Equilibrium Folding Transitions Research Articles

Intrinsic vs Environment Driven Equilibrium Folding Transitions in GTPase Effector Domain of Dynamin: NMR Insights

Relative importance of the intrinsic properties of the polypeptide chain vis-a-vis the environmental influences, in driving the folding of a protein, has been a subject of extensive debate and investigation. Folding/misfolding lead to self association in many systems, which have biological functional significance. We compare here, the NMR derived equilibrium folding transitions driven under different environmental conditions in the GTPase Effector Domain of dynamin, which self-associates into megadalton size species. We conclude that though hierarchy of folding and association of GED is substantially influenced by the solvents, these properties, to a good extent are also driven by intrinsic properties of the polypeptide chain, and the regions that form secondary structures, the types of secondary structures formed in those regions, and finally the regions that participate in the self-association are the same, indicating near neighbor interactions would have a telling effect on the final outcome of the folding process. These observations would open a new reliable frontier for elucidating the multiple folding trajectories and consequent self-association, by simulations in vacuum, for this protein.

Structural Rearrangements Linked to Global Folding Pathways of the Azoarcus Group I Ribozyme

Stable RNAs must fold into specific three-dimensional structures to be biologically active, yet many RNAs form metastable structures that compete with the native state. Our previous time-resolved footprinting experiments showed that Azoarcus group I ribozyme forms its tertiary structure rapidly (τ < 30 ms) without becoming significantly trapped in kinetic intermediates. Here, we use stopped-flow fluorescence spectroscopy to probe the global folding kinetics of a ribozyme containing 2-aminopurine in the loop of P9. The modified ribozyme was catalytically active and exhibited two equilibrium folding transitions centered at 0.3 and 1.6 mM Mg 2+, consistent with previous results. Stopped-flow fluorescence revealed four kinetic folding transitions with observed rate constants of 100, 34, 1, and 0.1 s − 1 at 37 °C. From comparison with time-resolved Fe(II)-ethylenediaminetetraacetic acid footprinting of the modified ribozyme under the same conditions, these folding transitions were assigned to formation of the I C intermediate, tertiary folding and docking of the nicked P9 tetraloop, reorganization of the P3 pseudoknot, and refolding of nonnative conformers, respectively. The footprinting results show that 50–60% of the modified ribozyme folds in less than 30 ms, while the rest of the RNA population undergoes slow structural rearrangements that control the global folding rate. The results show how small perturbations to the structure of the RNA, such as a nick in P9, populate kinetic folding intermediates that are not observed in the natural ribozyme.

The Folding Pathway of an FF domain: Characterization of an On-pathway Intermediate State Under Folding Conditions by 15N, 13C α and 13C-methyl Relaxation Dispersion and 1H/ 2H-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 15N, 13C α and methyl 13C Carr-Purcell-Meiboom-Gill (CPMG) NMR relaxation dispersion experiments and by 1H/ 2H-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 15N 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 15N, 13C α and side-chain methyl 13C chemical shifts, with further information from protection factors for the backbone amide groups from 1H/ 2H-exchange. Notably, helices H1–H3 are at least partially formed in I, while helix H4 is largely disordered. Chemical shift differences for the methyl 13C nuclei suggest a paucity of stable, native-like hydrophobic interactions in I. These data are consistent with Φ-analysis of the rate-limiting transition state between I and F. The combination of relaxation dispersion and Φ data can elucidate whole experimental folding pathways.

Residue-level NMR View of the Urea-driven Equilibrium Folding Transition of SUMO-1 (1-97): Native Preferences Do Not Increase Monotonously

