pH effects on the stability and dimerization of procaspase-3

pH-dependent conformational changes are known to occur in dimeric procaspase-3, and they have been shown to affect the rate of automaturation. We studied the equilibrium unfolding of procaspase-3(C163S) as a function of pH (between pH 8.5 and pH 4) in order to examine these changes in the context of folding and stability. The data show that the procaspase dimer undergoes a pH-dependent dissociation below pH 5, so that the protein is mostly monomeric at pH 4. Consistent with this, the dimer unfolds via a four-state process between pH 8.5 and pH 4.75, in which the native dimer isomerizes to a dimeric intermediate, and the dimeric intermediate dissociates to a monomer, which then unfolds. In contrast, a small protein concentration dependence was observed by circular dichroism, but not by fluorescence emission, at pH 4.5 and pH 4.2. There was no protein-concentration dependence to the data collected at pH 4. Overall, the results are consistent with the redistribution of the population of native dimer (N(2)) to dimeric intermediate (I(2)) to monomeric intermediate (I), as the pH is lowered so that at pH 4, the "native" ensemble resembles the monomeric intermediate (I) observed during unfolding at higher pH. An emerging picture of the monomeric procaspase is discussed. Procaspase-3 is most stable at pH approximately 7 (24-26 kcal/mol), and while the stability decreased with pH, it was observed that dimerization contributes the majority (>70%) of the conformational free energy.     

Folding of a de novo designed native-like four-helix bundle protein

The folding of a model native-like dimeric four-helix bundle protein, (alpha(2))(2), was investigated using guanidine hydrochloride, hydrostatic pressure, and low temperature. Unfolding by guanidine hydrochloride followed by circular dichroism and intrinsic fluorescence spectroscopy revealed a highly cooperative transition between the native-like and unfolded states, with free energy of unfolding determined from CD data, DeltaG(unf) = 14.3 +/- 0.8 kcal/mol. However, CD and intrinsic fluorescence data were not superimposable, indicating the presence of an intermediate state during the folding transition. To stabilize the folding intermediate, we used hydrostatic pressure and low temperature. In both cases, dissociation of the dimeric native-like (alpha(2))(2) into folded monomers (alpha(2)) was observed. van't Hoff analysis of the low temperature experiments, assuming a two-state dimer 171-monomer transition, yielded a free energy of dissociation of (alpha(2))(2) of DeltaG(diss) = 11.4 +/- 0.4 kcal/mol, in good agreement with the free energy determined from pressure dissociation experiments (DeltaG(diss) = 10.5 +/- 0.1 kcal/mol). Binding of the hydrophobic fluorescent probe 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) to the pressure- and cold-dissociated states of (alpha(2))(2) indicated the existence of molten-globule monomers. In conclusion, we demonstrate that the folding pathway of (alpha(2))(2) can be described by a three-state transition including a monomeric molten globule-like state.     

The folding energy landscape of the dimerization domain of Escherichia coli Trp repressor: a joint experimental and theoretical investigation

Enhanced structural insights into the folding energy landscape of the N-terminal dimerization domain of Escherichia coli tryptophan repressor, [2-66]2 TR, were obtained from a combined experimental and theoretical analysis of its equilibrium folding reaction. Previous studies have shown that the three intertwined helices in [2-66]2 TR are sufficient to drive the formation of a stable dimer for the full-length protein, [2-107]2 TR. The monomeric and dimeric folding intermediates that appear during the folding reactions of [2-66]2 TR have counterparts in the folding mechanism of the full-length protein. The equilibrium unfolding energy surface on which the folding and dimerization reactions occur for [2-66]2 TR was examined with a combination of native-state hydrogen exchange analysis, pepsin digestion and matrix-assisted laser/desorption mass spectrometry performed at several concentrations of protein and denaturant. Peptides corresponding to all three helices in [2-66]2 TR show multi-layered protection patterns consistent with the relative stabilities of the dimeric and monomeric folding intermediates. The observation of protection exceeding that offered by the dimeric intermediate in segments from all three helices implies that a segment-swapping mechanism may be operative in the monomeric intermediate. Protection greater than that expected from the global stability for a single amide hydrogen in a peptide from the C-helix possibly and another from the A-helix may reflect non-random structure, possibly a precursor for segment swapping, in the urea-denatured state. Native topology-based model simulations that correspond to a funnel energy landscape capture both the monomeric and dimeric intermediates suggested by the HX MS data and provide a rationale for the progressive acquisition of secondary structure in their conformational ensembles.     

