Structural analysis of the rate-limiting transition states in the folding of Im7 and Im9: similarities and differences in the folding of homologous proteins

The bacterial immunity proteins Im7 and Im9 fold with mechanisms of different kinetic complexity. Whilst Im9 folds in a two-state transition at pH 7.0 and 10 degrees C, Im7 populates an on-pathway intermediate under these conditions. In order to assess the role of sequence versus topology in the folding of these proteins, and to analyse the effect of populating an intermediate on the landscape for folding, we have determined the conformational properties of the rate-limiting transition state for Im9 folding/unfolding using Phi(F)-value analysis and have compared the results with similar data obtained previously for Im7. The data show that the rate-limiting transition states for Im9 and Im7 folding/unfolding are similar: both are compact (beta(T)=0.94 and 0.89, respectively) and contain three of the four native helices docked around a specific hydrophobic core. Significant differences are observed, however, in the magnitude of the Phi(F)-values obtained for the two proteins. Of the 20 residues studied in both proteins, ten have Phi(F)-values in Im7 that exceed those in Im9 by more than 0.2, and of these five differ by more than 0.4. The data suggest that the population of an intermediate in Im7 results in folding via a transition state ensemble that is conformationally restricted relative to that of Im9. The data are consistent with the view that topology is an important determinant of folding. Importantly, however, they also demonstrate that while the folding transition state may be conserved in homologous proteins that fold with two and three-state kinetics, the population of an intermediate can have a significant effect on the breadth of the transition state ensemble.     

Rapid folding with and without populated intermediates in the homologous four-helix proteins Im7 and Im9

The kinetics and thermodynamics of the folding of the homologous four-helix proteins Im7 and Im9 have been characterised at pH 7.0 and 10 degrees C. These proteins are 60 % identical in sequence and have the same three-dimensional structure, yet appear to fold by different kinetic mechanisms. The logarithm of the folding and unfolding rates of Im9 change linearly as a function of urea concentration and fit well to an equation describing a two-state mechanism (with a folding rate of 1500 s-1, an unfolding rate of 0. 01 s-1, and a highly compact transition state that has approximately 95 % of the native surface area buried). By contrast, there is clear evidence for the population of an intermediate during the refolding of Im7, as indicated by a change in the urea dependence of the folding rate and the presence of a significant burst phase amplitude in the refolding kinetics. Under stabilising conditions (0.25 M Na2SO4, pH 7.0 and 10 degrees C) the folding of Im9 remains two-state, whilst under similar conditions (0.4 M Na2SO4, pH 7.0 and 10 degrees C) the intermediate populated during Im7 refolding is significantly stabilised (KUI=125). Equilibrium denaturation experiments, under the conditions used in the kinetic measurements, show that Im7 is significantly less stable than Im9 (DeltaDeltaG 9.3 kJ/mol) and the DeltaG and m values determined accord with those obtained from the fit to the kinetic data. The results show, therefore, that the population of an intermediate in the refolding of the immunity protein structure is defined by the precise amino acid sequence rather than the global stability of the protein. We discuss the possibility that the intermediate of Im7 is populated due to differences in helix propensity in Im7 and Im9 and the relevance of these data to the folding of helical proteins in general.     

Sulfate-induced effects in the on-pathway intermediate of the bacterial immunity protein Im7

Intermediates have now been identified in the folding of a number of small, single-domain proteins. Here we describe experiments to determine the effect of Na(2)SO(4) on the properties of the on-pathway intermediate formed early during the folding of the four-helical protein, Im7. This intermediate, studied previously in 0.4 M Na(2)SO(4), contains three of the four native helices and is fascinating in that several residues in helices I, II, and IV make non-native interactions that stabilize this state. Whether these contacts form as a consequence of the presence of Na(2)SO(4), however, remained unresolved. Using kinetic analysis of the effect of Na(2)SO(4) on the unfolding and refolding kinetics of Im7*, combined with detailed analysis of the resulting chevron plots, we show that decreasing the concentration of Na(2)SO(4) from 0.4 to 0 M destabilizes the intermediate and rate-limiting transition (TS2) states by 7 and 10 kJ mol(-)(1), respectively, and has little effect on the relative compactness of these states compared with that of the unfolded ensemble (beta(I) approximately 0.8, beta(TS2) approximately 0.9 in 0 to 0.4 M Na(2)SO(4)). Analysis of 10 variants of the protein in 0.2 M Na(2)SO(4) using Phi-values showed that the structural properties of the intermediate and TS2 are not altered significantly by the concentration of the kosmotrope. The data demonstrate that the rapid formation of a compact intermediate stabilized by non-native interactions during Im7* folding is not induced by high concentrations of the stabilizing salt, but is a generic feature of the folding of this protein.     

