pH dependence of folding of iso-2-cytochrome c

Starting from a standard unfolded state (3.0 M guanidine hydrochloride, pH 7.2), the kinetics of refolding of iso-2-cytochrome c have been investigated as a function of final pH between pH 3 and pH 10. Absorbance in the ultraviolet and visible spectral regions and tryptophan fluorescence are used to monitor folding. Over most of the pH range, fast and slow folding phases are detected by both fluorescence and absorbance probes. Near neutral pH, the rate of fast folding appears to be the same when monitored by absorbance and fluorescence probes. At higher and lower pH, there are two fast folding reactions, with absorbance-detected fast folding occurring in a slightly faster time range than fluorescence-detected fast folding. The rates of both fast folding reactions pass through broad minima near neutral pH, indicating involvement of ionizable groups in rate-limiting steps. The rates of slow folding also depend on the final pH. At acid pH, there appears to be a single slow folding phase for both fluorescence and absorbance probes. At neutral pH, the absorbance-detected and fluorescence-detected slow folding phases separate into distinct kinetic processes which differ in rate and relative amplitude. At high pH, absorbance-detected slow folding is no longer observed, while fluorescence-detected slow folding is decreased in amplitude. In contrast, the equilibrium and kinetic properties of proline imide bond isomerization, believed to be involved in the slow folding reactions, are largely independent of pH. The results suggest that the pH dependence of slow folding involves coupling of pH-sensitive structure to proline imide bond isomerization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics and importance of the dimerization step in the folding pathway of the beta 2 subunit of Escherichia coli tryptophan synthase

During its folding, the polypeptide chain of the beta 2 subunit of Escherichia coli tryptophan synthase (L-serine hydrolyase (adding indole) EC 4.2.1.20) undergoes dimerization. To decide whether this dimerization precedes or follows the formation of the native, functional, tertiary structure of the polypeptide chain, the kinetics of renaturation of beta 2 are studied by monitoring both the regain of native conformation and the dimerization. Dimer formation is followed by the increase of the fluorescence polarization, or by energy transfer between a fluorescence donor and a fluorescence acceptor, which occur upon association of adequately labelled beta chains. Renaturation is followed by the regain of functional properties of beta 2, i.e. its ability to bind pyridoxal-5'-phosphate or to form a fluorescent ternary complex with this coenzyme and L-serine. It is shown that for beta 2 the dimerization obeys first-order kinetics, presumably because it occurs rapidly after a rate-limiting isomerization of the monomer. The dimerization is followed by another isomerization, taking place within the dimer, which leads to the formation of the pyridoxal-5'-phosphate binding site. Still another, slow, isomerization reaction involving the F1 (N-terminal) domain completes the renaturation. With a modified form of beta 2 (trypsin-nicked beta 2) where this isomerization of F1 can be made to occur before the dimerization, the dimer is also shown to appear before the functional properties. It is concluded that a non-native dimer indeed exists as an obligatory intermediate on the folding pathway of nicked beta 2 and of beta 2, and that interdomain interactions are needed to force the polypeptide chains into their native conformations.     

Observation of multistate kinetics during the slow folding and unfolding of barstar.

The kinetics of the slow folding and unfolding reactions of barstar, a bacterial ribonuclease inhibitor protein, have been studied at 23(+/-1) degrees C, pH 8, by the use of tryptophan fluorescence, far-UV circular dichroism (CD), near-UV CD, and transient mixing (1)H nuclear magnetic resonance (NMR) spectroscopic measurements in the 0-4 M range of guanidine hydrochloride (GdnHCl) concentration. The denaturant dependences of the rates of folding and unfolding processes, and of the initial and final values of optical signals associated with these kinetic processes, have been determined for each of the four probes of measurement. Values determined for rates as well as amplitudes are shown to be very much probe dependent. Significant differences in the intensities and rates of appearance and disappearance of several resolved resonances in the real-time one-dimensional NMR spectra have been noted. The NMR spectra also show increasing dispersion of chemical shifts during the slow phase of refolding. The denaturant dependences of rates display characteristic folding chevrons with distinct rollovers under strongly native as well as strongly unfolding conditions. Analyses of the data and comparison of the results obtained with different probes of measurement appear to indicate the accumulation of a myriad of intermediates on parallel folding and unfolding pathways, and suggest the existence of an ensemble of transition states. The energetic stabilities of the intermediates estimated from kinetic data suggest that they are approximately half as stable as the fully folded protein. The slowness of the folding and unfolding processes (tau = 10-333 s) and values of 20.5 (+/-1.4) and 18 (+/-0.5) kcal mol(-)(1) for the activation energies of the slow refolding and unfolding reactions suggest that proline isomerization is involved in these reactions, and that the intermediates accumulate and are therefore detectable because the slow proline isomerization reaction serves as a kinetic trap during folding.     