Physico-chemical properties of molten dimer ascorbate oxidase

The possible presence of dimeric unfolding intermediates might offer a clue to understanding the relationship between tertiary and quaternary structure formation in dimers. Ascorbate oxidase is a large dimeric enzyme that displays such an intermediate along its unfolding pathway. In this study the combined effect of high pressure and denaturing agents gave new insight on this intermediate and on the mechanism of its formation. The transition from native dimer to the dimeric intermediate is characterized by the release of copper ions forming the tri-nuclear copper center located at the interface between domain 2 and 3 of each subunit. This transition, which is pH-dependent, is accompanied by a decrease in volume, probably associated to electrostriction due to the loosening of intra-subunit electrostatic interactions. The dimeric species is present even at 3 x 10(8) Pa, providing evidence that mechanically or chemically induced unfolding lead to a similar intermediate state. Instead, dissociation occurs with an extremely large and negative volume change (DeltaV approximately -200 mL.mol(-1)) by pressurization in the presence of moderate amounts of denaturant. This volume change can be ascribed to the elimination of voids at the subunit interface. Furthermore, the combination of guanidine and high pressure uncovers the presence of a marginally stable (DeltaG approximately 2 kcal.mol(-1)) monomeric species (which was not observed in previous equilibrium unfolding measurements) that might be populated in the early folding steps of ascorbate oxidase. These findings provide new aspects of the protein folding pathway, further supporting the important role of quaternary interactions in the folding strategy of large dimeric enzymes.     

Folding and assembly kinetics of procaspase‐3

Caspases are vital to apoptosis and exist in the cell as inactive zymogens. Dimerization is central to procaspase activation because the active sites are comprised of loops from both monomers. Although initiator procaspases are stable monomers until activated on cell death scaffolds, the effector caspases, such as procaspase-3, are stable dimers. The activation mechanisms are reasonably well understood in terms of polypeptide chain cleavage and subsequent active site rearrangements in the dimer, but the mechanisms that govern dimer assembly are not known. To further understand procaspase dimerization, we examined the folding and assembly of procaspase-3 by fluorescence emission, circular dichroism, differential quenching by acrylamide, anisotropy, and enzyme activity assays. Single-mixing stopped-flow refolding studies showed a complex burst phase in which multiple monomeric species form rapidly. At longer times, the monomer folds through several intermediates, some of which appear to be off-pathway or misfolded, before eventually forming a dimerization-competent species. Enzyme activity studies demonstrated a slow rate of dimerization (∼70 M⁻¹ s⁻¹). In addition, single-mixing stopped-flow unfolding studies revealed a complex unfolding process with a slow rate of dimer dissociation. Interestingly, multiple dimeric species were observed in the burst phase for unfolding, suggesting that the native ensemble consists of at least two major conformations. Collectively, these results demonstrate complex folding and unfolding behavior for procaspase-3 and suggest that slow dimerization results from the lack of stabilizing native contacts in the initial encounter complex.     

Folding mechanism of FIS, the intertwined, dimeric factor for inversion stimulation

