Biomedical subjects

Christopher M Dobson

Publications and source records attributed to Christopher M Dobson.

113 records · Page 7Linked to original sources

PubMed2002

Kinetic partitioning of protein folding and aggregation.

We have systematically studied the effects of 40 single point mutations on the conversion of the denatured form of the alpha/beta protein acylphosphatase (AcP) into insoluble aggregates. All the mutations that significantly perturb the rate of aggregation are located in two regions of the protein sequence, residues 16-31 and 87-98, each of which has a relatively high hydrophobicity and propensity to form beta-sheet structure. The measured changes in aggregation rate upon mutation correlate with changes in the hydrophobicity and beta-sheet propensity of the regions of the protein in which the mutations are located. The two regions of the protein sequence that determine the aggregation rate are distinct from those parts of the sequence that determine the rate of protein folding. Dissection of the protein into six peptides corresponding to different regions of the sequence indicates that the kinetic partitioning between aggregation and folding can be attributed to the intrinsic conformational preferences of the denatured polypeptide chain.

Acid Anhydride Hydrolases↗

PubMed2002

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 beta-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 alpha-helices that form the core of the alpha-domain still have native-like structure, whereas the beta-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.

Genetic Variation↗

PubMed2002

Unfolding and aggregation during the thermal denaturation of streptokinase.

The thermal denaturation of streptokinase from Streptococcus equisimilis (SK) together with that of a set of fragments encompassing each of its three domains has been investigated using differential scanning calorimetry (DSC). Analysis of the effects of pH, sample concentration and heating rates on the DSC thermograms has allowed us to find conditions where thermal unfolding occurs unequivocally under equilibrium. Under these conditions, pH 7.0 and a sample concentration of less than approximately 1.5 mg x mL(-1), or pH 8.0, the heat capacity curves of intact SK can be quantitatively described by three independent two-state transitions, each of which compares well with the two-state transition observed for the corresponding isolated SK domain. The results indicate that each structural domain of SK behaves as a single cooperative unfolding unit under equilibrium conditions. At pH 7.0 and high sample concentration, or at pH 6.0 at any concentration investigated, the thermal unfolding of domain A was accompanied by the time-dependent formation of aggregates of SK. This produces a severe deformation of the DSC curves, which become concentration dependent and kinetically controlled, and thus precludes their proper analysis by standard deconvolution methods. A simple model involving time-dependent, high-order aggregation may account for the observed effects. Limited-proteolysis experiments suggest that in the aggregates the N-terminal segment 1-63 and the whole of SK domain C are at least partially structured, while domain B is highly unstructured. Unfolding of domain A, under conditions where the N-terminal segment 1-63 has a high propensity for beta sheet structure and a partially formed hydrophobic core, gives rise to rapid aggregation. It is likely that this region is able to act as a nucleus for the aggregation of the full-length protein.

Anilino Naphthalenesulfonates↗

PubMed2002

A kinetic study of beta-lactoglobulin amyloid fibril formation promoted by urea.

The formation of fibrillar aggregates by beta-lactoglobulin in the presence of urea has been monitored by using thioflavin T fluorescence and transmission electron microscopy (TEM). Large quantities of aggregated protein were formed by incubating beta-lactoglobulin in 3-5 M urea at 37 degrees C and pH 7.0 for 10-30 days. The TEM images of the aggregates in 3-5 M urea show the presence of fibrils with diameters of 8-10 nm, and increases in thioflavin T fluorescence are indicative of the formation of amyloid structures. The kinetics of spontaneous fibrillogenesis detected by thioflavin T fluorescence show sigmoidal behavior involving a clear lag phase. Moreover, addition of preformed fibrils into protein solutions containing urea shows that fibril formation can be accelerated by seeding processes that remove the lag phase. Both of these findings are indicative of nucleation-dependent fibril formation. The urea concentration where fibril formation is most rapid, both for seeded and unseeded solutions, is approximately 5.0 M, close to the concentration of urea corresponding to the midpoint of unfolding (5.3 M). This result indicates that efficient fibril formation involves a balance between the requirement of a significant population of unfolded or partially unfolded molecules and the need to avoid conditions that strongly destabilize intermolecular interactions.

Amyloid↗

PubMedDate not supplied

Probing the origins, diagnosis and treatment of amyloid diseases using antibodies.

The deposition of proteins in the form of amyloid fibrils is the characteristic feature of more than 20 medical conditions affecting the central nervous system or a variety of peripheral tissues. These disorders, which include Alzheimer's disease, the prion diseases and type II diabetes, are of enormous importance in the context of present-day human health and welfare. Extensive research is therefore being carried out to define the molecular details of the mechanism of the pathological conversion of amyloidogenic proteins from their soluble forms into fibrillar structures. This review focuses on recent studies that demonstrate the power of using antibodies or antibody fragments to probe the process of fibril formation, and discusses the emerging potential of these species as diagnostic and therapeutic agents.

Amyloid↗