Acidic conditions stabilise intermediates populated during the folding of Im7 and Im9

The helical bacterial immunity proteins Im7 and Im9 have been shown to fold via kinetic mechanisms of differing complexity, despite having 60 % sequence identity. At pH 7.0 and 10 degrees C, Im7 folds in a three-state mechanism involving an on-pathway intermediate, while Im9 folds in an apparent two-state transition. In order to examine the folding mechanisms of these proteins in more detail, the folding kinetics of both Im7 and Im9 (at 10 degrees C in 0.4 M sodium sulphate) have been examined as a function of pH. Kinetic modelling of the folding and unfolding data for Im7 between pH 5.0 and 8.0 shows that the on-pathway intermediate is stabilised by more acidic conditions, whilst the native state is destabilised. The opposing effect of pH on the stability of these states results in a significant population of the intermediate at equilibrium at pH 6.0 and below. At pH 7.0, the folding and unfolding kinetics for Im9 can be fitted adequately by a two-state model, in accord with previous results. However, under acidic conditions there is a clear change of slope in the plot of the logarithm of the folding rate constant versus denaturant concentration, consistent with the population of one or more intermediate(s) early during folding. The kinetic data for Im9 at these pH values can be fitted to a three-state model, where the intermediate ensemble is stabilised and the native state destabilised as the pH is reduced, rationalising previous results that showed that an intermediate is not observed experimentally at pH 7.0. The data suggest that intermediate formation is a general step in immunity protein folding and demonstrate that it is necessary to explore a wide range of refolding conditions in order to show that intermediates do not form in the folding of other small, single-domain proteins.     

Tuning lambda6-85 towards downhill folding at its melting temperature

The five-helix bundle lambda6-85* is a fast two-state folder. Several stabilized mutants have been reported to fold kinetically near-downhill or downhill. These mutants undergo a transition to two-state folding kinetics when heated. It has been suggested that this transition is caused by increased hydrophobicity at higher temperature. Here we investigate two histidine-containing mutants of lambda6-85* to see if a weaker hydrophobic core can extend the temperature range of downhill folding. The very stable lambdaHA is the fastest-folding lambda repressor to date (k(f)(-1) approximately k(obs)(-1)=2.3 micros at 44 degrees C). It folds downhill at low temperature, but transits back to two-state folding at its unfolding midpoint. lambdaHG has a weakened hydrophobic core. It is less stable than some slower folding mutants of lambda6-85*, and it has more exposed hydrophobic surface area in the folded state. This mutant nonetheless folds very rapidly, and has the non-exponential folding kinetics of an incipient downhill folder even at the unfolding midpoint (k(m)(-1) approximately 2 micros, k(a)(-1)=15 micros at 56 degrees C). We also compare the thermodynamic melting transition of lambdaHG with the nominal two-state folding mutant lambdaQG, which has a similar melting temperature. Unlike lambdaQG, lambdaHG yields fluorescence wavelength-dependent cooperativities and probe-dependent melting temperatures. This result combined with previous work shows that the energy landscapes of lambda repressor mutants support all standard folding mechanisms.     

Sequential barriers and an obligatory metastable intermediate define the apparent two-state folding pathway of the ubiquitin-like PB1 domain of NBR1

