Rate-limiting Step In Folding Research Articles

Early Events During Folding of Wild-type Staphylococcal Nuclease and a Single-tryptophan Variant Studied by Ultrarapid Mixing

A continuous-flow mixing device with a dead time of 100 μs coupled with intrinsic tryptophan and 1-anilinonaphthalene-8-sulfonate (ANS) fluorescence was used to monitor structure formation during early stages of the folding of staphylococcal nuclease (SNase). A variant with a unique tryptophan fluorophore in the N-terminal β-barrel domain (Trp76 SNase) was obtained by replacing the single Trp140 in wild-type SNase with His in combination with Trp substitution of Phe76. A common background of P47G, P117G and H124L mutations was chosen in order to stabilize the protein and prevent accumulation of cis proline isomers under native conditions. In contrast to WT ∗ SNase, which shows no changes in tryptophan fluorescence prior to the rate-limiting folding step (∼100 ms), the F76W/W140H variant shows additional changes (enhancement) during an early folding phase with a time constant of 75 μs. Both proteins exhibit a major increase in ANS fluorescence and identical rates for this early folding event. These findings are consistent with the rapid accumulation of an ensemble of states containing a loosely packed hydrophobic core involving primarily the β-barrel domain while the specific interactions in the α-helical domain involving Trp140 are formed only during the final stages of folding. The fact that both variants exhibit the same number of kinetic phases with very similar rates confirms that the folding mechanism is not perturbed by the F76W/W140H mutations. However, the Trp at position 76 reports on the rapid formation of a hydrophobic cluster in the N-terminal β-sheet region while the wild-type Trp140 is silent during this early stage of folding. Quantitative modeling of the (un)folding kinetics and thermodynamics of these two proteins versus urea concentration revealed that the F76W/W140H mutation selectively destabilizes the native state relative to WT ∗ SNase while the stability of transient intermediates remains unchanged, leading to accumulation of intermediates under equilibrium conditions at moderate denaturant concentrations.

Rapid Collapse Precedes the Fast Two-state Folding of the Cold Shock Protein

The cold shock protein Bc-Csp folds very rapidly in a reaction that is well described by a kinetic two-state mechanism without intermediates. We measured the shortening of six intra-protein distances during folding by Förster resonance energy transfer (FRET) in combination with stopped-flow experiments. Single tryptophan residues were engineered into the protein as the donors, and single 5-(((acetylamino)ethyl)amino)naphthalene-1-sulfonate (AEDANS) residues were placed as the acceptors at solvent-exposed sites of Bc-Csp. Their R 0 value of about 22 Å was well suited for following distance changes during the folding of this protein with a high sensitivity. The mutagenesis and the labeling did not alter the refolding kinetics. The changes in energy transfer during folding were monitored by both donor and acceptor emission and reciprocal effects were found. In two cases the donor–acceptor distances were similar in the unfolded and the folded state and, as a consequence, the kinetic changes in energy transfer upon folding were very small. For four donor/acceptor pairs we found that ≥50% of the increase in energy transfer upon folding occurred prior to the rate-limiting step of folding. This reveals that about half of the shortening of the intra-molecular distances upon folding has occurred already before the rate-limiting step and suggests that the fast two-state folding reaction of Bc-Csp is preceded by a very rapid collapse.

Ab initio Folding Simulation of the Trp-cage Mini-protein Approaches NMR Resolution

Here, we report a 100ns molecular dynamics simulation of the folding process of a recently designed autonomous-folding mini-protein designated as tc5b with a new AMBER force field parameter set developed based on condensed-phase quantum mechanical calculations and a Generalized Born continuum solvent model. Starting from its fully extended conformation, our simulation has produced a final structure resembling that of NMR native structure to within 1Å main-chain root mean square deviation. Remarkably, the simulated structure stayed in the native state for most part of the simulation after it reached the state. Of greater significance is that our simulation has not only reached the correct main-chain conformation, but also a very high degree of accuracy in side-chain packing conformation. This feat has traditionally been a challenge for ab initio simulation studies. In addition to characterization of the trajectory, comparison of our results to experimental data is also presented. Analysis of the trajectory suggests that the rate-limiting step of folding of this mini-protein is the packing of the Trp side-chain.

