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Alan R Fersht

Publications and source records attributed to Alan R Fersht.

At least 37 records · Page 2Linked to original sources
PubMed2005

The structure of the major transition state for folding of an FF domain from experiment and simulation.

We have analysed the transition state of folding of the four-helix FF domain from HYPA/FBP11 by high-resolution experiment and simulation as part of a continuing effort to understand the principles of folding and the refinement of predictive methods. The major transition state for folding was subjected to a Phi-value analysis utilising 50 mutants. The transition state contained a nucleus for folding centred around the end of helix 1 (H1) and the beginning of helix 2 (H2). Secondary structure in this region was fully formed (PhiF=0.9-1) and tertiary interactions were well developed. Interactions in the distal part of the native structure were weak (PhiF=0-0.2). The hydrophobic core and other parts of the protein displayed intermediate Phi-values, suggesting that interactions coalesce as the end of H1 and beginning of H2 are in the process of being formed. The distribution of Phi-values resembled that of barnase, which folds via an intermediate, rather than that of CI2 which folds by a concerted nucleation-condensation mechanism. The overall picture of the transition state structure identified in molecular dynamics simulations is in qualitative agreement, with the turn connecting H1 and H2 being formed, a loosened core, and H4 partially unfolded and detached from the core. There are some differences in the details and interpretation of specific Phi-values.

Carrier Proteins↗
PubMed2005

Comparative binding of p53 to its promoter and DNA recognition elements.

Tumor suppressor p53 is a transcription factor that transactivates a wide range of genes, including those in DNA repair, cell cycle arrest, apoptosis and its own degradation. To estimate the role of selectivity in binding to its promoters, we measured the binding affinities of a tetrameric p53 construct (p53CT) in vitro with 20 of its recognition elements from a variety of representative genes. The binding of full length p53 to four representative sequences exactly paralleled the affinities to p53CT. The binding of p53 to different recognition elements was co-operative and the affinities varied by up to 50-fold. p53 bound with high affinity to the recognition elements of all the genes involved in cell cycle arrest and some of the genes in apoptosis. All of the lower affinity-binding sites were in genes involved in apoptosis. Our quantitative-binding data were in agreement with published cell-based assays. The regulation of p53 activity is in part determined through the specificity of its DNA-binding interactions.

Apoptosis↗
PubMed2005

Simulation and experiment at high temperatures: ultrafast folding of a thermophilic protein by nucleation-condensation.

We used Phi-value analysis to characterise the transition state for folding of a thermophilic protein at the relatively high temperature of 325 K. PhiF values for the folding of the three-helix bundle, peripheral subunit binding domain from Bacillus stearothermophilus (E3BD) were determined by temperature-jump experiments in the absence of chemical denaturants. E3BD folded in microseconds through a highly diffuse transition state. Excellent agreement was observed between experiment and the results from eight (independent) molecular dynamics simulations of unfolding at 373 K. We used a combination of heteronuclear NMR experiments and molecular dynamics simulations to characterise the denatured ensemble, and found that it contained very little persistent, residual structure. However, those regions that adopt helical structure in the native state were found by simulation to be poised for helix formation in the denatured state. These regions also had significant structure in the transition state for folding. The overall folding pathway appears to be nucleation-condensation.

Bacterial Proteins↗
PubMed2005

Proteins of the S100 family regulate the oligomerization of p53 tumor suppressor.

S100B protein is elevated in the brains of patients with early stages of Alzheimer's disease and Down's syndrome. S100A4 is correlated with the development of metastasis. Both proteins bind to p53 tumor suppressor. We found that both S100B and S100A4 bind to the tetramerization domain of p53 (residues 325-355) only when exposed in lower oligomerization states and so they disrupt the tetramerization of p53. In addition, S100B binds to the negative regulatory and nuclear localization domains, which results in a very tight binding to p53 protein sequences that exposed the tetramerization domain in their C terminus. Because the trafficking of p53 depends on its oligomerization state, we suggest that S100B and S100A4 could regulate the subcellular localization of p53. But, the differences in the way these proteins bind to p53 could result in S100B and S1004 having different effects on p53 function in cell-cycle control.

Amino Acid Sequence↗
PubMed2005

Structures of p53 cancer mutants and mechanism of rescue by second-site suppressor mutations.

