Biomedical subjects

A R Fersht

Publications and source records attributed to A R Fersht.

At least 37 records · Page 2Linked to original sources

PubMed2000

Bacterial and yeast chaperones reduce both aggregate formation and cell death in mammalian cell models of Huntington's disease.

Huntington's disease (HD) is an autosomal dominant neurodegenerative condition caused by expansions of more than 35 uninterrupted CAG repeats in exon 1 of the huntingtin gene. The CAG repeats in HD and the other seven known diseases caused by CAG codon expansions are translated into long polyglutamine tracts that confer a deleterious gain of function on the mutant proteins. Intraneuronal inclusions comprising aggregates of the relevant mutant proteins are found in the brains of patients with HD and related diseases. It is crucial to determine whether the formation of inclusions is directly pathogenic, because a number of studies have suggested that aggregates may be epiphenomena or even protective. Here, we show that fragments of the bacterial chaperone GroEL and the full-length yeast heat shock protein Hsp104 reduce both aggregate formation and cell death in mammalian cell models of HD, consistent with a causal link between aggregation and pathology.

Amino Acid Substitution↗

PubMed2000

Protein folding transition states: elicitation of Hammond effects by 2,2,2-trifluoroethanol.

Adaptation of the techniques of classical physical-organic chemistry to the study of protein folding has led to our current detailed understanding of the transition states. Here, we have applied a series of structure--activity relationships to analyse the effects on protein folding transition states of 2,2,2-trifluoroethanol (TFE), a reagent that is usually assumed to act by stabilising secondary structure. The folding and unfolding of the highly alpha-helical tetramerisation domain of p53 provides a useful paradigm for analysing its effects on kinetics: The first step of its folding consists of an association reaction with little, if any, formation of secondary structure in the transition state; and the final step of the folding reaction involves just the formation of bonds at subunit interfaces, with the alpha-helical structure being completely formed. We have systematically measured the effects of TFE on two sets of structure--activity relationships. The first is for Phi values, which measure the degree of non-covalent bond formation at nearly every position in the transition state. The second is for relative effects of the denaturant, guanidinium chloride, on kinetics and equilibria, which measure the gross position of the transition state on the reaction co-ordinate. We find that TFE modulated the kinetics by a variety of effects other than that on secondary structure. In particular, there were Hammond effects, movement of the position of the transition state along the reaction co-ordinate, which either significantly speeded up or slowed down protein unfolding, depending on the particular mutant examined. The gross effects of TFE on protein folding kinetics are thus not a reliable guide to the structures of transition states.

Circular Dichroism↗

PubMed2000

Stabilization of GroEL minichaperones by core and surface mutations.

We report the crystal structures of two hexa-substituted mutants of a GroEL minichaperone that are more stable than wild-type by 7.0 and 6.1 kcal mol(-1). Their structures imply that the increased stability results from multiple factors including improved hydrophobic packing, optimised hydrogen bonding and favourable structural rearrangements. It is commonly believed that protein core residues are immutable and generally optimized for energy, while on the contrary, surface residues are variable and hence unimportant for stability. But, it is now becoming clear that mutations of both core and surface residues can increase protein stability, and that protein cores are more flexible and thus more tolerant to mutation than expected. Sequence comparison of homologous proteins has provided a way to pinpoint the residues that contribute constructively to stability and to guide the engineering of protein stability. Stabilizing mutations identified by this approach are most frequently located at protein surfaces but with a few found in protein cores. In the latter case, local flexibility in the hydrophobic core is the key factor that allows the energetically favourable burial of larger hydrophobic side-chains without undue energetic penalties from steric clashes.

Amino Acid Substitution↗

PubMed2000

Towards a complete description of the structural and dynamic properties of the denatured state of barnase and the role of residual structure in folding.

