Protein misfolding, evolution and disease.
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Alzheimer Disease↗
Biomedical subjects
C M Dobson
Publications and source records attributed to C M Dobson.
Alpha-lactalbumin forms a compact molten globule in the absence of disulfide bonds.
Human alpha-lactalbumin (alpha-LA) is a four disulfide-bonded protein that adopts partially structured conformations under a variety of mildly denaturing conditions. At low pH, the protein is denatured but compact, with a high degree of secondary structure and a native-like fold. This is commonly referred to as a molten globule. A variant of alpha-LA, in which all eight cysteines have been mutated to alanine (all-Ala alpha-LA), has been studied using NMR spectroscopy. At low pH all-Ala alpha-LA is nearly as compact as wild type alpha-LA. Urea-induced unfolding experiments reveal that the residues that remain compact in the absence of disulfide bonds are those that are most resistant to unfolding in the wild-type alpha-LA molten globule. This is particularly remarkable because this stable core is formed by segments of the polypeptide chain from both the N- and C-termini. These results show that the overall architecture of the protein fold of alpha-LA is determined by the polypeptide sequence itself, and not as the result of cross-linking by disulfide bonds, and provide insight into the way in which the sequence codes for the fold.
Amino Acid Substitution↗
Mutational analysis of acylphosphatase suggests the importance of topology and contact order in protein folding.
Muscle acylphosphatase (AcP) is a small protein that folds very slowly with two-state behavior. The conformational stability and the rates of folding and unfolding have been determined for a number of mutants of AcP in order to characterize the structure of the folding transition state. The results show that the transition state is an expanded version of the native protein, where most of the native interactions are partially established. The transition state of AcP turns out to be remarkably similar in structure to that of the activation domain of procarboxypeptidase A2 (ADA2h), a protein having the same overall topology but sharing only 13% sequence identity with AcP. This suggests that transition states are conserved between proteins with the same native fold. Comparison of the rates of folding of AcP and four other proteins with the same topology, including ADA2h, supports the concept that the average distance in sequence between interacting residues (that is, the contact order) is an important determinant of the rate of protein folding.
Acid Anhydride Hydrolases↗
Acceleration of the folding of acylphosphatase by stabilization of local secondary structure.
The addition of trifluoroethanol or hexafluoroisopropanol converts the apparent two-state folding of acylphosphatase, a small alpha/beta protein, into a multistate mechanism where secondary structure accumulates significantly in the denatured state before folding to the native state. This results in a marked acceleration of folding as revealed by following the intrinsic fluorescence and circular dichroism changes upon folding. The folding rate is at a maximum when the secondary-structure content of the denatured state corresponds to that of the native state, while further stabilization of secondary structure decreases the folding rate. These findings indicate that stabilization of intermediate structure can either enhance or retard folding depending on its nature and content of native-like interactions.
Acid Anhydride Hydrolases↗
Expression and characterization of the intact N-terminal domain of streptokinase.
Proteolytic studies have enabled two of the three putative domains of the fibrinolytic protein streptokinase to be isolated and characterized (Conejero-Lara F et al., 1996, Protein Sci 5:2583-2591). The N-terminal domain, however, could not be isolated in these experiments because of its susceptibility to proteolytic cleavage. To complete the biophysical characterization of the domain structure of streptokinase we have overexpressed, purified, and characterized the N-terminal region of the protein, residues 1-146. The results show this is cooperatively folded with secondary structure content and overall stability closely similar to those of the equivalent region in the intact protein.
Calorimetry, Differential Scanning↗
Formation of amyloid fibrils by peptides derived from the bacterial cold shock protein CspB.
Three peptides covering the sequence regions corresponding to the first two (CspB-1), the first three (CspB-2), and the last two (CspB-3) beta-strands of CspB, the major cold shock protein of Bacillus subtilis, have been synthesized and analyzed for their conformations in solution and for their precipitation behavior. The peptides are nearly insoluble in water, but highly soluble in aqueous solutions containing 50% acetonitrile (pH 4.0). Upon shifts of the solvent condition toward lower or higher acetonitrile concentrations, the peptides all form fibrils resembling those observed in amyloid associated diseases. These fibrils have been identified and characterized by electron microscopy, binding of the dye congo red, and X-ray fiber diffraction. Characterization of the peptides in solution by circular dichroism and NMR spectroscopy shows that the formation of these fibrils does not require specific preformed secondary structure in the solution state species. While the majority of the soluble fraction of each peptide is monomeric and unstructured, different types of structures including alpha-helical, beta-sheet, and random coil conformations are observed under conditions that eventually lead to fibril formation. We conclude that the absence of tertiary contacts under solution conditions where binding interactions between peptide units are still favorable is a crucial requirement for amyloid formation. Thus, fragmentation of a sequence, like partial chemical denaturation or mutation, can enhance the capacity of specific protein sequences to form such fibrils.
