Topology and sequence in the folding of a TIM barrel protein: global analysis highlights partitioning between transient off-pathway and stable on-pathway folding intermediates in the complex folding mechanism of a (betaalpha)8 barrel of unknown function from B. subtilis

The relative contributions of chain topology and amino acid sequence in directing the folding of a (betaalpha)(8) TIM barrel protein of unknown function encoded by the Bacillus subtilis iolI gene (IOLI) were assessed by reversible urea denaturation and a combination of circular dichroism, fluorescence and time-resolved fluorescence anisotropy spectroscopy. The equilibrium reaction for IOLI involves, in addition to the native and unfolded species, a stable intermediate with significant secondary structure and stability and self-associated forms of both the native and intermediate states. Global kinetic analysis revealed that the unfolded state partitions between an off-pathway refolding intermediate and the on-pathway equilibrium intermediate early in folding. Comparisons with the folding mechanisms of two other TIM barrel proteins, indole-3-glycerol phosphate synthase from the thermophile Sulfolobus solfataricus (sIGPS) and the alpha subunit of Escherichia coli tryptophan synthase (alphaTS), reveal striking similarities that argue for a dominant role of the topology in both early and late events in folding. Sequence-specific effects are apparent in the magnitudes of the relaxation times and relative stabilities, in the presence of additional monomeric folding intermediates for alphaTS and sIGPS and in rate-limiting proline isomerization reactions for alphaTS.     

Folding mechanism of indole-3-glycerol phosphate synthase from Sulfolobus solfataricus: a test of the conservation of folding mechanisms hypothesis in (beta(alpha))(8) barrels

As a test of the hypothesis that folding mechanisms are better conserved than sequences in TIM barrels, the equilibrium and kinetic folding mechanisms of indole-3-glycerol phosphate synthase (sIGPS) from the thermoacidophilic archaebacterium Sulfolobus solfataricus were compared to the well-characterized models of the alpha subunit of tryptophan synthase (alphaTS) from Escherichia coli. A multifaceted approach combining urea denaturation and far-UV circular dichroism, tyrosine fluorescence total intensity, and tyrosine fluorescence anisotropy was employed. Despite a sequence identity of only 13%, a stable intermediate (I) in sIGPS was found to be similar to a stable intermediate in alphaTS in terms of its thermodynamic properties and secondary structure. Kinetic experiments revealed that the fastest detectable folding event for sIGPS involves a burst-phase (<5ms) reaction that leads directly to the stable intermediate. The slower of two subsequent phases reflects the formation/disruption of an off-pathway dimeric form of I. The faster phase reflects the conversion of I to the native state and is limited by folding under marginally stable conditions and by isomerization or rearrangement under strongly folding conditions. By contrast, alphaTS is thought to fold via an off-pathway burst-phase intermediate whose unfolding controls access to a set of four on-pathway intermediates that comprise the stable equilibrium intermediate. At least three proline isomerization reactions are known to limit their interconversions and lead to a parallel channel mechanism. The simple sequential mechanism deduced for sIGPS reflects the dominance of the on-pathway burst-phase intermediate and the absence of prolyl residues that partition the stable intermediate into kinetically distinguishable species. Comparison of the results for sIGPS and alphaTS demonstrates that the thermodynamic properties and the final steps of the folding reaction are better conserved than the early events. The initial events in folding appear to be more sensitive to the sequence differences between the two TIM barrel proteins.     

