Proceedings of the National Academy of Sciences, 2017
Significance Destabilized mutants of the tumor suppressor p53 are inactivated by self-aggregation... more Significance Destabilized mutants of the tumor suppressor p53 are inactivated by self-aggregation in a substantial number of tumors and may also coaggregate with and inactivate WT p53 and family members. We found in vitro that self-aggregation proceeded via a network of multiple aggregation-prone sites in p53, and inhibition of an individual site did not inhibit aggregation. Nevertheless, peptides designed to be complementary to various aggregation sequences and inhibit their polymerization can specifically kill cancer cells and be potential anticancer drugs. We found that those peptides can also function by p53-independent routes in cancer cell cultures, implying further therapeutic targets.
Background: Chymotrypsin inhibitor 2 (CI2) is a member of the class of fast-folding small protein... more Background: Chymotrypsin inhibitor 2 (CI2) is a member of the class of fast-folding small proteins, which is very suitable for testing theories of folding. CI2 folds around a diffuse extended nucleus consisting of the single α helix and a set of hydrophobic residues. In particular, Ala16 has been predicted and independently found to interact with Leu49 and Ile57, hydrophobic residues that are highly conserved among homologues. We have characterised in detail the interactions between these residues in the folding nucleus of the protein by using double-mutant cycles. Results: Surprisingly, we find that there is some destabilising strain in the transition state for folding of the wild-type protein between Ala16 and Ile57. Further, we find that the strain is larger in the native state of the protein. This is shown directly in the unfolding kinetics, which clearly show a release of strain. The net result of this is that the presence of both residues speeds up folding. Ala16 and Leu49 interact favourably in the transition state, but have no net interaction energy in the native state. Conclusions: Part of the folding nucleus of the protein fits together more snugly in the transition state than it does in the native state. Interactions between some of the closely packed residues in the folding nucleus of CI2 may perhaps be optimised for the rate of folding and not for stability.
We have analyzed the folding pathway of the tetramerization domain of the tumor suppressor protei... more We have analyzed the folding pathway of the tetramerization domain of the tumor suppressor protein p53. Structures of transition states were determined from phi-values for 25 mutations, including leucine to norvaline, and the analysis encompassed nearly every residue in the domain. Denatured monomers fold and dimerize, through a transition state with little native structure, to form a transient, highly structured dimeric intermediate. The intermediate dimerizes, through a native-like transition state with the primary dimers fully folded but with interdimer interactions only partially formed, to form the native tetramer as a 'dimer of dimers'.
Background: Single-module proteins, such as chymotrypsin inhibitor 2 (CI2), fold as a single coop... more Background: Single-module proteins, such as chymotrypsin inhibitor 2 (CI2), fold as a single cooperative unit. To solve its folding pathway, we must characterize, under conditions that favour folding, its denatured state, its transition state, and its final folded structure. To obtain a 'denatured state' that can readily be thus characterized, we have used a trick of cleaving CI2 into two complementary fragments that associate and fold in a similar way to intact protein. Results: Fragment CI2(1-40)-which contains the sequence of the single ␣-helix, spanning residues 12-24-and CI2(41-64), and mutants thereof, were analyzed by NMR spectroscopy, the transition state for association/folding was characterized by the protein engineering method, and the structure of the complex was solved by NMR and X-ray crystallography. Both isolated fragments are largely disordered. The transition state for association/folding is structured around a nucleus of a nearly fully formed ␣-helix, as is the transition state for the folding of intact CI2, from residues Ser12 to Leu21. Ala16, a residue from the helix whose sidechain is buried in the hydrophobic core, makes interactions with Leu49 and Ile57 in the other fragment. Ala16 makes its full interaction energy in the transition state for the association/folding reaction, just as found during the folding of the intact protein. Conclusions: The specific contacts in the transition state form a nucleus that extends from one fragment to the next, but the nucleus is only 'flickeringly' present in the denatured state. This is direct evidence for the nucleationcondensation mechanism in which the nucleus is only weakly formed in the ground state and develops in the transition state. The low conformational preferences in the denatured state are not enough to induce significant local secondary structure, but are reinforced by tertiary interactions during the rapid condensation around the nucleus.