Amino Acid Sequence↗
Selective association of protein molecules followed by mass spectrometry.
Nanoflow electrospray mass spectrometry was used to monitor the formation of protein heterodimers of HU proteins from Bacillus stearothermophilus and Bacillus subtilis. This has enabled us to analyze both thermodynamic and kinetic features associated with the dissociation of homodimeric HU proteins. The results obtained correlate well with the kinetics of the protein dissociation process and the free energy difference between homo- and heterodimeric species anticipated from other studies. We suggest that this approach will have general applicability in studying protein association and dissociation under near-equilibrium conditions and will be relevant to a wide range of biological systems.
Bacillus subtilis↗
Structural and dynamical characterization of a biologically active unfolded fibronectin-binding protein from Staphylococcus aureus.
A 130-residue fragment (D1-D4) taken from a fibronectin-binding protein of Staphylococcus aureus, which contains four fibronectin-binding repeats and is unfolded but biologically active at neutral pH, has been studied extensively by NMR spectroscopy. Using heteronuclear multidimensional techniques, the conformational properties of D1-D4 have been defined at both a global and a local level. Diffusion studies give an average effective radius of 26.2 +/- 0.1 A, approximately 75% larger than that expected for a globular protein of this size. Analysis of chemical shift, 3JHNalpha coupling constant, and NOE data show that the experimental parameters agree well overall with values measured in short model peptides and with predictions from a statistical model for a random coil. Sequences where specific features give deviations from these predictions for a random coil have however been identified. These arise from clustering of hydrophobic side chains and electrostatic interactions between charged groups. 15N relaxation studies demonstrate that local fluctuations of the chain are the dominant motional process that gives rise to relaxation of the 15N nuclei, with a persistence length of approximately 7-10 residues for the segmental motion. The consequences of the structural and dynamical properties of this unfolded protein for its biological role of binding to fibronectin have been considered. It is found that the regions of the sequence involved in binding have a high propensity for populating extended conformations, a feature that would allow a number of both charged and hydrophobic groups to be presented to fibronectin for highly specific binding.
Adhesins, Bacterial↗
Slow folding of muscle acylphosphatase in the absence of intermediates.
The folding of a 98 residue protein, muscle acylphosphatase (AcP), has been studied using a variety of techniques including circular dichroism, fluorescence and NMR spectroscopy following transfer of chemically denatured protein into refolding conditions. A low-amplitude phase, detected in concurrence with the main kinetic phase, corresponds to the folding of a minor population (13%) of molecules with one or both proline residues in a cis conformation, as shown from the sensitivity of its rate to peptidyl prolyl isomerase. The major phase of folding has the same kinetic characteristics regardless of the technique employed to monitor it. The plots of the natural logarithms of folding and unfolding rate constants versus urea concentration are linear over a broad range of urea concentrations. Moreover, the initial state formed rapidly after the initiation of refolding is highly unstructured, having a similar circular dichroism, intrinsic fluorescence and NMR spectrum as the protein denatured at high concentrations of urea. All these results indicate that AcP folds in a two-state manner without the accumulation of intermediates. Despite this, the folding of the protein is extremely slow. The rate constant of the major phase of folding in water, kfH2O, is 0.23 s-1 at 28 degreesC and, at urea concentrations above 1 M, the folding process is slower than the cis-trans proline isomerisation step. The slow refolding of this protein is therefore not the consequence of populated intermediates that can act as kinetic traps, but arises from a large intrinsic barrier in the folding reaction.
Acid Anhydride Hydrolases↗
Structural characterization of the transition state for folding of muscle acylphosphatase.
The transition state for folding of a small protein, muscle acylphosphatase, has been studied by measuring the rates of folding and unfolding under a variety of solvent conditions. A strong dependence of the folding rate on the concentration of urea suggests the occurrence in the transition state of a large shielding of those groups that are exposed to interaction with the denaturant in the unfolded state (mainly hydrophobic moieties and groups located on the polypeptide backbone). The heat capacity change upon moving from the unfolded state to the transition state is small and is indicative of a substantial solvent exposure of hydrophobic groups. The solvent-accessibility of such groups in the transition state has also been found to be significant by measuring the rates of folding and unfolding in the presence of sugars. These rates have also been found to be accelerated by the addition of small quantities of alcohols. Trifluoroethanol and hexafluoroisopropanol were particularly effective, suggesting that stabilisation of local hydrogen bonds lowers the energy of the transition state relative to the folded and unfolded states. Finally, a study with a competitive inhibitor of acylphosphatase has provided evidence for the complete loss of ligand binding affinity in the transition state, indicating that specific long-range interactions at the level of the active site are not yet formed at this stage of the folding reaction. A model of the transition state for acylphosphatase folding, in which beta-turns and one or both alpha-helices are formed to a significant extent but in which the persistent long-range interactions characteristic of the folded state are largely absent, accounts for all our data. These results are broadly consistent with models of the transition states for folding of other small proteins derived from mutagenesis studies, and suggest that solvent perturbation methods can provide complementary information about the transition region of the energy surfaces for protein folding.
