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Christopher M Dobson

Publications and source records attributed to Christopher M Dobson.

At least 73 records · Page 4Linked to original sources
PubMed2004

Prediction of the absolute aggregation rates of amyloidogenic polypeptide chains.

Protein aggregation is associated with a variety of pathological conditions, including Alzheimer's and Creutzfeldt-Jakob diseases and type II diabetes. Such degenerative disorders result from the conversion of the normal soluble state of specific proteins into aggregated states that can ultimately form the characteristic amyloid fibrils found in diseased tissue. Under appropriate conditions it appears that many, perhaps all, proteins can be converted in vitro into amyloid fibrils. The aggregation propensities of different polypeptide chains have, however, been observed to vary substantially. Here, we describe an approach that uses the knowledge of the amino acid sequence and of the experimental conditions to reproduce, with a correlation coefficient of 0.92 and over five orders of magnitude, the in vitro aggregation rates of a wide range of unstructured peptides and proteins. These results indicate that the formation of protein aggregates can be rationalised to a considerable extent in terms of simple physico-chemical parameters that describe the properties of polypeptide chains and their environment.

Amino Acid Sequence↗
PubMed2004

Low-populated folding intermediates of Fyn SH3 characterized by relaxation dispersion NMR.

Many biochemical processes proceed through the formation of functionally significant intermediates. Although the identification and characterization of such species can provide vital clues about the mechanisms of the reactions involved, it is challenging to obtain information of this type in cases where the intermediates are transient or present only at low population. One important example of such a situation involves the folding behaviour of small proteins that represents a model for the acquisition of functional structure in biology. Here we use relaxation dispersion nuclear magnetic resonance (NMR) spectroscopy to identify, for two mutational variants of one such protein, the SH3 domain from Fyn tyrosine kinase, a low-population folding intermediate in equilibrium with its unfolded and fully folded states. By performing the NMR experiments at different temperatures, this approach has enabled characterization of the kinetics and energetics of the folding process as well as providing structures of the intermediates. A general strategy emerges for an experimental determination of the energy landscape of a protein by applying this methodology to a series of mutants whose intermediates have differing degrees of native-like structure.

Binding Sites↗
PubMed2004

A highly amyloidogenic region of hen lysozyme.

Amyloid fibrils obtained after incubating hen egg-white lysozyme (HEWL) at pH 2.0 and 65 degrees C for extended periods of time have been found to consist predominantly of fragments of the protein corresponding to residues 49-100, 49-101, 53-100 and 53-101, derived largely from the partial acid hydrolysis of Asp-X peptide bonds. These internal fragments of HEWL encompass part of the beta-domain and all the residues forming the C-helix in the native protein, and contain two internal disulfide bridges Cys64-Cys80 and Cys76-Cys94. The complementary protein fragments, including helices A, B and D of the native protein, are not significantly incorporated into the network of fibrils, but remain largely soluble, in agreement with their predicted lower propensities to aggregate. Further analysis of the properties of different regions of HEWL to form amyloid fibrils was carried out by studying fragments produced by limited proteolysis of the protein by pepsin. Here, we show that only fragment 57-107, but not fragment 1-38/108-129, is able to generate well-defined amyloid fibrils under the conditions used. This finding is of particular importance, as the beta-domain and C-helix of the highly homologous human lysozyme have been shown to unfold locally in the amyloidogenic variant D67H, which is associated with the familial cases of systemic amyloidosis linked to lysozyme deposition. The identification of the highly amyloidogenic character of this region of the polypeptide chain provides strong support for the involvement of partially unfolded species in the initiation of the aggregation events that lead to amyloid deposition in clinical disease.

Amino Acid Sequence↗
PubMed2004

Heteronuclear NMR investigations of dynamic regions of intact Escherichia coli ribosomes.

15N-(1)H NMR spectroscopy has been used to probe the dynamic properties of uniformly (15)N labeled Escherichia coli ribosomes. Despite the high molecular weight of the complex ( approximately 2.3 MDa), [(1)H-(15)N] heteronuclear single-quantum correlation spectra contain approximately 100 well resolved resonances, the majority of which arise from two of the four C-terminal domains of the stalk proteins, L7/L12. Heteronuclear pulse-field gradient NMR experiments show that the resonances arise from species with a translational diffusion constant consistent with that of the intact ribosome. Longitudinal relaxation time (T(1)) and T(1 rho) (15)N-spin relaxation measurements show that the observable domains tumble anisotropically, with an apparent rotational correlation time significantly longer than that expected for a free L7/L12 domain but much shorter than expected for a protein rigidly incorporated within the ribosomal particle. The relaxation data allow the ribosomally bound C-terminal domains to be oriented relative to the rotational diffusion tensor. Binding of elongation factor G to the ribosome results in the disappearance of the resonances of the L7/L12 domains, indicating a dramatic reduction in their mobility. This result is in agreement with cryoelectron microscopy studies showing that the ribosomal stalk assumes a single rigid orientation upon elongation factor G binding. As well as providing information about the dynamical properties of L7/L12, these results demonstrate the utility of heteronuclear NMR in the study of mobile regions of large biological complexes and form the basis for further NMR studies of functional ribosomal complexes in the context of protein synthesis.