Partial NMR assignments and secondary structure mapping of the isolated alpha subunit of Escherichia coli tryptophan synthase,a 29-kD TIM barrel protein

The alpha subunit of tryptophan synthase (alphaTS) from S. typhimurium belongs to the triosephosphate isomerase (TIM) or the (beta/alpha)(8) barrel fold, one of the most common structures in biology. To test the conservation of the global fold in the isolated Escherichia coli homolog, we have obtained a majority of the backbone assignments for the 29-kD alphaTS by using standard heteronuclear multidimensional NMR methods on uniformly (15)N- and (15)N/(13)C-labeled protein and on protein selectively (15)N-labeled at key hydrophobic residues. The secondary structure mapped by chemical shift index, nuclear Overhauser enhancements (NOEs), and hydrogen-deuterium (H-D) exchange, and several abnormal chemical shifts are consistent with the conservation of the global TIM barrel fold of the isolated E. coli alphaTS. Because most of the amide protons that are slow to exchange with solvent correspond to the beta-sheet residues, the beta-barrel is likely to play an important role in stabilizing the previously detected folding intermediates for E. coli alphaTS. A similar combination of uniform and selective labeling can be extended to other TIM barrel proteins to obtain insight into the role of the motif in stabilizing what appear to be common partially folded forms.     

Structural analysis of kinetic folding intermediates for a TIM barrel protein, indole-3-glycerol phosphate synthase, by hydrogen exchange mass spectrometry and Gō model simulation

The structures of partially folded states appearing during the folding of a (βα)₈ TIM barrel protein, the indole-3-glycerol phosphate synthase from Sulfolobus solfataricus (sIGPS), was assessed by hydrogen exchange mass spectrometry (HX-MS) and Gō model simulations. HX-MS analysis of the peptic peptides derived from the pulse-labeled product of the sub-millisecond folding reaction from the urea-denatured state revealed strong protection in the (βα)₄ region, modest protection in the neighboring (βα)_1-3 and (βα)₅β₆ segments and no significant protection in the remaining N and C-terminal segments. These results demonstrate that this species is not a collapsed form of the unfolded state under native-favoring conditions nor is it the native state formed via fast-track folding. However, the striking contrast of these results with the strong protection observed in the (βα)_2-5β₆ region after 5 s of folding demonstrates that these species represent kinetically distinct folding intermediates that are not identical as previously thought. A re-examination of the kinetic folding mechanism by chevron analysis of fluorescence data confirmed distinct roles for these two species: the burst-phase intermediate is predicted to be a misfolded, off-pathway intermediate, while the subsequent 5 s intermediate corresponds to an on-pathway equilibrium intermediate. Comparison with the predictions using a C^α Gō model simulation of the kinetic folding reaction for sIGPS shows good agreement with the core of the structure offering protection against exchange in the on-pathway intermediate(s). Because the native-centric Gō model simulations do not explicitly include sequence-specific information, the simulation results support the hypothesis that the topology of TIM barrel proteins is a primary determinant of the folding free energy surface for the productive folding reaction. The early misfolding reaction must involve aspects of non-native structure not detected by the Gō model simulation.     

An obligatory intermediate controls the folding of the alpha-subunit of tryptophan synthase, a TIM barrel protein

The proposed kinetic folding mechanism of the alpha-subunit of tryptophan synthase (alphaTS), a TIM barrel protein, displays multiple unfolded and intermediate forms which fold through four parallel pathways to reach the native state. To obtain insight into the secondary structure that stabilizes a set of late, highly populated kinetic intermediates, the refolding of urea-denatured alphaTS from Escherichia coli was monitored by pulse-quench hydrogen exchange mass spectrometry. Following dilution from 8 M urea, the protein was pulse-labeled with deuterium, quenched with acid and mass analyzed by electrospray ionization mass spectrometry (ESI-MS). Hydrogen bonds that form prior to the pulse of deuterium offer protection against exchange and, therefore, retain protons at the relevant amide bonds. Consistent with the proposed refolding model, an intermediate builds up rapidly and decays slowly over the first 100 seconds of folding. ESI-MS analysis of the peptic fragments derived from alphaTS mass-labeled and quenched after two seconds of refolding indicates that the pattern of protection of the backbone amide hydrogens in this transient intermediate is very similar to that observed previously for the equilibrium intermediate of alphaTS highly populated at 3 M urea. The protection observed in a contiguous set of beta-strands and alpha-helices in the N terminus implies a significant role for this sub-domain in directing the folding of this TIM barrel protein.     