structural framework. The INK4 family members, p16 [16], p15 [17], p18, and Lensfield Road Cambri... more structural framework. The INK4 family members, p16 [16], p15 [17], p18, and Lensfield Road Cambridge CB2 1EW p19 [18], regulate the progression of the cell cycle by binding to CDK4/6, thereby inducing G 1 phase cell cycle United Kindgdom arrest. They are biochemically indistinguishable from one another with respect to their interactions with CDK4/6 [14, 18]. p16 consists of four ANK repeats, Summary whereas p18 and p19 each contain five repeats, making p16 a minimal model for analyzing the properties of the The ANK repeat is a ubiquitous 33-residue motif that adopts a  hairpin helix-loop-helix fold. Multiple tan-motif. Further, alterations in p16 in tumors are common, with an occurrence of about 80% in certain tumor types dem repeats stack in a linear manner to produce an elongated structure that is stabilized predominantly [19-21], resulting in the classification of the protein as a tumor suppressor protein and adding further interest by short-range interactions between residues close in sequence. The tumor suppressor p16 INK4 consists of to the study of its folding behavior. The ANK motif is one of a number of tandem repeat four repeats and represents the minimal ANK folding unit. We found from ⌽ value analysis that p16 unfolded motifs that have recently emerged from the wealth of genomic data [22-26]. The fundamentally different na-sequentially. The two N-terminal ANK repeats, which are distorted from the canonical ANK structure in all ture of these structures compared with globular domains makes them a novel and highly pertinent target INK4 proteins and which are important for functional specificity, were mainly unstructured in the rate-lim-for protein folding studies and for testing experimental and theoretical views that have emerged from the study iting transition state for folding/unfolding, while the two C-terminal repeats were fully formed. A sequential un-of globular proteins. Here, we analyze the folding pathway of the ANK repeat p16 using ⌽ values [27, 28]. The folding mechanism could have implications for the cellular fate of wild-type and cancer-associated mutant method monitors the formation of specific interactions of side chains in the folding reaction and thereby allows p16 proteins. the structures of the rate-determining transition states and folding intermediates to be described at the residue Introduction level and, even, at the atomic level. The folding mechanism of a protein with a linearly repeated structure, The ankyrin repeat (ANK) is an -33فresidue protein motif [1, 2] that was originally discovered in two yeast cell rather than a globular fold, has not previously been resolved in any detail. We found that the p16 ankyrin recycle regulators, Cdc10 and Swi6, and, subsequently, in the cytoskeletal protein ankyrin [3]. It is found in proteins peats folded sequentially. The C-terminal repeats had native structure in the rate-determining transition state, with diverse functions, including cyclin-dependent kinase (CDK) inhibitors, transcriptional and develop-whereas the N-terminal repeats were largely unstructured. mental regulators, cytoskeletal proteins, and toxins in various classes of organisms, ranging from viruses to the human. Many ANK repeat proteins consist solely Results and Discussion of ANK repeats, while others contain repeats that are inserted between unrelated sequences. The number of p16 Structure and Design of Mutations One major difference between the ANK repeat struc-tandem copies in a protein is variable, but the proteins with fewer than four tandem repeats are uncommon. tures that have been determined to date is the extent of  sheet structure that is formed in the loops. This The crystal structure of the CDK4/CDK6 inhibitor (INK4) p16 shows an L-shaped structural scaffold of four region forms a continuous antiparallel  sheet in p18, whereas regular structure is present only between loops ANK repeats, with each repeat forming a helix-turn-helix motif [4] (Figure 1). The helices of each motif pack 2 and 3, not between loops 1 and 2, in p16 and p19. Thus, while the second and third loops are stabilized against each other in an antiparallel fashion via hydrophobic interactions to form helical bundles in the by  turns and by intraloop hydrogen bonds involving strictly conserved residues, the absence of this hydro-long arm of the L shape. The helix-turn-helix motifs are connected to the neighboring repeats by long loops gen bonding network between loops 1 and 2 in p16 and p19 means that they are held apart and have a "splayed" that are solvent exposed and fold back onto the helical region by forming  turns. The loop axis is perpendicular arrangement [15]. The other difference is the very short first helix of ANK II in all of the INK4 proteins in compari-to the helical axis, thus forming the base of the L shape. Structures of other ANK repeat proteins, 53BP2 [5], IB␣ son with the canonical ANK helices [4, 12, 14]. This
GroEL minichaperones have potential in the biotechnology industry for the refolding of recombinan... more GroEL minichaperones have potential in the biotechnology industry for the refolding of recombinant proteins. With the aim of enhancing and widening their use, we have created two highly stable functional variants of minichaperone GroEL~193-345!. A sequence alignment of 130 members of the chaperonin 60~Cpn60! family was used to design 37 single mutations. Two small-to-large mutations, A223T, A223V and one similar-size mutation, M233L, all located in the hydrophobic core were found to stabilize the protein by more than 1 kcal mol Ϫ1 each. Six stabilizing mutations were combined, yielding two multiple mutants that were 6.99 and 6.15 kcal mol Ϫ1 more stable than wild-type protein. Even though some of the substituted residue pairs are close to each other in the protein structure, the energetic effects of mutation are approximately additive. In particular, the stabilizing substitution A223T is unexpected and would have been missed by purely structural analysis. In the light of previously reported successes employing similar methods with several other proteins, our results show that a homology based approach is a simple and efficient method of increasing the stability of a protein.
