wound around a hollow core, with a helical crossover repeat of ∼600 Å and an axial subunit repeat of ∼27 Å. The native SH3 domain is too compact to fit into the fibril density, and must unfold to adopt a longer, thinner shape in the amyloid form. The 20×40‐Å protofilaments can only accommodate one pair of flat β‐sheets stacked against each other, with very little inter‐strand twist. We propose a model for the polypeptide packing as a basis for understanding the structure of amyloid fibrils in general.

From the globular to the fibrous state: protein structure and structural conversion in amyloid formation

The term ‘amyloid’ was used originally to describe certain deposits found post- mortem in organs and tissues, which gave a positive reaction when stained with iodine (Virchow, 1854). Only later was it realized that the material was in fact predominantly proteinaceous, although it is known to be associated with carbohydrates, particularly glucosoaminoglycans, when obtained from many ex vivo sources. With the increasing precision in the definition of amyloid, initially from its characteristic green birefringence when stained with the dye Congo Red (Missmahl & Hartwig, 1953), and later from its particular appearance under the electron microscope (Cohen & Calkins, 1959) and its X-ray diffraction pattern (Eanes & Glenner, 1968), it has become evident that it is a specific fibrillar protein state, which can also be formed by some proteins when denatured in vitro (Burke & Rougvie, 1972), and by synthetic oligopeptides (Bradbury et al . 1960) that may form amyloid spontaneously when placed in pure aqueous medium (Serpell, 1996). Although these latter may form useful experimental systems for the study of amyloid, its major interest at present is that it is associated with a number of prominent lethal diseases (Benson & Wallace, 1989; Pepys, 1994).

Mutations in Cardiac T-Box Factor Gene TBX20 Are Associated with Diverse Cardiac Pathologies, Including Defects of Septation and Valvulogenesis and Cardiomyopathy

The T-box family transcription factor gene TBX20 acts in a conserved regulatory network, guiding heart formation and patterning in diverse species. Mouse Tbx20 is expressed in cardiac progenitor cells, differentiating cardiomyocytes, and developing valvular tissue, and its deletion or RNA interference-mediated knockdown is catastrophic for heart development. TBX20 interacts physically, functionally, and genetically with other cardiac transcription factors, including NKX2-5, GATA4, and TBX5, mutations of which cause congenital heart disease (CHD). Here, we report nonsense (Q195X) and missense (I152M) germline mutations within the T-box DNA-binding domain of human TBX20 that were associated with a family history of CHD and a complex spectrum of developmental anomalies, including defects in septation, chamber growth, and valvulogenesis. Biophysical characterization of wild-type and mutant proteins indicated how the missense mutation disrupts the structure and function of the TBX20 T-box. Dilated cardiomyopathy was a feature of the TBX20 mutant phenotype in humans and mice, suggesting that mutations in developmental transcription factors can provide a sensitized template for adult-onset heart disease. Our findings are the first to link TBX20 mutations to human pathology. They provide insights into how mutation of different genes in an interactive regulatory circuit lead to diverse clinical phenotypes, with implications for diagnosis, genetic screening, and patient follow-up.

Zinc Fingers - Folds for Many Occasions

Zinc finger domains (ZnFs) are common, relatively small protein motifs that fold around one or more zinc ions. In addition to their role as a DNA‐binding module, ZnFs have recently been shown to mediate protein:protein and protein:lipid interactions. This small zinc‐ligating domain, often found in clusters containing fingers with different binding specificities, can facilitate multiple, often independent intermolecular interactions between nucleic acids and proteins. Classical ZnFs, typified by TFIIIA, ligate zinc via pairs of cysteine and histidine residues but there are at least 14 different classes of Zn fingers, which differ in the nature and arrangement of their zinc‐binding residues. Some GATA‐type ZnFs can bind to both DNA and a variety of other proteins. Thus proteins with multiple GATA‐type fingers can play a complex role in regulating transcription through the interplay of these different binding selectivities and affinities. Other ZnFs have more specific functions, such as DNA‐binding ZnFs in the nuclear hormone receptor proteins and small‐molecule‐binding ZnFs in protein kinase C. Some classes of ZnFs appear to act exclusively in protein‐only interactions. These include the RING family of ZnFs that are involved in ubiquitination processes and in the assembly of large protein complexes, LIM, TAZ, and PHD domains. We review the similarities and differences in structure and functions of different ZnF classes and highlight the versatility of this fold.

