Effect of a single point mutation on the stability,residual structure and dynamics in the denatured state of GED: Relevance to self-assembly

The GTPase effector domain (GED) of dynamin forms large soluble oligomers in vitro, while its mutant – I697A – lacks this property at low concentrations. With a view to understand the intrinsic structural characteristics of the polypeptide chain, the global unfolding characteristics of GED wild type (WT) and I697A were compared using biophysical techniques. Quantitative analysis of the CD and fluorescence denaturation profiles revealed that unfolding occurred by a two-state process and the mutant was less stable than the WT. Even in the denatured state, the mutation caused chemical shift perturbations and significant differences were observed in the ¹⁵N transverse relaxation rates (R₂), not only at the mutation site but all around. These results demonstrate that the hydrophobic change associated with the mutation perturbs the structural and motional preferences locally, which are then relayed via different folding pathways along the chain and the property of oligomerization in the native state is affected.     

Exploring the Denatured State Ensemble by Single-Molecule Chemo-Mechanical Unfolding: The Effect of Force,Temperature, and Urea

While it is widely appreciated that the denatured state of a protein is a heterogeneous conformational ensemble, there is still debate over how this ensemble changes with environmental conditions. Here, we use single-molecule chemo-mechanical unfolding, which combines force and urea using the optical tweezers, together with traditional protein unfolding studies to explore how perturbants commonly used to unfold proteins (urea, force, and temperature) affect the denatured-state ensemble. We compare the urea m-values, which report on the change in solvent accessible surface area for unfolding, to probe the denatured state as a function of force, temperature, and urea. We find that while the urea- and force-induced denatured states expose similar amounts of surface area, the denatured state at high temperature and low urea concentration is more compact. To disentangle these two effects, we use destabilizing mutations that shift the T_m and C_m. We find that the compaction of the denatured state is related to changing temperature as the different variants of acyl-coenzyme A binding protein have similar m-values when they are at the same temperature but different urea concentration. These results have important implications for protein folding and stability under different environmental conditions.     

1H, 15N, 13C resonance assignment of 9.7 M urea-denatured state of the GTPase effector domain (GED) of dynamin

The GTPase effector domain (GED) of dynamin, a multi-domain protein involved in endocytosis, forms a megadalton-sized self-assembly (even at micromolar concentrations) in native conditions in vitro. While such large assemblies have remained inaccessible to detailed NMR structural characterization, till date, a significant recent achievement has been the elucidation of the GED association pathway starting from a Gdn-HCl denatured monomer. Since, the nature of the denaturant has a strong influence on the conformational preferences in the denatured states, and hence on the association pathways, or even on the final assembly, we report here the NMR resonance assignment of 9.7 M urea-denatured GED from Homo sapiens. This will form the basis for the characterization of the association pathways and the final assembly driven by urea dilution. Jeetender Chugh and Shilpy Sharma have contributed equally.     

Understanding the mechanism of beta-hairpin folding via phi-value analysis

The folding kinetics of a 16-residue beta-hairpin (trpzip4) and five mutants were studied by a laser-induced temperature-jump infrared method. Our results indicate that mutations which affect the strength of the hydrophobic cluster lead to a decrease in the thermal stability of the beta-hairpin, as a result of increased unfolding rates. For example, the W45Y mutant has a phi-value of approximately zero, implying a folding transition state in which the native contacts involving Trp45 are not yet formed. On the other hand, mutations in the turn or loop region mostly affect the folding rate. In particular, replacing Asp46 with Ala leads to a decrease in the folding rate by roughly 9 times. Accordingly, the phi-value for D46A is determined to be approximately 0.77, suggesting that this residue plays a key role in stabilizing the folding transition state. This is most likely due to the fact that the main chain and side chain of Asp46 form a characteristic hydrogen bond network with other residues in the turn region. Taken together, these results support the folding mechanism we proposed before, which suggests that the turn formation is the rate-limiting step in beta-hairpin folding and, consequently, a stronger turn-promoting sequence increases the stability of a beta-hairpin primarily by increasing its folding rate, whereas a stronger hydrophobic cluster increases the stability of a beta-hairpin primarily by decreasing its unfolding rate. In addition, we have examined the compactness of the thermally denatured and urea-denatured states of another 16-residue beta-hairpin, using the method of fluorescence resonance energy transfer. Our results show that the thermally denatured state of this beta-hairpin is significantly more compact than the urea-denatured state, suggesting that the very first step in beta-hairpin folding, when initiated from an extended conformation, probably corresponds to a process of hydrophobic collapse.     