The detailed characterization of denatured proteins remains elusive due to their mobility and conformational heterogeneity. NMR studies are beginning to provide clues regarding residual structure in the denatured state but the resulting data are too sparse to be transformed into molecular models using conventional techniques. Molecular dynamics simulations can complement NMR by providing detailed structural information for components of the denatured ensemble. Here, we describe three independent 4 ns high-temperature molecular dynamics simulations of barnase in water. The simulated denatured state was conformationally heterogeneous with respect to the conformations populated both within a single simulation and between simulations. Nonetheless, there were some persistent interactions that occurred to varying degrees in all simulations and primarily involved the formation of fluid hydrophobic clusters with participating residues changing over time. The region of the beta(3-4) hairpin contained a particularly high degree of such side-chain interactions but it lacked beta-structure in two of the three denatured ensembles: beta(3-4) was the only portion of the beta-structure to contain significant residual structure in the denatured state. The two principal alpha-helices (alpha1 and alpha2) adopted dynamic helical structure. In addition, there were persistent contacts that pinched off core 2 from the body of the protein. The rest of the protein was unstructured, aside from transient and mostly local side-chain interactions. Overall, the simulated denatured state contains residual structure in the form of dynamic, fluctuating secondary structure in alpha1 and alpha2, as well as fluctuating tertiary contacts in the beta(3-4) region, and between alpha1 and beta(3-4), in agreement with previous NMR studies. Here, we also show that these regions containing residual structure display impaired mobility by both molecular dynamics and NMR relaxation experiments. The residual structure was important in decreasing the conformational states available to the chain and in repairing disrupted regions. For example, tertiary contacts between beta(3-4) and alpha1 assisted in the refolding of alpha1. This contact-assisted helix formation was confirmed in fragment simulations of beta(3-4) and alpha1 alone and complexed, and, as such, alpha1 and beta(3-4) appear to be folding initiation sites. The role of these sites in folding was investigated by working backwards and considering the simulation in reverse, noting that earlier time-points from the simulations provide models of the major intermediate and transition states in quantitative agreement with data from both unfolding and refolding experiments. Both beta(3-4) and alpha1 are dynamic in the denatured state but when they collide and make enough contacts, they provide a loose structural scaffold onto which further beta-strands pack. The beta-structure condenses about beta(3-4), while alpha1 aids in stabilizing beta(3-4) and maintaining its orientation. The resulting beta-structure is relatively planar and loose in the major intermediate. Further packing ensues, and as a result the beta-sheet twists, leading to the major transition state. The structure is still expanded and loops are not well formed at this point. Fine-tuning of the packing interactions and the final condensation of the structure then occurs to yield the native state.

Amino Acid Sequence↗

PubMed2000

Quantitative analysis of residual folding and DNA binding in mutant p53 core domain: definition of mutant states for rescue in cancer therapy.

The tumour suppressor p53 is mutated in half of all human cancers, most frequently with missense substitutions in its core domain. We present a new assessment of the mutation database based on quantitative folding and DNA-binding studies of the isolated core domain. Our data identify five distinct mutant classes that correlate with four well-defined regions of the core domain structure. On extrapolation to 37 degrees C the wild-type protein has a stability of 3.0 kcal/mol. This also emerges as an oncogenic threshold: all beta-sandwich mutants destabilized by this amount (50% denatured) are expected to promote cancer. Other weakly destabilizing mutations are restricted to loop 3 in the DNA-binding region. Drugs that stabilize mutant p53 folding have the potential to reactivate apoptotic signalling pathways in tumour cells either by transactivation-dependent or independent pathways. Using an affinity ligand as a proof of principle we have recovered the thermodynamic stability of the hotspot G245S. With reference states for the five mutant classes as a guide, future therapeutic strategies may similarly stabilize partially structured or binding states of mutant p53 that restore limited p53 pathways to tumour suppression.

Apoptosis↗

PubMed2000

Transition-state structure as a unifying basis in protein-folding mechanisms: contact order, chain topology, stability, and the extended nucleus mechanism.

I attempt to reconcile apparently conflicting factors and mechanisms that have been proposed to determine the rate constant for two-state folding of small proteins, on the basis of general features of the structures of transition states. Phi-Value analysis implies a transition state for folding that resembles an expanded and distorted native structure, which is built around an extended nucleus. The nucleus is composed predominantly of elements of partly or well-formed native secondary structure that are stabilized by local and long-range tertiary interactions. These long-range interactions give rise to connecting loops, frequently containing the native loops that are poorly structured. I derive an equation that relates differences in the contact order of a protein to changes in the length of linking loops, which, in turn, is directly related to the unfavorable free energy of the loops in the transition state. Kinetic data on loop extension mutants of CI2 and alpha-spectrin SH3 domain fit the equation qualitatively. The rate of folding depends primarily on the interactions that directly stabilize the nucleus, especially those in native-like secondary structure and those resulting from the entropy loss from the connecting loops, which vary with contact order. This partitioning of energy accounts for the success of some algorithms that predict folding rates, because they use these principles either explicitly or implicitly. The extended nucleus model thus unifies the observations of rate depending on both stability and topology.

Algorithms↗

PubMed2000

Directed evolution of new catalytic activity using the alpha/beta-barrel scaffold.

