Structural characterization of partially folded intermediates of apomyoglobin H64F

We present a detailed investigation of unfolded and partially folded states of a mutant apomyoglobin (apoMb) where the distal histidine has been replaced by phenylalanine (H64F). Previous studies have shown that substitution of His64, located in the E helix of the native protein, stabilizes the equilibrium molten globule and native states and leads to an increase in folding rate and a change in the folding pathway. Analysis of changes in chemical shift and in backbone flexibility, detected via [1H]-15N heteronuclear nuclear Overhauser effect measurements, indicates that the phenylalanine substitution has only minor effects on the conformational ensemble in the acid- and urea-unfolded states, but has a substantial effect on the structure, dynamics, and stability of the equilibrium molten globule intermediate formed near pH 4. In H64F apomyoglobin, additional regions of the polypeptide chain are recruited into the compact core of the molten globule. Since the phenylalanine substitution has negligible effect on the unfolded ensemble, its influence on folding rate and stability comes entirely from interactions within the compact folded or partly folded states. Replacement of His64 with Phe leads to favorable hydrophobic packing between the helix E region and the molten globule core and leads to stabilization of helix E secondary structure and overall thermodynamic stabilization of the molten globule. The secondary structure of the equilibrium molten globule parallels that of the burst phase kinetic intermediate; both intermediates contain significant helical structure in regions of the polypeptide that comprise the A, B, E, G, and H helices of the fully folded protein.     

pH-dependent stability of the human alpha-lactalbumin molten globule state: contrasting roles of the 6 - 120 disulfide and the beta-subdomain at low and neutral pH

Many proteins are capable of populating partially folded states known as molten globule states. Alpha-lactalbumin forms a molten globule under a range of conditions including low pH (the A-state) and at neutral pH in the absence of Ca(2+) with modest amounts of denaturant. The A-state is the most thoroughly characterized and thought to mimic a kinetic intermediate populated during refolding at neutral pH. We demonstrate that the properties and interactions that stabilize the A-state and the pH 7 molten globule of human alpha-lactalbumin differ. The unfolding of the wild-type protein is compared to the unfolding of a variant that lacks the 6 - 120 disulfide bond and to an autonomously folded peptide construct that we have previously shown represents the minimum core structure of the A-state of human alpha-lactalbumin. Studies conducted at pH 2 and 7 show that the disulfide makes little contribution to the stability of the molten globule at pH 7 but is important at pH 2. In contrast, the beta-subdomain of the protein is less important at pH 2 than at pH 7. The role of helix propensity in stabilizing the different forms of the molten globule state is examined and it is shown that it cannot account for the differences. The strikingly different behavior observed at pH 2 and 7 indicates that the A-state may not be a rigorous mimic of the folding intermediate populated at pH 7.     

Conformational and dynamic characterization of the molten globule state of an apomyoglobin mutant with an altered folding pathway.

Kinetic and equilibrium studies of apomyoglobin folding pathways and intermediates have provided important insights into the mechanism of protein folding. To investigate the role of intrinsic helical propensities in the apomyoglobin folding process, a mutant has been prepared in which Asn132 and Glu136 have been substituted with glycine to destabilize the H helix. The structure and dynamics of the equilibrium molten globule state formed at pH 4.1 have been examined using NMR spectroscopy. Deviations of backbone (13)C(alpha) and (13)CO chemical shifts from random coil values reveal high populations of helical structure in the A and G helix regions and in part of the B helix. However, the H helix is significantly destabilized compared to the wild-type molten globule. Heteronuclear [(1)H]-(15)N NOEs show that, although the polypeptide backbone in the H helix region is more flexible than in the wild-type protein, its motions are restricted by transient hydrophobic interactions with the molten globule core. Quench flow hydrogen exchange measurements reveal stable helical structure in the A and G helices and part of the B helix in the burst phase kinetic intermediate and confirm that the H helix is largely unstructured. Stabilization of structure in the H helix occurs during the slow folding phases, in synchrony with the C and E helices and the CD region. The kinetic and equilibrium molten globule intermediates formed by N132G/E136G are similar in structure. Although both the wild-type apomyoglobin and the mutant fold via compact helical intermediates, the structures of the intermediates and consequently the detailed folding pathways differ. Apomyoglobin is therefore capable of compensating for mutations by using alternative folding pathways within a common basic framework. Tertiary hydrophobic interactions appear to play an important role in the formation and stabilization of secondary structure in the H helix of the N132G/E136G mutant. These studies provide important insights into the interplay between secondary and tertiary structure formation in protein folding.     