Multi-state unfolding of the alpha subunit of tryptophan synthase, a TIM barrel protein: insights into the secondary structure of the stable equilibrium intermediates by hydrogen exchange mass spectrometry

The urea-induced unfolding of the alpha subunit of tryptophan synthase (alphaTS) from Escherichia coli, an eight-stranded (beta/alpha)(8) TIM barrel protein, has been shown to involve two stable equilibrium intermediates, I1 and I2, well populated at approximately 3 M and 5 M urea, respectively. The characterization of the I1 intermediate by circular dichroism (CD) spectroscopy has shown that I1 retains a significant fraction of the native ellipticity; the far-UV CD signal for the I2 species closely resembles that of the fully unfolded form. To obtain detailed insight into the disruption of secondary structure in the urea-induced unfolding process, a hydrogen exchange-mass spectrometry study was performed on alphaTS. The full-length protein was destabilized in increasing concentration of urea, the amide hydrogen atoms were pulse-labeled with deuterium, the labeled samples were quenched in acid and the products were analyzed by electrospray ionization mass spectrometry. Consistent with the CD results, the I1 intermediate protects up to approximately 129 amide hydrogen atoms against exchange while the I2 intermediate offers no protection. Electrospray ionization mass spectrometry analysis of the peptic fragments derived from alphaTS labeled at 3 M urea indicates that most of the region between residues 12-130, which constitutes the first four beta strands and three alpha helices, (beta/alpha)(1-3)beta(4), is structured. The (beta/alpha)(1-3)beta(4) module appears to represent the minimum sub-core of stability of the I1 intermediate. A 4+2+2 folding model is proposed as a likely alternative to the earlier 6+2 folding mechanism for alphaTS.     

Equilibrium and kinetic folding pathways of a TIM barrel with a funneled energy landscape

The role of native contact topology in the folding of a TIM barrel model based on the alpha-subunit of tryptophan synthase (alphaTS) from Salmonella typhimurium (Protein Data Bank structure 1BKS) was studied using both equilibrium and kinetic simulations. Equilibrium simulations of alphaTS reveal the population of two intermediate ensembles, I1 and I2, during unfolding/refolding at the folding temperature, Tf = 335 K. Equilibrium intermediate I1 demonstrates discrete structure in regions alpha0-beta6 whereas intermediate I2 is a loose ensemble of states with N-terminal structure varying from at least beta1-beta3 (denoted I2A) to alpha0-beta4 at most (denoted I2B). The structures of I1 and I2 match well with the two intermediate states detected in equilibrium folding experiments of Escherichia coli alphaTS. Kinetic folding simulations of alphaTS reveal the sequential population of four intermediate ensembles, I120Q, I200Q, I300Q, and I360Q, during refolding. Kinetic intermediates I120Q, I200Q, and I300Q are highly similar to equilibrium alphaTS intermediates I2A, I2B, and I1, respectively, consistent with kinetic experiments on alphaTS from E. coli. A small population (approximately 10%) of kinetic trajectories are trapped in the I120Q intermediate ensemble and require a slow and complete unfolding step to properly refold. Both the on-pathway and off-pathway I120Q intermediates show structure in beta1-beta3, which is also strikingly consistent with kinetic folding experiments of alphaTS. In the off-pathway intermediate I(120Q), helix alpha2 is wrapped in a nonnative chiral arrangement around strand beta3, sterically preventing the subsequent folding step between beta3 and beta4. These results demonstrate the success of combining kinetic and equilibrium simulations of minimalist protein models to explore TIM barrel folding and the folding of other large proteins.     