Peripheral subunit binding domains (PSBDs) are integral parts of large multienzyme complexes invo... more Peripheral subunit binding domains (PSBDs) are integral parts of large multienzyme complexes involved in carbohydrate metabolism. PSBDs facilitate shuttling of prosthetic groups between different catalytic subunits. Their protein surface is characterized by a high density of positive charges required for binding to subunits within the complex. Here, we investigated folding thermodynamics and kinetics of the human PSBD (HSBD) using circular dichroism and tryptophan fluorescence experiments. HSBD was only marginally stable under physiological solvent conditions but folded within microseconds via a barrier‐limited apparent two‐state transition, analogous to its bacterial homologues. The high positive surface‐charge density of HSBD leads to repulsive Coulomb forces that modulate protein stability and folding kinetics, and appear to even induce native‐state movement. The electrostatic strain was alleviated at high solution‐ionic‐strength by Debye‐Hückel screening. Differences in ionic‐st...
Site-directed mutagenesis, including double-mutant cycles, is used routinely for studying protein... more Site-directed mutagenesis, including double-mutant cycles, is used routinely for studying protein-protein interactions. We now present a case analysis of chymotrypsin inhibitor 2 (CI2) and subtilisin BPNЈ using (i) a residue in CI2 that is known to interact directly with subtilisin (Tyr42) and (ii) two CI2 residues that do not have direct contacts with subtilisin (Arg46 and Arg48). We find that there are similar changes in binding energy on mutation of these two sets of residues. It can thus be difficult to interpret mutagenesis data in the absence of structural information.
The core domain of the tumour suppressor p53 is of inherently low thermodynamic stability and als... more The core domain of the tumour suppressor p53 is of inherently low thermodynamic stability and also low kinetic stability, which leads to rapid irreversible denaturation. Some oncogenic mutations of p53 act by just making the core domain thermosensitive, and so it is the target of novel anti-cancer drugs that bind to and stabilise the protein. Increasing the stability of the unstable core domain has also been crucial for biophysical and structural studies, in which a stabilised quadruple mutant (QM) is currently used. We generated an even more stabilised hexamutant (HM) by making two additional substitutions, Y236F and T253I, to the QM. The residues are found in the more stable paralogs p63 and p73 and stabilise the wild-type p53 core domain. We solved the structure of the HM core domain by X-ray crystallography at 1.75 Å resolution. It has minimal structural changes from QM that affect the packing of hydrophobic core residues of the b-sandwich. The full-length HM was also fully functional in DNA binding. HM was more stable than QM at 378 8 8 8 8C. Anomalies in biophysics and spectroscopy in urea-mediated denaturation curves of HM implied the accumulation of a folding intermediate, which may be related to those detected in kinetic experiments. The two additional mutations over-stabilise an unfolding intermediate. These results should be taken into consideration in drug design strategies for increasing the stability of temperature-sensitive mutants of p53.
Proceedings of the National Academy of Sciences, 1999
We have measured the stability and stoichiometry of variants of the human p53 tetramerization dom... more We have measured the stability and stoichiometry of variants of the human p53 tetramerization domain to assess the effects of mutation on homo- and hetero-oligomerization. The residues chosen for mutation were those in the hydrophobic core that we had previously found to be critical for its stability but are not conserved in human p73 or p51 or in p53-related proteins from invertebrates or vertebrates. The mutations introduced were either single natural mutations or combinations of mutations present in p53-like proteins from different species. Most of the mutations were substantially destabilizing when introduced singly. The introduction of multiple mutations led to two opposite effects: some combinations of mutations that have occurred during the evolution of the hydrophobic core of the domain in p53-like proteins had additive destabilizing effects, whereas other naturally occurring combinations of mutations had little or no net effect on the stability, there being mutually compens...
Proceedings of the National Academy of Sciences, 1998
We have designed a p53 DNA binding domain that has virtually the same binding affinity for the ga... more We have designed a p53 DNA binding domain that has virtually the same binding affinity for the gadd45 promoter as does wild-type protein but is considerably more stable. The design strategy was based on molecular evolution of the protein domain. Naturally occurring amino acid substitutions were identified by comparing the sequences of p53 homologues from 23 species, introducing them into wild-type human p53, and measuring the changes in stability. The most stable substitutions were combined in a multiple mutant. The advantage of this strategy is that, by substituting with naturally occurring residues, the function is likely to be unimpaired. All point mutants bind the consensus DNA sequence. The changes in stability ranged from +1.27 (less stable Q165K) to −1.49 (more stable N239Y) kcal mol −1 , respectively. The changes in free energy of unfolding on mutation are additive. Of interest, the two most stable mutants (N239Y and N268D) have been known to act as suppressors and restored ...