Probing subtle differences in the hydrogen exchange behavior of variants of the human alpha-lactalbumin molten globule using mass spectrometry

The hydrogen-exchange behavior of the low-pH molten globule of human alpha-lactalbumin, containing all four disulfides, has been examined and compared with that of a single disulfide variant, [28-111] alpha-lactalbumin, and of a series of proline variants of [28-111] alpha-lactalbumin. The small differences in hydrogen-exchange protection exhibited by these partially folded species were compared by mixing two or more proteins and monitoring their exchange simultaneously using mass spectrometry. The effect of single proline mutations within each alpha-domain helix on hydrogen-exchange protection has been investigated using six proline variants of [28-111] alpha-lactalbumin, L11P, L12P, M30P, I95P, K108P and Q117P. The results show that proline mutations in the A, B, C and D alpha-helices lead to a loss of hydrogen-exchange protection for residues in the local helix without perturbing hydrogen-exchange protection in other regions of the protein. Thus, local unfolding of the A, B, C and D helices does not significantly alter the packing and solvent accessibility of other regions of the molten globule. By contrast, introduction of a proline residue in the C-terminal 3(10) helix produces a larger and more widespread loss of hydrogen-exchange protection, demonstrating that longer-range perturbations of the molten globule have occurred. Thus, residues in this C-terminal region must be involved in contacts that are critical for the stabilisation of the compact molten globule structure.     

Putative interhelix ion pairs involved in the stability of myoglobin.

An earlier theoretical study predicted that specific ion pair interactions between neighboring helices should be important in stabilizing myoglobin. To measure these interactions in sperm whale myoglobin, single mutations were made to disrupt them. To obtain reliable DeltaG values, conditions were found in which the urea induced unfolding of holomyoglobin is reversible and two-state. The cyanomet form of myoglobin satisfies this condition at pH 5, 25 degrees C. The unfolding curves monitored by far-UV CD and Soret absorbance are superimposable and reversible. None of the putative ion pairs studied here makes a large contribution to the stability of native myoglobin. The protein stability does decrease somewhat between 0 and 0.1 M NaCl, however, indicating that electrostatic interactions contribute favorably to myoglobin stability at pH 5.0. A previous mutational study indicated that the net positive charge of the A[B]GH subdomain of myoglobin is an important factor affecting the stability of the pH 4 folding intermediate and potential ion pairs within the subdomain do not contribute significantly to its stability. One of the assumptions made in that study is tested here: replacement of either positively or negatively charged residues outside the A[B]GH subdomain has no significant effect on the stability of the pH 4 molten globule.     

Absence of a stable intermediate on the folding pathway of protein A.

The B-domain of protein A has one of the simplest protein topologies, a three-helix bundle. Its folding has been studied as a model for elementary steps in the folding of larger proteins. Earlier studies suggested that folding might occur by way of a helical hairpin intermediate. Equilibrium hydrogen exchange measurements indicate that the C-terminal helical hairpin could be a potential folding intermediate. Kinetic refolding experiments were performed using stopped-flow circular dichroism and NMR hydrogen-deuterium exchange pulse labeling. Folding of the entire molecule is essentially complete within the 6 ms dead time of the quench-flow apparatus, indicating that the intermediate, if formed, progresses rapidly to the final folded state. Site-directed mutagenesis of the isoleucine residue at position 16 was used to generate a variant protein containing tryptophan (the 116 W mutant). The formation of the putative folding intermediate was expected to be favored in this mutant at the expense of the native folded form, due to predicted unfavorable steric interactions of the bulky tryptophan side chain in the folded state. The 116 W mutant refolds completely within the dead time of a stopped-flow fluorescence experiment. No partly folded intermediate could be detected by either kinetic or equilibrium measurements. Studies of peptide fragments suggest that the protein A sequence has an intrinsic propensity to form a helix II/helix III hairpin. However, its stability appears to be marginal (of the order of 1/2 kT) and it could not be an obligatory intermediate on a defined folding pathway. These results explicitly demonstrate that the protein A B domain folds extremely rapidly by an apparent two-state mechanism without formation of stable partly folded intermediates. Similar mechanisms may also be involved in the rapid folding of subdomains of larger proteins to form the compact molten globule intermediates that often accumulate during the folding process.     