FIS, the factor for inversion stimulation, from Escherichia coli and other enteric bacteria, is an interwined alpha-helical homodimer. Size exclusion chromatography and static light scattering measurements demonstrated that FIS is predominately a stable dimer at the concentrations (1-10 microM monomer) and buffer conditions employed in this study. The folding and unfolding of FIS were studied with both equilibrium and kinetic methods by circular dichroism using urea and guanidinium chloride (GdmCl) as the perturbants. The equilibrium folding is reversible and well-described by a two-state folding model, with stabilities at 10 degrees C of 15.2 kcal mol(-1) in urea and 13.5 kcal mol(-1) in GdmCl. The kinetic data are consistent with a two-step folding reaction where the two unfolded monomers associate to a dimeric intermediate within the mixing time for the stopped-flow instrument (<5 ms), and a slower, subsequent folding of the dimeric intermediate to the native dimer. Fits of the burst phase amplitudes as a function of denaturant showed that the free energy for the formation of the dimeric intermediate constitutes the majority of the stability of the folding (9.6 kcal mol(-1) in urea and 10.5 kcal mol(-1) in GdmCl). Folding-to-unfolding double jump kinetic experiments were also performed to monitor the formation of native dimer as a function of folding delay times. The data here demonstrate that the dimeric intermediate is obligatory and on-pathway. The folding mechanism of FIS, when compared to other intertwined, alpha-helical, homodimers, suggests that a transient kinetic dimeric intermediate may be a common feature of the folding of intertwined, segment-swapped, alpha-helical dimers.     

Thermal unfolding of triosephosphate isomerase from Entamoeba histolytica: dimer dissociation leads to extensive unfolding

In mesophiles, triosephosphate isomerase (TIM) is an obligated homodimer. We have previously shown that monomeric folding intermediates are common in the chemical unfolding of TIM, where dissociation provides 75% of the overall conformational stability of the dimer. However, analysis of the crystallographic structure shows that, during unfolding, intermonomeric contacts contribute to only 5% of the overall increase in accessible surface area. In this work several methodologies were used to characterize the thermal dissociation and unfolding of the TIM from Entamoeba histolytica (EhTIM) and a monomeric variant obtained by chemical derivatization (mEhTIM). During EhTIM unfolding, sequential transitions corresponding to dimer dissociation into a compact monomeric intermediate followed by unfolding and further aggregation of the intermediate occurred. In the case of mEhTIM, a single transition, analogous to the second transition of EhTIM, was observed. Calorimetric, spectroscopic, hydrodynamic, and functional evidence shows that dimer dissociation is not restricted to localized interface reorganization. Dissociation represents 55% (DeltaH(Diss) = 146.8 kcal mol(-1)) of the total enthalpy change (DeltaH(Tot) = 266 kcal mol(-1)), indicating that this process is linked to substantial unfolding. We propose that, rather than a rigid body process, subunit assembly is best represented by a fly-casting mechanism. In TIM, catalysis is restricted to the dimer; therefore, the interface can be viewed as the final nucleation motif that directs assembly, folding, and function.     

Flipping the switch from monomeric to dimeric CV-N has little effect on antiviral activity

Cyanovirin-N can exist in solution in monomeric and domain-swapped dimeric forms, with HIV-antiviral activity being reported for both. Here we present results for CV-N variants that form stable solution dimers: the obligate dimer [DeltaQ50]CV-N and the preferential dimer [S52P]CV-N. These variants exhibit comparable DeltaG values (10.6 +/- 0.5 and 9.4 +/- 0.5 kcal.mol(-1), respectively), similar to that of stabilized, monomeric [P51G]CV-N (9.8 +/- 0.5 kcal.mol(-1)), but significantly higher than wild-type CV-N (4.1 +/- 0.2 kcal.mol(-1)). During folding/unfolding, no stably folded monomer was observed under any condition for the obligate dimer [DeltaQ50]CV-N, whereas two monomeric, metastable species were detected for [S52P]CV-N at low concentrations. This is in contrast to our previous results for [P51G]CV-N and wild-type CV-N, for which the dimeric forms were found to be the metastable species. The dimeric mutants exhibit comparable antiviral activity against HIV and Ebola, similar to that of wild-type CV-N and the stabilized [P51G]CV-N variant.     