Proceedings of the National Academy of Sciences, 2007
Conventional cooperative protein folding invokes discrete ensembles of native and denatured state... more Conventional cooperative protein folding invokes discrete ensembles of native and denatured state structures in separate free-energy wells. Unimodal noncooperative (“downhill”) folding, however, proposes an ensemble of states occupying a single free-energy well for proteins folding at ≥4 × 10 4 s −1 at 298 K. It is difficult to falsify unimodal mechanisms for such fast folding proteins by standard equilibrium experiments because both cooperative and unimodal mechanisms can present the same time-averaged structural, spectroscopic, and thermodynamic properties when the time scale used for observation is longer than for equilibration. However, kinetics can provide the necessary evidence. Chevron plots with strongly sloping linear refolding arms are very difficult to explain by downhill folding and are a signature for cooperative folding via a transition state ensemble. The folding kinetics of the peripheral subunit binding domain POB and its mutants fit to strongly sloping chevrons at ...
Proceedings of the National Academy of Sciences, 2000
A series of studies on the small protein barnase in the 1990s established it as a paradigm for pr... more A series of studies on the small protein barnase in the 1990s established it as a paradigm for protein folding in which there is a kinetically important intermediate. But, a recent study in PNAS claims that there are no stable intermediates on the folding pathway. I summarize the evidence that proves that the folding kinetics of barnase is inconsistent with the absence of a folding intermediate. I reinterpret the major evidence presented against the intermediate (an inflection in the unfolding limb of a chevron plot) and show that the inflection is precisely what is predicted from the energy diagram for a three-state reaction with a kinetically significant on-pathway intermediate. The inflection is indicative of a change of rate determining step from the formation to breakdown of an intermediate on unfolding. Other evidence presented against the intermediate is, in fact, consistent with a kinetically important intermediate. I show how the complexities in the kinetics provide a means...
Proceedings of the National Academy of Sciences, 1998
Experimental data from protein engineering studies and NMR spectroscopy have been used by theoret... more Experimental data from protein engineering studies and NMR spectroscopy have been used by theoreticians to develop algorithms for helix propensity and to benchmark computer simulations of folding pathways and energy landscapes. Molecular dynamic simulations of the unfolding of chymotrypsin inhibitor 2 (CI2) have provided detailed structural models of the transition state ensemble for unfolding/folding of the protein. We now have used the simulated transition state structures to design faster folding mutants of CI2. The models pinpoint a number of unfavorable local interactions at the carboxyl terminus of the single α-helix and in the protease-binding loop region of CI2. By removing these interactions or replacing them with stabilizing ones, we have increased the rate of folding of the protein up to 40-fold (τ = 0.4 ms). This correspondence, and other examples of agreement between experiment and theory in general, Φ-values and molecular dynamics simulations, in particular, suggest th...
Proceedings of the National Academy of Sciences, 1997
We have documented the folding pathway of the 10-kDa protein barstar from the first few microseco... more We have documented the folding pathway of the 10-kDa protein barstar from the first few microseconds at the resolution of individual residues from its well characterized denatured state. The denatured state had been shown from NMR to have flickering native-like structure in the first two of its four α-helices. Φ-value analysis shows that the first helix becomes substantially consolidated as the intermediate is formed in a few hundred microseconds, as does the second to a lesser extent. A native-like structure then is formed in a few hundred milliseconds as the whole structure consolidates. Peptide fragments corresponding to sequences containing the first two helices separately and together as a helix-loop-helix motif have little helical structure under conditions that favor folding. The early stages of folding fit the nucleation–condensation model that was proposed for the smaller chymotrypsin inhibitor 2, which is a single module of structure and folds by two-state kinetics. The ea...
Proceedings of the National Academy of Sciences, 2004
We have assessed the published predictions of the pathway of folding of the B domain of protein A... more We have assessed the published predictions of the pathway of folding of the B domain of protein A, the pathway most studied by computer simulation. We analyzed the transition state for folding of the three-helix bundle protein, by using experimental Φ values on some 70 suitable mutants. Surprisingly, the third helix, which has the most stable α-helical structure as a peptide fragment, is poorly formed in the transition state, especially at its C terminus. The protein folds around a nearly fully formed central helix, which is stabilized by extensive hydrophobic side chain interactions. The turn connecting the poorly structured first helix to the central helix is unstructured, but the turn connecting the central helix to the third is in the process of being formed as the N-terminal region of the third helix begins to coalesce. The transition state is inconsistent with a classical framework mechanism and is closer to nucleation–condensation. None of the published atomistic simulations ...
