Search PubMed⌕ Search

Biomedical subjects

Christopher M Dobson

Publications and source records attributed to Christopher M Dobson.

At least 55 records · Page 3Linked to original sources
PubMed2005

Multiple subsets of side-chain packing in partially folded states of alpha-lactalbumins.

Photochemically induced dynamic nuclear polarization NMR pulse-labeling techniques have been used to obtain detailed information about side-chain surface accessibilities in the partially folded (molten globule) states of bovine and human alpha-lactalbumin prepared under a variety of well defined conditions. Pulse labeling involves generating nuclear polarization in the partially folded state, rapidly refolding the protein within the NMR sample tube, then detecting the polarization in the well dispersed native-state spectrum. Differences in the solvent accessibility of specific side chains in the various molten globule states indicate that the hydrophobic clusters involved in stabilizing the alpha-lactalbumin fold can be formed from interactions between a variety of different hydrophobic residues in both native and non-native environments. The multiple subsets of hydrophobic clusters are likely to result from the existence of distinct but closely related local minima on the free-energy landscape of the protein and show that the fold and topology of a given protein may be formed from degenerate groups of side chains.

Amino Acid Sequence↗
PubMed2005

High hydrostatic pressure dissociates early aggregates of TTR105-115, but not the mature amyloid fibrils.

A range of disorders such as Alzheimer's disease and type II diabetes have been linked to protein misfolding and aggregation. Transthyretin is an amyloidogenic protein which is involved in familial amyloid polyneuropathy, the most common form of systemic amyloid disease. A peptide fragment of this protein, TTR105-115, has been shown to form well-defined amyloid fibrils in vitro. In this study, the stability of amyloid fibrils towards high hydrostatic pressure has been investigated by Fourier transform infrared spectroscopy. Information on the morphology of the species exposed to high hydrostatic pressure was obtained by atomic force microscopy. The species formed early in the aggregation process were found to be dissociated by relatively low hydrostatic pressure (220 MPa), whereas mature fibrils are pressure insensitive up to 1.3 GPa. The pressure stability of the mature fibrils is consistent with a fibril structure in which there is an extensive hydrogen bond network in a tightly packed environment from which water is excluded. The fact that early aggregates can be dissociated by low pressure suggests, however, that hydrophobic and electrostatic interactions are the dominant factors stabilizing the species formed in the early stages of fibril formation.

Amyloid↗
PubMed2005

Towards complete descriptions of the free-energy landscapes of proteins.

In recent years increasingly detailed information about the structures and dynamics of protein molecules has been obtained by innovative applications of experimental techniques, in particular nuclear magnetic resonance spectroscopy and protein engineering, and theoretical methods, notably molecular dynamics simulations. In this article we discuss how such approaches can be combined by incorporating a wide range of different types of experimental data as restraints in computer simulations to provide unprecedented detail about the ensembles of structures that describe proteins in a wide variety of states from the native structure to highly unfolded species. Knowledge of these ensembles is beginning to enable the complete free-energy landscapes of individual proteins to be defined at atomic resolution. This strategy has provided new insights into the mechanism by which proteins are able to fold into their native states, or by which they fail to do so and give rise to harmful aggregates that are associated with a wide range of debilitating human diseases.

Computer Simulation↗
PubMed2005

Formation of native and non-native interactions in ensembles of denatured ACBP molecules from paramagnetic relaxation enhancement studies.

Paramagnetic relaxation enhancement measurements in the denatured state of ACBP have provided distance restraints that have been used in computer simulations to determine the conformational ensembles representing the denatured states of ACBP under a variety of conditions. A detailed comparison of the residual structure in the denatured state of ACBP under these different conditions has enabled us to infer that regions in the N and C-terminal parts of the protein sequence have a high tendency to interact in the unfolded state under physiological conditions. By comparing the structural features in the denatured states with those in the transition state for folding we also provided new insights into the mechanism of formation of the native state of this protein.

Animals↗
PubMed2005

Amyloid formation from HypF-N under conditions in which the protein is initially in its native state.