Conformational analysis of intermediates involved in the in vitro folding pathways of recombinant human macrophage colony stimulating factor beta by sulfhydryl group trapping and hydrogen/deuterium pulsed labeling

In vitro oxidative folding of reduced recombinant human macrophage colony stimulating factor beta (rhm-CSFbeta) involves two major events: disulfide isomerization in the monomeric intermediates and disulfide-mediated dimerization. Kinetic analysis of rhm-CSFbeta folding indicated that monomer isomerization is slower than dimerization and is, in fact, the rate-determining step. A time-dependent determination of the number of free cysteines remaining was made after refolding commence. The folding intermediates revealed that rhm-CSFbeta folds systematically, forming disulfide bonds via multiple pathways. Mass spectrometric evidence indicates that native as well as non-native intrasubunit disulfide bonds form in monomeric intermediates. Initial dimerization is assumed to involve formation of disulfide bonds, Cys 157/159-Cys' 157/159. Among six intrasubunit disulfide bonds, Cys 48-Cys 139 and Cys' 48-Cys' 139 are assumed to be the last to form, while Cys 31-Cys' 31 is the last intersubunit disulfide bond that forms. Conformational properties of the folding intermediates were probed by H/D exchange pulsed labeling, which showed the coexistence of noncompact dimeric and monomeric species at early stages of folding. As renaturation progresses, the noncompact dimer undergoes significant structural rearrangement, forming a native-like dimer while the monomer maintains a noncompact conformation.     

Mapping the folding free energy surface for metal-free human Cu,Zn superoxide dismutase

Mutations at many different sites in the gene encoding human Cu,Zn superoxide dismutase (SOD) are known to be causative agents in amyotrophic lateral sclerosis (ALS). One explanation for the molecular basis of this pathology is the aggregation of marginally soluble, partially structured states whose populations are enhanced in the protein variants. As a benchmark for testing this hypothesis, the equilibrium and kinetic properties of the reversible folding reaction of a metal-free variant of SOD were investigated. Reversibility was achieved by replacing the two non-essential cysteine residues with non-oxidizable analogs, C6A/C111S, to produce apo-AS-SOD. The metal-free pseudo-wild-type protein is folded and dimeric in the absence of chemical denaturants, and its equilibrium folding behavior is well described by an apparent two-state mechanism involving the unfolded monomer and the native dimer. The apparent free energy of folding in the absence of denaturant and at standard state is -20.37(+/- 1.04) kcal (mol dimer)(-1). A global analysis of circular dichroism kinetic traces for both unfolding and refolding reactions, combined with results from small angle X-ray scattering and time-resolved fluorescence anisotropy measurements, supports a sequential mechanism involving the unfolded monomer, a folded monomeric intermediate, and the native dimer. The rate-limiting monomer folding reaction is followed by a near diffusion-limited self-association reaction to form the native dimer. The relative population of the folded monomeric intermediate is predicted not to exceed 0.5% at micromolar concentrations of protein under equilibrium and both strongly unfolding and refolding conditions for metal-free pseudo-wild-type SOD.     

Closing the gate to the active site: effect of the inhibitor methoxyarachidonyl fluorophosphonate on the conformation and membrane binding of fatty acid amide hydrolase

Fatty acid amide hydrolase (FAAH) is a dimeric, membranebound enzyme that degrades neuromodulatory fatty acid amides and esters and is expressed in mammalian brain and peripheral tissues. The cleavage of approximately 30 amino acids from each subunit creates an FAAH variant that is soluble and homogeneous in detergent-containing buffers, opening the avenue to the in vitro mechanistic and structural studies. Here we have studied the stability of FAAH as a function of guanidinium hydrochloride concentration and of hydrostatic pressure. The unfolding transition was observed to be complex and required a fitting procedure based on a three-state process with a monomeric intermediate. The first transition was characterized by dimer dissociation, with a free energy change of approximately 11 kcal/mol that accounted for approximately 80% of the total stabilization energy. This process was also paralleled by a large change in the solvent-accessible surface area, because of the hydration occurring both at the dimeric interface and within the monomers. As a consequence, the isolated subunits were found to be much less stable (DeltaG approximately 3 kcal/mol). The addition of methoxyarachidonyl fluorophosphonate, an irreversible inhibitor of FAAH activity, enhanced the stability of the dimer by approximately 2 kcal/mol, toward denaturant- and pressure-induced unfolding. FAAH inhibition by methoxyarachidonyl fluorophosphonate also reduced the ability of the protein to bind to the membranes. These findings suggest that local conformational changes at the level of the active site might induce a tighter interaction between the subunits of FAAH, affecting the enzymatic activity and the interaction with membranes.     