Proceedings of the National Academy of Sciences, 2017
Significance Destabilized mutants of the tumor suppressor p53 are inactivated by self-aggregation... more Significance Destabilized mutants of the tumor suppressor p53 are inactivated by self-aggregation in a substantial number of tumors and may also coaggregate with and inactivate WT p53 and family members. We found in vitro that self-aggregation proceeded via a network of multiple aggregation-prone sites in p53, and inhibition of an individual site did not inhibit aggregation. Nevertheless, peptides designed to be complementary to various aggregation sequences and inhibit their polymerization can specifically kill cancer cells and be potential anticancer drugs. We found that those peptides can also function by p53-independent routes in cancer cell cultures, implying further therapeutic targets.
Background: Chymotrypsin inhibitor 2 (CI2) is a member of the class of fast-folding small protein... more Background: Chymotrypsin inhibitor 2 (CI2) is a member of the class of fast-folding small proteins, which is very suitable for testing theories of folding. CI2 folds around a diffuse extended nucleus consisting of the single α helix and a set of hydrophobic residues. In particular, Ala16 has been predicted and independently found to interact with Leu49 and Ile57, hydrophobic residues that are highly conserved among homologues. We have characterised in detail the interactions between these residues in the folding nucleus of the protein by using double-mutant cycles. Results: Surprisingly, we find that there is some destabilising strain in the transition state for folding of the wild-type protein between Ala16 and Ile57. Further, we find that the strain is larger in the native state of the protein. This is shown directly in the unfolding kinetics, which clearly show a release of strain. The net result of this is that the presence of both residues speeds up folding. Ala16 and Leu49 interact favourably in the transition state, but have no net interaction energy in the native state. Conclusions: Part of the folding nucleus of the protein fits together more snugly in the transition state than it does in the native state. Interactions between some of the closely packed residues in the folding nucleus of CI2 may perhaps be optimised for the rate of folding and not for stability.
We have analyzed the folding pathway of the tetramerization domain of the tumor suppressor protei... more We have analyzed the folding pathway of the tetramerization domain of the tumor suppressor protein p53. Structures of transition states were determined from phi-values for 25 mutations, including leucine to norvaline, and the analysis encompassed nearly every residue in the domain. Denatured monomers fold and dimerize, through a transition state with little native structure, to form a transient, highly structured dimeric intermediate. The intermediate dimerizes, through a native-like transition state with the primary dimers fully folded but with interdimer interactions only partially formed, to form the native tetramer as a 'dimer of dimers'.
Background: Single-module proteins, such as chymotrypsin inhibitor 2 (CI2), fold as a single coop... more Background: Single-module proteins, such as chymotrypsin inhibitor 2 (CI2), fold as a single cooperative unit. To solve its folding pathway, we must characterize, under conditions that favour folding, its denatured state, its transition state, and its final folded structure. To obtain a 'denatured state' that can readily be thus characterized, we have used a trick of cleaving CI2 into two complementary fragments that associate and fold in a similar way to intact protein. Results: Fragment CI2(1-40)-which contains the sequence of the single ␣-helix, spanning residues 12-24-and CI2(41-64), and mutants thereof, were analyzed by NMR spectroscopy, the transition state for association/folding was characterized by the protein engineering method, and the structure of the complex was solved by NMR and X-ray crystallography. Both isolated fragments are largely disordered. The transition state for association/folding is structured around a nucleus of a nearly fully formed ␣-helix, as is the transition state for the folding of intact CI2, from residues Ser12 to Leu21. Ala16, a residue from the helix whose sidechain is buried in the hydrophobic core, makes interactions with Leu49 and Ile57 in the other fragment. Ala16 makes its full interaction energy in the transition state for the association/folding reaction, just as found during the folding of the intact protein. Conclusions: The specific contacts in the transition state form a nucleus that extends from one fragment to the next, but the nucleus is only 'flickeringly' present in the denatured state. This is direct evidence for the nucleationcondensation mechanism in which the nucleus is only weakly formed in the ground state and develops in the transition state. The low conformational preferences in the denatured state are not enough to induce significant local secondary structure, but are reinforced by tertiary interactions during the rapid condensation around the nucleus.
