Mutational Studies Uncover Non-Native Structure in the Dimeric Kinetic Intermediate of the H2A-H2B Heterodimer

The folding pathway of the histone H2A-H2B heterodimer minimally includes an on-pathway, dimeric, burst-phase intermediate, I₂. The partially folded H2A and H2B monomers populated at equilibrium were characterized as potential monomeric kinetic intermediates. Folding kinetics were compared for initiation from isolated, folded monomers and the heterodimer unfolded in 4 M urea. The observed rates were virtually identical above 0.4 M urea, exhibiting a log-linear relationship on the final denaturant concentration. Below ∼ 0.4 M urea (concentrations inaccessible from the  4-M urea unfolded state), a rollover in the rates was observed; this suggests that a component of the I₂ ensemble contains non-native structure that rearranges/isomerizes to a more native-like species. The contribution of helix propensity to the stability of the I₂ ensemble was assessed with a set of H2A-H2B mutants containing Ala and Gly replacements at nine sites, focusing mainly on the long, central α2 helix. Equilibrium and kinetic folding/unfolding data were collected to determine the effects of the mutations on the stability of I₂ and the transition state between I₂ and N₂. This limited mutational study indicated that residues in the α2 helices of H2A and H2B as well as α1 of H2B and both the C-terminus of α3 and the short αC helix of H2A contribute to the stability of the I₂ burst-phase species. Interestingly, at least eight of the nine targeted residues stabilize I₂ by interactions that are non-native to some extent. Given that destabilizing I₂ and these non-native interactions does not accelerate folding, it is concluded that the native and non-native structures present in the I₂ ensemble enable efficient folding of H2A-H2B.     

Temperature‐induced partially unfolded state of hUBF HMG Box‐5: Conformational and dynamic investigations of the Box‐5 thermal intermediate ensemble

Human upstream binding factor (hUBF) HMG Box‐5 is a highly conserved protein domain, containing 84 amino acids and belonging to the family of the nonspecific DNA‐binding HMG boxes. Its native structure adopts a twisted L shape, which consists of three α‐helices and two hydrophobic cores: the major wing and the minor wing. In this article, we report a reversible three‐state thermal unfolding equilibrium of hUBF HMG Box‐5, which is investigated by differential scanning calorimetry (DSC), circular dichroism spectroscopy, fluorescence spectroscopy, and NMR spectroscopy. DSC data show that Box‐5 unfolds reversibly in two separate stages. Spectroscopic analyses suggest that different structural elements exhibit noncooperative transitions during the unfolding process and that the major form of the Box‐5 thermal intermediate ensemble at 55°C shows partially unfolded characteristics. Compared with previous thermal stability studies of other boxes, it appears that Box‐5 possesses a more stable major wing and two well separated subdomains. NMR chemical shift index and sequential ¹H^N_i‐¹H^N_i+1 NOE analyses indicate that helices 1 and 2 are native‐like in the thermal intermediate ensemble, while helix 3 is partially unfolded. Detailed NMR relaxation dynamics are compared between the native state and the intermediate ensemble. Our results implicate a fluid helix‐turn‐helix folding model of Box‐5, where helices 1 and 2 potentially form the helix 1‐turn‐helix 2 motif in the intermediate, while helix 3 is consolidated only as two hydrophobic cores form to stabilize the native structure. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Amino Acid Insertion Reveals a Necessary Three-Helical Intermediate in the Folding Pathway of the Colicin E7 Immunity Protein Im7

The small (87-residue) α-helical protein Im7 (an inhibitor protein for colicin E7 that provides immunity to cells producing colicin E7) folds via a three-state mechanism involving an on-pathway intermediate. This kinetic intermediate contains three of four native helices that are oriented in a non-native manner so as to minimise exposed hydrophobic surface area at this point in folding. The short (6-residue) helix III has been shown to be unstructured in the intermediate ensemble and does not dock onto the developing hydrophobic core until after the rate-limiting transition state has been traversed. After helix III has docked, it adopts an α-helical secondary structure, and the side chains of residues within this region provide contacts that are crucial to native-state stability. In order to probe further the role of helix III in the folding mechanism of Im7, we created a variant that contains an eight-amino-acid polyalanine-like helix stabilised by a Glu-Arg salt bridge and an Asn-Pro-Gly capping motif, juxtaposed C-terminal to the natural 6-residue helix III. The effect of this insertion on the structure of the native protein and its folding mechanism were studied using NMR and ?-value analysis, respectively. The results reveal a robust native structure that is not perturbed by the presence of the extended helix III. Mutational analysis performed to probe the folding mechanism of the redesigned protein revealed a conserved mechanism involving the canonical three-helical intermediate. The results suggest that folding via a three-helical species stabilised by both native and non-native interactions is an essential feature of Im7 folding, independent of the helical propensity of helix III.     