Escherichia coli↗
PubMed2004

The importance of loop length in the folding of an immunoglobulin domain.

Immunoglobulin (Ig)-like proteins have been shown to fold following formation of a nucleus comprising interactions between residues that are distant in the primary sequence. What role do the loops connecting these nucleus residues play? Here, the importance of loops connecting beta-strands in different sheets of the Ig fold is investigated, by insertion of five glycine residues into the B-C loop of an Ig domain from human titin, TI I27. The folding pathway of this elongated 'pseudo wild-type' TI I27 is probed using protein engineering and Phi-value analysis. The Phi-values calculated for mutants within the pseudo wild-type protein indicate that the folding nucleus in wild-type TI I27 is conserved, supporting the hypothesis that the inter-sheet loop is not critical to the formation of a long-range folding nucleus.

Amino Acid Sequence↗
PubMed2004

Formation of amyloid aggregates from human lysozyme and its disease-associated variants using hydrostatic pressure.

Formation of amyloid deposits from the Ile56Thr or Asp67His variants of human lysozyme is a hallmark of autosomal hereditary systemic amyloidosis. It has recently been shown that amyloid fibrils can be formed in vitro from wild-type (WT), I56T, or D67H lysozyme variants upon prolonged incubation at acidic pH and elevated temperatures (1). Here, we have used hydrostatic pressure as a tool to generate amyloidogenic states of WT and variant lysozymes at physiological pH. WT or variant lysozyme samples were initially compressed to 3.5 kbar (at 57 degrees C, pH 7.4). Decompression led to the formation of amyloid fibrils, protofibrils, or globular aggregates, as indicated by light scattering, thioflavin T fluorescence, and transmission electron microscopy analysis. Increased 1-anilinonaphthalene-8-sulfonate binding to the proteins was also observed, indicating exposure of hydrophobic surface area. Thus, pressure appears to induce a conformational state of lysozyme that aggregates readily upon decompression. These results support the notion that amyloid aggregation results from the formation of partially unfolded protein conformations and suggest that pressure may be a useful tool for the generation of the amyloidogenic conformations of lysozyme and other proteins.

Amino Acid Substitution↗
PubMed2004

Prefibrillar amyloid protein aggregates share common features of cytotoxicity.

The intracellular free Ca(2+) concentration and redox status of murine fibroblasts exposed to prefibrillar aggregates of the HypF N-terminal domain have been investigated in vitro and in vivo using a range of fluorescent probes. Aggregate entrance into the cytoplasm is followed by an early rise of reactive oxygen species and free Ca(2+) levels and eventually by cell death. Such changes correlate directly with the viability of the cells and are not observed when cell are cultured in the presence of reducing agents or in Ca(2+)-free media. In addition, moderate cell stress following exposure to the aggregates was found to be fully reversible. The results show that the cytotoxicity of prefibrillar aggregates of HypF-N, a protein not associated with clinical disease, has the same fundamental origin as that produced by similar types of aggregates of proteins linked with specific amyloidoses. These findings suggest that misfolded proteinaceous aggregates stimulate generic cellular responses as a result of the exposure of regions of the structure (such as hydrophobic residues and the polypeptide main chain) that are buried in the normally folded proteins. They also support the idea that a higher number of degenerative pathologies than previously known might be considered as protein deposition diseases.

Amyloid↗
PubMed2004

Short amino acid stretches can mediate amyloid formation in globular proteins: the Src homology 3 (SH3) case.

Protein misfolding and deposition underlie an increasing number of debilitating human disorders. We have shown that model proteins unrelated to disease, such as the Src homology 3 (SH3) domain of the p58alpha subunit of bovine phosphatidyl-inositol-3'-kinase (PI3-SH3), can be converted in vitro into assemblies with structural and cytotoxic properties similar to those of pathological aggregates. By contrast, homologous proteins, such as alpha-spectrin-SH3, lack the capability of forming amyloid fibrils at a measurable rate under any of the conditions we have so far examined. However, transplanting a small sequence stretch (6 aa) from PI3-SH3 to alpha-spectrin-SH3, comprising residues of the diverging turn and adjacent RT loop, creates an amyloidogenic protein closely similar in its behavior to the original PI3-SH3. Analysis of specific PI3-SH3 mutants further confirms the involvement of this region in conferring amyloidogenic properties to this domain. Moreover, the inclusion in this stretch of two consensus residues favored in SH3 sequences substantially inhibits aggregation. These findings show that short specific amino acid stretches can act as mediators or facilitators in the incorporation of globular proteins into amyloid structures, and they support the suggestion that natural protein sequences have evolved in part to code for structural characteristics other than those included in the native fold, such as avoidance of aggregation.