Energetic Frustration of Apomyoglobin Folding: Role of the B Helix

Apomyoglobin folds by a sequential mechanism in which the A, G, and H helix regions undergo rapid collapse to form a compact intermediate onto which the central portion of the B helix subsequently docks. To investigate the factors that frustrate folding, we have made mutations in the N-terminus of the B helix to stabilize helical structure (in the mutant G23A/G25A) and to promote native-like hydrophobic packing interactions with helix G (in the mutant H24L/H119F). The kinetic and equilibrium intermediates of G23A/G25A and H24L/H119F were studied by hydrogen exchange pulse labeling and interrupted hydrogen/deuterium exchange combined with NMR. For both mutants, stabilization of helical structure in the N-terminal region of the B helix is confirmed by increased exchange protection in the equilibrium molten globule states near pH 4. Increased protection is also observed in the GH turn region in the G23A/G25A mutant, suggesting that stabilization of the B helix facilitates native-like interactions with the C-terminal region of helix G. These interactions are further enhanced in H24L/H119F. The kinetic burst phase intermediates of both mutants show increased protection, relative to wild-type protein, of amides in the N-terminus of the B helix and in part of the E helix. Stabilization of the E helix in the intermediate is attributed to direct interactions between E helix residues and the newly stabilized N-terminus of helix B. Stabilization of native packing between the B and G helices in H24L/H119F also favors formation of native-like interactions in the GH turn and between the G and H helices in the ensemble of burst phase intermediates. We conclude that instability at the N-terminus of the B helix of apomyoglobin contributes to the energetic frustration of folding by preventing docking and stabilization of the E helix.     

Structural and thermodynamic consequences of removal of a conserved disulfide bond from equine beta-lactoglobulin

A disulfide bond between cysteine 66 and cysteine 160 of equine beta-lactoglobulin was removed by substituting cysteine residues with alanine. This disulfide bond is conserved across the lipocalin family. The conformation and stability of the disulfide-deleted mutant protein was investigated by circular dichroism. The mutant protein assumes a native-like structure under physiological conditions and assumes a helix-rich molten globule structure at acid pH or at moderate concentrations of urea as the wild-type protein does. The urea-induced unfolding experiment shows that the stability of the native conformation was reduced but that of the molten globule intermediate is not significantly changed at pH 4 by removal of the disulfide bond. On the other hand, the molten globule at acid pH was destabilized by removal of the disulfide bond. This difference in the stabilizing effect of the disulfide bond was interpreted by the effect of the disulfide in keeping the molecule compact against the electrostatic repulsion at acid pH. In contrast to the wild-type protein, the circular dichroism spectrum in the molten globule state at acid pH depends on anion concentration, suggesting that the expansion of the molecule through electrostatic repulsion induces alpha-helices as observed in the cold denatured state of the wild-type protein.     

Biophysical characterization of Z(SPA-1)--a phage-display selected binder to protein A

Affibodies are a novel class of binding proteins selected from phagemid libraries of the Z domain from staphylococcal protein A. The Z(SPA-1) affibody was selected as a binder to protein A, and it binds the parental Z domain with micromolar affinity. In earlier work we determined the structure of the Z:Z(SPA-1) complex and noted that Z(SPA-1) in the free state exhibits several properties characteristic of a molten globule. Here we present a more detailed biophysical investigation of Z(SPA-1) and four Z(SPA-1) mutants with the objective to understand these properties. The characterization includes thermal and chemical denaturation profiles, ANS binding assays, size exclusion chromatography, isothermal titration calorimetry, and an investigation of structure and dynamics by NMR. The NMR characterization of Z(SPA-1) was facilitated by the finding that trimethylamine N-oxide (TMAO) stabilizes the molten globule conformation in favor of the fully unfolded state. All data taken together lead us to conclude the following: (1) The topology of the molten globule conformation of free Z(SPA-1) is similar to that of the fully folded structure in the Z-bound state; (2) the extensive mutations in helices 1 and 2 destabilize these without affecting the intrinsic stability of helix 3; (3) stabilization and reduced aggregation can be achieved by replacing mutated residues in Z(SPA-1) with the corresponding wild-type Z residues. This stabilization is better correlated to changes in helix propensity than to an expected increase in polar versus nonpolar surface area of the fully folded state.     