Protein Folding Mechanism of the Dimeric AmphiphysinII/Bin1 N-BAR Domain

The human AmphyphisinII/Bin1 N-BAR domain belongs to the BAR domain superfamily, whose members sense and generate membrane curvatures. The N-BAR domain is a 57 kDa homodimeric protein comprising a six helix bundle. Here we report the protein folding mechanism of this protein as a representative of this protein superfamily. The concentration dependent thermodynamic stability was studied by urea equilibrium transition curves followed by fluorescence and far-UV CD spectroscopy. Kinetic unfolding and refolding experiments, including rapid double and triple mixing techniques, allowed to unravel the complex folding behavior of N-BAR. The equilibrium unfolding transition curve can be described by a two-state process, while the folding kinetics show four refolding phases, an additional burst reaction and two unfolding phases. All fast refolding phases show a rollover in the chevron plot but only one of these phases depends on the protein concentration reporting the dimerization step. Secondary structure formation occurs during the three fast refolding phases. The slowest phase can be assigned to a proline isomerization. All kinetic experiments were also followed by fluorescence anisotropy detection to verify the assignment of the dimerization step to the respective folding phase. Based on these experiments we propose for N-BAR two parallel folding pathways towards the homodimeric native state depending on the proline conformation in the unfolded state.     

Equilibrium unfolding of dimeric and engineered monomeric forms of lambda Cro (F58W) repressor and the effect of added salts: evidence for the formation of folded monomer induced by sodium perchlorate

The equilibrium unfolding transitions of Cro repressor variants, dimeric variant Cro F58W and monomer Cro K56[DGEVK]F58W, have been studied by urea and guanidine hydrochloride to probe the folding mechanism. The unfolding transitions of a dimeric variant are well described by a two state process involving native dimer and unfolded monomer with a free energy of unfolding, DeltaG(0,un)(0), of approximately 10-11 kcal/mol. The midpoint of transition curves is dependent on total protein concentration and DeltaG(0,un)(0) is independent of protein concentration, as expected for this model. Unfolding of Cro monomer is well described by the standard two state model. The stability of both forms of protein increases in the presence of salt but decreases with the decrease in pH. Because of the suggested importance of a N2<-->2F dimerization process in DNA binding, we have also studied the effect of sodium perchlorate, containing the chaotropic perchlorate anion, on the conformational transition of Cro dimer by CD, fluorescence and NMR (in addition to urea and guanidine hydrochloride) in an attempt both to characterize the thermodynamics of the process and to identify conditions that lead to an increase in the population of the folded monomers. Data suggest that sodium perchlorate stabilizes the protein at low concentration (<1.5 M) and destabilizes the protein at higher perchlorate concentration with the formation of a "significantly folded" monomer. The tryptophan residue in the "significantly folded" monomer induced by perchlorate is more exposed to the solvent than in native dimer.     

Structure of a rapidly formed intermediate in ribonuclease T1 folding.