SUMO-1 (1-97) is a crucial protein in the machinery of post-translational modifications. We observed by circular dichroism and fluorescence spectroscopy that urea-induced unfolding of this protein is a complex process with the possibility of occurrence of detectable intermediates along the way. The tertiary structure is completely lost around ∼4.5 M urea with a transition mid-point at 2.53 M urea, while the secondary structure unfolding seems to show two transitions, with mid-points at 2.42 M and 5.69 M urea. We have elucidated by systematic urea titration, the equilibrium residue level structural and dynamics changes along the entire folding/unfolding transition by multidimensional NMR. With urea dilution, the protein is seen to progressively lose most of the broad β-domain structural preferences present at 8 M urea, acquire some helical propensities at 5 M urea, and lose some of them again on further dilution of urea. Between 3 M and 2 M urea, the protein starts afresh to acquire native structural features. These observations are contrary to the conventional notion that proteins fold with monotonously increasing native-type preferences. For folding below ∼3 M urea, the region around the α1 helix appears to be a potential folding initiation site. The folding seems to start with a collapse into native-like topologies, at least in parts, and is followed by formation of secondary and tertiary structure, perhaps by cooperative rearrangements. The motional characteristics of the protein show sequence-dependent variation as the concentration of urea is progressively reduced. At the sub-nanosecond level, the features are extremely unusual for denatured states, and only certain segments corresponding to the flexible regions in the native protein display these motions at the different concentrations of urea.

Folding of the Tetrahymena ribozyme by polyamines: importance of counterion valence and size.

Polyamines are abundant metabolites that directly influence gene expression. Although the role of polyamines in DNA condensation is well known, their role in RNA folding is less understood. Non-denaturing gel electrophoresis was used to monitor the equilibrium folding transitions of the Tetrahymena ribozyme in the presence of polyamines. All of the polyamines tested induce near-native structures that readily convert to the native conformation in Mg(2+). The stability of the folded structure increases with the charge of the polyamine and decreases with the size of the polyamine. When the counterion excluded volume becomes large, the transition to the native state does not go to completion even under favorable folding conditions. Brownian dynamics simulations of a model polyelectrolyte suggest that the kinetics of counterion-mediated collapse and the dimensions of the collapsed RNA chains depend on the structure of the counterion. The results are consistent with delocalized condensation of polyamines around the RNA. However, the effective charge of the counterions is lowered by their excluded volume. The stability of the folded RNA is enhanced when the spacing between amino groups matches the distance between adjacent phosphate groups. These results show how changes in intracellular polyamine concentrations could alter RNA folding pathways.

Folding of an abridged beta-lactamase.

The effects of C-terminal truncation on the equilibrium folding transitions and folding kinetics of B. licheniformis exo small beta-lactamase (ES-betaL) have been measured. ES-betaL lacking 19 residues (ES-betaL(C)(Delta)(19)) has no enzymic activity. Deletion of the last 14 residues produces ES-betaL(C)(Delta)(14), which is 0.1% active. The enzyme lacking nine residues (ES-betaL(C)(Delta)(9)) is nearly fully active, has native optical and hydrodynamic properties, and is protease resistant, a distinguishing feature of the wild-type enzyme. Although ES-betaL(C)(Delta)(9) folds properly, it does so 4 orders of magnitude slower than ES-betaL, making possible the isolation and characterization of a compact intermediate state (I(P) ES-betaL(C)(Delta)(9)). Based on the analysis of folding rates and equilibrium constants, we propose that equilibrium between I(P) ES-betaL(C)(Delta)(9) and other intermediate slow folding. Residues removed in ES-betaL(C)(Delta)(9) and ES-betaL(C)(Delta)(14) are helical and firmly integrated into the enzyme body through many van der Waals interactions involving residues distant in sequence. The results suggest that the deleted residues play a key role in the folding process and also the existence of a modular organization of the protein matrix, at the subdomain level. The results are compared with other examples of this kind in the folding literature.

SecA folds via a dimeric intermediate.

Though many proteins in the cell are large and multimeric, their folding has not been extensively studied. We have chosen SecA as a folding model because it is a large, homodimeric protein (monomer molecular mass of 102 kDa) with multiple folding domains. SecA is the ATPase for the Sec-dependent preprotein translocase of many bacteria. SecA is a soluble protein that can penetrate into the membrane during preprotein translocation. Because SecA may partially unfold prior to its insertion into the membrane, studies of its stability and folding pathway are important for understanding how it functions in vivo. Kinetic folding transitions in the presence of urea were monitored using circular dichroism and tryptophan fluorescence, while equilibrium folding transitions were monitored using the same techniques as well as a fluorescent ATP analogue. The reversible equilibrium folding transition exhibited a plateau, indicating the presence of an intermediate. Based on the data presented here, we propose a three-state model, N(2) if I(2) if 2U, where the native protein unfolds to a dimeric intermediate which then dissociates into two unfolded monomers. The SecA dimer was determined to have an overall stability (DeltaG) of -22.5 kcal/mol. We also investigated the stability of SecA using analytical ultracentrifugation equilibrium and velocity sedimentation, which again indicated that native or refolded SecA was a stable dimer. The rate-limiting step in the folding pathway was conversion of the dimeric intermediate to the native dimer. Unfolding of native, dimeric SecA was slow with a relaxation time in H(2)O of 3.3 x 10(4) s. Since SecA is a stable dimer, dissociation to monomeric subunits during translocation is unlikely.