Amino Acid Sequence↗
PubMed2004

Transition states for protein folding have native topologies despite high structural variability.

We present a structural analysis of the folding transition states of three SH3 domains. Our results reveal that the secondary structure is not yet fully formed at this stage of folding and that the solvent is only partially excluded from the interior of the protein. Comparison of the members of the transition state ensemble with a database of native folds shows that, despite substantial local variability, the transition state structures can all be classified as having the topology characteristic of an SH3 domain. Our results suggest a mechanism for folding in which the formation of a network of interactions among a subset of hydrophobic residues ensures that the native topology is generated. Such a mechanism enables high fidelity in folding while minimizing the need to establish a large number of specific interactions in the conformational search.

Amino Acid Sequence↗
PubMed2004

Local and global cooperativity in the human alpha-lactalbumin molten globule.

NMR spectroscopy has been used to follow the urea-induced unfolding of the low pH molten globule states of a single-disulfide variant of human alpha-lactalbumin ([28-111] alpha-LA) and of two mutants, each with a single proline substitution in a helix. [28-111] alpha-LA forms a molten globule very similar to that formed by the wild-type four-disulfide protein, and this variant has been used as a model for the alpha-lactalbumin (alpha-LA) molten globule in a number of studies. The urea-induced unfolding behavior of [28-111] alpha-LA is similar to that of the four-disulfide form of the protein, except that [28-111] alpha-LA is less stable and has greater cooperativity in the loss of different elements of structure. For one mutant, L11P, the helix containing the mutation is highly destabilized such that it is completely unfolded even in the absence of urea. By contrast, for the other mutant, Q117P, the helix containing the mutation retains its compact structure. Both mutations, however, show significant long-range destabilization of the overall fold showing that the molten globule state has a degree of global cooperativity. The results reveal that different permutations of three of the four major alpha-helices of the protein can form a stable, locally cooperative, compact structural core. Taken together, these findings demonstrate that the molten globule state of alpha-LA is an ensemble of conformations, with different subsets of structures linked by a range of long-range interactions.

Amino Acid Substitution↗
PubMed2004

Three-dimensional structures of translating ribosomes by Cryo-EM.

Cryo-electron microscopy and image reconstruction techniques have been used to obtain three-dimensional maps for E. coli ribosomes stalled following translation of three representative proteins. Comparisons of these electron density maps, at resolutions of between 13 and 16 A, with that of a nontranslating ribosome pinpoint specific structural differences in stalled ribosomes and identify additional material, including tRNAs and mRNA. In addition, the tunnel through the large subunit, the anticipated exit route of newly synthesized proteins, is partially occluded in all the stalled ribosome structures. This observation suggests that significant segments of the nascent polypeptide chains examined here could be located within an expanded tunnel, perhaps in a rudimentary globular conformation. Such behavior could be an important aspect of the folding of at least some proteins in the cellular environment.

Cryoelectron Microscopy↗
PubMed2004

Determination of an ensemble of structures representing the denatured state of the bovine acyl-coenzyme a binding protein.

The denatured state of a protein contains important information about the determinants of the folding process. By combining site-directed spin-labeling NMR experiments and restrained computer simulations, we have determined ensembles of conformations that represent the denatured state of the bovine acyl-coenzyme A binding protein (ACBP) at three different concentrations of guanidine hydrochloride. As the experimentally determined distance information corresponds to weighted averages over a broad ensemble of structures, we applied the experimental restraints to a system of noninteracting replicas of the protein by using a Monte Carlo sampling scheme. This procedure permits us to sample ensembles of conformations that are compatible with the experimental data and thus to obtain information regarding the distribution of structures in the denatured state. Our results show that the denatured state of ACBP is highly heterogeneous. The high sensitivity of the computational method that we present, however, enabled us to identify long-range interactions between two regions, located near the N- and C-termini, that include both native and non-native elements. The preferential formation of these contacts suggests that the sequence-dependent patterns of helical propensity and hydrophobicity are important determinants of the structure in the denatured state of ACBP.

Acyl Coenzyme A↗
PubMed2004

Exploring amyloid formation by a de novo design.