structural framework. The INK4 family members, p16 [16], p15 [17], p18, and Lensfield Road Cambri... more structural framework. The INK4 family members, p16 [16], p15 [17], p18, and Lensfield Road Cambridge CB2 1EW p19 [18], regulate the progression of the cell cycle by binding to CDK4/6, thereby inducing G 1 phase cell cycle United Kindgdom arrest. They are biochemically indistinguishable from one another with respect to their interactions with CDK4/6 [14, 18]. p16 consists of four ANK repeats, Summary whereas p18 and p19 each contain five repeats, making p16 a minimal model for analyzing the properties of the The ANK repeat is a ubiquitous 33-residue motif that adopts a  hairpin helix-loop-helix fold. Multiple tan-motif. Further, alterations in p16 in tumors are common, with an occurrence of about 80% in certain tumor types dem repeats stack in a linear manner to produce an elongated structure that is stabilized predominantly [19-21], resulting in the classification of the protein as a tumor suppressor protein and adding further interest by short-range interactions between residues close in sequence. The tumor suppressor p16 INK4 consists of to the study of its folding behavior. The ANK motif is one of a number of tandem repeat four repeats and represents the minimal ANK folding unit. We found from ⌽ value analysis that p16 unfolded motifs that have recently emerged from the wealth of genomic data [22-26]. The fundamentally different na-sequentially. The two N-terminal ANK repeats, which are distorted from the canonical ANK structure in all ture of these structures compared with globular domains makes them a novel and highly pertinent target INK4 proteins and which are important for functional specificity, were mainly unstructured in the rate-lim-for protein folding studies and for testing experimental and theoretical views that have emerged from the study iting transition state for folding/unfolding, while the two C-terminal repeats were fully formed. A sequential un-of globular proteins. Here, we analyze the folding pathway of the ANK repeat p16 using ⌽ values [27, 28]. The folding mechanism could have implications for the cellular fate of wild-type and cancer-associated mutant method monitors the formation of specific interactions of side chains in the folding reaction and thereby allows p16 proteins. the structures of the rate-determining transition states and folding intermediates to be described at the residue Introduction level and, even, at the atomic level. The folding mechanism of a protein with a linearly repeated structure, The ankyrin repeat (ANK) is an -33فresidue protein motif [1, 2] that was originally discovered in two yeast cell rather than a globular fold, has not previously been resolved in any detail. We found that the p16 ankyrin recycle regulators, Cdc10 and Swi6, and, subsequently, in the cytoskeletal protein ankyrin [3]. It is found in proteins peats folded sequentially. The C-terminal repeats had native structure in the rate-determining transition state, with diverse functions, including cyclin-dependent kinase (CDK) inhibitors, transcriptional and develop-whereas the N-terminal repeats were largely unstructured. mental regulators, cytoskeletal proteins, and toxins in various classes of organisms, ranging from viruses to the human. Many ANK repeat proteins consist solely Results and Discussion of ANK repeats, while others contain repeats that are inserted between unrelated sequences. The number of p16 Structure and Design of Mutations One major difference between the ANK repeat struc-tandem copies in a protein is variable, but the proteins with fewer than four tandem repeats are uncommon. tures that have been determined to date is the extent of  sheet structure that is formed in the loops. This The crystal structure of the CDK4/CDK6 inhibitor (INK4) p16 shows an L-shaped structural scaffold of four region forms a continuous antiparallel  sheet in p18, whereas regular structure is present only between loops ANK repeats, with each repeat forming a helix-turn-helix motif [4] (Figure 1). The helices of each motif pack 2 and 3, not between loops 1 and 2, in p16 and p19. Thus, while the second and third loops are stabilized against each other in an antiparallel fashion via hydrophobic interactions to form helical bundles in the by  turns and by intraloop hydrogen bonds involving strictly conserved residues, the absence of this hydro-long arm of the L shape. The helix-turn-helix motifs are connected to the neighboring repeats by long loops gen bonding network between loops 1 and 2 in p16 and p19 means that they are held apart and have a "splayed" that are solvent exposed and fold back onto the helical region by forming  turns. The loop axis is perpendicular arrangement [15]. The other difference is the very short first helix of ANK II in all of the INK4 proteins in compari-to the helical axis, thus forming the base of the L shape. Structures of other ANK repeat proteins, 53BP2 [5], IB␣ son with the canonical ANK helices [4, 12, 14]. This
GroEL minichaperones have potential in the biotechnology industry for the refolding of recombinan... more GroEL minichaperones have potential in the biotechnology industry for the refolding of recombinant proteins. With the aim of enhancing and widening their use, we have created two highly stable functional variants of minichaperone GroEL~193-345!. A sequence alignment of 130 members of the chaperonin 60~Cpn60! family was used to design 37 single mutations. Two small-to-large mutations, A223T, A223V and one similar-size mutation, M233L, all located in the hydrophobic core were found to stabilize the protein by more than 1 kcal mol Ϫ1 each. Six stabilizing mutations were combined, yielding two multiple mutants that were 6.99 and 6.15 kcal mol Ϫ1 more stable than wild-type protein. Even though some of the substituted residue pairs are close to each other in the protein structure, the energetic effects of mutation are approximately additive. In particular, the stabilizing substitution A223T is unexpected and would have been missed by purely structural analysis. In the light of previously reported successes employing similar methods with several other proteins, our results show that a homology based approach is a simple and efficient method of increasing the stability of a protein.