The kinetic and equilibrium molten globule intermediates of apoleghemoglobin differ in structure

An important question in protein folding is whether molten globule states formed under equilibrium conditions are good structural models for kinetic folding intermediates. The structures of the kinetic and equilibrium intermediates in the folding of the plant globin apoleghemoglobin have been compared at high resolution by quench-flow pH-pulse labeling and interrupted hydrogen/deuterium exchange analyzed in dimethyl sulfoxide. Unlike its well studied homolog apomyoglobin, where the equilibrium and kinetic intermediates are quite similar, there are striking structural differences between the intermediates formed by apoleghemoglobin. In the kinetic intermediate, formed during the burst phase of the quench-flow experiment, protected amides and helical structure are found mainly in the regions corresponding to the G and H helices of the folded protein, and in parts of the E helix and CE loop regions, whereas in the equilibrium intermediate, amide protection and helical structure are seen in parts of the A and B helix regions, as well as in the G and H regions, and the E helix remains largely unfolded. These results suggest that the structure of the molten globule intermediate of apoleghemoglobin is more plastic than that of apomyoglobin, so that it is readily transformed depending on the solution conditions, particularly pH. Thus, in the case of apoleghemoglobin at least, the equilibrium molten globule formed under destabilizing conditions at acid pH is not a good model for the compact intermediate formed during kinetic refolding experiments. Our high-precision kinetic analysis also reveals an additional slow phase during the folding of apoleghemoglobin, which is not observed for apomyoglobin. Hydrogen exchange pulse-labeling experiments show that the slow-folding phase is associated with residues in the CE loop, which probably forms non-native structure in the intermediate that must be resolved before folding can proceed to completion.     

Characterization of acid-induced unfolding intermediates of glucose/xylose isomerase.

Acid-induced unfolding of the tetrameric glucose/xylose isomerase (GXI) from Streptomyces sp. NCIM 2730 has been investigated using intrinsic fluorescence, fluorescence quenching, second derivative spectroscopy, hydrophobic dye (1-anilino-8-naphthalene-sulfonate) binding and CD techniques. The pH dependence of tryptophanyl fluorescence of GXI at different temperatures indicated the presence of two stable intermediates at pH 5.0 and pH 3.0. The pH 3.2 intermediate was a dimer and exhibited molten globule-like characteristics, such as the presence of native-like secondary structure, loss of tertiary structure, increased exposure of hydrophobic pockets, altered microenvironment of tyrosine residues and increased accessibility to quenching by acrylamide. Fluorescence and CD studies on GXI at pH 5.0 suggested the involvement of a partially folded intermediate state in the native to molten globule state transition. The partially folded intermediate state retained considerable secondary and tertiary structure compared to the molten globule state. This state was characterized by its hydrophobic dye binding capacity, which is smaller than the molten globule state, but was greater than that of the native state. This state shared the dimeric status of the molten globule state but was prone to aggregate formation as evident by the Rayleigh light scattering studies. Based on these results, the unfolding pathway of GXI can be illustrated as: N-->PFI-->MG-->U; where N is the native state at pH 7.5; PFI is the partially folded intermediate state at pH 5.0; MG is the molten globule state at pH 3.2 and U is the monomeric unfolded state of GXI obtained in the presence of 6 M GdnHCl. Our results demonstrate the existence of a partially folded state and molten globule state on the unfolding pathway of a multimeric alpha/beta barrel protein.     

Defining the core structure of the alpha-lactalbumin molten globule state.