Urea-induced sequential unfolding of fibronectin: a fluorescence spectroscopy and circular dichroism study

Fibronectin (FN) is an extracellular matrix (ECM) protein found soluble in corporal fluids or as an insoluble fibrillar component incorporated in the ECM. This phenomenon implicates structural changes that expose FN binding sites and activate the protein to promote intermolecular interactions with other FN. We have investigated, using fluorescence and circular dichroism spectroscopy, the unfolding process of human fibronectin induced by urea in different ionic strength conditions. At any ionic strength, the equilibrium unfolding data are well described by a four-state equilibrium model N <= => I(1) <= =>I(2) <= => U. Fitting this model to experimental values, we have determined the free energy change for the different steps. We found that the N <= => I(1) transition corresponds to a free energy of 10.5 +/- 0.4 kcal/mol. Comparable values of free energy change are generally associated with a partial unfolding of the type III domain. For the I(1) <= => I(2) transition, the free energy change is 7.6 +/- 0.4 kcal/mol at low ionic strength but is twice as low at high ionic strength. This result is consistent with observations indicating that the complete unfolding of the type III domain from partially unfolded forms necessitates about 5 kcal/mol. The third step, I(2) <= => U, which leads to the complete unfolding of fibronectin, corresponds to a free energy change of 14.4 +/- 0.9 kcal/mol at low ionic strength whereas this energy is again twice as low under high ionic strength conditions. This hierarchical unfolding of fibronectin, as well as the stability of the different intermediates controlled by ionic strength demonstrated here, could be important for the understanding of activation of the matrix assembly.     

Kinetic traps in the folding/unfolding of procaspase-1 CARD domain

We have examined the folding and unfolding of the caspase recruitment domain of procaspase-1 (CP1-CARD), a member of the alpha-helical Greek key protein family. The equilibrium folding/unfolding of CP1-CARD is described by a two-state mechanism, and the results show CP1-CARD is marginally stable with a DeltaG(H2O) of 1.1 +/- 0.2 kcal/mole and an m-value of 0.65 +/- 0.06 kcal/mole/M (10 mM Tris-HCl at pH 8.0, 1 mM DTT, 25 degrees C). Consistent with the equilibrium folding data, CP1-CARD is a monomer in solution when examined by size exclusion chromatography. Single-mixing stopped-flow refolding and unfolding studies show that CP1-CARD folds and unfolds rapidly, with no detectable slow phases, and the reactions appear to reach equilibrium within 10 msec. However, double jump kinetic experiments demonstrate the presence of an unfolded-like intermediate during unfolding. The intermediate converts to the fully unfolded conformation with a half-time of 10 sec. Interrupted refolding studies demonstrate the presence of one or more nativelike intermediates during refolding, which convert to the native conformation with a half-time of about 60 sec. Overall, the data show that both unfolding and refolding processes are slow, and the pathways contain kinetically trapped species.     

Folding and stability of trp aporepressor from Escherichia coli

Equilibrium and kinetic studies of the urea-induced unfolding of trp aporepressor from Escherichia coli were performed to probe the folding mechanism of this intertwined, dimeric protein. The equilibrium unfolding transitions at pH 7.6 and 25 degrees C monitored by difference absorbance, fluorescence, and circular dichroism spectroscopy are coincident within experimental error. All three transitions are well described by a two-state model involving the native dimer and the unfolded monomer; the free energy of folding in the absence of denaturant and under standard-state conditions is estimated to be 23.3 +/- 0.9 kcal/mol of dimer. The midpoint of the equilibrium unfolding transition increases with increasing protein concentration in the manner expected from the law of mass action for the two-state model. We find no evidence for stable folding intermediates. Kinetic studies reveal that unfolding is governed by a single first-order reaction whose relaxation time decreases exponentially with increasing urea concentration and also decreases with increasing protein concentration in the transition zone. Refolding involves at least three phases that depend on both the protein concentration and the final urea concentration in a complex manner. The relaxation time of the slowest of these refolding phases is identical with that for the single phase in unfolding in the transition zone, consistent with the results expected for a reaction that is kinetically reversible. The two faster refolding phases are presumed to arise from slow isomerization reactions in the unfolded form and reflect parallel folding channels.     