Titration Properties and Thermodynamics of the Transition State for Folding: Comparison of Two-state and Multi-state Folding Pathways

CI2 folds and unfolds as a single cooperative unit by simple two-state kinetics, which enables the properties of the transition state to be measured from both the forward and backward rate constants. We have examined how the free energy of the transition state for the folding of chymotrypsin inhibitor 2 (CI2) changes with pH and temperature. In addition to the standard thermodynamic quantities, we have measured the overall acid-titration properties of the transition state and its heat capacity relative to both the denatured and native states. We were able to determine the latter by a method analogous to a well-established procedure for measuring the change in heat capacity for equilibrium unfolding: the enthalpy of activation of unfolding at different values of acid pH were plotted against the average temperature of each determination. Our results show that the transition state of CI2 has lost most of the electrostatic and van der Waals' interactions that are found in the native state, but it remains compact and this prevents water molecules from entering some parts of the hydrophobic core. The properties of the transition state of CI2 are then compared with the major folding transition state of the larger protein barnase, which folds by a multi-state mechanism, with the accumulation of a partly structured intermediate (D^physor I). CI2 folds from a largely unstructured denatured state under physiological conditionsviaa transition state which is compact but relatively uniformly unstructured, with tertiary and secondary structure being formed in parallel. We term this an expanded pathway. Conversely, barnase folds from a largely structured denatured state in which elements of structure are well formed through a transition state that has islands of folded elements of structure. We term this a compact pathway. These two pathways may correspond to the two extreme ends of a continuous spectrum of protein folding mechanisms. Although the properties of the two transition states are very different, the activation barrier for folding (D^phys→3 ) is very similar for both proteins.     

Comparison of temperature effect on the guanidine hydrochloride and urea denaturations of lysozyme

Guanidine hydrochloride (GdnHCl) and urea denaturations of lysozyme have been observed at various temperatures by measuring changes in fluorescence. Both transitions appear to be two state, with GdnHCl almost twice as effecitve a denaturant as urea for this protein. By plotting the denaturant concentrations at midpoint of the transition vs. the experimental temperature, it can be demonstrated that urea-denatured lysozyme does not obtain the degree of unfolding found in lysozyme denatured by GdnHCl.     

Osmolyte effects on kinetics of FKBP12 C22A folding coupled with prolyl isomerization

Unfolding and refolding kinetics of human FKBP12 C22A were monitored by fluorescence emission over a wide range of urea concentration in the presence and absence of protecting osmolytes glycerol, proline, sarcosine and trimethylamine-N-oxide (TMAO). Unfolding is well described by a mono-exponential process, while refolding required a minimum of two exponentials for an adequate fit throughout the urea concentration range considered. The bi-exponential behavior resulted from complex coupling between protein folding, and prolyl isomerization in the denatured state in which the urea-dependent rate constant for folding was greater than, equal to, and less than the rate constants for prolyl isomerization within the urea concentration range of zero to five molar. Amplitudes and the observed folding and unfolding rate constants were fitted to a reversible three-state model composed of two sequential steps involving the native state and a folding-competent denatured species thermodynamically linked to a folding-incompetent denatured species. Excellent agreement between thermodynamic parameters for FKBP12 C22A folding calculated from the kinetic parameters and those obtained directly from equilibrium denaturation assays provides strong support for the applicability of the mechanism, and provides evidence that FKBP12 C22A folding/unfolding is two-state, with prolyl isomer heterogeneity in the denatured ensemble. Despite the chemical diversity of the protecting osmolytes, they all exhibit the same kinetic behavior of increasing the rate constant of folding and decreasing the rate constant for unfolding. Osmolyte effects on folding/unfolding kinetics are readily explained in terms of principles established in understanding osmolyte effects on protein stability. These principles involve the osmophobic effect, which raises the Gibbs energy of the denatured state due to exposure of peptide backbone, thereby increasing the folding rate. This effect also plays a key role in decreasing the unfolding rate when, as is often the case, the activated complex exposes more backbone than is exposed in the native state.     