Specific structure appears at the N terminus in the sub-millisecond folding intermediate of the alpha subunit of tryptophan synthase, a TIM barrel protein

Competing views of the products of sub-millisecond folding reactions observed in many globular proteins have been ascribed either to the formation of discrete, partially folded states or to the random collapse of the unfolded chain under native-favoring conditions. To test the validity of these alternative interpretations for the stopped-flow burst-phase reaction in the (betaalpha)8, TIM barrel motif, a series of alanine replacements were made at five different leucine or isoleucine residues in the alpha subunit of tryptophan synthase (alphaTS) from Escherichia coli. This protein has been proposed to fold, in the sub-millisecond time range, to an off-pathway intermediate with significant stability and approximately 50% of the far-UV circular dichroism (CD) signal of the native conformation. Individual alanine replacements at any of three isoleucine or leucine residues in either alpha1, beta2 or beta3 completely eliminate the off-pathway species. These variants, within 5 ms, access an intermediate whose properties closely resemble those of an on-pathway equilibrium intermediate that is highly populated at moderate urea concentrations in wild-type alphaTS. By contrast, alanine replacements for leucine residues in either beta4 or beta6 destabilize but preserve the off-pathway, burst-phase species. When considered with complementary thermodynamic and kinetic data, this mutational analysis demonstrates that the sub-millisecond appearance of CD signal for alphaTS reflects the acquisition of secondary structure in a distinct thermodynamic state, not the random collapse of an unfolded chain. The contrasting results for replacements in the contiguous alpha1/beta2/beta3 domain and the C-terminal beta4 and beta6 strands imply a heterogeneous structure for the burst-phase species. The alpha1/beta2/beta3 domain appears to be tightly packed, and the C terminus appears to behave as a molten-globule-like structure whose folding is tightly coupled to that of the alpha1/beta2/beta3 domain.     

NMR analysis of partially folded states and persistent structure in the alpha subunit of tryptophan synthase: implications for the equilibrium folding mechanism of a 29-kDa TIM barrel protein

Structural insights into the equilibrium folding mechanism of the alpha subunit of tryptophan synthase (αTS) from Escherichia coli, a (βα)₈ TIM barrel protein, were obtained with a pair of complementary nuclear magnetic resonance (NMR) spectroscopic techniques. The secondary structures of rare high-energy partially folded states were probed by native-state hydrogen-exchange NMR analysis of main-chain amide hydrogens. 2D heteronuclear single quantum coherence NMR analysis of several ¹⁵N-labeled nonpolar amino acids was used to probe the side chains involved in stabilizing a highly denatured intermediate that is devoid of secondary structure. The dynamic broadening of a subset of isoleucine and leucine side chains and the absence of protection against exchange showed that the highest energy folded state on the free-energy landscape is stabilized by a hydrophobic cluster lacking stable secondary structure. The core of this cluster, centered near the N-terminus of αTS, serves as a nucleus for the stabilization of what appears to be nonnative secondary structure in a marginally stable intermediate. The progressive decrease in protection against exchange from this nucleus toward both termini and from the N-termini to the C-termini of several β-strands is best described by an ensemble of weakly coupled conformers. Comparison with previous data strongly suggests that this ensemble corresponds to a marginally stable off-pathway intermediate that arises in the first few milliseconds of folding and persists under equilibrium conditions. A second, more stable intermediate, which has an intact β-barrel and a frayed α-helical shell, coexists with this marginally stable species. The conversion of the more stable intermediate to the native state of αTS entails the formation of a stable helical shell and completes the acquisition of the tertiary structure.     

Long-range side-chain-main-chain interactions play crucial roles in stabilizing the (betaalpha)8 barrel motif of the alpha subunit of tryptophan synthase