The 90-residue N-terminal Phox and Bem1p (PB1) domain of NBR1 forms an α/β ubiquitin-like fold. Kinetic analysis using stopped-flow fluorescence reveals two-state kinetics; however, nonlinear effects in the denaturant dependence of the unfolding data demonstrate changes in the position of the rate-limiting barrier along the folding coordinate as the folding conditions change. The kinetics of wt-PB1 and several mutants show that this curvature is consistent with a single-pathway mechanism involving sequential transition states (TS1 and TS2) separated by a transiently populated high-energy intermediate, rather than movement of the transition state on a broad energy plateau. We show that the two transition states within the sequential model represent structurally and thermodynamically distinct species. TS1 is a collapsed state (α_TS1 = 0.71) with a large enthalpic barrier to formation that is rate-limiting under conditions that strongly favour folding. TS2 is highly native-like (α_TS2 = 0.93) and represents a late entropic barrier to formation of the native state. In support of the sequential transition state mechanism, we show that the G62A helix 2 substitution stabilises TS1 and the intermediate to such an extent that the latter becomes significantly populated, leading to the observation of a fast kinetic phase representing the initial U → I transition, with TS2 (α_TS2 = 0.87) becoming rate-limiting. The folding rate is not retarded by populating an intermediate, which would be expected for a misfold state, but is accelerated, suggesting that the I state is productive and on-pathway. The results show that the apparent two-state folding of the wt-PB1 domain occurs along a well-defined pathway involving structurally and thermodynamically distinct sequential transition states and an obligatory metastable intermediate that represents a productive local minimum in the energy landscape that increases the efficiency of barrier crossing through favourable effects on the entropy of activation.     

The nature of the free energy barriers to two-state folding

We introduce a simple procedure to analyze the temperature dependence of the folding and unfolding rates of two-state proteins. We start from the simple transition-state-like rate expression: k = D(eff)exp(-DeltaG(TS)/RT), in which upper and lower bounds for the intra-chain effective diffusion coefficient (D(eff)) are obtained empirically using the timescales of elementary processes in protein folding. From the changes in DeltaG(TS) as a function of temperature, we calculate enthalpies and heat capacities of activation, together with the more elusive entropies of activation. We then estimate the conformational entropy of the transition state by extrapolation to the temperature at which the solvation entropy vanishes by cancellation between polar and apolar terms. This approach is based on the convergence temperatures for the entropy of solvating apolar (approximately 385 K) and polar groups (approximately 335 K), the assumption that the structural properties of the transition state are somewhere in between the unfolded and folded states, and the established relationship between observed heat capacity and solvent accessibility.1 To circumvent the lack of structural information about transition states, we use the empirically determined heat capacities of activation as constraints to identify the extreme values of the transition state conformational entropy that are consistent with experiment. The application of this simple approach to six two-state folding proteins for which there is temperature-dependent data available in the literature provides important clues about protein folding. For these six proteins, we obtain an average equilibrium cost in conformational entropy of -4.3 cal x mol(-1)K(-1)per residue, which is in close agreement to previous empirical and computational estimates of the same quantity. Furthermore, we find that all these proteins have a conformationally diverse transition state, with more than half of the conformational entropy of the unfolded state. In agreement with predictions from theory and computer simulations, the transition state signals the change from a regime dominated by loss in conformational entropy to one driven by the gain in stabilization free energy (i.e., including protein interactions and solvation effects). Moreover, the height of the barrier is determined by how much stabilization free energy is realized at that point, which is related to the relative contribution of local versus non-local interactions. A remarkable observation is that the fraction of conformational entropy per residue that is present in the transition state is very similar for the six proteins in this study. Based on this commonality, we propose that the observed change in thermodynamic regime is connected to a change in the pattern of structure formation: from one driven by formation of pairwise interactions to one dominated by coupling of the networks of interactions involved in forming the protein core. In this framework, the barrier to two-state folding is crossed when the folding protein reaches a "critical native density" that allows expulsion of remaining interstitial water and consolidation of the core. The principle of critical native density should be general for all two-state proteins, but can accommodate different folding mechanisms depending on the particularities of the structure and sequence.     

The folding pathway of barnase: the rate-limiting transition state and a hidden intermediate under native conditions

