Protein-GAG interactions: new surface-based techniques, spectroscopies and nanotechnology probes.

New approaches, rooted in the physical sciences, have been developed to gain a more fundamental understanding of protein-GAG (glycosaminoglycan) interactions. DPI (dual polarization interferometry) is an optical technique, which measures real-time changes in the mass of molecules bound at a surface and the geometry of the bound molecules. QCM-D (quartz crystal microbalance-dissipation), an acoustic technique, measures the mass and the viscoelastic properties of adsorbates. The FTIR (Fourier-transform IR) amide bands I, II and III, resulting from the peptide bond, provide insight into protein secondary structure. Synchrotron radiation CD goes to much shorter wavelengths than laboratory CD, allowing access to chromophores that provide insights into the conformation of the GAG chain and of beta-strand structures of proteins. To tackle the diversity of GAG structure, we are developing noble metal nanoparticle probes, which can be detected at the level of single particles and so enable single molecule biochemistry and analytical chemistry. These new approaches are enabling new insights into structure-function relationships in GAGs and together they will resolve many of the outstanding problems in this field.

Human lysozyme gene mutations cause hereditary systemic amyloidosis.

Hereditary non-neuropathic systemic amyloidosis (Ostertag-type) is a rare autosomal dominant disease in which amyloid deposition in the viscera is usually fatal by the fifth decade. In some families it is caused by mutations in the apolipoprotein AI gene but in two unrelated English families under our care the amyloid deposits did not contain apoAI, despite a report that this may have been the case in one of them. Lysozyme is a ubiquitous bacteriolytic enzyme present in external secretions and in polymorphs and macrophages, but its physiological role is not always clear. Here we report that in these two families, lysozyme is the amyloid fibril protein. Affected individuals are heterozygous for point mutations in the lysozyme gene that cause substitution of highly conserved residues, namely threonine for isoleucine at position 56 in one family, and histidine for aspartic acid at residue 67 in the other. Amyloid fibrils from one individual were composed of the full-length Thr-56 variant lysozyme molecule. To our knowledge, this is the first report of naturally occurring variants of human lysozyme and of lysozyme-associated disease. As the structures of human and hen egg-white lysozyme are known to atomic resolution and their folding and structure-function relationships have been exhaustively analysed, our observations should provide a powerful model for understanding amyloidogenesis.

Structure of Met30 variant of transthyretin and its amyloidogenic implications.

Familial amyloidotic polyneuropathy (FAP) is an autosomal dominant hereditary type of lethal amyloidosis involving single (or double) amino acid substitutions in the amyloidogenic protein transthyretin (TTR). The most common type of FAP (Type I, or Portuguese) is characterized by a Val-->Met substitution at position 30. The Met30 variant of TTR has been produced by recombinant methods, crystallized in a form isomorphous with native TTR, subjected to X-ray analysis and compared structurally with the wild-type protein. The comparison shows that the effect of the substitution at position 30 is transmitted through the protein core to Cys10, the only thiol group in the TTR subunit, which becomes slightly more exposed. The variant TTR molecule is otherwise in a near-native state. Use of computer graphics has shown that it is possible to model a linear aggregate of TTR molecules, each linked to the next by a pair of disulphide bonds involving Cys10 residues. Formation of these disulphide bonds involves a small number of slightly short molecular contacts with native TTR molecules, most of which are relieved in the Met30 variant. We propose this model as a possible basis for a molecular description of the FAP amyloid fibrils.

Comparison of the modelled thyroxine binding site in TBG with the experimentally determined site in transthyretin.

The structure of cleaved thyroxine-binding globulin (TBG) has been modelled on the crystal structure of cleaved alpha 1-antitrypsin (a member of the serine proteinase inhibitor, serpin, superfamily) based on the high sequence homology exhibited by the two proteins. Particular attention was paid to the identification and modelled characteristics of the thyroxine binding site. The primary aim of the study was to compare the site qualitatively with the crystallographically determined binding site of transthyretin, the other major transporter of thyroxine, in an attempt to explain the higher binding affinity of the site compared with the known thyroxine binding site in transthyretin (10(10) versus 10(8) M-1). The proposed binding site shares some similar characteristics with the transthyretin binding site but also includes a cluster of aromatic residues which are entirely absent in transthyretin. It is proposed that this might account for the substantial difference in binding affinities.