The role of hither-to-fore unrecognized long-range hydrogen bonds between main-chain amide hydrogens and polar side chains on the stability of a well-studied (betaalpha)8, TIM barrel protein, the alpha subunit of tryptophan synthase (alphaTS), was probed by mutational analysis. The F19-D46 and I97-D124 hydrogen bonds link the N terminus of a beta-strand with the C terminus of the succeeding antiparallel alpha-helix, and the A103-D130 hydrogen bond links the N terminus of an alpha-helix with the C terminus of the succeeding antiparallel beta-strand, forming clamps for the respective betaalpha or alphabeta hairpins. The individual replacement of these aspartic acid side chains with alanine leads to what appear to be closely related partially folded structures with significantly reduced far-UV CD ellipticity and thermodynamic stability. Comparisons with the effects of eliminating another main-chain-side-chain hydrogen bond, G26-S33, and two electrostatic side-chain-side-chain hydrogen bonds, D38-H92 and D112-H146, all in the same N-terminal folding unit of alphaTS, demonstrated a unique role for the clamp interactions in stabilizing the native barrel conformation. Because neither the asparagine nor glutamic acid variant at position 46 can completely reproduce the spectroscopic, thermodynamic, or kinetic folding properties of aspartic acid, both size and charge are crucial to its unique role in the clamp hydrogen bond. Kinetic studies suggest that the three clamp hydrogen bonds act in concert to stabilize the transition state leading to the fully folded TIM barrel motif.     

Molecular dissection of the folding mechanism of the alpha subunit of tryptophan synthase: an amino-terminal autonomous folding unit controls several rate-limiting steps in the folding of a single domain protein.

The alpha subunit of tryptophan synthase (alphaTS) from Escherichia coli is a 268-residue 8-stranded beta/alpha barrel protein. Two autonomous folding units, comprising the first six strands (residues 1-188) and the last two strands (residues 189-268), have been previously identified in this single structural domain protein by tryptic digestion [Higgins, W., Fairwell, T., and Miles, E. W. (1979) Biochemistry 18, 4827-4835]. The larger, amino-terminal fragment, alphaTS(1-188), was overexpressed and independently purified, and its equilibrium and kinetic folding properties were studied by absorbance, fluorescence, and near- and far-UV circular dichroism spectroscopies. The native state of the fragment unfolds cooperatively in an apparent two-state transition with a stability of 3.98 +/- 0.19 kcal mol(-1) in the absence of denaturant and a corresponding m value of 1.07 +/- 0.05 kcal mol(-1) M(-1). Similar to the full-length protein, the unfolding of the fragment shows two kinetic phases which arise from the presence of two discrete native state populations. Additionally, the fragment exhibits a significant burst phase in unfolding, indicating that a fraction of the folded state ensemble under native conditions has properties similar to those of the equilibrium intermediate populated at 3 M urea in full-length alphaTS. Refolding of alphaTS(1-188) is also complex, exhibiting two detectable kinetic phases and a burst phase that is complete within 5 ms. The two slowest isomerization phases observed in the refolding of the full-length protein are absent in the fragment, suggesting that these phases reflect contributions from the carboxy-terminal segment. The folding mechanism of alphaTS(1-188) appears to be a simplified version of the mechanism for the full-length protein [Bilsel, O., Zitzewitz, J. A., Bowers, K.E, and Matthews, C. R.(1999) Biochemistry 38, 1018-1029]. Four parallel channels in the full-length protein are reduced to a pair of channels that most likely reflect a cis/trans proline isomerization reaction in the amino-terminal fragment. The off- and on-pathway intermediates that exist for both full-length alphaTS and alphaTS(1-188) may reflect the preponderance of local interactions in the beta/alpha barrel motif.     

A tightly packed hydrophobic cluster directs the formation of an off-pathway sub-millisecond folding intermediate in the alpha subunit of tryptophan synthase, a TIM barrel protein