An isolated helix persists in a sparsely populated form of KIX under native conditions

NMR relaxation dispersion techniques were used to investigate conformational exchange of the three-helix bundle protein KIX under native conditions. These experiments provide site-resolved kinetic information about microsecond-to-millisecond time scale motions along with structural (chemical shift) information without requiring a perturbation of the equilibrium. All kinetic data are consistent with an apparent two-state transition between natively folded KIX and a partially unfolded high-energy state that is populated to 3.0 +/- 0.2% at 27 degrees C. By combining (13)C- and (15)N-based experiments that probe specific structural aspects, we show that the sparsely populated high-energy state displays a strong conformational preference. An isolated secondary structural element, C-terminal helix alpha3, is highly populated, while the hydrophobic core of the domain and the remainder of the protein backbone, including helices alpha1 and alpha2, are disordered and devoid of specific interactions. This high-energy state presumably represents the equilibrium analogue of a folding intermediate that is transiently populated in stopped-flow kinetic experiments [Horng, J. C., Tracz, S. M., Lumb, K. J., and Raleigh, D. P. (2002) Biochemistry 44, 627-634].     

Single-Molecule Studies of the Im7 Folding Landscape

Under appropriate conditions, the four-helical Im7 (immunity protein 7) folds from an ensemble of unfolded conformers to a highly compact native state via an on-pathway intermediate. Here, we investigate the unfolded, intermediate, and native states populated during folding using diffusion single-pair fluorescence resonance energy transfer by measuring the efficiency of energy transfer (or proximity or P ratio) between pairs of fluorophores introduced into the side chains of cysteine residues placed in the center of helices 1 and 4, 1 and 3, or 2 and 4. We show that while the native states of each variant give rise to a single narrow distribution with high P values, the distributions of the intermediates trapped at equilibrium (denoted I^eqm) are fitted by two Gaussian distributions. Modulation of the folding conditions from those that stabilize the intermediate to those that destabilize the intermediate enabled the distribution of lower P value to be assigned to the population of the unfolded ensemble in equilibrium with the intermediate state. The reduced stability of the I^eqm variants allowed analysis of the effect of denaturant concentration on the compaction and breadth of the unfolded state ensemble to be quantified from 0 to 6 M urea. Significant compaction is observed as the concentration of urea is decreased in both the presence and absence of sodium sulfate, as previously reported for a variety of proteins. In the presence of Na₂SO₄ in 0 M urea, the P value of the unfolded state ensemble approaches that of the native state. Concurrent with compaction, the ensemble displays increased peak width of P values, possibly reflecting a reduction in the rate of conformational exchange among iso-energetic unfolded, but compact conformations. The results provide new insights into the initial stages of folding of Im7 and suggest that the unfolded state is highly conformationally constrained at the outset of folding.     

Conformational properties of the unfolded state of Im7 in nondenaturing conditions

The unfolded ensemble in aqueous solution represents the starting point of protein folding. Characterisation of this species is often difficult since the native state is usually predominantly populated at equilibrium. Previous work has shown that the four-helix protein, Im7 (immunity protein 7), folds via an on-pathway intermediate. While the transition states and folding intermediate have been characterised in atomistic detail, knowledge of the unfolded ensemble under the same ambient conditions remained sparse. Here, we introduce destabilising amino acid substitutions into the sequence of Im7, such that the unfolded state becomes predominantly populated at equilibrium in the absence of denaturant. Using far- and near-UV CD, fluorescence, urea titration and heteronuclear NMR experiments, we show that three amino acid substitutions (L18A–L19A–L37A) are sufficient to prevent Im7 folding, such that the unfolded state is predominantly populated at equilibrium. Using measurement of chemical shifts, ¹⁵N transverse relaxation rates and sedimentation coefficients, we show that the unfolded species of L18A–L19A–L37A deviates significantly from random-coil behaviour. Specifically, we demonstrate that this unfolded species is compact (R_h = 25 Å) relative to the urea-denatured state (R_h ≥ 30 Å) and contains local clusters of hydrophobic residues in regions that correspond to the four helices in the native state. Despite these interactions, there is no evidence for long-range stabilising tertiary interactions or persistent helical structure. The results reveal an unfolded ensemble that is conformationally restricted in regions of the polypeptide chain that ultimately form helices I, II and IV in the native state.     