Pressure and denaturants in the unfolding of triosephosphate isomerase: the monomeric intermediates of the enzymes from Saccharomyces cerevisiae and Entamoeba histolytica

Triosephosphate isomerase (TIM) is a dimeric enzyme formed by two identical (beta/alpha)8 barrels. In this work, we compare the unfolding and refolding of the TIMs from Entamoeba histolytica (EhTIM) and baker's yeast (yTIM). A monomeric intermediate was detected in the GdnHCl-induced unfolding of EhTIM. The thermodynamic, spectroscopic, catalytic, and hydrodynamic properties of this intermediate were found to be very similar to those previously described for a monomeric intermediate of yTIM observed in GdnHCl. Monomer unfolding was reversible for both TIMs; however, the dissociation step was reversible in yTIM and irreversible in EhTIM. Monomer unfolding induced by high pressure in the presence of GdnHCl was a reversible process. DeltaGUnf, DeltaVUnf, and P1/2 were obtained for the 0.7-1.2 M GdnHCl range. The linear extrapolation of these thermodynamic parameters to the absence of denaturant showed the same values for both intermediates. The DeltaVUnfH2O values calculated for EhTIM and yTIM monomeric intermediates are the same within experimental error (-57 +/- 10 and -76 +/- 14 mL/mol, respectively). These DeltaVUnf H2O values are smaller than those reported for the unfolding of monomeric proteins of similar size, suggesting that TIM intermediates are only partially hydrated. |DeltaVUnf| increased with denaturant concentration; this behavior is probably related to structural changes in the unfolded state induced by GdnHCl and pressure. From the thermodynamic parameters that were obtained, it is predicted that in the absence of denaturants, pressure would induce monomer unfolding (P1/2 approximately 140 MPa) prior to dimer dissociation (P1/2 approximately 580 MPa). Therefore, dimerization prevents the pressure unfolding of the monomer.     

Rough energy landscapes in protein folding: dimeric E. coli Trp repressor folds through three parallel channels

The folding mechanism of the dimeric Escherichia coli Trp repressor (TR) is a kinetically complex process that involves three distinguishable stages of development. Following the formation of a partially folded, monomeric ensemble of species, within 5 ms, folding to the native dimer is controlled by three kinetic phases. The rate-limiting step in each phase is either a non-proline isomerization reaction or a dimerization reaction, depending on the final denaturant concentration. Two approaches have been employed to test the previously proposed folding mechanism of TR through three parallel channels: (1) unfolding double-jump experiments demonstrate that all three folding channels lead directly to native dimer; and (2) the differential stabilization of the transition state for the final step in folding and the native dimer, by the addition of salt, shows that all three channels involve isomerization of a dimeric species. A refined model for the folding of Trp repressor is presented, in which all three channels involve a rapid dimerization reaction between partially folded monomers followed by the isomerization of the dimeric intermediates to yield native dimer. The ensemble of partially folded monomers can be captured at equilibrium by low pH; one-dimensional proton NMR spectra at pH 2.5 demonstrate that monomers exist in two distinct, slowly interconverting conformations. These data provide a potential structural explanation for the three-channel folding mechanism of TR: random association of two different monomeric forms, which are distinguished by alternative packing modes of the core dimerization domain and the DNA-binding, helix-turn-helix, domain. One, perhaps both, of these packing modes contains non-native contacts.     

Relationship between stability of folding intermediates and amyloid formation for the yeast prion Ure2p: a quantitative analysis of the effects of pH and buffer system