Protein misfolding is now recognized as playing a crucial role in both normal and pathogenic folding reactions. An interesting example of misfolding at the earliest state of a natural folding reaction is provided by the alpha-subunit of tryptophan synthase, a (beta/alpha)(8) TIM barrel protein. The molecular basis for the formation of this off-pathway misfolded intermediate, I(BP), and a subsequent on-pathway intermediate, I1, was probed by mutational analysis of 20 branched aliphatic side-chains distributed throughout the sequence. The elimination of I(BP) and the substantial destabilization of I1 by replacement of a selective set of the isoleucine, leucine or valine residues (ILV) with alanine in a large ILV cluster external-to-the-barrel and spanning the N and C termini (cluster 2) implies tight-packing at most sites in both intermediates. Differential effects on I(BP) and I1 for replacements in alpha3, beta4 and alpha8 at the boundaries of cluster 2 suggest that their incorporation into I1 but not I(BP) reflects non-native folds at the edges of the crucial (beta/alpha)(1-2)beta(3) core in I(BP). The retention of I(BP) and the smaller and consistent destabilization of both I(BP) and I1 by similar replacements in an internal-to-the-barrel ILV cluster (cluster 1) and a second external-to-the-barrel ILV cluster (cluster 3) imply molten globule-like packing. The tight packing inferred, in part, for I(BP) or for all of I1 in cluster 2, but not in clusters 1 and 3, may reflect the larger size of cluster 2 and/or the enhanced number of isoleucine, leucine and valine self-contacts in and between contiguous elements of secondary structure. Tightly packed ILV-dominated hydrophobic clusters could serve as an important driving force for the earliest events in the folding and misfolding of the TIM barrel and other members of the (beta/alpha)(n) class of proteins.     

Detecting equilibrium cytochrome c folding intermediates by electrospray ionisation mass spectrometry: two partially folded forms populate the molten-globule state

Nanoelectrospray ionization mass spectrometry (nano-ESI-MS) is applied to the characterization of ferric cytochromec (cytc) conformational states under different solvent conditions. The methanol-induced molten-globule state in the pH range 2.6-3.0 is found to be populated by two distinct, partially folded conformers I(A) and I(B). The more compact intermediate I(B) resembles that induced by glycerol in acid-unfolded cytc. The less compact one, I(A), also can be induced by destabilization of the native structure by trifluoroethanol. I(A) and I(B) can be detected, in the absence of additives, around the midpoint of the acid-induced unfolding transition, providing direct evidence for involvement of equilibrium folding intermediates in cytc conformational transitions at low pH. This study shows that mass spectrometry can contribute to the characterization of molten-globule states of proteins by detection of distinct, although poorly populated, conformations involved in a dynamic equilibrium.     

Kinetic traps in the folding of beta alpha-repeat proteins: CheY initially misfolds before accessing the native conformation

The βα-repeat class of proteins, represented by the (βα)₈ barrel and the α/β/α sandwich, are among the most common structural platforms in biology. Previous studies on the folding mechanisms of these motifs have revealed or suggested that the initial event involves the submillisecond formation of a kinetically trapped species that must at least partially unfold before productive folding to the respective native conformation can occur. To test the generality of these observations, CheY, a bacterial response regulator, was subjected to an extensive analysis of its folding reactions. Although earlier studies had proposed the formation of an off-pathway intermediate, the data available were not sufficient to rule out an alternative on-pathway mechanism. A global analysis of single- and double-jump kinetic data, combined with equilibrium unfolding data, was used to show that CheY folds and unfolds through two parallel channels defined by the state of isomerization of a prolyl peptide bond in the active site. Each channel involves a stable, highly structured folding intermediate whose kinetic properties are better described as the properties of an off-pathway species. Both intermediates subsequently flow through the unfolded state ensemble and adopt the native cis-prolyl isomer prior to forming the native state. Initial collapse to off-pathway folding intermediates is a common feature of the folding mechanisms of βα-repeat proteins, perhaps reflecting the favored partitioning to locally determined substructures that cannot directly access the native conformation. Productive folding requires the dissipation of these prematurely folded substructures as a prelude to forming the larger-scale transition state that leads to the native conformation. Results from Gō-modeling studies in the accompanying paper elaborate on the topological frustration in the folding free-energy landscape of CheY.     