NMR characterisation of the relationship between frustration and the excited state of Im7

Previous work shows that Im9 folds in a two-state transition while its homologue Im7 folds in a three-state transition via an on-pathway kinetic intermediate state (KIS), with this difference being related to frustration in the structure of Im7. We have used NMR spectroscopy to study conformational dynamics connected to the frustration. A combination of equilibrium peptide N¹H/N²H exchange, model-free analyses of backbone NH relaxation data and relaxation dispersion (RD)-NMR shows that the native state of Im7 is in equilibrium with an intermediate state that is lowly populated [equilibrium intermediate state (EIS)]. Comparison of kinetic and thermodynamic parameters describing the EIS native-state equilibrium obtained by RD-NMR with previously reported parameters describing the KIS native-state equilibrium obtained from stopped-flow fluorescence studies of refolding His-tagged Im7 shows that the KIS and the EIS are the same species. ¹⁵N chemical shifts of the EIS obtained from the RD-NMR analysis show that residues forming helix III in the native state are unstructured in the EIS while other residues experiencing frustration in the native state are in structured regions of the EIS. We show that binding of Im7 and its L53A/I54A variant (which resembles the EIS as shown in previous work) to the cognate partner for Im7, the DNase domain of colicin E7, causes the dynamic processes associated with the frustration to be dampened.     

Characterisation of the conformational properties of urea-unfolded Im7: implications for the early stages of protein folding

The colicin immunity protein Im7 folds from its unfolded state in 6 M urea to its native four-helix structure through an on-pathway intermediate that lacks one of the helices of the native structure (helix III). In order to further characterize the folding mechanism of Im7, we have studied the conformational properties of the protein unfolded in 6 M urea in detail using heteronuclear NMR. Triple-resonance experiments with 13C/15N-labelled Im7 in 6 M urea provided almost complete resonance assignments for the backbone nuclei, and measurement of backbone 15N relaxation parameters allowed dynamic ordering of the unfolded polypeptide chain to be investigated. Reduced spectral density mapping and fitting backbone R2 relaxation rates to a polymer dynamics model identified four clusters of interacting residues, each predicted by the average area buried upon folding for each residue. Chemical shift analyses and measurement of NOEs detected with a long mixing-time 1H-1H-15N NOESY-HSQC spectrum confirmed the formation of four clusters. Each cluster of interacting side-chains in urea-unfolded Im7 occurs in a region of the protein that forms a helix in the protein, with the largest clusters being associated with the three long helices that are formed in the on-pathway folding intermediate, whilst the smallest cluster forms a helix only in the native state. NMR studies of a Phe15Ala Im7 variant and a protein in which residues 51-56 are replaced by three glycine residues (H3G3 Im7*), indicated that the clusters do not interact with each other, possibly because they are solvated by urea, as indicated by analysis of NOEs between the protein and the solvent. Based on these data, we suggest that dilution of the chaotrope to initiate refolding will result in collapse of the clusters, leading to the formation of persistent helical structure and the generation of the three-helix folding intermediate.     

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.     

Mutational analysis of the stability of the H2A and H2B histone monomers

The eukaryotic histone heterodimer H2A-H2B folds through an obligatory dimeric intermediate that forms in a nearly diffusion-limited association reaction in the stopped-flow dead time. It is unclear whether there is partial folding of the isolated monomers before association. To address the possible contributions of structure in the monomers to the rapid association, we characterized H2A and H2B monomers in the absence of their heterodimeric partner. By far-UV circular dichroism, the H2A and H2B monomers are 15% and 31% helical, respectively—significantly less than observed in X-ray crystal structures. Acrylamide quenching of the intrinsic Tyr fluorescence was indicative of tertiary structure. The H2A and H2B monomers exhibit free energies of unfolding of 2.5 and 2.9 kcal mol^− 1, respectively; at 10 μM, the sum of the stability of the monomers is ∼ 60% of the stability of the native dimer. The helical content, stability, and m values indicate that H2B has a more stable, compact structure than H2A. The monomer m values are larger than expected for the extended histone fold motif, suggesting that the monomers adopt an overly collapsed structure. Stopped-flow refolding—initiated from urea-denatured monomers or the partially folded monomers populated at low denaturant concentrations—yielded essentially identical rates, indicating that monomer folding is productive in the rapid association and folding of the heterodimer. A series of Ala and Gly mutations were introduced into H2A and H2B to probe the importance of helix propensity on the structure and stability of the monomers. The mutational studies show that the central α-helix of the histone fold, which makes extensive intermonomer contacts, is structured in H2B but only partially folded in H2A.     