In biological systems, enzymes catalyse the efficient synthesis of complex molecules under benign conditions, but widespread industrial use of these biocatalysts depends crucially on the development of new enzymes with useful catalytic functions. The evolution of enzymes in biological systems often involves the acquisition of new catalytic or binding properties by an existing protein scaffold. Here we mimic this strategy using the most common fold in enzymes, the alpha/beta-barrel, as the scaffold. By combining an existing binding site for structural elements of phosphoribosylanthranilate with a catalytic template required for isomerase activity, we are able to evolve phosphoribosylanthranilate isomerase activity from the scaffold of indole-3-glycerol-phosphate synthase. We find that targeting the catalytic template for in vitro mutagenesis and recombination, followed by in vivo selection, results in a new phosphoribosylanthranilate isomerase that has catalytic properties similar to those of the natural enzyme, with an even higher specificity constant. Our demonstration of divergent evolution and the widespread occurrence of the alpha/beta-barrel suggest that this scaffold may be a fold of choice for the directed evolution of new biocatalysts.

Aldose-Ketose Isomerases↗

PubMed2000

Mechanism of rescue of common p53 cancer mutations by second-site suppressor mutations.

The core domain of p53 is extremely susceptible to mutations that lead to loss of function. We analysed the stability and DNA-binding activity of such mutants to understand the mechanism of second-site suppressor mutations. Double-mutant cycles show that N239Y and N268D act as 'global stability' suppressors by increasing the stability of the cancer mutants G245S and V143A-the free energy changes are additive. Conversely, the suppressor H168R is specific for the R249S mutation: despite destabilizing wild type, H168R has virtually no effect on the stability of R249S, but restores its binding affinity for the gadd45 promoter. NMR structural comparisons of R249S/H168R and R249S/T123A/H168R with wild type and R249S show that H168R reverts some of the structural changes induced by R249S. These results have implications for possible drug therapy to restore the function of tumorigenic mutants of p53: the function of mutants such as V143A and G245S is theoretically possible to restore by small molecules that simply bind to and hence stabilize the native structure, whereas R249S requires alteration of its mutant native structure.

DNA↗

PubMed2000

Stability and folding of the protein complexes of barnase.

Native-like complexes of proteins, formed by the association of two complementary fragments comprising the entire sequence of the protein, can be used to gain insight into the stability and folding of the intact protein. We have studied the structural, thermodynamic and kinetic properties of four barnase complexes, with the cleavage site at different positions of the amino-acid chain (CB36, at position 36; CB56, at position 56; CB68, at position 68; and CB79, at position 79). The four barnase complexes have native-like structure as shown by fluorescence, far-and near-UV CD, size-exclusion chromatography and NMR. The NMR characterization indicated that the structural changes were mainly located in regions close to the cleavage site. The main core of the protein was fully formed and the overall structure was similar to that of intact barnase. The thermal and chemical denaturation showed that all complexes were substantially destabilized. CB56 displayed two denaturation transitions, probably because of the presence of partially folded conformations around the cleavage site. The rate constant for the association/folding of fragments decreased with the decreasing length of the C-terminal fragment. Thus, the larger the fragment (and, consequently, the larger the amount of residual native-like structure), the faster the association. These findings are consistent with the proposed model of barnase folding.

Bacterial Proteins↗

PubMed2000

The effects of disulfide bonds on the denatured state of barnase.

The effects of engineered disulfide bonds on protein stability are poorly understood because they can influence the structure, dynamics, and energetics of both the native and denatured states. To explore the effects of two engineered disulfide bonds on the stability of barnase, we have conducted a combined molecular dynamics and NMR study of the denatured state of the two mutants. As expected, the disulfide bonds constrain the denatured state. However, specific extended beta-sheet structure can also be detected in one of the mutant proteins. This mutant is also more stable than would be predicted. Our study suggests a possible cause of the very high stability conferred by this disulfide bond: the wild-type denatured ensemble is stabilized by a nonnative hydrophobic cluster, which is constrained from occurring in the mutant due to the formation of secondary structure.

Bacterial Proteins↗

PubMed1999

Interdomain interactions within the gene 3 protein of filamentous phage.

Infection of Escherichia coli by filamentous phage fd is mediated by the phage gene 3 protein (g3p). The g3p consists of three domains (g3p-D1, D2 and D3) linked by flexible glycine-rich linkers. All three domains are indispensable for phage infectivity; the g3p-D1 domain binds to the TolA receptor presumably at the inner face of the outer membrane, the g3p-D2 domain to the F-pilus and the g3p-D3 domain anchors g3p to the phage coat. The N-terminal domains g3p-D1 and D2 interact with each other; this interaction is abrogated by binding of g3p-D2 to the F-pilus leading to the release of g3p-D1 to bind to TolA. Here, using phages with deletions in g3p, we have discovered a specific interaction between the two N-terminal domains and g3p-D3, the C-terminal domain of g3p. We propose that these interdomain interactions within g3p lead to a compact and stable organisation when displayed on the phage tip, but that during infection, this compact state must be unraveled.