The high-resolution NMR structure of the early folding intermediate of the Thermus thermophilus ribonuclease H

Elucidation of the high-resolution structures of folding intermediates is a necessary but difficult step toward the ultimate understanding of the mechanism of protein folding. Here, using hydrogen-exchange-directed protein engineering, we populated the folding intermediate of the Thermus thermophilus ribonuclease H, which forms before the rate-limiting transition state, by removing the unfolded regions of the intermediate, including an α-helix and two β-strands (51 folded residues). Using multidimensional NMR, we solved the structure of this intermediate mimic to an atomic resolution (backbone rmsd, 0.51 Å). It has a native-like backbone topology and shows some local deviations from the native structure, revealing that the structure of the folded region of an early folding intermediate can be as well defined as the native structure. The topological parameters calculated from the structures of the intermediate mimic and the native state predict that the intermediate should fold on a millisecond time scale or less and form much faster than the native state. Other factors that may lead to the slow folding of the native state and the accumulation of the intermediate before the rate-limiting transition state are also discussed.     

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.     

Time-resolved fluorescence anisotropy study of the refolding reaction of the alpha-subunit of tryptophan synthase reveals nonmonotonic behavior of the rotational correlation time

Time-resolved fluorescence anisotropy of a bound extrinsic probe was studied in an effort to characterize dynamic properties of the transient partially folded forms that appear during the folding of the alpha-subunit of tryptophan synthase (alphaTS) from Escherichia coli. Previous studies have shown that alphaTS, a single structural domain, can be cleaved into autonomously folding amino- and carboxy-folding units comprising residues 1-188 and 189-268, respectively [Higgins, W., Fairwell, T., and Miles, E. W. (1979) Biochemistry 18, 4827-4835]. By use of a double-kinetic approach [Jones, B. E., Beechem, J. M., and Matthews, C. R. (1995) Biochemistry 34, 1867-1877], the rotational correlation time of 1-anilino-8-naphthalene sulfonate bound to nonpolar surfaces of folding intermediates was measured by time-correlated single photon counting at varying time delays following initiation of folding from the urea-denatured form by stopped-flow techniques. Comparison of the rotational correlation times for the full-length alphaTS and the amino-terminal fragment suggests that folding of the amino-terminal fragment and carboxy-terminal fragment is coordinated, not autonomous, on the milliseconds to seconds time scale. If a spherical shape is assumed, the apparent hydrodynamic radius of alphaTS after 5 ms is 26.8 A. The radius increases to 28.5 A by 1 s before decreasing to the radius for native alphaTS, 24.7 A, on a longer time scale (>25 s). Viewed within the context of the kinetic folding model of alphaTS [Bilsel, O., Zitzewitz, J. A., Bowers, K. E. , and Matthews, C. R. (1999) Biochemistry 38, 1018-1029], the initial collapse reflects the formation of an off-pathway burst-phase intermediate in which at least part of the carboxy folding unit interacts with the amino folding unit. The subsequent increase in rotational correlation time corresponds to the formation of an on-pathway intermediate that leads to the native conformation. The apparent increase in the radius for the on-pathway intermediate may reflect a change in the interaction of the two-folding units, thereby forming a direct precursor for the alpha/beta barrel structure.     

The progressive development of structure and stability during the equilibrium folding of the alpha subunit of tryptophan synthase from Escherichia coli.