Mechanisms of the multiphasic kinetics in the folding and unfolding of globular proteins

The multiphasic kinetics of the protein folding and unfolding processes are examined for a “cluster model” with only two thermodynamically stable macroscopic states, native (N) and denatured (D), which are essentially distributions of microscopic states. The simplest kinetic schemes consistent with the model are: N-(fast) → I-(slow) → D for unfolding and N ← (fast)-D₂ ← (slow)-D₁ for refolding. The fast phase during the unfolding process can be visualized as the redistribution of the native population N to I within its free energy valley. Then, this population crosses over the free energy barrier to the denatured state D in the slow phase. Therefore, the macrostate I is a kinetic intermediate which is not stable at equilibrium. For the refolding process, the initial equilibrium distribution of the denatured state D appears to be separated into D₁ and D₂ in the final condition because of the change in position of the free energy barrier. The fast refolding species D₂ is due to the “leak” from the broadly distributed D state, while the rest is the slow refolding species D₁, which must overpass the free energy barrier to reach N. At an early stage of the folding process the amino acid chain is considered to be composed of several locally ordered regions, which we call clusters, connected by random coil chain parts. Thus, the denatured state contains different sizes and distributions of clusters depending on the external condition. A later stage of the folding process is the association of smaller clusters. The native state is expressed by a maximum-size cluster with possible fluctuation sites reflecting this association. A general discussion is given of the correlation between the kinetics and thermodynamics of proteins from the overall shape of the free energy function. The cluster model provides a conceptual link between the folding kinetics and the structural patterns of globular proteins derived from the X-ray crystallographic data.     

Complete Reversible Refolding of a G-Protein Coupled Receptor on a Solid Support

The factors defining the correct folding and stability of integral membrane proteins are poorly understood. Folding of only a few select membrane proteins has been scrutinised, leaving considerable deficiencies in knowledge for large protein families, such as G protein coupled receptors (GPCRs). Complete reversible folding, which is problematic for any membrane protein, has eluded this dominant receptor family. Moreover, attempts to recover receptors from denatured states are inefficient, yielding at best 40–70% functional protein. We present a method for the reversible unfolding of an archetypal family member, the β₁-adrenergic receptor, and attain 100% recovery of the folded, functional state, in terms of ligand binding, compared to receptor which has not been subject to any unfolding and retains its original, folded structure. We exploit refolding on a solid support, which could avoid unwanted interactions and aggregation that occur in bulk solution. We determine the changes in structure and function upon unfolding and refolding. Additionally, we employ a method that is relatively new to membrane protein folding; pulse proteolysis. Complete refolding of β₁-adrenergic receptor occurs in n-decyl-β-D-maltoside (DM) micelles from a urea-denatured state, as shown by regain of its original helical structure, ligand binding and protein fluorescence. The successful refolding strategy on a solid support offers a defined method for the controlled refolding and recovery of functional GPCRs and other membrane proteins that suffer from instability and irreversible denaturation once isolated from their native membranes.     

Cooperative Unfolding of Residual Structure in Heat Denatured Proteins by Urea and Guanidinium Chloride

The denatured states of proteins have always attracted our attention due to the fact that the denatured state is the only experimentally achievable state of a protein, which can be taken as initial reference state for considering the in vitro folding and defining the native protein stability. It is known that heat and guanidinium chloride (GdmCl) give structurally different states of RNase-A, lysozyme, α-chymotrypsinogen A and α-lactalbumin. On the contrary, differential scanning calorimetric (DSC) and isothermal titration calorimetric measurements, reported in the literature, led to the conclusion that heat denatured and GdmCl denatured states are thermodynamically and structurally identical. In order to resolve this controversy, we have measured changes in the far-UV CD (circular dichroism) of these heat-denatured proteins on the addition of different concentrations of GdmCl. The observed sigmoidal curve of each protein was analyzed for Gibbs free energy change in the absence of the denaturant (ΔG ⁰ _X→D) associated with the process heat denatured (X) state ↔ GdmCl denatured (D) state. To confirm that this thermodynamic property represents the property of the protein alone and is not a manifestation of salvation effect, we measured urea-induced denaturation curves of these heat denatured proteins under the same experimental condition in which GdmCl-induced denaturation was carried out. In this paper we report that (a) heat denatured proteins contain secondary structure, and GdmCl (or urea) induces a cooperative transition between X and D states, (b) for each protein at a given pH and temperature, thermodynamic cycle connects quantities, ΔG ⁰ _N→X (native (N) state ↔ X state), ΔG ⁰ _X→D and ΔG ⁰ _N→D (N state ↔ D state), and (c) there is not a good enthalpy difference between X and D states, which is the reason for the absence of endothermic peak in DSC scan for the transition, X state ↔ D state.     