The nature of the rate-limiting transition state at zero denaturant (TS(1)) and whether there are hidden intermediates are the two major unsolved problems in defining the folding pathway of barnase. In earlier studies, it was shown that TS(1) has small phi values throughout the structure of the protein, suggesting that the transition state has either a defined partially folded secondary structure with all side chains significantly exposed or numerous different partially unfolded structures with similar stability. To distinguish the two possibilities, we studied the effect of Gly mutations on the folding rate of barnase to investigate the secondary structure formation in the transition state. Two mutations in the same region of a beta-strand decreased the folding rate by 20- and 50-fold, respectively, suggesting that the secondary structures in this region are dominantly formed in the rate-limiting transition state. We also performed native-state hydrogen exchange experiments on barnase at pD 5.0 and 25 degrees C and identified a partially unfolded state. The structure of the intermediate was investigated using protein engineering and NMR. The results suggest that the intermediate has an omega loop unfolded. This intermediate is more folded than the rate-limiting transition state previously characterized at high denaturant concentrations (TS(2)). Therefore, it exists after TS(2) in folding. Consistent with this conclusion, the intermediate folds with the same rate and denaturant dependence as the wild-type protein, but unfolds faster with less dependence on the denaturant concentration. These and other results in the literature suggest that barnase folds through partially unfolded intermediates that exist after the rate-limiting step. Such folding behavior is similar to those of cytochrome c and Rd-apocyt b(562). Together, we suggest that other small apparently two-state proteins may also fold through hidden intermediates.     

The effect of increasing the stability of non-native interactions on the folding landscape of the bacterial immunity protein Im9

How stabilising non-native interactions influence protein folding energy landscapes is currently not well understood: such interactions could speed folding by reducing the conformational search to the native state, or could slow folding by increasing ruggedness. Here, we examine the influence of non-native interactions in the folding process of the bacterial immunity protein Im9, by exploiting our ability to manipulate the stability of the intermediate and rate-limiting transition state (TS) in the folding of this protein by minor alteration of its sequence or changes in solvent conditions. By analysing the properties of these species using Phi-value analysis, and exploration of the structural properties of the TS ensemble using molecular dynamics simulations, we demonstrate the importance of non-native interactions in immunity protein folding and demonstrate that the rate-limiting step involves partial reorganisation of these interactions as the TS ensemble is traversed. Moreover, we show that increasing the contribution to stability made by non-native interactions results in an increase in Phi-values of the TS ensemble without altering its structural properties or solvent-accessible surface area. The data suggest that the immunity proteins fold on multiple, but closely related, micropathways, resulting in a heterogeneous TS ensemble that responds subtly to mutation or changes in the solvent conditions. Thus, altering the relative strength of native and non-native interactions influences the search to the native state by restricting the pathways through the folding energy landscape.     

Is there an en route folding intermediate for cold shock proteins?

Cold shock proteins (Csps) play an important role in cold shock response of a diverse number of organisms ranging from bacteria to humans. Numerous studies of the Csp from various species showed that a two-state folding mechanism is conserved and the transition state (TS) appears to be very compact. However, the atomic details of the folding mechanism of Csp remain unclear. This study presents the folding mechanism of Csp in atomic detail using an all-atom Go model-based simulations. Our simulations predict that there may exist an en route intermediate, in which β strands 1-2-3 are well ordered and the contacts between β1 and β4 are almost developed. Such an intermediate might be too unstable to be detected in the previous fluorescence energy transfer experiments. The transition state ensemble has been determined from the P(fold) analysis and the TS appears even more compact than the intermediate state.     

Folding thermodynamics and kinetics of the leucine-rich repeat domain of the virulence factor Internalin B

Although the folding of alpha-helical repeat proteins has been well characterized, much less is known about the folding of repeat proteins containing beta-sheets. Here we investigate the folding thermodynamics and kinetics of the leucine-rich repeat (LRR) domain of Internalin B (InlB), an extracellular virulence factor from the bacterium Lysteria monocytogenes. This domain contains seven tandem leucine-rich repeats, of which each contribute a single beta-strand that forms a continuous beta-sheet with neighboring repeats, and an N-terminal alpha-helical capping motif. Despite its modular structure, InlB folds in an equilibrium two-state manner, as reflected by the identical thermodynamic parameters obtained by monitoring its sigmoidal urea-induced unfolding transition by different spectroscopic probes. Although equilibrium two-state folding is common in alpha-helical repeat proteins, to date, InlB is the only beta-sheet-containing repeat protein for which this behavior is observed. Surprisingly, unlike other repeat proteins exhibiting equilibrium two-state folding, InlB also folds by a simple two-state kinetic mechanism lacking intermediates, aside from the effects of prolyl isomerization on the denatured state. However, like other repeat proteins, InlB also folds significantly more slowly than expected from contact order. When plotted against urea, the rate constants for the fast refolding and single unfolding phases constitute a linear chevron that, when fitted with a kinetic two-state model, yields thermodynamic parameters matching those observed for equilibrium folding. Based on these kinetic parameters, the transition state is estimated to comprise 40% of the total surface area buried upon folding, indicating that a large fraction of the native contacts are formed in the rate-limiting step to folding.     