The topomer search model: A simple, quantitative theory of two-state protein folding kinetics.

Most small, single-domain proteins fold with the uncomplicated, single-exponential kinetics expected for diffusion on a smooth energy landscape. Despite this energetic smoothness, the folding rates of these two-state proteins span a remarkable million-fold range. Here, we review the evidence in favor of a simple, mechanistic description, the topomer search model, which quantitatively accounts for the broad scope of observed two-state folding rates. The model, which stipulates that the search for those unfolded conformations with a grossly correct topology is the rate-limiting step in folding, fits observed rates with a correlation coefficient of approximately 0.9 using just two free parameters. The fitted values of these parameters, the pre-exponential attempt frequency and a measure of the difficulty of ordering an unfolded chain, are consistent with previously reported experimental constraints. These results suggest that the topomer search process may dominate the relative barrier heights of two-state protein-folding reactions.

Multiple methionine substitutions are tolerated in T4 lysozyme and have coupled effects on folding and stability

In order to further explore the tolerance of proteins to amino acid substitutions within the interior, a series of core residues was replaced by methionine within the C-terminal domain of T4 lysozyme. By replacing leucine, isoleucine, valine and phenylalanine residues a total of 10 methionines could be introduced, which corresponds to a third of the residues that are buried in this domain. As more methionines are incorporated the protein gradually loses stability. This is attributed in part to a reduction in hydrophobic stabilization, in part to the increased entropic cost of localizing the long, flexible methionine sidechains, and in part to steric clashes. The changes in structure of the mutants relative to the wildtype protein are modest but tend to increase in an additive fashion as more methionines are included. In the most extreme case, namely the 10-methionine mutant, much of the C-terminal domain remains quite similar to wildtype (root-mean-square backbone shifts of 0.56 Å), while the F and G helices undergo rotations of approximately 20° and center-of-mass shifts of approximately 1.4 Å. For up to six methionine substitutions the changes in stability are additive. Beyond this point, however, the multiple mutants are somewhat more stable than suggested from the sum of their constituents, especially for those including the replacement Val111→Met. This is interpreted in terms of the larger structural changes associated with this substitution. The substituted sidechains in the mutant structures have somewhat higher crystallographic thermal factors than their counterparts in WT*. Nevertheless, the interiors of the mutant proteins retain a well-defined structure with little suggestion of molten-globule characteristics. Lysozymes in which selenomethionine has been incorporated rather than methionine tend to have increased stability. At the same time they also fold faster. This provides further evidence that, at the rate-limiting step in folding, the structure of the C-terminal domain of T4 lysozyme is similar to that of the fully folded protein.

Characterization of the nucleation step and folding of a collagen triple-helix peptide.

Peptide T1-892 is a triple-helical peptide designed to include two distinct domains: a C-terminal (Gly-Pro-Hyp)(4) sequence, together with an N-terminal 18-residue sequence from the alpha1(I) chain of type I collagen. Folding experiments of T1-892 using CD spectroscopy were carried out at varying concentrations and temperatures, and fitting of kinetic models to the data was used to obtain information about the folding mechanism and to derive rate constants. Proposed models include a heterogeneous population of monomers with respect to cis-trans isomerization and a third-order folding reaction from competent monomer to the triple helix. Fitting results support a nucleation domain composed of all or most of the (Gly-Pro-Hyp)(4) sequence, which must be in trans form before the monomer is competent to initiate triple-helix formation. The folding of competent monomer to a triple helix is best described by an all-or-none third-order reaction. The temperature dependence of the third-order rate constant indicates a negative activation energy and provides information about the thermodynamics of the trimerization step. These CD studies complement NMR studies carried out on the same peptide at high concentrations, illustrating how the rate-limiting folding step is affected by changes in concentration. This sequence preference of repeating Gly-Pro-Hyp units for the initiation of triple-helix formation in peptide T1-892 may be related to features in the triple-helix folding of collagens.