Multy-state protein: Determination of carbonic anhydrase free-energy landscape

Studies of the folding pathway of large proteins whose kinetics is complicated due to the formation of several intermediate states are most frequently impeded or totally impossible because of rapid folding phase occurring during instrument dead time. In this paper the obtaining of energy characteristics of one of such proteins—carbonic anhydrase B—is reported. Tryptophan fluorescence and absorption methods have been used to measure the folding and unfolding kinetics of carbonic anhydrase B at different urea concentrations. In spite of the fact that the formation of the initial intermediate state of this protein takes place during the instrument dead time, the population of this state has been estimated in a wide range of urea concentrations. The use of the population of the rapidly formed intermediate state and the effective rates of slow phases of the protein folding/unfolding permitted us to calculate free energies of all the protein states and the height of energy barriers between them. It has been shown that folding of carbonic anhydrase B can be described by a consecutive reaction scheme. The possibility to obtain energy characteristics of carbonic anhydrase would allow studying structural characteristics of both intermediate and transition states via site-directed mutations.     

Atomic-level structure characterization of an ultrafast folding mini-protein denatured state

Atomic-level analyses of non-native protein ensembles constitute an important aspect of protein folding studies to reach a more complete understanding of how proteins attain their native form exhibiting biological activity. Previously, formation of hydrophobic clusters in the 6 M urea-denatured state of an ultrafast folding mini-protein known as TC5b from both photo-CIDNP NOE transfer studies and FCS measurements was observed. Here, we elucidate the structural properties of this mini-protein denatured in 6 M urea performing (15)N NMR relaxation studies together with a thorough NOE analysis. Even though our results demonstrate that no elements of secondary structure persist in the denatured state, the heterogeneous distribution of R(2) rate constants together with observing pronounced heteronuclear NOEs along the peptide backbone reveals specific regions of urea-denatured TC5b exhibiting a high degree of structural rigidity more frequently observed for native proteins. The data are complemented with studies on two TC5b point mutants to verify the importance of hydrophobic interactions for fast folding. Our results corroborate earlier findings of a hydrophobic cluster present in urea-denatured TC5b comprising both native and non-native contacts underscoring their importance for ultra rapid folding. The data assist in finding ways of interpreting the effects of pre-existing native and/or non-native interactions on the ultrafast folding of proteins; a fact, which might have to be considered when defining the starting conditions for molecular dynamics simulation studies of protein folding.     

Sequence specific 1H, 13C and 15N resonance assignments of hahellin in 8 M urea

The sequence specific ¹H, ¹³C and ¹⁵N resonance assignments of hahellin in 8 M urea-denatured state have been accomplished by NMR spectroscopy. Secondary chemical shift analysis reveals the native-like propensities for β-rich conformation in the denatured state.     

Denaturant dependence of equilibrium unfolding intermediates and denatured state structure of horse ferricytochrome c

Nuclear magnetic resonance spectroscopy is employed to characterize unfolding intermediates and the denatured state of horse ferricytochrome c in guanidine hydrochloride. Unfolded and partially unfolded species with non-native heme ligation are detected by analysis of hyperfine-shifted (1)H resonances. Two equilibrium unfolding intermediates with His-Lys heme axial ligation are detected, as are two unfolded species with bis-His heme ligation. These results are contrasted with previous results on horse ferricytochrome c denaturation by urea, for which only one unfolding intermediate and one unfolded species were detected by NMR spectroscopy. Urea and guanidine hydrochloride are often used interchangeably in protein denaturation studies, but these results and those of others indicate that unfolded and intermediate states in these two denaturants may have substantially different properties. Implications of these results for folding studies and the biological function of mitochondrial cytochromes c are discussed.     

Investigation of folding/unfolding kinetics of apomyoglobin

Apomyoglobin kinetic and equilibrium unfolding and folding processes were studied at pH 6.2, 11 degrees C by stopped-flow tryptophan fluorescence. There are two distinct consecutive processes in apomyoglobin folding process, namely, the protein fast transition between the unfolded (U) and an intermediate (I) states (U <----> I) and slow transition between the intermediate and the native (N) states (I <----> N). Accumulation of the intermediate state was observed in the wide range of urea concentrations. The presence of the intermediate state was shown even beyond the middle transition on the unfolding limb. The dependence of observed folding/unfolding rates on urea concentration (chevron plot) was obtained. The shape of this dependence was compared with that of two-state proteins, folding from the U to N state.     