Characterization of the Oligomeric States of Insulin in Self-Assembly and Amyloid Fibril Formation by Mass Spectrometry

The self-assembly and aggregation of insulin molecules has been investigated by means of nanoflow electrospray mass spectrometry. Hexamers of insulin containing predominantly two, but up to four, Zn(2+) ions were observed in the gas phase when solutions at pH 4.0 were examined. At pH 3.3, in the absence of Zn(2+), dimers and tetramers are observed. Spectra obtained from solutions of insulin at millimolar concentrations at pH 2.0, conditions under which insulin is known to aggregate in solution, showed signals from a range of higher oligomers. Clusters containing up to 12 molecules could be detected in the gas phase. Hydrogen exchange measurements show that in solution these higher oligomers are in rapid equilibrium with monomeric insulin. At elevated temperatures, under conditions where insulin rapidly forms amyloid fibrils, the concentration of soluble higher oligomers was found to decrease with time yielding insoluble high molecular weight aggregates and then fibrils. The fibrils formed were examined by electron microscopy and the results show that the amorphous aggregates formed initially are converted to twisted, unbranched fibrils containing several protofilaments. Fourier transform infrared spectroscopy shows that both the soluble form of insulin and the initial aggregates are predominantly helical, but that formation of beta-sheet structure occurs simultaneously with the appearance of well-defined fibrils.

Local cooperativity in the unfolding of an amyloidogenic variant of human lysozyme.

Hydrogen exchange experiments monitored by NMR and mass spectrometry reveal that the amyloidogenic D67H mutation in human lysozyme significantly reduces the stability of the β-domain and the adjacent C-helix in the native structure. In addition, mass spectrometric data reveal that transient unfolding of these regions occurs with a high degree of cooperativity. This behavior results in the occasional population of a partially structured intermediate in which the three α-helices that form the core of the α-domain still have native-like structure, whereas the β-domain and C-helix are simultaneously substantially unfolded. This finding suggests that the extensive intermolecular interactions that will be possible in such a species are likely to initiate the aggregation events that ultimately lead to the formation of the well-defined fibrillar structures observed in the tissues of patients carrying this mutation in the lysozyme gene.

A Systematic Investigation into the Effect of Protein Destabilisation on Beta 2-Microglobulin Amyloid Formation

Beta-2-microglobulin (beta(2)m) has been shown to form amyloid fibrils with distinct morphologies under acidic conditions in vitro. Short, curved fibrils (<600 nm in length), form rapidly without a lag phase, with a maximum rate at pH 3.5. By contrast, fibrils with a long (approximately 1 microm), straight morphology are produced by incubation of the protein at pH< or =3.0. Both fibril types display Congo red birefringence, bind Thioflavin-T and have X-ray fibre diffraction patterns consistent with a cross-beta structure. In order to investigate the role of different partially folded states in generating fibrils of each type, and to probe the effect of protein stability on amyloid formation, we have undertaken a detailed mutagenesis study of beta(2)m. Thirteen variants containing point mutations in different regions of the native protein were created and their structure, stability and fibril forming propensities were investigated as a function of pH. By altering the stability of the native protein in this manner, we show that whilst destabilisation of the native state is important in the generation of amyloid fibrils, population of specific denatured states is a pre-requisite for amyloid formation from this protein. Moreover, we demonstrate that the formation of fibrils with different morphologies in vitro correlates with the relative population of different precursor states.

Hydrophobins--unique fungal proteins.

Microorganisms are often covered by a proteinaceous surface layer that serves as a sieve for external molecular influx, as a shield to protect microbes from external aggression, or as an aid to help microbial dispersion. In bacteria, the latter is called the S-layer, in Actinomycetes, the rod-like fibrillar layer, and in fungi, the rodlet layer [1]. The self-assembly properties and remarkable structural and physicochemical characteristics of hydrophobin proteins underlie the multiple roles played by these unique proteins in fungal biology.

X-ray fiber diffraction of amyloid fibrils

Publisher Summary Amyloid is an ordered structure generated by the polymerization of amyloidogenic proteins. It is a high molecular weight, insoluble material and therefore the atomic structure cannot be investigated by conventional X-ray crystallography or nuclear magnetic resonance (NMR). However, information about its overall fibrillar structure can be obtained by X-ray fiber diffraction, particularly if high-resolution data can be collected. The earliest reported use of this technique, investigating serum amyloid A and light chain amyloid, reoported meridional reflections at 4.68 A and equatorial reflections at 9.8 A. These diffraction patterns are consistent with fibrils composed of polypeptide chains extended in the so-called cross-β conformation, a structure that had earlier been identified as a possible conformation for polypeptide chains on the grounds of model building by Pauling and Corey and which was described for insect silk (Crysopa) by Geddes and co-workers. The meridional reflection indicates a regular structural repeat of 4.68 A along the fibril axis, and the equatorial reflection indicates a structural spacing of 9.8 A perpendicular to the fibril axis.