Kinetic intermediates in protein folding are short-lived and therefore difficult to detect and to characterize. In the folding of polypeptide chains with incorrect isomers of Xaa-Pro peptide bonds the final rate-limiting transition to the native state is slow, since it is coupled to prolyl isomerization. Incorrect prolyl isomers thus act as effective traps for folding intermediates and allow their properties to be studied more easily. We employed this strategy to investigate the mechanism of slow folding of ribonuclease T1. In our experiments we use a mutant form of this protein with a single cis peptide bond at proline 39. During refolding, protein chains with an incorrect trans proline 39 can rapidly form extensive secondary structure. The CD signal in the amide region is regained within the dead-time of stopped-flow mixing (15 ms), indicating a fast formation of the single alpha-helix of ribonuclease T1. This step is correlated with partial formation of a hydrophobic core, because the fluorescence emission maximum of tryptophan 59 is shifted from 349 nm to 325 nm within less than a second. After about 20 s of refolding an intermediate is present that shows about 40% enzymatic activity compared to the completely refolded protein. In addition, the solvent accessibility of tryptophan 59 is drastically reduced in this intermediate and comparable to that of the native state as determined by acrylamide quenching of the tryptophan fluorescence. Activity and quenching measurements have long dead-times and therefore we do not know whether enzymatic activity and solvent accessibility also change in the time range of milliseconds. At this stage of folding at least part of the beta-sheet structure is already present, since it hosts the active site of the enzyme. The trans to cis isomerization of the tyrosine 38-proline 39 peptide bond in the intermediate and consequently the formation of native protein is very slow (tau = 6,500 s at pH 5.0 and 10 degrees C). It is accompanied by an additional increase in tryptophan fluorescence, by the development of the fine structure of the tryptophan emission spectrum, and by the regain of the full enzymatic activity. This indicates that the packing of the hydrophobic core, which involves both tryptophan 59 and proline 39, is optimized in this step. Apparently, refolding polypeptide chains with an incorrect prolyl isomer can very rapidly form partially folded intermediates with native-like properties.     

FAD insertion is essential for attaining the assembly competence of the dihydrolipoamide dehydrogenase (E3) monomer from Escherichia coli

Dihydrolipoamide dehydrogenase (E3) from Escherichia coli, an FAD-linked homodimer, can be fully reconstituted in vitro following denaturation in 6 m guanidinium chloride. Complete restoration of activity occurs within 1-2 h in the presence of FAD, dithiothreitol, and bovine serum albumin. In the absence of FAD, the dihydrolipoamide dehydrogenase monomer forms a stable folding intermediate, which is incapable of dimerization. This intermediate displays a similar tryptic resistance to the native enzyme but is less heat-stable, because its ability to form native E3 is lost after incubation at 65 degrees C for 15 min. The presence of FAD promotes slow, additional conformational rearrangements of the E3 subunit as observed by cofactor-dependent decreases in intrinsic tryptophan fluorescence. However, after 2 h, the tryptophan fluorescence spectrum and far UV CD spectrum of E3, refolded in the absence of FAD, are similar to that of the native enzyme, and full activity can still be recovered on addition of FAD. Cross-linking studies show that FAD insertion is necessary for the monomeric folding intermediate to attain an assembly competent state leading to dimerization. Thus cofactor insertion represents a key step in the assembly of this enzyme, although its initial presence appears not to be required to promote the correct folding pathway.     

Detection of a hidden folding intermediate of the third domain of PDZ

The folding pathway of the third domain of PDZ from the synaptic protein PSD-95 was characterized using kinetic and equilibrium methods by monitoring the fluorescence signal from a Trp residue that is incorporated at a near-surface position. Kinetic folding of this domain showed multiple exponential phases, whereas unfolding showed a single exponential phase. The slow kinetic phases were attributed to isomerization of proline residues, since there are five proline residues in this domain. We found that the logarithms of the rate constants for the fast phase of folding and unfolding are linearly dependent on the concentrations of denaturant. The unfolding free energy derived from these rate constants at zero denaturant was close to the value measured using the equilibrium method, suggesting the absence of detectable sub-millisecond folding intermediates. However, native-state hydrogen exchange experiments detected a partially unfolded intermediate under native conditions. It was further confirmed by a protein engineering study. These data suggest that a hidden intermediate exists after the rate-limiting step in the folding of the third domain of PDZ.     