Acid Anhydride Hydrolases↗
Protein folding and misfolding inside and outside the cell.
The workshop was held at St Catherine's College, Oxford, from March 25-28, 1998, and attracted participants from 32 nations. Protein folding is one of the most important processes in biology since it adds functional flesh to the bare bones of genes, but it has traditionally been studied by people separated both intellectually and physically because they are training in different disciplines. The aim of the meeting was to bring together chemists and structural biologists studying how pure, denatured proteins refold spontaneously in the test tube, with biochemists and cell biologists who are concerned with how proteins fold inside living cells and medical scientists interested in the diseases that result when this process goes wrong. In this report we concentrate on general concepts and themes rather than on detailing every contribution.
Amyloid↗
Protein subunit interactions and structural integrity of amyloidogenic transthyretins: evidence from electrospray mass spectrometry.
Wild-type and variant transthyretins form amyloid fibrils in two different diseases. The biologically active form of transthyretin is a tetramer but there is evidence that a monomeric species is the amyloidogenic intermediate. Using mass spectrometry we have developed an approach to monitor the proportions of monomer and tetramer in wild-type and variant transthyretins, and found a strong correlation between the instability of the tetramer in the gas phase and the amyloidogenicity of the protein variant. The presence of water molecules in the central channel has been found to be critical for maintaining intact the complex in the gas phase, with additional stability observed in the presence of excess thyroxine. The solution structure of monomeric transthyretin under fibril-forming conditions was studied using hydrogen exchange monitored by mass spectrometry. The results show that Val30Met transthyretin, the commonest amyloidogenic variant, exhibits loss of hydrogen exchange protection substantially more rapidly than the wild-type protein, suggesting partial unfolding of the beta-sheet structure. These results provide new insights into the correlation between tetramer stability and amyloidogenicity as well as supporting a possible route to fibril formation via transient unfolding of the transthyretin monomer.
Amyloid↗
Molecular dynamics simulations of peptide fragments from hen lysozyme: insight into non-native protein conformations.
Molecular dynamics simulations of four peptides taken from the hen lysozyme sequence have been used to generate models for non-native protein conformations. Comparisons between the different peptides and with experimental data for denatured lysozyme and peptide fragments provides insight into the characteristics of the conformational ensembles populated in these non-native states and the dependence of their structural features on the amino acid sequence. For the denatured conformers populated local contacts dominate in determining the properties observed in the trajectories, all four peptides showing similar characteristics. These include a significant increase in the number of main-chain O(i)-NH(i+2) hydrogen bonds and hydrogen bonds involving side-chain groups, this increase compensating to a large extent for the loss of hydrogen bonds involved in helical or beta-sheet secondary structure in the native fold, and the generation of a population of collapsed states with local clusterings of hydrophobic groups. The hydrophobic clusters enable at least partial burial of many side-chains exposed by the loss of tertiary contacts on denaturation and provide models that may explain the experimentally observed protection of amides from hydrogen exchange and the existence of residual secondary structure in non-native species of lysozyme. The results suggest that this approach has an important role to play in aiding the interpretation of experimental data for conformationally disordered non-native states of proteins.
Animals↗
Mass spectrometry of ribosomes and ribosomal subunits.
Nanoflow electrospray ionization has been used to introduce intact Escherichia coli ribosomes into the ion source of a mass spectrometer. Mass spectra of remarkable quality result from a partial, but selective, dissociation of the particles within the mass spectrometer. Peaks in the spectra have been assigned to individual ribosomal proteins and to noncovalent complexes of up to five component proteins. The pattern of dissociation correlates strongly with predicted features of ribosomal protein-protein and protein-RNA interactions. The spectra allow the dynamics and state of folding of specific proteins to be investigated in the context of the intact ribosome. This study demonstrates a potentially general strategy to probe interactions within complex biological assemblies.
Escherichia coli↗
Characterization of collapsed states in the early stages of the refolding of hen lysozyme.