Peripheral subunit binding domains (PSBDs) are integral parts of large multienzyme complexes invo... more Peripheral subunit binding domains (PSBDs) are integral parts of large multienzyme complexes involved in carbohydrate metabolism. PSBDs facilitate shuttling of prosthetic groups between different catalytic subunits. Their protein surface is characterized by a high density of positive charges required for binding to subunits within the complex. Here, we investigated folding thermodynamics and kinetics of the human PSBD (HSBD) using circular dichroism and tryptophan fluorescence experiments. HSBD was only marginally stable under physiological solvent conditions but folded within microseconds via a barrier‐limited apparent two‐state transition, analogous to its bacterial homologues. The high positive surface‐charge density of HSBD leads to repulsive Coulomb forces that modulate protein stability and folding kinetics, and appear to even induce native‐state movement. The electrostatic strain was alleviated at high solution‐ionic‐strength by Debye‐Hückel screening. Differences in ionic‐st...
Site-directed mutagenesis, including double-mutant cycles, is used routinely for studying protein... more Site-directed mutagenesis, including double-mutant cycles, is used routinely for studying protein-protein interactions. We now present a case analysis of chymotrypsin inhibitor 2 (CI2) and subtilisin BPNЈ using (i) a residue in CI2 that is known to interact directly with subtilisin (Tyr42) and (ii) two CI2 residues that do not have direct contacts with subtilisin (Arg46 and Arg48). We find that there are similar changes in binding energy on mutation of these two sets of residues. It can thus be difficult to interpret mutagenesis data in the absence of structural information.
The core domain of the tumour suppressor p53 is of inherently low thermodynamic stability and als... more The core domain of the tumour suppressor p53 is of inherently low thermodynamic stability and also low kinetic stability, which leads to rapid irreversible denaturation. Some oncogenic mutations of p53 act by just making the core domain thermosensitive, and so it is the target of novel anti-cancer drugs that bind to and stabilise the protein. Increasing the stability of the unstable core domain has also been crucial for biophysical and structural studies, in which a stabilised quadruple mutant (QM) is currently used. We generated an even more stabilised hexamutant (HM) by making two additional substitutions, Y236F and T253I, to the QM. The residues are found in the more stable paralogs p63 and p73 and stabilise the wild-type p53 core domain. We solved the structure of the HM core domain by X-ray crystallography at 1.75 Å resolution. It has minimal structural changes from QM that affect the packing of hydrophobic core residues of the b-sandwich. The full-length HM was also fully functional in DNA binding. HM was more stable than QM at 378 8 8 8 8C. Anomalies in biophysics and spectroscopy in urea-mediated denaturation curves of HM implied the accumulation of a folding intermediate, which may be related to those detected in kinetic experiments. The two additional mutations over-stabilise an unfolding intermediate. These results should be taken into consideration in drug design strategies for increasing the stability of temperature-sensitive mutants of p53.
Proceedings of the National Academy of Sciences, 1999
We have measured the stability and stoichiometry of variants of the human p53 tetramerization dom... more We have measured the stability and stoichiometry of variants of the human p53 tetramerization domain to assess the effects of mutation on homo- and hetero-oligomerization. The residues chosen for mutation were those in the hydrophobic core that we had previously found to be critical for its stability but are not conserved in human p73 or p51 or in p53-related proteins from invertebrates or vertebrates. The mutations introduced were either single natural mutations or combinations of mutations present in p53-like proteins from different species. Most of the mutations were substantially destabilizing when introduced singly. The introduction of multiple mutations led to two opposite effects: some combinations of mutations that have occurred during the evolution of the hydrophobic core of the domain in p53-like proteins had additive destabilizing effects, whereas other naturally occurring combinations of mutations had little or no net effect on the stability, there being mutually compens...
Proceedings of the National Academy of Sciences, 1998
We have designed a p53 DNA binding domain that has virtually the same binding affinity for the ga... more We have designed a p53 DNA binding domain that has virtually the same binding affinity for the gadd45 promoter as does wild-type protein but is considerably more stable. The design strategy was based on molecular evolution of the protein domain. Naturally occurring amino acid substitutions were identified by comparing the sequences of p53 homologues from 23 species, introducing them into wild-type human p53, and measuring the changes in stability. The most stable substitutions were combined in a multiple mutant. The advantage of this strategy is that, by substituting with naturally occurring residues, the function is likely to be unimpaired. All point mutants bind the consensus DNA sequence. The changes in stability ranged from +1.27 (less stable Q165K) to −1.49 (more stable N239Y) kcal mol −1 , respectively. The changes in free energy of unfolding on mutation are additive. Of interest, the two most stable mutants (N239Y and N268D) have been known to act as suppressors and restored ...