Capsid Proteins↗

PubMed1999

Formation of short-lived protein aggregates directly from the coil in two-state folding.

Recent results on the 102 residue protein U1A show that protein aggregation is not always slow and irreversible but may take place transiently in refolding studies on a millisecond time scale. In this study we observe a similar aggregation behavior with the classical two-state protein CI2. Since both U1A and CI2 appear to fold directly from the coil at low protein concentrations, it is likely that the aggregates also form directly from the coil. This is in contrast to the behavior of larger multistate proteins where aggregation occurs in connection to "sticky" intermediates.

Chymotrypsin↗

PubMed1999

Equilibria and kinetics of folding of gelsolin domain 2 and mutants involved in familial amyloidosis-Finnish type.

Mutations D187N and D187Y in domain 2 of the actin-regulating protein gelsolin cause familial amyloidosis-Finnish type (FAF). We have constructed and expressed a recombinant version of gelsolin domain 2 that is sufficiently stable for kinetic and equilibrium measurements. The wild-type domain and the two amyloidogenic mutants fold via simple two-state kinetics without the accumulation of an intermediate. Unfolding kinetics exhibits significant curvature with increasing urea concentration, indicating that the transition state for unfolding becomes more native-like under increasingly denaturing conditions in accordance with the Hammond postulate. Mutations D187N and D187Y destabilize gelsolin domain 2 by 1.22 and 2.16 kcal. mol(-1) (1 kcal = 4.18 kJ) respectively. The mutations do not prevent disulfide bond formation despite their direct contiguity with a cysteine residue involved in disulfide linkage. The destabilization conferred on gelsolin domain 2 by the FAF mutations is sufficient to predict that an appreciable fraction is unfolded and, therefore, extremely susceptible to proteolysis at body temperature.

Amyloidosis↗

PubMed1999

GroEL recognises sequential and non-sequential linear structural motifs compatible with extended beta-strands and alpha-helices.

The chaperonin GroEL binds a variety of polypeptides that share no obvious sequence similarity. The precise structural, chemical and dynamic features that are recognised remain largely unknown. Structural models of the complex between GroEL and its co-chaperonin GroES, and of the isolated apical domain of GroEL (minichaperone; residues 191-376) with a 17 residue N-terminal tag show that a linear sequential sequence (extended beta-strand) can be bound. We have analysed characteristics of the motifs that bind to GroEL by using affinity panning of immobilised GroEL minichaperones for a library of bacteriophages that display the fungal cellulose-binding domain of the enzyme cellobiohydrolase I. This protein has seven non-sequential residues in its binding site that form a linear binding motif with similar dimensions and characteristics to the peptide tag that was bound to the minichaperone GroEL(191-376). The seven residues thus form a constrained scaffold. We find that GroEL does bind suitable mutants of these seven residues. The side-chains recognised do not have to be totally hydrophobic, but polar and positively charged chains can be accommodated. Further, the spatial distribution of the side-chains is also compatible with those in an alpha-helix. This implies that GroEL can bind a wide range of structures, from extended beta-strands and alpha-helices to folded states, with exposed side-chains. The binding site can accommodate substrates of approximately 18 residues when in a helical or seven when in an extended conformation. The data support two activities of GroEL: the ability to act as a temporary parking spot for sticky intermediates by binding many motifs; and an unfolding activity of GroEL by binding an extended sequential conformation of the substrate.

Bacteriophages↗

PubMed1999

Identification of substrate binding site of GroEL minichaperone in solution.

It is difficult to obtain high-resolution structural information on the substrate-binding site of intact GroEL. But minichaperones, domains containing the peptide-binding site of GroEL, do constitute tractable systems for detailed studies. A peptide-binding site was located in crystals of a minichaperone and proposed to constitute a model for substrate-binding. We have now located the substrate binding site of the minichaperone GroEL(193-335) in solution by labelling it at various positions with a fluorescent probe and detecting which positions are perturbed on binding a denatured substrate. The fluorescence of a probe attached to a cysteine residue engineered at position 228 (N terminus of helix H8), 241 (helix H8), 261 (helix H9), or 267 (helix H9) was affected significantly by binding of substrate. But there was little change for a label at positions 193, 212, 217 or 293. The dissociation constants between substrates and minichaperone were evaluated from fluorescence anisotropy assays. The effects of salt and temperature were the same as those with intact GroEL. These results indicate that the region around helices H8 and H9 is the substrate-binding site for the apical domain fragment. Intriguingly, the same site is involved in the binding of GroES. Thus, an important function of GroES in the regulation of the activity of GroEL for substrates is to displace the bound substrate by competing for its binding site.