D/H amide kinetic isotope effects reveal when hydrogen bonds form during protein folding

We have exploited a procedure to identify when hydrogen bonds (H-bonds) form under two-state folding conditions using equilibrium and kinetic deuterium/hydrogen amide isotope effects. Deuteration decreases the stability of equine cytochrome c and the dimeric and crosslinked versions of the GCN4-p1 coiled coil by approximately 0. 5 kcal mol-1. For all three systems, the decrease in equilibrium stability is reflected by a decrease in refolding rates and a near equivalent increase in unfolding rates. This apportionment indicates that approximately 50% of the native H-bonds are formed in the transition state of these helical proteins. In contrast, an alpha/beta protein, mammalian ubiquitin, exhibits a small isotope effect only on unfolding rates, suggesting its folding pathway may be different. These four proteins recapitulate the general trend that approximately 50% of the surface buried in the native state is buried in the transition state, leading to the hypothesis that H-bond formation in the transition state is cooperative, with alpha-helical proteins forming a number of H-bonds proportional to the amount of surface buried in the transition state.     

Calorimetric study of a series of designed repeat proteins: modular structure and modular folding

Repeat proteins comprise tandem arrays of a small structural motif. Their structure is defined and stabilized by interactions between residues that are close in the primary sequence. Several studies have investigated whether their structural modularity translates into modular thermodynamic properties. Tetratricopeptide repeat proteins (TPRs) are a class in which the repeated unit is a 34 amino acid helix-turn-helix motif. In this work, we use differential scanning calorimetry (DSC) to study the equilibrium stability of a series of TPR proteins with different numbers of an identical consensus repeat, from 2 to 20, CTPRa2 to CTPRa20. The DSC data provides direct evidence that the folding/unfolding transition of CTPR proteins does not fit a two-state folding model. Our results confirm and expand earlier studies on TPR proteins, which showed that apparent two-state unfolding curves are better fit by linear statistical mechanics models: 1D Ising models in which each repeat is treated as an independent folding unit.     

Folding pathway of FKBP12 and characterisation of the transition state.

The folding pathway of human FKBP12, a 12 kDa FK506-binding protein (immunophilin), has been characterised. Unfolding and refolding rate constants have been determined over a wide range of denaturant concentrations and data are shown to fit to a two-state model of folding in which only the denatured and native states are significantly populated, even in the absence of denaturant. This simple model for folding, in which no intermediate states are significantly populated, is further supported from stopped-flow circular dichroism experiments in which no fast "burst" phases are observed. FKBP12, with 107 residues, is the largest protein to date which folds with simple two-state kinetics in water (kF=4 s(-1)at 25 degrees C). The topological crossing of two loops in FKBP12, a structural element suggested to cause kinetic traps during folding, seems to have little effect on the folding pathway.The transition state for folding has been characterised by a series of experiments on wild-type FKBP12. Information on the thermodynamic nature of, the solvent accessibility of, and secondary structure in, the transition state was obtained from experiments measuring the unfolding and refolding rate constants as a function of temperature, denaturant concentration and trifluoroethanol concentration. In addition, unfolding and refolding studies in the presence of ligand provided information on the structure of the ligand-binding pocket in the transition state. The data suggest a compact transition state relative to the unfolded state with some 70 % of the surface area buried. The ligand-binding site, which is formed mainly by two loops, is largely unstructured in the transition state. The trifluoroethanol experiments suggest that the alpha-helix may be formed in the transition state. These results are compared with results from protein engineering studies and molecular dynamics simulations (see the accompanying paper).     