Aggregation of the N-terminal domain of the Escherichia coli HypF (HypF-N) was investigated in mild denaturing conditions, generated by addition of 6-12% (v/v) trifluoroethanol (TFE). Atomic force microscopy indicates that under these conditions HypF-N converts into the same type of protofibrillar aggregates previously shown to be highly toxic to cultured cells. These convert subsequently, after some weeks, into well-defined fibrillar structures. The rate of protofibril formation, monitored by thioflavin T (ThT) fluorescence, depends strongly on the concentration of TFE. Prior to aggregation the protein has far-UV circular dichroism (CD) and intrinsic fluorescence spectra identical with those observed for the native protein in the absence of co-solvent; the quenching of the intrinsic tryptophan fluorescence by acrylamide and the ANS binding properties are also identical in the two cases. These findings indicate that HypF-N is capable of forming amyloid protofibrils and fibrils under conditions in which the protein is initially in a predominantly native-like conformation. The rate constants for folding and unfolding of HypF-N, determined in 10% TFE using the stopped-flow technique, indicate that a partially folded state is in rapid equilibrium with the native state and populated to ca 1%. A kinetic analysis reveals that aggregation results from molecules accessing such a partially folded state. The approach described here shows that it is possible to probe the mechanism of aggregation of a specific protein under conditions in which the protein is initially native and hence relevant to a physiological environment. In addition, the results indicate that toxic protofibrils can be formed from globular proteins under conditions that are only marginally destabilising and in which the large majority of molecules have the native fold. This conclusion emphasises the importance for cells to constantly combat the propensity for even the most stable of these proteins to aggregate.

Amyloid↗
PubMed2005

Heat shock protein 70 inhibits alpha-synuclein fibril formation via preferential binding to prefibrillar species.

Parkinson's disease (PD) is a neurodegenerative disorder affecting an estimated 4 million people worldwide. Intracellular proteinaceous inclusions called Lewy bodies are the histological hallmarks of PD and are primarily composed of aggregated alpha-synuclein (alphaSyn). Although the detailed mechanisms remain unclear, mounting evidence suggests that the misfolding of alphaSyn into prefibrillar and fibrillar species is the driving force responsible for cellular toxicity. We show here that the molecular chaperone heat shock protein (Hsp) 70 strongly inhibits alphaSyn fibril formation via preferential binding to prefibrillar species. Moreover, our studies reveal that Hsp70 alters the characteristics of toxic alphaSyn aggregates and indicate that cellular toxicity arises from the prefibrillar forms of alphaSyn. This work therefore elucidates a specific role of Hsp70 in the pathogenesis of PD and supports the general concept that chaperone action is a crucial aspect in protecting against the otherwise damaging consequences of protein misfolding.

Benzothiazoles↗
PubMed2005

Mapping long-range interactions in alpha-synuclein using spin-label NMR and ensemble molecular dynamics simulations.

The intrinsically disordered protein alpha-synuclein plays a key role in the pathogenesis of Parkinson's disease (PD). We show here that the native state of alpha-synuclein consists of a broad distribution of conformers with an ensemble-averaged hydrodynamic radius significantly smaller than that expected for a random coil structure. This partial condensation is driven by interactions between the highly charged C-terminus and a large hydrophobic central region of the protein sequence. We suggest that this structure could inhibit the formation of alpha-synuclein aggregates, which are thought to be the cytotoxic species responsible for neurodegeneration in PD.

Computer Simulation↗
PubMed2005

Simultaneous determination of protein structure and dynamics.

We present a protocol for the experimental determination of ensembles of protein conformations that represent simultaneously the native structure and its associated dynamics. The procedure combines the strengths of nuclear magnetic resonance spectroscopy--for obtaining experimental information at the atomic level about the structural and dynamical features of proteins--with the ability of molecular dynamics simulations to explore a wide range of protein conformations. We illustrate the method for human ubiquitin in solution and find that there is considerable conformational heterogeneity throughout the protein structure. The interior atoms of the protein are tightly packed in each individual conformation that contributes to the ensemble but their overall behaviour can be described as having a significant degree of liquid-like character. The protocol is completely general and should lead to significant advances in our ability to understand and utilize the structures of native proteins.

Computer Simulation↗
PubMed2005

Characterisation of disulfide-bond dynamics in non-native states of lysozyme and its disulfide deletion mutants by NMR.