Temperature dependence of the folding rate in a simple protein model: Search for a “glass” transition

Monte Carlo simulation of model proteins on a cubic lattice are used to study the thermodynamics and kinetics of protein folding over a wide range of temperatures. Both random sequences and sequences designed to have a pronounced minimum of energy are examined. There is no indication in the kinetics of a “glass” transition at low temperature, i.e., below the temperature of the equilibrium folding transition, the kinetics of folding is described by the Arrhenius law at all temperatures that were examined. The folding kinetics is single-exponential in the whole range of studied temperatures for random sequences. The general implications of the temperature dependence of the folding rate are discussed and related to certain properties of the energy spectrum. The results obtained in the simulations are in qualitative disagreement with the conclusions of a theoretical analysis of protein folding kinetics based on certain kinetics assumptions introduced in the Random Energy Model. The origins of the discrepancies are analyzed and a simple phenomenological theory is presented to describe the temperature dependence of the folding time for random sequences.

Cooperativity in protein folding: from lattice models with sidechains to real proteins

Over the past few years novel folding mechanisms of globular proteins have been proposed using minimal lattice and off-lattice models. The factors determining the cooperativity of folding in these models and especially their explicit relation to experiments have not been fully established, however. We consider equilibrium folding transitions in lattice models with and without sidechains. A dimensionless measure, omega c, is introduced to quantitatively assess the degree of cooperativity in lattice models and in real proteins. We show that larger values of omega c resembling the values seen in proteins are obtained in lattice models with sidechains. The enhanced cooperativity of such models results from possible denser packing of sidechains in the interior of the model polypeptide chain. We also establish that omega c correlates extremely well with sigma T = (T o - T f) /T o, where T o and T f are collapse and folding transition temperatures, respectively. These theoretical ideas are used to analyze folding transitions in two-state folders (RNase A, chymotrypsin inhibitor 2, fibronectin type III modules and tendamistat) and three-state folders (apomyoglobin and lysozyme). The values of omega c extracted from experiments show a correlation with sigma T (suitably generalized when folding is induced by denaturants or acid). A quantitative description of the cooperative transition of real proteins can be made by lattice models with sidechains. The degree of cooperativity in minimal models and real proteins can be expressed in terms of the single parameter sigma, which can be estimated from experimental data.

The collapse transition in the HP model

We studied the collapse transition on small homopolymeric and heteropolymeric sequences in the HP model on a two-dimensional square lattice both with exact enumerations and Monte Carlo simulations. For heteropolymers, our analysis is restricted to native sequences (sequences which present one single minimal energy configuration) with a fixed fraction of hydrophobic residues ( ρ= N H/ N=0.5). In the homopolymer limit ( ρ=1) we were able to reproduce several known results such as the exponent ν and the critical temperature for the collapse transition. For the heteropolymer case, we devised a combinatorial extrapolation scheme based on the method used for homopolymers which enabled us to study the dependence of the collapse critical temperature on the monomer sequence in the thermodynamic limit. For the small chain lengths studied we find that the θ-point is strongly dependent on the monomer sequence, our extrapolation scheme suggests that even in the thermodynamic limit, the critical temperature of the collapse transition in the HP model in the square lattice depends on the specific location of the hydrophobic residues in the chain. We have also studied the folding transition, our preliminary results for the chain lengths studied do not indicate the existence of an equilibrium folding transition occurring at a temperature different from that of the collapse transition.