Apo-azurin folds via an intermediate that resembles the molten-globule

The folding of Pseudomonas aeruginosa apo-azurin was investigated with the intent of identifying putative intermediates. Two apo-mutants were constructed by replacing the main metal-binding ligand C112 with a serine (C112S) and an alanine (C112A). The guanidinium-induced unfolding free energies (DeltaG(U-N)(H2O)) of the C112S and C112A mutants were measured to 36.8 +/- 1 kJ mole(-1) and 26.1 +/- 1 kJ mole(-1), respectively, and the m-value of the transition to 23.5 +/- 0.7 kJ mole(-1) M(-1). The difference in folding free energy (DeltaDeltaG(U-N)(H2O)) is largely attributed to the intramolecular hydrogen bonding properties of the serine Ogamma in the C112S mutant, which is lacking in the C112A structure. Furthermore, only the unfolding rates differ between the two mutants, thus pointing to the energy of the native state as the source of the observed Delta DeltaG(U-N)(H2O). This also indicates that the formation of the hydrogen bonds present in C112S but absent in C112A is a late event in the folding of the apo-protein, thus suggesting that formation of the metal-binding site occurs after the rate-limiting formation of the transition state. In both mutants we also noted a burst-phase intermediate. Because this intermediate was capable of binding 1-anilinonaphtalene-8-sulfonate (ANS), as were an acid-induced species at pH 2.6, we ascribe it molten globule-like status. However, despite the presence of an intermediate, the folding of apo-azurin C112S is well approximated by a two-state kinetic mechanism.     

Presence of the cofactor speeds up folding of Desulfovibrio desulfuricans flavodoxin

Flavodoxin is an alpha/beta protein with a noncovalently bound flavin-mononucleotide (FMN) cofactor. The apo-protein adopts a structure identical to that of the holo-form, although there is more dynamics in the FMN-binding loops. The equilibrium unfolding processes of Azotobacter vinelandii apo-flavodoxin, and Desulfovibrio desulfuricans ATCC strain 27774 apo- and holo-flavodoxins involve rather stable intermediates. In contrast, we here show that both holo- and apo-forms of flavodoxin from D. desulfuricans ATCC strain 29577 (75% sequence similarity with the strain 27774 protein) unfold in two-state equilibrium processes. Moreover, the FMN cofactor remains bound to the unfolded holo-protein. The folding and unfolding kinetics for holo-flavodoxin exhibit two-state behavior, albeit an additional slower phase is present at very low denaturant concentrations. The extrapolated folding time in water for holo-flavodoxin, approximately 280 microsec, is in excellent agreement with that predicted from the protein's native-state topology. Unlike the holo-protein behavior, the folding and unfolding reactions for apo-flavodoxin are best described by two kinetic phases, with rates differing approximately 15-fold, suggesting the presence of a kinetic intermediate. Both folding phases for apo-flavodoxin are orders of magnitude slower (40- and 530-fold, respectively) than that for the holo-protein. We conclude that polypeptide-cofactor interactions in the unfolded state of D. desulfuricans strain 29577 flavodoxin alter the kinetic-folding path towards two-state and speed up the folding reaction.     

Cytochrome c folds through a smooth funnel

A dominant feature of folding of cytochrome c is the presence of nonnative His-heme kinetic traps, which either pre-exist in the unfolded protein or are formed soon after initiation of folding. The kinetically trapped species can constitute the majority of folding species, and their breakdown limits the rate of folding to the native state. A temperature jump (T-jump) relaxation technique has been used to compare the unfolding/folding kinetics of yeast iso-2 cytochrome c and a genetically engineered double mutant that lacks His-heme kinetic traps, H33N,H39K iso-2. The results show that the thermodynamic properties of the transition states are very similar. A single relaxation time tau(obs) is observed for both proteins by absorbance changes at 287 nm, a measure of solvent exclusion from aromatic residues. At temperatures near Tm, the midpoint of the thermal unfolding transitions, tau(obs) is four to eight times faster for H33N,H39K iso-2 (tau(obs) approximately 4-10 ms) than for iso-2 (tau(obs) approximately 20-30 ms). T-jumps show that there are no kinetically unresolved (tau < 1-3 micros T-jump dead time) "burst" phases for either protein. Using a two-state model, the folding (k(f)) and unfolding (k(u)) rate constants and the thermodynamic activation parameters standard deltaGf, standard deltaGu, standard deltaHf, standard deltaHu, standard deltaSf, standard deltaSu are evaluated by fitting the data to a function describing the temperature dependence of the apparent rate constant k(obs) (= tau(obs)(-1)) = k(f) + k(u). The results show that there is a small activation enthalpy for folding, suggesting that the barrier to folding is largely entropic. In the "new view," a purely entropic kinetic barrier to folding is consistent with a smooth funnel folding landscape.     