Molten globules are partially folded states of proteins which are generally believed to mimic structures formed during the folding process. In order to determine the minimal requirements for the formation of a molten globule state, we have prepared a set of peptide models of the molten globule state of human alpha-lactalbumin (alphaLA). A peptide consisting of residues 1-38 crosslinked, via the native 28-111 disulfide bond, to a peptide corresponding to residues 95-120 forms a partially folded state at pH 2.8 which has all of the characteristics of the molten globule state of alphaLA as judged by near and far UV CD, fluorescence, ANS binding and urea denaturation experiments. The structure of the peptide construct is the same at pH 7.0. Deletion of residues 95-100 from the construct has little effect. Thus, less than half the sequence is required to form a molten globule. Further truncation corresponding to the selective deletion of the A (residues 1-19) or D (residues 101-110) helices or the C-terminal 310 helix (residues 112-120) leads to a significant loss of structure. The loss of structure which results from the deletion of any of these three regions is much greater than that which would be expected based upon the non-cooperative loss of local helical structure. Deletion of residues corresponding to the region of the D helix or C-terminal 310 helix region results in a peptide construct which is largely unfolded and contains no more helical structure than is expected from the sum of the helicity of the two reduced peptides. These experiments have defined the minimum core structure of the alphaLA molten globule state.     

The molten globule state of a chimera of human alpha-lactalbumin and equine lysozyme.

The molten globule state of equine lysozyme is more stable than that of alpha-lactalbumin and is stabilized by non-specific hydrophobic interactions and native-like hydrophobic interactions. We constructed a chimeric protein which is produced by replacing the flexible loop (residues 105-110) in human alpha-lactalbumin with the helix D (residues 109-114) in equine lysozyme to investigate the possible role of the helix D for the high stability and native-like packing interaction in the molten globule state of equine lysozyme. The stability of the molten globule state formed by the chimeric protein to guanidine hydrochloride-induced unfolding is the same as that of equine lysozyme and is substantially greater than that of human alpha-lactalbumin, although only six residues come from equine lysozyme. Our results also suggest that the non-native interaction in the molten globule state of alpha-lactalbumin changes to the native-like packing interaction due to helix substitution. The solvent-accessibility of the Trp residues in the molten globule state of the chimeric protein is similar to that in the molten globule state of equine lysozyme in which packing interaction around the Trp residues in the native state is partially preserved. Therefore, the helix D in equine lysozyme is one of the contributing factors to the high stability and native-like packing interaction in the molten globule state of equine lysozyme. Our results indicate that the native-like packing interaction can stabilize the rudimentary intermediate which is stabilized by the non-specific hydrophobic interactions.     

On the role of some conserved and nonconserved amino acid residues in the transitional state and intermediate of apomyoglobin folding

The contributions of some amino acid residues in the A, B, G, and H helices to the formation of the folding nucleus and folding intermediate of apomyoglobin were estimated. The effects of point substitutions of Ala for hydrophobic amino acid residues on the structural stability of the native (N) protein and its folding intermediate (I), as well as on the folding/unfolding rates for four mutant apomyoglobin forms, were studied. The equilibrium and kinetic studies of the folding/unfolding rates of these mutant proteins in a wide range of urea concentrations demonstrated that their native state was considerably destabilized as compared with the wild-type protein, whereas the stability of the intermediate state changed moderately. It was shown that the amino acid residues in the A, G, and H helices contributed insignificantly to the stabilization of the apomyoglobin folding nucleus in the rate-limiting I ? N transition, taking place after the formation of the intermediate, whereas the residue of the B helix was of great importance in the formation of the folding nucleus in this transition.     