Dynamic Monte Carlo simulations of globular protein folding/unfolding pathways: II. α-Helical motifs

Dynamic Monte Carlo simulations of the folding pathways of α-helical protein motifs have been undertaken in the context of a diamond lattice model of globular proteins. The first question addressed is the nature of the assembly process of an α-helical hairpin. While the hairpin could, in principle, be formed via the diffusion-collision-adhesion of isolated performed helices, this is not the dominant mechanism of assembly found in the simulations. Rather, the helices that form native hairpins are constructed on-site, with folding initiating at or near the turn in almost all cases. Next, the folding/unfolding pathways of four-helix bundles having tight bends and one and two long loops in the native state are explored. Once again, an on-site construction mechanism of folding obtains, with a hairpin forming first, followed by the formation of a three-helix bundle, and finally the fourth helix of the native bundle assembles. Unfolding is essentially the reverse of folding. A simplified analytic theory is developed that reproduces the equilibrium folding transitions obtained from the simulations remarkably well and, for the dominant folding pathway, correctly identifies the intermediates seen in the simulations. The analytic theory provides the free energy along the reaction co-ordinate and identifies the transition state for all three motifs as being quite close to the native state, with three of the four helices assembled, and approximately one turn of the fourth helix in place. The transition state is separated from the native conformation by a free-energy barrier of mainly energetic origin and from the denatured state by a barrier of mainly entropic origin. The general features of the folding pathway seen in all variants of the model four-helix bundles are similar to those observed in the folding of β-barrel, Greek key proteins; this suggests that many of the qualitative aspects of folding are invariant to the particular native state topology and secondary structure.

Secondary structure in ribonuclease: I. Equilibrium folding transitions seen by amide circular dichroism

The amide circular dichroism (c.d.) spectra of RNAase A, RNAase S and S-protein were measured and analyzed in the wavelength region from 200 to 260 nm. The range is sufficient to quantitate reliably the apparent fractional contents of α-helix and antiparallel β-sheet. Extraction of additional structural information was not attempted (e.g. the apparent fraction of β-turns). Various decomposition procedures were tested and gave the same results. Three spectroscopically well-distinct denaturation transitions were detected and characterized: (1) acid denaturation; (2) thermal denaturation; (3) denaturant induced unfolding. 1. (1) Lowering the pH from 6.8 to 1.7 (at 10 °C) results in the loss of some 9% helical content for RNAase S; no change in structural composition was detected for RNAase A. The 9% loss in helix is consistent with the disruption of the S-peptide α-helix of residues 3 to 12. It is known that S-peptide dissociates at low pH values (Richards & Logue, 1962). 2. (2) Thermal denaturation at pH 6.8 or 1.7 destroys the β-structure in RNAase A, RNAase S or S-protein. A fractional change of 36 to 38% is detected, which agrees favorably with the entire β-sheet content (40%) (Wyckoff et al., 1970). In addition, thermal denaturation of RNAase A at pH 6.8 or 1.7 results in a fractional loss of 9% α-helix. No change in apparent helical content is detected for S-protein in the same conditions, although the folded S-protein moiety accounts for almost two-thirds of the helical content of ribonuclease. RNAase S behaves during thermal denaturation: at low pH the same as S-protein; at neutral pH like RNAase A, with a lower, concentration-dependent melting temperature ( t m), however. 3. (3) Random coil c.d. spectra are generated by subjecting thermally denatured or reduced and carboxymethylated RNAase A, RNAase S or S-protein to proteolytic digestion by trypsin (at neutral pH) or pepsin (at pH 1.7). A c.d. spectrum very similar to that of digested material is obtained with undigested material in 9 m-urea or 6 m-guanidine hydrochloride (GuHCl). The difference between the spectra of thermally denatured material and material denatured by 6 m-GuHCl is consistent in shape with an α-helix to coil difference spectrum, and in magnitude with a fraction of 10 to 12% of the residues being involved. The latter figure and the 9% change in helical content assigned to the denaturation of the S-peptide add up satisfactorily to the 21% helical content of RNAase S in the crystal structure (Wyckoff et al., 1970). Comparing the changes in apparent structural composition during denaturation under various conditions obtained for RNAase A, RNAase S and S-protein allows one to analyze the mutual stabilization of the different secondary structural elements. (1) The folded β-sheet is necessary in order to observe folding of the S-peptide helix. It has been shown earlier that (low affinity) fragment association is not sufficient (Labhardt, 1981). (2) Folding of the β-sheet alone cannot stabilize the entire helical content of the S-protein moiety in the presence of GuHCl. (3) The folded helical S-peptide stabilizes both, the β-sheet and the entire S-protein moiety helix content.