Comparison of the transition state ensembles for folding of Im7 and Im9 determined using all-atom molecular dynamics simulations with phi value restraints

Delineation of the structural properties of transition states is key to deriving models for protein folding. Here we describe the structures of the transition states of the bacterial immunity proteins Im7 and Im9 obtained by all-atom molecular dynamics simulations with phi value restraints derived from protein engineering experiments. This pair of proteins is of special interest because, at pH 7 and 10 degrees C, Im7 folds via an intermediate while Im9 folds with a two-state transition. The structures of the transition states for Im7 and Im9, together with their radii of gyration and distances from the native state, are similar. The typical distance between any two members of the transition state ensemble of both proteins is large, with that of Im9 nearly twice that of Im7. Thus, a broad range of structures make up the transition state ensembles of these proteins. The ensembles satisfy the set of rather low phi values and yet are consistent with high beta(T) values (> 0.85 for both proteins). For both Im7 and Im9 the inter-helical angles are highly variable in the transition state ensembles, although the native contacts between helices I and IV are well conserved. By measuring the distribution of the accessible surface area for each residue we show that the hydrophobic residues that are buried in the native state remain buried in the transition state, corresponding to a hydrophobic collapse to a relatively ordered globule. The data provide new insights into the structural properties of the transition states of these proteins at an atomic level of detail and show that molecular dynamics simulations with phi value restraints can significantly enhance the knowledge of the transition state ensembles (TSE) provided by the experimental phi values alone.     

Test for cooperativity in the early kinetic intermediate in lysozyme folding

During the folding of many proteins, collapsed globular states are formed prior to the native structure. The role of these states for the folding process has been widely discussed. Comparison with properties of synthetic homo and heteropolymers had suggested that the initial collapse represented a shift of the ensemble of unfolded conformations to more compact states without major energy barriers. We investigated the folding/unfolding transition of a collapsed state, which transiently populates early in lysozyme folding. This state forms within the dead-time of stopped-flow mixing and it has been shown to be significantly more compact and globular than the denaturant-induced unfolded state. We used the GdmCl-dependence of the dead-time signal change to characterize the unfolding transition of the burst phase intermediate. Fluorescence and far-UV CD give identical unfolding curves, arguing for a cooperative two-state folding/unfolding transition between unfolded and collapsed lysozyme. These results show that collapse leads to a distinct state in the folding process, which is separated from the ensemble of unfolded molecules by a significant energy barrier. NMR, fluorescence and small angle X-ray scattering data further show that some local interactions in unfolded lysozyme exist at denaturant concentrations above the coil-collapse transition. These interactions might play a crucial role in the kinetic partitioning between fast and slow folding pathways.     

Unfolding and refolding of porcine odorant binding protein in guanidinium hydrochloride: equilibrium studies at neutral pH

Unfolding and refolding studies on porcine odorant binding protein (pOBP) have been performed at pH 7 in the presence of guanidinium hydrochloride (GdnHCl). Unfolding, monitored by following changes of protein fluorescence and circular dichroism (CD), was found to be a reversible process, in terms of recovered structure and function. The equilibrium transition data were fitted by a simple two-state sigmoidal function of denaturant concentration and the thermodynamic folding parameters, derived from the two techniques, were very similar (average values: C(1/2) approximately 2.4 M, m approximately 2 kcal mol(-1) M(-1), DeltaG(unf,w)(0) approximately 4.7 kcal mol(-1)). The transition was independent of protein concentration, indicating that only monomeric species are involved. Only a minor protective effect by the fluorescent ligand 1-amino-anthracene (AMA) against protein unfolding was detected, whereas dihydromyrcenol (DHM) stabilised the protein to a larger extent (DeltaC(1/2) approximately 0.5 M). Refolding was complete, when the protein, denatured with GdnHCl, was diluted with buffer. On the other hand, refolding by dialysis was largely prevented by concomitant aggregation. The present results on pOBP are compared with those on bovine OBP (bOBP) [Biochim. Biophys. Acta 1599 (2002) 90], where subunit folding is accompanied by domain swapping. We finally suggest that the generally observed two-state folding of many lipocalins is probably favoured by their beta-barrel topology.