The E-box DNA binding protein Sgc1p suppresses the gcr2 mutation, which is involved in transcriptional activation of glycolytic genes in Saccharomyces cerevisiae

Maltose binding protein (MBP) exhibits a slow phase of folding at pH 7.4, 298 K. The kinetics of this phase has been characterized as a function of denaturant concentration and temperature. Denaturant double-jump experiments and the activation energy for folding indicate that the slow phase involves processes other than proline isomerization. Although the first five N-terminal residues are disordered in the MBP crystal structure, mutations in this region slow down folding and destabilize the native structure. This is the first report showing that disordered N-terminal residues can affect folding kinetics and stability.     

Effect of multiple prolyl isomerization reactions on the stability and folding kinetics of the notch ankyrin domain: experiment and theory

Studies on the folding kinetics of the Notch ankyrin domain have demonstrated that the major refolding phase is slow, the minor refolding phase is limited by the isomerization of prolyl peptide bonds, and that unfolding is multiexponential. Here, we explore the relationship between prolyl isomerization and folding heterogeneity using a combination of experiment and simulation. Proline residues were replaced with alanine, both singly and in various combinations. These destabilizing substitutions combine to eliminate the minor refolding phase, although unfolding heterogeneity persists even when all seven proline residues are replaced. To test whether prolyl isomerization influences the major refolding phase, we modeled folding and prolyl isomerization as a system of sequential reactions. Simulations that use rate constants of the major folding phase of the Notch ankyrin domain to represent intrinsic folding indicate that even with seven prolyl isomerization reactions, only two significant phases should be observed, and that the fast observed phase provides a good approximation of the intrinsic folding in the absence of prolyl isomerization. These results indicate that the major refolding phase of the Notch ankyrin domain reflects an intrinsically slow folding transition, rather than coupling of fast folding events with slow prolyl isomerization steps. This is consistent with the observation that the single observed refolding phase of a construct in which all proline residues are replaced remains slow. Finally, the simulation fails to produce a second unfolding phase at high urea concentrations, indicating that prolyl isomerization does not play a role in the three-state mechanism that leads to this heterogeneity.     

Trigger factor-assisted folding of bovine carbonic anhydrase II

Spontaneous refolding of GdnHCl denatured bovine carbonic anhydrase II (BCA II) shows at least three phases: a burst phase, a fast phase, and a slow phase. The fast and slow phases are both controlled by proline isomerization. However, we find that in trigger factor (TF)-assisted BCA II folding, only the fast phase is catalyzed by wild-type TF, suggesting that certain proline residues are accessible in folding intermediates. The refolding yields of BCA II assisted by wild-type TF and TF mutants which lack PPIase activity are about the same, which provides further experimental evidence that the PPIase and chaperone activities of TF are independent. The binding of TF to folding intermediates during BCA II refolding was characterized by chemical crosslinking and Western blotting. A scheme for TF-assisted BCA II folding is proposed and the possible role of the TF dimer as a "binding" chaperone in vivo is discussed.     

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.     

Folding kinetics of phage 434 Cro protein

Folding kinetics for phage 434 Cro protein are examined and compared with those reported for lambda(6-85), the N-terminal domain of the repressor of phage lambda. The two proteins have similar all-helical structures consisting of five helices but different stabilities. In contrast to lambda(6-85), sharp and distinct aromatic (1)H NMR signals without exchange broadening characterize the native and urea-denatured 434 Cro forms at equilibrium at 20 degrees C, indicating slow interconversion on the NMR time scale. Stopped-flow fluorescence data using the single 434 Cro tryptophan indicate strongly urea-dependent refolding rates and smaller urea dependencies of the unfolding rates, suggesting a native-like transition state ensemble. Refolding rates are slower and unfolding rates considerably faster at pH 4 than at pH 6. This accounts for the lower stability of 434 Cro at pH 4 and suggests the existence of pH-dependent, possibly salt bridge interactions that are more stabilizing at pH 6. At <2 M urea, decreased folding amplitudes and nonlinear urea dependencies that are apparent at pH 6 indicate deviation from two-state behavior and suggest the formation of an early folding intermediate. The folding behavior of 434 Cro and why it folds 2 orders of magnitude slower than lambda(6-85) are rationalized in terms of the lower intrinsic helix stabilities and putative charge interactions in 434 Cro.     