Folding Pathways of the Tetrahymena Ribozyme

Like many structured RNAs, the Tetrahymena group I intron ribozyme folds through multiple pathways and intermediates. Under standard conditions in vitro, a small fraction reaches the native state (N) with k_obs ≈ 0.6 min^− 1, while the remainder forms a long-lived misfolded conformation (M) thought to differ in topology. These alternative outcomes reflect a pathway that branches late in folding, after disruption of a trapped intermediate (I_trap). Here we use catalytic activity to probe the folding transitions from I_trap to the native and misfolded states. We show that mutations predicted to weaken the core helix P3 do not increase the rate of folding from I_trap but they increase the fraction that reaches the native state rather than forming the misfolded state. Thus, P3 is disrupted during folding to the native state but not to the misfolded state, and P3 disruption occurs after the rate-limiting step. Interestingly, P3-strengthening mutants also increase native folding. Additional experiments show that these mutants are rapidly committed to folding to the native state, although they reach the native state with approximately the same rate constant as the wild-type ribozyme (~ 1 min^− 1). Thus, the P3-strengthening mutants populate a distinct pathway that includes at least one intermediate but avoids the M state, most likely because P3 and the correct topology are formed early. Our results highlight multiple pathways in RNA folding and illustrate how kinetic competitions between rapid events can have long-lasting effects because the “choice” is enforced by energy barriers that grow larger as folding progresses.     

The folding pathway of an FF domain: characterization of an on-pathway intermediate state under folding conditions by (15)N, (13)C(alpha) and (13)C-methyl relaxation dispersion and (1)H/(2)H-exchange NMR spectroscopy

The FF domain from the human protein HYPA/FBP11 folds via a low-energy on-pathway intermediate (I). Elucidation of the structure of such folding intermediates and denatured states under conditions that favour folding are difficult tasks. Here, we investigated the millisecond time-scale equilibrium folding transition of the 71-residue four-helix bundle wild-type protein by (15)N, (13)C(alpha) and methyl(13)C Carr-Purcell-Meiboom-Gill (CPMG) NMR relaxation dispersion experiments and by (1)H/(2)H-exchange measurements. The relaxation data for the wild-type protein fitted a simple two-site exchange process between the folded state (F) and I. Destabilization of F in mutants A17G and Q19G allowed the detection of the unfolded state U by (15)N CPMG relaxation dispersion. The dispersion data for these mutants fitted a three-site exchange scheme, U<-->I<-->F, with I populated higher than U. The kinetics and thermodynamics of the folding reaction were obtained via temperature and urea-dependent relaxation dispersion experiments, along with structural information on I from backbone (15)N, (13)C(alpha) and side-chain methyl (13)C chemical shifts, with further information from protection factors for the backbone amide groups from (1)H/(2)H-exchange. Notably, helices H1-H3 are at least partially formed in I, while helix H4 is largely disordered. Chemical shift differences for the methyl (13)C nuclei suggest a paucity of stable, native-like hydrophobic interactions in I. These data are consistent with Phi-analysis of the rate-limiting transition state between I and F. The combination of relaxation dispersion and Phi data can elucidate whole experimental folding pathways.     

NMR analysis of partially folded states and persistent structure in the alpha subunit of tryptophan synthase: implications for the equilibrium folding mechanism of a 29-kDa TIM barrel protein