We have solved the crystal structures of three oncogenic mutants of the core domain of the human tumor suppressor p53. The mutations were introduced into a stabilized variant. The cancer hot spot mutation R273H simply removes an arginine involved in DNA binding without causing structural distortions in neighboring residues. In contrast, the "structural" oncogenic mutations H168R and R249S induce substantial structural perturbation around the mutation site in the L2 and L3 loops, respectively. H168R is a specific intragenic suppressor mutation for R249S. When both cancer mutations are combined in the same molecule, Arg(168) mimics the role of Arg(249) in wild type, and the wild type conformation is largely restored in both loops. Our structural and biophysical data provide compelling evidence for the mechanism of rescue of mutant p53 by intragenic suppressor mutations and reveal features by which proteins can adapt to deleterious mutations.

Arginine↗
PubMed2005

Binding of natively unfolded HIF-1alpha ODD domain to p53.

Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor that plays a crucial role in mediating oxygen response in the cell. Using biophysical techniques, we characterized two fragments of the HIF-1alpha subunit, one the full-length ODD domain (residues 403-603) and the second comprising the N-TAD (N-transactivation domain) and inhibitory domain (residues 530-698). Both were unstructured in solution under physiological conditions and so belong to the family of natively unfolded proteins. The HIF-1alpha ODD domain binds weakly to the isolated p53 core domain but tightly to full-length p53 to give a complex of one HIF-1alpha ODD domain with a p53 dimer. By being unstructured, the HIF-1alpha ODD domain can thread both its binding sites through the p53 multimer and bind tightly by the "chelate effect." These results support the idea that hypoxic p53-mediated apoptosis does involve the direct binding of HIF-1alpha to p53.

Apoptosis↗
PubMed2005

PFD: a database for the investigation of protein folding kinetics and stability.

We have developed a new database that collects all protein folding data into a single, easily accessible public resource. The Protein Folding Database (PFD) contains annotated structural, methodological, kinetic and thermodynamic data for more than 50 proteins, from 39 families. A user-friendly web interface has been developed that allows powerful searching, browsing and information retrieval, whilst providing links to other protein databases. The database structure allows visualization of folding data in a useful and novel way, with a long-term aim of facilitating data mining and bioinformatics approaches. PFD can be accessed freely at http://pfd.med.monash.edu.au.

Databases, Protein↗
PubMed2005

Modulation of binding of DNA to the C-terminal domain of p53 by acetylation.

The binding of nonspecific DNA to the C-terminal negative regulatory domain (CTD) of p53 modulates its activity. The CTD is a natively unfolded region, which is subject to acetylation and phosphorylation at several residues as part of control. To measure the effect of covalent modification on binding to DNA, we synthesized a series of fluorescein-labeled CTD peptides with single and multiple acetylations at lysine residues that we had identified by NMR as making contact with DNA, and developed an analytical ultracentrifugation method to study their binding to DNA. Binding depended on ionic strength, indicating an electrostatic contribution. Monoacetylation weakened DNA binding at physiological ionic strength 2- to 3-fold, diacetylations resulted in further 2- to 3-fold decrease in the affinity, and tri- and tetraacetylations rendered DNA binding undetectable. Phosphorylation at S392 did not affect DNA binding. NMR spectroscopy showed binding to DNA did not induce significant structure into CTD, apart possibly from local helix formation.

Acetylation↗
PubMed2005

Human full-length Securin is a natively unfolded protein.

Human Securin, also called PTTG1 (pituitary tumor transforming gene 1 product), is an estrogen-regulated proto-oncogene with multifunctional properties. We characterized human full-length Securin using a variety of biophysical techniques, such as nuclear magnetic resonance, circular dichroism, and size-exclusion chromatography. Under physiological conditions, Securin is devoid of tertiary and secondary structure except for a small amount of poly-(L-proline) type II helix and its hydrodynamic characteristics suggest it behaves as an extended polypeptide. These results suggest that Securin is unstructured in solution and so belongs to the family of natively unfolded proteins. In addition, to gain structural and quantitative insight, we investigated the binding of Securin to p53. Analytical ultracentrifugation and fluorescence anisotropy studies revealed no evidence of any direct interaction between unmodified recombinant Securin and p53 in vitro.

Humans↗
PubMed2004

Effects of heme on the structure of the denatured state and folding kinetics of cytochrome b562.

Heme-linked proteins, such as cytochromes, are popular subjects for protein folding studies. There is the underlying question of whether the heme affects the structure of the denatured state by cross-linking it and forming other interactions, which would perturb the folding pathway. We have studied wild-type and mutant cytochrome b562 from Escherichia coli, a 106 residue four-alpha-helical bundle. The holo protein apparently refolds with a half-life of 4 micros in its ferrous state. We have analysed the folding of the apo protein using continuous-flow fluorescence as well as stopped-flow fluorescence and CD. The apo protein folded much more slowly with a half-life of 270 micros that was unaffected by the presence of exogenous heme. We examined the nature of the denatured states of both holo and apo proteins by NMR methods over a range of concentrations of guanidine hydrochloride. The starting point for folding of the holo protein in concentrations of denaturant around the denaturation transition was a highly ordered native-like species with heme bound. Fully denatured holo protein at higher concentrations of denaturant consisted of denatured apo protein and free heme. Our results suggest that the very fast folding species of denatured holo protein is in a compact state, whereas the normal folding pathway from fully denatured holo protein consists of the slower folding of the apo protein followed by the binding of heme. These data should be considered in the analysis of folding of heme proteins.