Early conformational states in the refolding of hen lysozyme from guanidine hydrochloride have been characterized by measuring both the fluorescence and the solvent exchange properties of tryptophan side chains. The indole proton occupancies indicate that at pH 5.5, 25 degrees C, half the protection against pulse labeling occurs in the dead time (4 ms) of the experiment, with the remaining protection developing with a time constant of 55 ms. Comparison of these data with the protection kinetics of backbone amides and with the fluorescence data provides evidence for hydrophobic collapse involving incorporation of tryptophan residues in a solvent-excluded state in advance of stable secondary structure formation. Analysis of the pH dependence of the indole hydrogen exchange protection is consistent with two or more structurally distinct collapsed states, and indicates that the generation of a correctly folded compact hydrophobic core is a key precursor to the formation of persistent native-like structure during refolding.
Animals↗
The origin of the alpha-domain intermediate in the folding of hen lysozyme.
Stopped-flow fluorescence and circular dichroism spectroscopy have been used in conjunction with quenched-flow hydrogen exchange labelling, monitored by electrospray ionization mass spectrometry, to compare the refolding kinetics of hen egg-white lysozyme at 20 degrees C and 50 degrees C. At 50 degrees C there is clear evidence for distinct fast and slow refolding populations, as observed at 20 degrees C, although folding occurs significantly more rapidly. The folding process is, however, substantially more cooperative at the higher temperature. In particular, the transient intermediate on the major refolding pathway at 20 degrees C, having persistent native-like structure in the alpha-helical domain of the protein, is not detected by hydrogen exchange labelling at 50 degrees C. In addition, the characteristic maximum in negative ellipticity and the minimum in fluorescence intensity observed in far UV CD and intrinsic fluorescence experiments at 20 degrees C, respectively, are not seen at 50 degrees C. Addition of 2 M NaCl to the refolding buffer at 50 degrees C, however, regenerates both the hydrogen exchange and optical properties associated with the alpha-domain intermediate but has no significant effect on the overall refolding kinetics. Together with previous findings, these results indicate that non-native interactions within the alpha-domain intermediate are directly responsible for the unusual optical properties observed during refolding, and that this intermediate accumulates as a consequence of its intrinsic stability in a folding process where the formation of stable structure in the beta-domain constitutes the rate-limiting step for the majority of molecules.
Animals↗
Amyloid fibril formation by an SH3 domain.
The SH3 domain is a well characterized small protein module with a simple fold found in many proteins. At acid pH, the SH3 domain (PI3-SH3) of the p85alpha subunit of bovine phosphatidylinositol 3-kinase slowly forms a gel that consists of typical amyloid fibrils as assessed by electron microscopy, a Congo red binding assay, and x-ray fiber diffraction. The soluble form of PI3-SH3 at acid pH (the A state by a variety of techniques) from which fibrils are generated has been characterized. Circular dichroism in the far- and near-UV regions and 1H NMR indicate that the A state is substantially unfolded relative to the native protein at neutral pH. NMR diffusion measurements indicate, however, that the effective hydrodynamic radius of the A state is only 23% higher than that of the native protein and is 20% lower than that of the protein denatured in 3.5 M guanidinium chloride. In addition, the A state binds the hydrophobic dye 1-anilinonaphthalene-8-sulfonic acid, which suggests that SH3 in this state has a partially formed hydrophobic core. These results indicate that the A state is partially folded and support the hypothesis that partially folded states formed in solution are precursors of amyloid deposition. Moreover, that this domain aggregates into amyloid fibrils suggests that the potential for amyloid deposition may be a common property of proteins, and not only of a few proteins associated with disease.
Amyloid↗
Comparison of the conformational state and in vitro refolding of yeast chaperonin protein cpn10 with bacterial GroES.
Gel filtration studies demonstrate that the heptameric complex of yeast cpn10 (pI around 8.8) reversibly disassembles into monomers when lowering the pH to 4.5, whereas its secondary structure is retained as demonstrated by circular dichroism. Monomeric yeast cpn10 does not bind to GroEL in the presence of nucleotides, whereas under identical conditions E. coli cpn10 (GroES), having a strong sequence homology to the yeast form but a pI of 5.2, shows no pH-dependent dissociation and is able to complex with GroEL at both pH 7.5 and 4.5. Using circular dichroism it is shown that, unlike E. coli cpn10, yeast cpn10 is not able to refold spontaneously after first being fully unfolded in 8 M urea. However, refolding of yeast cpn10 to a complex that can be recognised by GroEL depends on the presence of a lipid-water interface with a specificity for negatively charged lipids. We suggest that the requirements for refolding of yeast cpn10 are related to its post-translational transport and subcellular localization.
Bacterial Proteins↗