Structural insights into the equilibrium folding mechanism of the alpha subunit of tryptophan synthase (αTS) from Escherichia coli, a (βα)₈ TIM barrel protein, were obtained with a pair of complementary nuclear magnetic resonance (NMR) spectroscopic techniques. The secondary structures of rare high-energy partially folded states were probed by native-state hydrogen-exchange NMR analysis of main-chain amide hydrogens. 2D heteronuclear single quantum coherence NMR analysis of several ¹⁵N-labeled nonpolar amino acids was used to probe the side chains involved in stabilizing a highly denatured intermediate that is devoid of secondary structure. The dynamic broadening of a subset of isoleucine and leucine side chains and the absence of protection against exchange showed that the highest energy folded state on the free-energy landscape is stabilized by a hydrophobic cluster lacking stable secondary structure. The core of this cluster, centered near the N-terminus of αTS, serves as a nucleus for the stabilization of what appears to be nonnative secondary structure in a marginally stable intermediate. The progressive decrease in protection against exchange from this nucleus toward both termini and from the N-termini to the C-termini of several β-strands is best described by an ensemble of weakly coupled conformers. Comparison with previous data strongly suggests that this ensemble corresponds to a marginally stable off-pathway intermediate that arises in the first few milliseconds of folding and persists under equilibrium conditions. A second, more stable intermediate, which has an intact β-barrel and a frayed α-helical shell, coexists with this marginally stable species. The conversion of the more stable intermediate to the native state of αTS entails the formation of a stable helical shell and completes the acquisition of the tertiary structure.     

Conformational dynamics is more important than helical propensity for the folding of the all α‐helical protein Im7

Im7 folds via an on‐pathway intermediate that contains three of the four native α‐helices. The missing helix, helix III, is the shortest and its failure to be formed until late in the pathway is related to frustration in the structure. Im7H3M3, a 94‐residue variant of the 87‐residue Im7 in which helix III is the longest of the four native helices, also folds via an intermediate. To investigate the structural basis for this we calculated the frustration in the structure of Im7H3M3 and used NMR to investigate its dynamics. We found that the native state of Im7H3M3 is highly frustrated and in equilibrium with an intermediate state that lacks helix III, similar to Im7. Model‐free analysis identified residues with chemical exchange contributions to their relaxation that aligned with the residues predicted to have highly frustrated interactions, also like Im7. Finally, we determined properties of urea‐denatured Im7H3M3 and identified four clusters of interacting residues that corresponded to the α‐helices of the native protein. In Im7 the cluster sizes were related to the lengths of the α‐helices with cluster III being the smallest but in Im7H3M3 cluster III was also the smallest, despite this region forming the longest helix in the native state. These results suggest that the conformational properties of the urea‐denatured states promote formation of a three‐helix intermediate in which the residues that form helix III remain non‐helical. Thus it appears that features of the native structure are formed early in folding linked to collapse of the unfolded state.     

Slow folding of a three-helix protein via a compact intermediate

The KIX domain of CREB binding protein (CBP) forms a small three-helix bundle which folds autonomously. Previous equilibrium unfolding experiments led to the suggestion that folding may not be strictly two-state. To investigate the folding mechanism in more detail, the folding kinetics of KIX have been studied by urea jump fluorescence-detected stopped-flow experiments. Clear evidence for an intermediate is obtained from the plot of the natural log of the observed rate constant versus denaturant concentration, the chevron plot, and from analysis of the initial fluorescence amplitudes of the stopped-flow experiments. The chevron plot exhibits a change in shape, rollover, at low denaturant concentrations, characteristic of the formation of an intermediate. The kinetic data can be fit to a three-state model involving a compact intermediate. An on-pathway model predicts that the position of the intermediate lies close to the native state. The folding rate in the absence of denaturant is 260 s(-)(1) at pH 7.5 and 25 degrees C. This is significantly slower than the rates of other helical proteins similar in size. The slow folding may be due to the necessity of forming a buried polar interaction in the native state. The potential functional significance of the folding intermediate is discussed.     

Conservation of transition state structure in fast folding peripheral subunit-binding domains

Φ-Value analysis was used to characterise the structure of the transition state (TS) for folding of POB L146A Y166W, a peripheral subunit-binding domain that folds in microseconds. Helix 2 was structured in the TS with consolidating interactions from the structured loop that connects the two α-helices. This distribution of Φ-values was very similar to that determined for E3BD F166W, a homologue with high sequence and structural similarity. The extrapolated folding rate constants in water at 298 K were 210,000 s^− 1 for POB and 27,500 s^− 1 for E3BD. A contribution to the faster folding of POB came from its having significantly greater helical propensity in helix 2, the folding nucleus. The folding rate also appeared to be influenced by differences in the sequence and structural properties of the loop connecting the two helices. Unimodal downhill folding has been proposed as a conserved, biologically important property of peripheral subunit-binding domains. POB folds five times faster and E3BD folds slower than a proposed limit of 40,000 s^− 1 for barrier-limited folding. However, experimental evidence strongly suggests that both POB L146A Y166W and E3BD F166W fold in a barrier-limited process through a very similar TS ensemble.