Proline replacements and the simplification of the complex,parallel channel folding mechanism for the alpha subunit of Trp synthase,a TIM barrel protein

The kinetic folding mechanism for the alpha subunit of tryptophan synthase (alphaTS) from Escherichia coli involves four parallel channels whose inter-conversions are controlled by three cis/trans prolyl isomerization reactions (tau(1), tau(2) and tau(3)). A previous mutational analysis of all 19 proline positions, including the unique cis Asp27-Pro28 peptide bond, revealed that the G(3)P28G, P78A or P96A mutations selectively eliminated the fast, tau(1) (ten seconds), folding phase, while the P217M and P261A mutations eliminated the medium, tau(2) (40 seconds) and the slow, tau(3) ( approximately 300 seconds) folding phases, respectively. To further elucidate the role of these proline residues and to simplify the folding mechanism, a series of double and triple mutants were constructed at these critical positions, and comprehensive kinetic and thermodynamic experiments were performed. Although it was not possible to construct a stable system that was free of proline isomerization constraints, a double mutant variant, G(3)P28G/P217M, in which the refolding of more than 90% of the unfolded protein is not limited by proline isomerization reactions was identified. Further, long-range interactions between several of these residues appear to be a crucial part of the cooperative network of structure that stabilizes the TIM barrel motif for alphaTS.     

Slow and Bimolecular Folding of a De Novo Designed Monomeric Protein DS119

De novo protein design offers a unique means to test and advance our understanding of how proteins fold. However, most current design methods are native structure eccentric and folding kinetics has rarely been considered in the design process. Here, we show that a de novo designed mini-protein DS119, which folds into a βαβ structure, exhibits unusually slow and concentration-dependent folding kinetics. For example, the folding time for 50 μM of DS119 was estimated to be ∼2 s. Stopped-flow fluorescence resonance energy transfer experiments further suggested that its folding was likely facilitated by a transient dimerization process. Taken together, these results highlight the need for consideration of the entire folding energy landscape in de novo protein design and provide evidence suggesting nonnative interactions can play a key role in protein folding.     

A trade between similar but nonequivalent intrasubunit and intersubunit contacts in Cro dimer evolution

The homodimeric lambda Cro protein has a "ball-and-socket" interface that includes insertion of an aromatic side chain, Phe 58, from each subunit into a cavity in the hydrophobic core of the other subunit. This overlap between the subunit core and dimer interface hypothetically explains the strong dimerization and weak monomer stability of lambda Cro in comparison to homologues. According to a model developed here and in a previous study [LeFevre, K. R., and Cordes, M. H. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 2345-2350], the socket cavity evolved in part by replacement of a buried tryptophan in an ancestral stable monomer with a smaller side chain (Ala 33 in lambda Cro). The resulting core defect was in effect repaired by insertion of a different side chain (Phe 58) from a second subunit, generating the ball and socket. Consistent with such an evolutionary trade between intrasubunit and intersubunit interactions, we showed in the previous study that restoration of the ancestral Trp 33 in lambda Cro stabilized the monomer and reduced the extent of dimerization. Here, we report the solution structure of a stable lambda Cro monomer containing the Ala33Trp mutation, which confirms that the restored tryptophan fulfills its ancestral role as a core side chain, filling part of the socket cavity occupied by Phe 58 in the wild-type dimer. The structure also reveals, however, that the cavity is not completely filled by Trp 33, suggesting that its formation could have involved multiple mutations that reduced side chain volume. We offer suggestive evidence of a role of mutations at a second position.     