Amino Acid Sequence↗
PubMed2004

Binding of Rad51 and other peptide sequences to a promiscuous, highly electrostatic binding site in p53.

Homologous recombination is repressed by the binding of p53 to Rad51. We identified by fluorescence and NMR spectroscopy that peptides corresponding to residues 179-190 of Rad51 bind to the core domain of p53 in a site that overlaps with its specific DNA binding site. The p53 site is quite promiscuous, since it also binds peptides derived from 53BP1, 53BP2, Hif-1alpha, and BCL-X(L) in overlapping regions. Binding is mediated mainly by a strong, nonspecific, electrostatic component and is fine tuned by specific interactions. Competition of the different proteins with each other and with specific DNA for a single site in p53 could be a factor in regulation of its activity.

Anisotropy↗
PubMed2004

One-state downhill versus conventional protein folding.

Classical protein folding invokes a cooperative transition between distinct thermodynamic states that are individually populated at equilibrium and separated by an energy barrier. It has been proposed, however, that the small protein, BBL, undergoes one-step downhill folding whereby it folds non-cooperatively to its native state without encountering an appreciable energy barrier. Only a single conformational ensemble is populated under given conditions, and so the denatured state ensemble progressively changes into the native structure. A wide dispersion of thermal denaturation midpoints that was observed for an extrinsically labelled fragment of BBL is proposed to be evidence for its one-state, downhill folding, a phenomenon that is also suggested to be functionally important for BBL and its homologues. We found, however, that thermal denaturation of unlabelled wild-type BBL was highly cooperative, with very similar transition midpoints for the melting of secondary and tertiary interactions, as well as for individual residues when monitored by NMR. Similar results were also observed for two other homologues, E3BD and POB. Further, the extrinsic fluorophores perturbed the unfolding energetics of labelled BBL, and complicated its equilibrium behaviour. One-step downhill folding may well occur for some proteins that do not have distinct folded states but not for BBL and its well-folded homologues.

Amino Acid Sequence↗
PubMed2004

Relationship of Leffler (Bronsted) alpha values and protein folding Phi values to position of transition-state structures on reaction coordinates.

The positions of transition states along reaction coordinates (r++) for simple chemical reactions are often estimated from Leffler alpha values, the slope of plots of DeltaG++ (activation energy) versus DeltaG(0) (equilibrium free energy) for a series of structural variants. Protein folding is more complex than simple chemical reactions and has a multitude of reaction coordinates. Phi-Value analysis measures degree of structure formation at individual residues in folding transition states from the ratio DeltaDeltaG++/DeltaDeltaG(0) for mutations. alpha values are now being used to analyze protein folding by lumping series of Phi values into single plots. But, there are discrepancies in the values of alpha for folding with more classical measures of the extent of structure formation, which I rationalize here. I show for chemical reactions with just a single reaction coordinate that alpha = r++ only for limiting cases, such as for reactants and products being in parabolic energy wells of identical curvature. Otherwise, alpha can differ radically from r++, with alpha being determined just by the angles of intersection of reactant and product energy surfaces. Phi is an index of the progress of a local, energy-based reaction coordinate at the global transition state: Phi <0.5 corresponds to <50% progress of the local coordinate at the global transition state and Phi >0.5 means >50%. Protein Leffler plots can force different local indexes to a single fit and give skewed underestimates of the extent of global structure formation in transition states that differ from other measures of structure formation.

Animals↗
PubMed2004

Regulation of DNA binding of p53 by its C-terminal domain.

The tumor suppressor p53 is a tetrameric multi-domain transcription factor. Its C-terminal domain is thought to regulate the binding of its core domain to specific recognition sequences in promoters. The mechanism of regulation by the C-terminal domain and the role of its post-translational modification are controversial. We have examined the binding of DNA in solution to a series of unmodified p53 constructs that lack various domains. The specific DNA sequences bind tightly to the core domain, irrespective of whether or not the C-terminal domain is part of the construct. Unmodified p53 is accordingly an active DNA binding protein. Non-specific DNA sequences do not inhibit directly the binding of the specific sequences to the core but bind to the C terminus and inhibit p53 via that binding mode. Using NMR, we identified the residues of the C terminus that interact with the non-specific DNA. They include residues that are known to be modified post-translationally. Our data provide direct support for the regulatory role of the C terminus in the activity of p53 and show that p53 containing the unmodified C terminus actively binds to short double-stranded DNA.