Binding Sites↗

PubMed1999

NMR analysis of the binding of a rhodanese peptide to a minichaperone in solution.

A detailed structural analysis of interactions between denatured proteins and GroEL is essential for an understanding of its mechanism. Minichaperones constitute an excellent paradigm for obtaining high-resolution structural information about the binding site and conformation of substrates bound to GroEL, and are particularly suitable for NMR studies. Here, we used transferred nuclear Overhauser effects to study the interaction in solution between minichaperone GroEL(193-335) and a synthetic peptide (Rho), corresponding to the N-terminal alpha-helix (residues 11 to 23) of the mitochondrial rhodanese, a protein whose in vitro refolding is mediated by minichaperones. Using a 60 kDa maltose-binding protein (MBP)-GroEL(193-335) fusion protein to increase the sensitivity of the transferred NOEs, we observed characteristic sequential and mid-range transferred nuclear Overhauser effects. The peptide adopts an alpha-helical conformation upon binding to the minichaperone. Thus the binding site of GroEL is compatible with binding of alpha-helices as well as extended beta-strands. To locate the peptide-binding site on GroEL(193-335), we analysed changes in its chemical shifts on adding an excess of Rho peptide. All residues with significant chemical shift differences are localised in helices H8 and H9. Non-specific interactions were not observed. This indicates that the peptide Rho binds specifically to minichaperone GroEL(193-335). The binding region identified by NMR in solution agrees with crystallographic studies with small peptides and with fluorescence quenching studies with denatured proteins.

ATP-Binding Cassette Transporters↗

PubMed1999

Hot-spot mutants of p53 core domain evince characteristic local structural changes.

Most of the oncogenic mutations in the tumor suppressor p53 map to its DNA-binding (core) domain. It is thus a potential target in cancer therapy for rescue by drugs. To begin to understand how mutation inactivates p53 and hence to provide a structural basis for drug design, we have compared structures of wild-type and mutant p53 core domains in solution by NMR spectroscopy. Structural changes introduced by five hot-spot mutations (V143A, G245S, R248Q, R249S, and R273H) were monitored by chemical-shift changes. Only localized changes are observed for G245S, R248Q, R249S, and R273H, suggesting that the overall tertiary folds of these mutant proteins are similar to that of wild type. Structural changes in R273H are found mainly in the loop-sheet-helix motif and the loop L3 of the core domain. Mutations in L3 (G245S, R248Q, and R249S) introduce structural changes in the loop L2 and L3 as well as terminal residues of strands 4, 9, and 10. It is noteworthy that R248Q, which is often regarded as a contact mutant that affects only interactions with DNA, introduces structural changes as extensive as the other loop L3 mutations (G245S and R249S). These changes suggest that R248Q is also a structural mutant that perturbs the structure of loop L2-L3 regions of the p53 core domain. In contrast to other mutants, replacement of the core residue valine 143 to alanine causes chemical-shift changes in almost all residues in the beta-sandwich and the DNA-binding surface. Long-range effects of V143A mutation may affect the specificity of DNA binding.

Binding Sites↗

PubMed1999

The solution structure of oxidized Escherichia coli cytochrome b562.

The solution structure of the oxidized, paramagnetic form of cytochrome b562 from Escherichia coli (106 amino acids) is here reported as obtained from 1653 meaningful NOEs (from a total of 2051 unique NOEs), 33 (3)JHNHalpha values, and 339 pseudocontact shifts. The structure displays the typical four-helix bundle motif, and a disordered loop between helices alpha2 and alpha3, as found in the solid state. The solution structure has a conformation intermediate between the two independent solid-state molecules, although different orientations are observed for a few residues. The magnetic susceptibility tensor is similar to that of cytochrome c, which has the same ligands, although the anisotropy is somewhat smaller. This difference in the electronic structure is consistent with the thermal accessibility in cytochrome b562 of states with S > 1/2. The structure is also compared with the solution structure of the apoprotein, and some information on the role of the cofactor on the protein folding and mobility is obtained. Helix alpha4 seems to be the most sensitive to the chemical environment in terms of structure and mobility. The pKa values affecting the hyperfine-shifted signals are also discussed. Quite intriguing is the comparison of the structure of cytochrome b562 with the available structures of cytochromes c' which display a similar folding motif and similar pKa values but very little sequence similarity.

Amino Acid Sequence↗