Parallel channels and rate-limiting steps in complex protein folding reactions: prolyl isomerization and the alpha subunit of Trp synthase,a TIM barrel protein

A kinetic folding mechanism for the alpha subunit of tryptophan synthase (alphaTS) from Escherichia coli, involving four parallel channels with multiple native, intermediate and unfolded forms, has recently been proposed. The hypothesis that cis/trans isomerization of several Xaa-Pro peptide bonds is the source of the multiple folding channels was tested by measuring the sensitivity of the three rate-limiting phases (tau(1), tau(2), tau(3)) to catalysis by cyclophilin, a peptidyl-prolyl isomerase. Although the absence of catalysis for the tau(1) (fast) phase leaves its assignment ambiguous, our previous mutational analysis demonstrated its connection to the unique cis peptide bond preceding proline 28. The acceleration of the tau(2) (medium) and tau(3) (slow) refolding phases by cyclophilin demonstrated that cis/trans prolyl isomerization is also the source of these phases. A collection of proline mutants, which covered all of the remaining 18 trans proline residues of alphaTS, was constructed to obtain specific assignments for these phases. Almost all of the mutant proteins retained the complex equilibrium and kinetic folding properties of wild-type alphaTS; only the P217A, P217G and P261A mutations caused significant changes in the equilibrium free energy surface. Both the P78A and P96A mutations selectively eliminated the tau(1) folding phase, while the P217M and P261A mutations eliminated the tau(2) and tau(3) folding phases, respectively. The redundant assignment of the tau(1) phase to Pro28, Pro78 and Pro96 may reflect their mutual interactions in non-random structure in the unfolded state. The non-native cis isomers for Pro217 and Pro261 may destabilize an autonomous C-terminal folding unit, thereby giving rise to kinetically distinct unfolded forms. The nature of the preceding amino acid, the solvent exposure, or the participation in specific elements of secondary structure in the native state, in general, are not determinative of the proline residues whose isomerization reactions can limit folding.     

Proline isomerization and the slow folding reactions of the alpha subunit of tryptophan synthase from Escherichia coli

Previous studies on the refolding of the alpha subunit of tryptophan synthase from Escherichia coli assigned two slow refolding phases to rate-limiting isomerizations of two 'essential' proline residues, one in each of the two domains of the protein (Matthews, C.R., Crisanti, M.M., Manz, J.T. and Gepner, G.L. (1983) Biochemistry 22, 1445-1452). The double-jump experiment (Brandts, J.F., Halvorson, H.R. and Brennan, M. (1975) Biochemistry 14, 4953-4963) was used to further investigate this phenomenon. The reaction assigned to the carboxyl domain is consistent with the proline isomerization hypothesis. The amino domain process is more rapid than expected for proline isomerization and may reflect another type of slow folding reaction. The results permit a further refinement of the folding model for the alpha subunit and demonstrate the existence of a third unfolded species whose folding is not limited by either of these two reactions.     

N15 Cro and lambda Cro: orthologous DNA-binding domains with completely different but equally effective homodimer interfaces

Bacteriophage Cro proteins bind to target DNA as dimers but do not all dimerize with equal strength, and differ in fold in the region of the dimer interface. We report the structure of the Cro protein from Enterobacteria phage N15 at 1.05 A resolution. The subunit fold contains five alpha-helices and is closely similar to the structure of P22 Cro (1.3 A backbone room mean square difference over 52 residues), but quite different from that of lambda Cro, a structurally diverged member of this family with a mixed alpha-helix/beta-sheet fold. N15 Cro crystallizes as a biological dimer with an extensive interface (1303 A(2) change in accessible surface area per dimer) and also dimerizes in solution with a K(d) of 5.1 +/- 1.5 microM. Its dimerization is much stronger than that of its structural homolog P22 Cro, which does not self-associate detectably in solution. Instead, the level of self-association and interfacial area for N15 Cro is similar to that of lambda Cro, even though these two orthologs do not share the same fold and have dimer interfaces that are qualitatively different in structure. The common Cro ancestor is thought to be an all-helical monomer similar to P22 Cro. We propose that two Cro descendants independently developed stronger dimerization by entirely different mechanisms.