The dimeric yeast protein Ure2 shows prion-like behaviour in vivo and forms amyloid fibrils in vitro. A dimeric intermediate is populated transiently during refolding and is apparently stabilized at lower pH, conditions suggested to favour Ure2 fibril formation. Here we present a quantitative analysis of the effect of pH on the thermodynamic stability of Ure2 in Tris and phosphate buffers over a 100-fold protein concentration range. We find that equilibrium denaturation is best described by a three-state model via a dimeric intermediate, even under conditions where the transition appears two-state by multiple structural probes. The free energy for complete unfolding and dissociation of Ure2 is up to 50 kcal mol(-1). Of this, at least 20 kcal mol(-1) is contributed by inter-subunit interactions. Hence the native dimer and dimeric intermediate are significantly more stable than either of their monomeric counterparts. The previously observed kinetic unfolding intermediate is suggested to represent the dissociated native-like monomer. The native state is stabilized with respect to the dimeric intermediate at higher pH and in Tris buffer, without significantly affecting the dissociation equilibrium. The effects of pH, buffer, protein concentration and temperature on the kinetics of amyloid formation were quantified by monitoring thioflavin T fluorescence. The lag time decreases with increasing protein concentration and fibril formation shows pseudo-first order kinetics, consistent with a nucleated assembly mechanism. In Tris buffer the lag time is increased, suggesting that stabilization of the native state disfavours amyloid nucleation.     

Unique fluorophores in the dimeric archaeal histones hMfB and hPyA1 reveal the impact of nonnative structure in a monomeric kinetic intermediate

Homodimeric archaeal histones and heterodimeric eukaryotic histones share a conserved structure but fold through different kinetic mechanisms, with a correlation between faster folding/association rates and the population of kinetic intermediates. Wild-type hMfB (from Methanothermus fervidus) has no intrinsic fluorophores; Met35, which is Tyr in hyperthermophilic archaeal histones such as hPyA1 (from Pyrococcus strain GB-3A), was mutated to Tyr and Trp. Two Tyr-to-Trp mutants of hPyA1 were also characterized. All fluorophores were introduced into the long, central alpha-helix of the histone fold. Far-UV circular dichroism (CD) indicated that the fluorophores did not significantly alter the helical content of the histones. The equilibrium unfolding transitions of the histone variants were two-state, reversible processes, with DeltaG degrees (H2O) values within 1 kcal/mol of the wild-type dimers. The hPyA1 Trp variants fold by two-state kinetic mechanisms like wild-type hPyA1, but with increased folding and unfolding rates, suggesting that the mutated residues (Tyr-32 and Tyr-36) contribute to transition state structure. Like wild-type hMfB, M35Y and M35W hMfB fold by a three-state mechanism, with a stopped-flow CD burst-phase monomeric intermediate. The M35 mutants populate monomeric intermediates with increased secondary structure and stability but exhibit decreased folding rates; this suggests that nonnative interactions occur from burial of the hydrophobic Tyr and Trp residues in this kinetic intermediate. These results implicate the long central helix as a key component of the structure in the kinetic monomeric intermediates of hMfB as well as the dimerization transition state in the folding of hPyA1.     

Accessing the global minimum conformation of stefin A dimer by annealing under partially denaturing conditions.

Stefin A folds as a monomer under strongly native conditions. We have observed that under partially denaturing conditions in the temperature range from 74 to 93 degrees C it folds into a dimer, while it is monomeric above the melting temperature of 95 degrees C. Below 74 degrees C the dimer is trapped and it does not dissociate. The dimer is a folded and structured protein as judged by CD and NMR, nevertheless it is no more functional as an inhibitor of cysteine proteases. The monomer-dimer transition proceeds at a slow rate and the activation energy of dimerization at 99 kcal/mol is comparable to the unfolding enthalpy. A large and negative dimerization enthalpy of -111(+/- 8) kcal/mol was calculated from the temperature dependence of the dissociation constant. An irreversible pretransition at 10-15 deg. below the global unfolding temperature has been observed previously by DSC and can now be assigned to the monomer-dimer transition. Backbone resonances of all the dimer residues were assigned using 15N isotopically enriched protein. The dimer is symmetric and the chemical shift differences between the monomer and dimer are localized around the tripartite hydrophobic wedge, which otherwise interacts with cysteine proteases. Hydrogen exchange protection factors of the residues affected by dimer formation are higher in the dimer than in the monomer. The monomer to dimer transition is accompanied by a rapid exchange of all of the amide protons which are protected in the dimer, indicating that the transition state is unfolded to a large extent. Our results demonstrate that the native monomeric state of stefin A is actually metastable but is favored by the kinetics of folding. The substantial energy barrier which separates the monomer from the more stable dimer traps each state under native conditions.