Base Sequence↗
PubMed2004

Cooperative binding of tetrameric p53 to DNA.

We analysed by analytical ultracentrifugation and fluorescence anisotropy the binding of p53 truncation mutants to sequence-specific DNA. The synthetic 30 base-pair DNA oligomers contained the 20 base-pair recognition elements for p53, consisting of four sites of five base-pairs per p53 monomer. We found that the binding at low ionic strengths was obscured by artifacts of non-specific binding and so made measurements at higher ionic strengths. Analytical ultracentrifugation of the construct p53CT (residues 94-360, containing the DNA-binding core and tetramerization domains) gave a dissociation constant of approximately 3 microM for its dimer-tetramer equilibrium, similar to that of full-length protein. Analytical ultracentrifugation and fluorescence anisotropy showed that p53CT formed a complex with the DNA constructs with 2:1 stoichiometry (dimer:DNA). The binding of p53CT (1-100 nm range) to DNA was highly cooperative, with a Hill coefficient of 1.8 (dimer:DNA). The dimeric L344A mutant of p53CT has impaired tetramerization. It bound to full-length DNA p53 recognition sequence, but with sixfold less affinity than wild-type protein. It did not form a detectable complex with a 30-mer DNA construct containing two specific five base-pair sites and two random sites, emphasizing the high co-operativity of the binding. The fundamental active unit of p53 appears to be the tetramer, which is induced by DNA binding, although it is a dimer at low concentrations.

Base Sequence↗
PubMed2004

Phi-value analysis and the nature of protein-folding transition states.

Phi values are used to map structures of protein-folding transition states from changes in free energies of denaturation (DeltaDeltaG(D-N)) and activation on mutation. A recent reappraisal proposed that Phi values for DeltaDeltaG(D-N) < 1.7 kcal/mol are artifactual. On discarding such derived Phi values from published studies, the authors concluded that there are no high Phi values in diffuse transition states, which are consequently uniformly diffuse with no evidence for nucleation. However, values of DeltaDeltaG(D-N) > 1.7 kcal/mol are often found for large side chains that make dispersed tertiary interactions, especially in hydrophobic cores that are in the process of being formed in the transition state. Conversely, specific local interactions that probe secondary structure tend to have DeltaDeltaG(D-N) approximately 0.5-2 kcal/mol. Discarding Phi values from lower-energy changes discards the crucial information about local interactions and makes transition states appear uniformly diffuse by overemphasizing the dispersed tertiary interactions. The evidence for the 1.7 kcal/mol cutoff was based on mutations that had been deliberately designed to be unsuitable for Phi-value analysis because they are structurally disruptive. We confirm that reliable Phi values can be derived from the recommended mutations in suitable proteins with 0.6 < DeltaDeltaG(D-N) < 1.7 kcal/mol, and there are many reliable high Phi values. Transition states vary from being rather diffuse to being well formed with islands of near-complete secondary structure. We also confirm that the structures of transition-state ensembles can be perturbed by mutations with DeltaDeltaG(D-N) >> 2 kcal/mol and that protein-folding transition states do move on the energy surface on mutation.

Alanine↗
PubMed2004

Demonstration of a low-energy on-pathway intermediate in a fast-folding protein by kinetics, protein engineering, and simulation.

It is controversial whether fast-folding proteins can form productive on-pathway intermediates that are more stable than the denatured state because noncovalent intermediates are usually evanescent. Here, we apply the classical criteria for the existence of intermediates: namely, the intermediates form and react rapidly enough to be on pathway and they can be isolated and characterized. The folding of the 71-residue, mainly alpha-helical FF domain from human HYPA/FBP11 fulfills these classical criteria, as was found for Im7. The FF domain folds in two phases, one on the micros and the other on the ms time scale. An engineered mutant folds only to a partly folded state, with some 20-40% of the native helical content. The kinetic properties of the mutant are identical to those found for the fast phase of the wild-type protein, and it is likely that the mutant folds just to the intermediate state. A full kinetic analysis of the folding of wild-type protein, using the amplitudes of its native and denatured states and the observed values for the mutant, rules out an off-pathway scheme but fits an on-pathway scheme, with a low energy intermediate that is modeled by the mutant. The experimental proof benchmarks a molecular dynamics method that identifies an obligatory intermediate observed in multiple simulations. The conformational space defining this intermediate is visited several times in the simulations, leading to high populations consistent with the presence of a low energy intermediate.

Kinetics↗