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

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

A protein dissection study demonstrates that two specific hydrophobic clusters play a key role in stabilizing the core structure of the molten globule state of human alpha-lactalbumin

The molten globule state of alpha-lactalbumin (alpha LA) has served as a paradigm for understanding the role of these partially folded states in protein folding. We previously showed that a peptide construct consisting of the A and B helices (residues 1-38) cross-linked to the D- and C-terminal 3(10) helices (residues 101-120) of alpha LA is capable of folding to a stable molten globule-like state. Here, we report the study of three peptide constructs that are designed to investigate the contribution two short hydrophobic sequences located near the C-terminus of alpha LA make to the structure and stability of the alpha LA molten globule state. These regions of the protein have been shown to form stable non-native structures in isolation. The three peptide constructs contain residues 1-38 cross-linked to three separate C-terminal peptides via the native 28-111 disulfide bond. The C-terminal peptides consist of residues 101-114, 106-120, and 106-114. The results of CD, fluorescence, ANS binding, and urea denaturation experiments indicate that constructs that lack either of the hydrophobic sequences (residues 101-105 and 115-120) are significantly less structured. These results highlight the importance of long-range, mutually stabilizing interactions within the molten globule state of the protein. Proteins 2001;42:237-242.     

Equilibrium phi-analysis of a molten globule: the 1-149 apoflavodoxin fragment

The apoflavodoxin fragment comprising residues 1-149 that can be obtained by chemical cleavage of the C-terminal alpha-helix of the full-length protein is known to populate a molten globule conformation that displays a cooperative behaviour and experiences two-state urea and thermal denaturation. Here, we have used a recombinant form of this fragment to investigate molten globule energetics and to derive structural information by equilibrium Phi-analysis. We have characterized 15 mutant fragments designed to probe the persistence of native interactions in the molten globule and compared their conformational stability to that of the equivalent full-length apoflavodoxin mutants. According to our data, most of the mutations analysed modify the stability of the molten globule fragment following the trend observed when the same mutations are implemented in the full-length protein. However, the changes in stability observed in the molten globule are much smaller and the Phi-values calculated are (with a single exception) below 0.4. This is consistent with an overall and significant debilitation of the native structure. Nevertheless, the fact that the molten globule fragment can be stabilised using as a guide the native structure of the full-length protein (by increasing helix propensity, optimising charge interactions and filling small cavities) suggests that the overall structure of the molten globule is still quite close to native, in spite of the lowered stability observed.     

Effects of the stabilization of the molten globule state on the folding mechanism of alpha-lactalbumin: a study of a chimera of bovine and human alpha-lactalbumin

The N-terminal half of the alpha-domain (residues 1 to 34) is more important for the stability of the acid-induced molten globule state of alpha-lactalbumin than the C-terminal half (residues 86 to 123). The refolding and unfolding kinetics of a chimera, in which the amino acid sequence of residues 1 to 34 was from human alpha-lactalbumin and the remainder of the sequence from bovine alpha-lactalbumin, were studied by stopped-flow tryptophan fluorescence spectroscopy. The chimeric protein refolded and unfolded substantially faster than bovine alpha-lactalbumin. The stability of the molten globule state formed by the chimera was greater than that of bovine alpha-lactalbumin, and the hydrophobic surface area buried inside of the molecule in the molten globule state was increased by the substitution of residues 1 to 34. Peptide fragments corresponding to the A- and B-helix of the chimera showed higher helix propensity than those of the bovine protein, indicating the contribution of local interactions to the high stability of the molten globule state of the chimera. Moreover, the substitution of residues 1-34 decreased the free energy level of the transition state and increased hydrophobic surface area buried inside of the molecule in the transition state. Our results indicate that local interactions as well as hydrophobic interactions formed in the molten globule state are important in guiding the subsequent structural formation of alpha-lactalbumin.     

Nm23-H1/NDP kinase folding intermediates and cancer: a hypothesis

The Nm23-H1/nucleoside diphosphate (NDP) kinase A is a metastasis suppressor, besides its enzymatic activity. The mutant S120G has been found in high-grade neuroblastomas. The mutant protein, once denatured in urea, is unable to refold in vitro. A size-exclusion chromatography analysis of the folding/association pathway showed that recombinant wild-type and S120G mutant human Nm23-H1/NDP kinase A unfold and refold passing through a molten globule state while typical hexameric NDP kinases unfold without dissociated species and refold through a native monomeric intermediate. A survey of the recent literature showed that several proteins involved in cancer, and their mutants, are marginally stable, like the wild-type Nm23-H1/NDP kinase A, or are misfolded, like its S120G mutant. We therefore suggest that the low thermodynamic stability and the folding intermediate of the Nm23-H1/NDP kinase A may be necessary for its regulatory properties.