Protein deposition as amyloid fibrils underlies many debilitating human disorders. The complexity and size of disease-related polypeptides, however, often hinders a detailed rational approach to study effects that contribute to the process of amyloid formation. We report here a simplified peptide sequence successfully designed de novo to fold into a coiled-coil conformation under ambient conditions but to transform into amyloid fibrils at elevated temperatures. We have determined the crystal structure of the coiled-coil form and propose a detailed molecular model for the peptide in its fibrillar state. The relative stabilities of the two structural forms and the kinetics of their interconversion were found to be highly sensitive to small sequence changes. The results reveal the importance of specific packing interactions on the kinetics of amyloid formation and show the potential of this exceptionally favorable system for probing details of the molecular origins of amyloid disease.

Amino Acid Sequence↗
PubMed2004

High-resolution molecular structure of a peptide in an amyloid fibril determined by magic angle spinning NMR spectroscopy.

Amyloid fibrils are self-assembled filamentous structures associated with protein deposition conditions including Alzheimer's disease and the transmissible spongiform encephalopathies. Despite the immense medical importance of amyloid fibrils, no atomic-resolution structures are available for these materials, because the intact fibrils are insoluble and do not form diffraction-quality 3D crystals. Here we report the high-resolution structure of a peptide fragment of the amyloidogenic protein transthyretin, TTR(105-115), in its fibrillar form, determined by magic angle spinning NMR spectroscopy. The structure resolves not only the backbone fold but also the precise conformation of the side chains. Nearly complete (13)C and (15)N resonance assignments for TTR(105-115) formed the basis for the extraction of a set of distance and dihedral angle restraints. A total of 76 self-consistent experimental measurements, including 41 restraints on 19 backbone dihedral angles and 35 (13)C-(15)N distances between 3 and 6 A were obtained from 2D and 3D NMR spectra recorded on three fibril samples uniformly (13)C, (15)N-labeled in consecutive stretches of four amino acids and used to calculate an ensemble of peptide structures. Our results indicate that TTR(105-115) adopts an extended beta-strand conformation in the amyloid fibrils such that both the main- and side-chain torsion angles are close to their optimal values. Moreover, the structure of this peptide in the fibrillar form has a degree of long-range order that is generally associated only with crystalline materials. These findings provide an explanation of the unusual stability and characteristic properties of this form of polypeptide assembly.

Amino Acid Sequence↗
PubMed2004

Principles of protein folding, misfolding and aggregation.

This review summarises our current understanding of the underlying and universal mechanism by which newly synthesised proteins achieve their biologically functional states. Protein molecules, however, all have a finite tendency either to misfold, or to fail to maintain their correctly folded states, under some circumstances. This article describes some of the consequences of such behaviour, particularly in the context of the aggregation events that are frequently associated with aberrant folding. It focuses in particular on the emerging links between protein aggregation and the increasingly prevalent forms of debilitating disease with which it is now known to be associated.

Amyloid↗
PubMed2004

Experimental investigation of protein folding and misfolding.

Newly synthesised proteins need to fold, often to intricate and close-packed structures, in order to function. The underlying mechanism by which this complex process takes place both in vitro and in vivo is now becoming understood, at least in general terms, as a result of the application of a wide range of biophysical and computational methods used in combination with the techniques of biochemistry and protein engineering. It is increasingly apparent, however, that folding is not only crucial for generating biological activity, but that it is also coupled to a wide range of processes within the cell, ranging from the trafficking of proteins to specific organelles to the regulation of cell growth and differentiation. Not surprisingly, therefore, the failure of proteins to fold appropriately, or to remain correctly folded, is associated with a large number of cellular malfunctions that give rise to disease. Misfolding, and its consequences such as aggregation, can be investigated by extending the types of techniques used to study the normal folding process. Application of these techniques is enabling the development of a unified description of the interconversion and regulation of the different conformational states available to proteins in living systems. Such a description proves a generic basis for understanding the fundamental links between protein misfolding and its associated clinical disorders, such as Alzheimer's disease and Type II diabetes, and for exploring novel therapeutic strategies directed at their prevention and treatment on a rational basis.

Alzheimer Disease↗
PubMed2004

Observation of sequence specificity in the seeding of protein amyloid fibrils.

It is well established that the rate of formation of fibrils by amyloidogenic proteins is enhanced by the addition of preformed fibrils, a phenomenon known as seeding. We show that the efficiency of seeding fibril formation from solutions of hen lysozyme by a series of other proteins depends strongly on the similarity of their sequences. This observation is consistent with the importance of long-range interactions in stabilizing the core structure of amyloid fibrils and may be associated with the existence of a species barrier observed in the transmissible spongiform encephalopathies. In addition, it is consistent with the observation of a single dominant type of protein in the deposits associated with each form of amyloid disease.

Animals↗