Copper binding traps the folded state of the SCO protein from Bacillus subtilis

The SCO protein from the aerobic bacterium Bacillus subtilis (BsSCO) is involved in the assembly of the cytochrome c oxidase complex, and specifically with the Cu_A center. BsSCO has been proposed to play various roles in Cu_A assembly including, the direct delivery of copper ions to the Cu_A site, and/or maintaining the appropriate redox state of the cysteine ligands during formation of Cu_A. BsSCO binds copper in both Cu(II) and Cu(I) redox states, but has a million-fold higher affinity for Cu(II). As a prerequisite to kinetic studies, we measured equilibrium stability of oxidized, reduced and Cu(II)-bound BsSCO by chemical and thermal induced denaturation. Oxidized and reduced apo-BsSCO exhibit two-state behavior in both chemical- and thermal-induced unfolding. However, the Cu(II) complex of BsSCO is stable in up to nine molar urea. Thermal or guanidinium-induced unfolding of BsSCO-Cu(II) ensues only as the Cu(II) species is lost. The effect of copper (II) on the folding of BsSCO is complicated by a rapid redox reaction between copper and reduced, denatured BsSCO. When denatured apo-BsSCO is refolded in the presence of copper (II) some of the population is recovered as the BsSCO-Cu(II) complex and some is oxidized indicating that refolding and oxidation are competing processes. The proposed functional roles for BsSCO in vivo require that its cysteine residues are reduced and the presence of copper during folding may be detrimental to BsSCO attaining its functional state.     

Conformational stability and thermodynamic characterization of the lipoic acid bearing domain of human mitochondrial branched chain alpha-ketoacid dehydrogenase

The lipoic acid bearing domain (hbLBD) of human mitochondrial branched chain alpha-ketoacid dehydrogenase (BCKD) plays important role of substrate channeling in oxidative decarboxylation of the branched chain alpha-ketoacids. Recently hbLBD has been found to follow two-step folding mechanism without detectable presence of stable or kinetic intermediates. The present study describes the conformational stability underlying the folding of this small beta-barrel domain. Thermal denaturation in presence of urea and isothermal urea denaturation titrations are used to evaluate various thermodynamic parameters defining the equilibrium unfolding. The linear extrapolation model successfully describes the two-step; native state <-->denatured state unfolding transition of hbLBD. The average temperature of maximum stability of hbLBD is estimated as 295.6 +/- 0.9 K. Cold denaturation of hbLBD is also predicted and discussed.     

Water and urea interactions with the native and unfolded forms of a beta-barrel protein

A fundamental understanding of protein stability and the mechanism of denaturant action must ultimately rest on detailed knowledge about the structure, solvation, and energetics of the denatured state. Here, we use (17)O and (2)H magnetic relaxation dispersion (MRD) to study urea-induced denaturation of intestinal fatty acid-binding protein (I-FABP). MRD is among the few methods that can provide molecular-level information about protein solvation in native as well as denatured states, and it is used here to simultaneously monitor the interactions of urea and water with the unfolding protein. Whereas CD shows an apparently two-state transition, MRD reveals a more complex process involving at least two intermediates. At least one water molecule binds persistently (with residence time >10 nsec) to the protein even in 7.5 M urea, where the large internal binding cavity is disrupted and CD indicates a fully denatured protein. This may be the water molecule buried near the small hydrophobic folding core at the D-E turn in the native protein. The MRD data also provide insights about transient (residence time <1 nsec) interactions of urea and water with the native and denatured protein. In the denatured state, both water and urea rotation is much more retarded than for a fully solvated polypeptide. The MRD results support a picture of the denatured state where solvent penetrates relatively compact clusters of polypeptide segments.     

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 approximately 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 beta-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 approximately 3 M urea, the region around the alpha1 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.     

Structural Characteristic of the Initial Unfolded State on Refolding Determines Catalytic Efficiency of the Folded Protein in Presence of Osmolytes

Osmolytes are low molecular weight organic molecules accumulated by organisms to assist proper protein folding, and to provide protection to the structural integrity of proteins under denaturing stress conditions. It is known that osmolyte-induced protein folding is brought by unfavorable interaction of osmolytes with the denatured/unfolded states. The interaction of osmolyte with the native state does not significantly contribute to the osmolyte-induced protein folding. We have therefore investigated if different denatured states of a protein (generated by different denaturing agents) interact differently with the osmolytes to induce protein folding. We observed that osmolyte-assisted refolding of protein obtained from heat-induced denatured state produces native molecules with higher enzyme activity than those initiated from GdmCl- or urea-induced denatured state indicating that the structural property of the initial denatured state during refolding by osmolytes determines the catalytic efficiency of the folded protein molecule. These conclusions have been reached from the systematic measurements of enzymatic kinetic parameters (K _m and k _cat), thermodynamic stability (T _m and ΔH _m) and secondary and tertiary structures of the folded native proteins obtained from refolding of various denatured states (due to heat-, urea- and GdmCl-induced denaturation) of RNase-A in the presence of various osmolytes.