This report describes NMR-spectroscopic investigations of the conformational dynamics of disulfide bonds in hen-egg-white lysozyme substitution mutants. The following four systems have been investigated: 2SS(alpha), a lysozyme variant that contains C64A, C76A, C80A and C94A substitutions, was studied in water at pH 2 and 3.8 and in urea (8 M, pH 2); 2SS(beta) lysozyme, which has C6S, C30A, C115A and C127A substitutions, was studied in water (pH 2) and urea (8 M, pH 2). The NMR analysis of heteronuclear 15N-relaxation rates shows that the barrier to disulfide-bond isomerisation can vary substantially in different lysozyme mutants and depends on the residual structure present in these states. The investigations reveal cooperativity in the modulation of micro- to millisecond dynamics that is due to the presence of multiple disulfide bridges in lysozyme. Mutation of cysteines in one of the two structural domains substantially diminishes the barrier to rotational isomerisation in the other domain. However, the interactions between hydrophobic clusters within and across the domains remains intact.

Animals↗
PubMed2005

Glycine residues appear to be evolutionarily conserved for their ability to inhibit aggregation.

Six glycine residues of human muscle acylphosphatase (AcP) are evolutionarily conserved across the three domains of life. We have generated six variants of AcP, each having a glycine substituted by an alanine (G15A, G19A, G37A, G45A, G53A, and G69A). Three additional variants had Gly45 replaced by serine, glutamate, and arginine, respectively. The mutational variants do not, on average, have a lower conformational stability than other variants with substitutions of nonconserved residues. In addition, only the G15A variant is enzymatically inactive. However, all variants, with the exception of the G15A mutant, form amyloid aggregates more rapidly than the wild-type. Dynamic light-scattering experiments carried out under conditions close to physiological confirm that aggregate formation is generally more pronounced for the glycine-substituted variants. Apart from the glycine at position 15, all other conserved glycine residues in this protein could have been maintained during evolution because of their ability to inhibit aggregation.

Acid Anhydride Hydrolases↗
PubMed2005

Protein folding and the organization of the protein topology universe.

The mechanism by which proteins fold to their native states has been the focus of intense research in recent years. The rate-limiting event in the folding reaction is the formation of a conformation in a set known as the transition-state ensemble. The structural features present within such ensembles have now been analysed for a series of proteins using data from a combination of biochemical and biophysical experiments together with computer-simulation methods. These studies show that the topology of the transition state is determined by a set of interactions involving a small number of key residues and, in addition, that the topology of the transition state is closer to that of the native state than to that of any other fold in the protein universe. Here, we review the evidence for these conclusions and suggest a molecular mechanism that rationalizes these findings by presenting a view of protein folds that is based on the topological features of the polypeptide backbone, rather than the conventional view that depends on the arrangement of different types of secondary-structure elements. By linking the folding process to the organization of the protein structure universe, we propose an explanation for the overwhelming importance of topology in the transition states for protein folding.

Animals↗
PubMed2004

Reversal of protein aggregation provides evidence for multiple aggregated States.

Observations that prefibrillar aggregates from different amyloidogenic proteins can be solubilised under some conditions have raised questions as to the generality of this phenomenon and the nature of the factors that influence it. By studying aggregates formed from human muscle acylphosphatase (AcP) under mild denaturing conditions, and by using a battery of techniques, we demonstrate that disaggregation is possible under conditions close to physiological where the protein is stable in its native state. In the presence of 25% (v/v) trifluoroethanol (TFE) AcP undergoes partial unfolding and globular aggregates (60-200 nm in diameter) that can assemble further into clusters (400-800 nm in diameter) develop progressively. Yet larger superstructures (>5 microm) are formed when the concentration of the globular aggregates exceeds a critical concentration. After diluting the sample to give a solution containing 5% TFE, the fraction of partially unfolded monomeric protein refolds very rapidly, with a rate constant of approximately 1s(-1). The 60-200 nm globular aggregates disaggregate with an apparent rate constant of approximately 2.5 x 10(-3)s(-1) while the 400-800 nm clusters disassembly more slowly with a rate constant of approximately 3.1 x 10(-4)s(-1). The larger (>5 microm) superstructures are not disrupted under the conditions used here. These results suggest that amyloid formation occurs in discrete steps whose reversibility is increasingly difficult, and dependent on the size of the aggregates, and that disaggregation experiments can provide a powerful method of detecting different types of species within the complex process of aggregation. In addition, our work suggests that destabilization of amyloid aggregates resulting in the conversion of misfolded proteins back to their native states could be an important factor in both the onset and treatment of diseases associated with protein aggregation.

Acid Anhydride Hydrolases↗
PubMed2004

Chemical space and biology.