The urea-induced equilibrium unfolding of the alpha subunit of tryptophan synthase (alphaTS), a single domain alpha/beta barrel protein, displays a stable intermediate at approximately 3.2 M urea when monitored by absorbance and circular dichroism (CD) spectroscopy (Matthews CR, Crisanti MM, 1981, Biochemistry 20:784-792). The same experiment, monitored by one-dimensional proton NMR, shows another cooperative process between 5 and 9 M urea that involves His92 (Saab-Rincón G et al., 1993, Biochemistry 32:13,981-13,990). To further test and quantify the implied four-state model, N <--> I1 <--> I2 <--> U, the urea-induced equilibrium unfolding process was followed by tyrosine fluorescence total intensity, tyrosine fluorescence anisotropy and far-UV CD. All three techniques resolve the four stable states, and the transitions between them when the FL total intensity and CD spectroscopy data were analyzed by the singular value decomposition method. Relative to U, the stabilities of the N, I1, and I2 states are 15.4, 9.4, and 4.9 kcal mol(-1), respectively. I2 partially buries one or more of the seven tyrosines with a noticeable restriction of their motion; it also recovers approximately 6% of the native CD signal. This intermediate, which is known to be stabilized by the hydrophobic effect, appears to reflect the early coalescence of nonpolar side chains without significant organization of the backbone. I1 recovers an additional 43% of the CD signal, further sequesters tyrosine residues in nonpolar environments, and restricts their motion to an extent similar to N. The progressive development of a higher order structure as the denaturant concentration decreases implies a monotonic contraction in the ensemble of conformations that represent the U, I2, I1, and N states of alphaTS.     

Absence of a stable intermediate on the folding pathway of protein A.

The B-domain of protein A has one of the simplest protein topologies, a three-helix bundle. Its folding has been studied as a model for elementary steps in the folding of larger proteins. Earlier studies suggested that folding might occur by way of a helical hairpin intermediate. Equilibrium hydrogen exchange measurements indicate that the C-terminal helical hairpin could be a potential folding intermediate. Kinetic refolding experiments were performed using stopped-flow circular dichroism and NMR hydrogen-deuterium exchange pulse labeling. Folding of the entire molecule is essentially complete within the 6 ms dead time of the quench-flow apparatus, indicating that the intermediate, if formed, progresses rapidly to the final folded state. Site-directed mutagenesis of the isoleucine residue at position 16 was used to generate a variant protein containing tryptophan (the 116 W mutant). The formation of the putative folding intermediate was expected to be favored in this mutant at the expense of the native folded form, due to predicted unfavorable steric interactions of the bulky tryptophan side chain in the folded state. The 116 W mutant refolds completely within the dead time of a stopped-flow fluorescence experiment. No partly folded intermediate could be detected by either kinetic or equilibrium measurements. Studies of peptide fragments suggest that the protein A sequence has an intrinsic propensity to form a helix II/helix III hairpin. However, its stability appears to be marginal (of the order of 1/2 kT) and it could not be an obligatory intermediate on a defined folding pathway. These results explicitly demonstrate that the protein A B domain folds extremely rapidly by an apparent two-state mechanism without formation of stable partly folded intermediates. Similar mechanisms may also be involved in the rapid folding of subdomains of larger proteins to form the compact molten globule intermediates that often accumulate during the folding process.     

Probing the folding intermediate of Rd-apocyt b562 by protein engineering and infrared T-jump

Small proteins often fold in an apparent two-state manner with the absence of detectable early-folding intermediates. Recently, using native-state hydrogen exchange, intermediates that exist after the rate-limiting transition state have been identified for several proteins. However, little is known about the folding kinetics from these post-transition intermediates to their corresponding native states. Herein, we have used protein engineering and a laser-induced temperature-jump (T-jump) technique to investigate this issue and have applied it to Rd-apocyt b(562) , a four-helix bundle protein. Previously, it has been shown that Rd-apocyt b(562) folds via an on-pathway hidden intermediate, which has only the N-terminal helix unfolded. In the present study, a double mutation (V16G/I17A) in the N-terminal helix of Rd-apocyt b(562) was made to further increase the relative population of this intermediate state at high temperature by selectively destabilizing the native state. In the circular dichroism thermal melting experiment, this mutant showed apparent two-state folding behavior. However, in the T-jump experiment, two kinetic phases were observed. Therefore, these results are in agreement with the idea that a folding intermediate is populated on the folding pathway of Rd-apocyt b(562) . Moreover, it was found that the exponential growth rate of the native state from this intermediate state is roughly (25 microsec)(-1) at 65 degrees C.