Proceedings of the National Academy of Sciences, 2007
Conventional cooperative protein folding invokes discrete ensembles of native and denatured state... more Conventional cooperative protein folding invokes discrete ensembles of native and denatured state structures in separate free-energy wells. Unimodal noncooperative (“downhill”) folding, however, proposes an ensemble of states occupying a single free-energy well for proteins folding at ≥4 × 10 4 s −1 at 298 K. It is difficult to falsify unimodal mechanisms for such fast folding proteins by standard equilibrium experiments because both cooperative and unimodal mechanisms can present the same time-averaged structural, spectroscopic, and thermodynamic properties when the time scale used for observation is longer than for equilibration. However, kinetics can provide the necessary evidence. Chevron plots with strongly sloping linear refolding arms are very difficult to explain by downhill folding and are a signature for cooperative folding via a transition state ensemble. The folding kinetics of the peripheral subunit binding domain POB and its mutants fit to strongly sloping chevrons at ...
Proceedings of the National Academy of Sciences, 2000
A series of studies on the small protein barnase in the 1990s established it as a paradigm for pr... more A series of studies on the small protein barnase in the 1990s established it as a paradigm for protein folding in which there is a kinetically important intermediate. But, a recent study in PNAS claims that there are no stable intermediates on the folding pathway. I summarize the evidence that proves that the folding kinetics of barnase is inconsistent with the absence of a folding intermediate. I reinterpret the major evidence presented against the intermediate (an inflection in the unfolding limb of a chevron plot) and show that the inflection is precisely what is predicted from the energy diagram for a three-state reaction with a kinetically significant on-pathway intermediate. The inflection is indicative of a change of rate determining step from the formation to breakdown of an intermediate on unfolding. Other evidence presented against the intermediate is, in fact, consistent with a kinetically important intermediate. I show how the complexities in the kinetics provide a means...
Proceedings of the National Academy of Sciences, 1998
Experimental data from protein engineering studies and NMR spectroscopy have been used by theoret... more Experimental data from protein engineering studies and NMR spectroscopy have been used by theoreticians to develop algorithms for helix propensity and to benchmark computer simulations of folding pathways and energy landscapes. Molecular dynamic simulations of the unfolding of chymotrypsin inhibitor 2 (CI2) have provided detailed structural models of the transition state ensemble for unfolding/folding of the protein. We now have used the simulated transition state structures to design faster folding mutants of CI2. The models pinpoint a number of unfavorable local interactions at the carboxyl terminus of the single α-helix and in the protease-binding loop region of CI2. By removing these interactions or replacing them with stabilizing ones, we have increased the rate of folding of the protein up to 40-fold (τ = 0.4 ms). This correspondence, and other examples of agreement between experiment and theory in general, Φ-values and molecular dynamics simulations, in particular, suggest th...
Proceedings of the National Academy of Sciences, 1997
We have documented the folding pathway of the 10-kDa protein barstar from the first few microseco... more We have documented the folding pathway of the 10-kDa protein barstar from the first few microseconds at the resolution of individual residues from its well characterized denatured state. The denatured state had been shown from NMR to have flickering native-like structure in the first two of its four α-helices. Φ-value analysis shows that the first helix becomes substantially consolidated as the intermediate is formed in a few hundred microseconds, as does the second to a lesser extent. A native-like structure then is formed in a few hundred milliseconds as the whole structure consolidates. Peptide fragments corresponding to sequences containing the first two helices separately and together as a helix-loop-helix motif have little helical structure under conditions that favor folding. The early stages of folding fit the nucleation–condensation model that was proposed for the smaller chymotrypsin inhibitor 2, which is a single module of structure and folds by two-state kinetics. The ea...
Proceedings of the National Academy of Sciences, 2004
We have assessed the published predictions of the pathway of folding of the B domain of protein A... more We have assessed the published predictions of the pathway of folding of the B domain of protein A, the pathway most studied by computer simulation. We analyzed the transition state for folding of the three-helix bundle protein, by using experimental Φ values on some 70 suitable mutants. Surprisingly, the third helix, which has the most stable α-helical structure as a peptide fragment, is poorly formed in the transition state, especially at its C terminus. The protein folds around a nearly fully formed central helix, which is stabilized by extensive hydrophobic side chain interactions. The turn connecting the poorly structured first helix to the central helix is unstructured, but the turn connecting the central helix to the third is in the process of being formed as the N-terminal region of the third helix begins to coalesce. The transition state is inconsistent with a classical framework mechanism and is closer to nucleation–condensation. None of the published atomistic simulations ...
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Papers by Alan Fersht