Chemical space--which encompasses all possible small organic molecules, including those present in biological systems--is vast. So vast, in fact, that so far only a tiny fraction of it has been explored. Nevertheless, these explorations have greatly enhanced our understanding of biology, and have led to the development of many of today's drugs. The discovery of new bioactive molecules, facilitated by a deeper understanding of the nature of the regions of chemical space that are relevant to biology, will advance our knowledge of biological processes and lead to new strategies to treat disease.

Biochemistry↗
PubMed2004

Investigating the effects of mutations on protein aggregation in the cell.

The conversion of peptides and proteins into highly ordered and intractable aggregates is associated with a range of debilitating human diseases and represents a widespread problem in biotechnology. Protein engineering studies carried out in vitro have shown that mutations promote aggregation when they either destabilize the native state of a globular protein or accelerate the conversion of unfolded or partially folded conformations into oligomeric structures. We have extended such studies to investigate protein aggregation in vivo where a number of additional factors able to modify dramatically the aggregation behavior of proteins are present. We have expressed, in Escherichia coli cells, an E. coli protein domain, HypF-N. The results for a range of mutational variants indicate that although mutants with a conformational stability similar to that of the wild-type protein are soluble in the E. coli cytosol, variants with single point mutations predicted to destabilize the protein invariably aggregate after expression. We show, however, that aggregation of destabilized variants can be prevented by incorporating multiple mutations designed to reduce the intrinsic propensity of the polypeptide chain to aggregate; in the cases discussed here, this is achieved by an increase in the net charge of the protein. These results suggest that the principles being established to rationalize aggregation behavior in vitro have general validity for situations in vivo where aggregation has both biotechnological and medical relevance.

Animals↗
PubMed2004

Reduced global cooperativity is a common feature underlying the amyloidogenicity of pathogenic lysozyme mutations.

One of the 20 or so human amyloid diseases is associated with the deposition in vital organs of full-length mutational variants of the antibacterial protein lysozyme. Here, we report experimental data that permit a detailed comparison to be made of the behaviour of two of these amyloidogenic variants, I56T and D67H, under identical conditions. Hydrogen/deuterium exchange experiments monitored by NMR and mass spectrometry reveal that, despite their different locations and the different effects of the two mutations on the structure of the native state of lysozyme, both mutations cause a cooperative destabilisation of a remarkably similar segment of the structure, comprising in both cases the beta-domain and the adjacent C-helix. As a result, both variant proteins populate transiently a closely similar, partially unstructured intermediate in which the beta-domain and the adjacent C-helix are substantially and simultaneously unfolded, whereas the three remaining alpha-helices that form the core of the alpha-domain still have their native-like structure. We show, in addition, that the binding of a camel antibody fragment, cAb-HuL6, which was raised against wild-type lysozyme, restores to both variant proteins the stability and cooperativity characteristic of the wild-type protein; as a consequence, it inhibits the formation of amyloid fibrils by both variants. These results indicate that the reduction in global cooperativity, and the associated ability to populate transiently a specific, partly unfolded intermediate state under physiologically relevant conditions, is a common feature underlying the behaviour of these two pathogenic mutations. The formation of intermolecular interactions between lysozyme molecules that are in this partially unfolded state is therefore likely to be the fundamental trigger of the aggregation process that ultimately leads to the formation and deposition in tissue of amyloid fibrils.

Amino Acid Substitution↗
PubMed2004

The formation of spherulites by amyloid fibrils of bovine insulin.

Bovine insulin has long been known to self-assemble in vitro into amyloid fibrils. We have observed a further higher-order self-association of the protein into spherical structures, with diameters typically around 50 microm but ranging from 10 to 150 microm. In a polarizing light microscope, these structures exhibit a "Maltese-cross" extinction pattern typical of spherulites. Spherical structures of a similar size distribution can be observed in the environmental scanning electron microscope, which also reveals the presence of significant amounts of water in the structures. The spherulites contain a large quantity of well defined amyloid fibrils, suggesting that they are formed at least in part as a consequence of the self-assembly of preformed fibrils. Similar structures also have been observed in the tissues of patients suffering from amyloid disorders. The ability of amyloid fibrils to form such higher-order assemblies supports the hypothesis that they represent a generic form of polypeptide structure with properties that are analogous to those of classical synthetic polymers.

Amyloid↗