Native state EX2 and EX1 hydrogen exchange of Escherichia coli CspA, a small beta-sheet protein.

Escherichia coli CspA is a small all-beta-sheet protein that folds fast (tau = 4 ms) via an apparent two-state mechanism. Our previous studies have shown that a large aromatic cluster on the surface of the protein participates in the rate-limiting step of folding and thus may be part of the folding nucleus of this protein. To obtain a more detailed picture of molecular events at the peptide backbone during unfolding and folding of CspA, we used native state hydrogen exchange and nuclear magnetic resonance spectroscopy (NMR). The experiments with native CspA were performed over a range of pH values from low pH, where exchange is governed by a rapid equilibrium before chemical exchange (EX2 exchange), to high pH, where exchange is dictated by the rate of unfolding (EX1 exchange). Rates of folding and unfolding were determined for 11 residues. The distribution of rates of folding within the structure of CspA suggests that hairpin turns, including one near the aromatic cluster, may nucleate the folding of CspA.

Productive folding to the native state by a group II intron ribozyme

Group II introns are large catalytic RNA molecules that fold into compact structures essential for the catalysis of splicing and intron mobility reactions. Despite a growing body of information on the folded state of group II introns at equilibrium, there is currently no information on the folding pathway and little information on the ionic requirements for folding. Folding isotherms were determined by hydroxyl radical footprinting for the 32 individual protections that are distributed throughout a group II intron ribozyme derived from intron ai5γ. The isotherms span a similar range of Mg 2+ concentrations and share a similar index of cooperativity. Time-resolved hydroxyl radical footprinting studies show that all regions of the ribozyme fold slowly and with remarkable synchrony into a single catalytically active structure at a rate comparable to those of other ribozymes studied thus far. The rate constants for the formation of tertiary contacts and recovery of catalytic activity are identical within experimental error. Catalytic activity analyses in the presence of urea provide no evidence that the slow folding of the ai5γ intron is attributable to the presence of unproductive kinetic traps along the folding pathway. Taken together, the data suggest that the rate-limiting step for folding of group II intron ai5γ occurs early along the reaction pathway. We propose that this behavior resembles protein folding that is limited in rate by high contact order, or the need to form key tertiary interactions from partners that are located far apart in the primary or secondary structure.

Interaction of the Periplasmic Peptidylprolylcis-trans Isomerase SurA with Model Peptides: THE N-TERMINAL REGION OF SurA IS ESSENTIAL AND SUFFICIENT FOR PEPTIDE BINDING

One of the rate-limiting steps in protein folding has been shown to be the cis-trans isomerization of proline residues, which is catalyzed by a range of peptidylprolyl cis-trans isomerases. To characterize the interaction between model peptides and the periplasmic peptidylprolyl cis-trans isomerase SurA from E. coli, we employed a chemical cross-linking strategy that has been used previously to elucidate the interaction of substrates with other folding catalysts. The interaction between purified SurA and model peptides was significant in that it showed saturation and was abolished by denaturation of SurA; however the interaction was independent of the presence of proline residues in the model peptides. From results obtained by limited proteolysis we conclude that an N-terminal fragment of SurA, comprising 150 amino acids that do not contain the active sites involved in the peptidylprolyl cis-trans isomerization, is essential for the binding of peptides by SurA. This was confirmed by probing the interaction of the model peptide with the recombinant N-terminal fragment, expressed in Escherichia coli. Hence we propose that, similar to protein disulfide isomerase and other folding catalysts, SurA exhibits a modular architecture composed of a substrate binding domain and distinct catalytically active domains.

An early transition state for folding of the P4-P6 RNA domain.

Tertiary folding of the 160-nt P4-P6 domain of the Tetrahymena group I intron RNA involves burying of substantial surface area, providing a model for the folding of other large RNA domains involved in catalysis. Stopped-flow fluorescence was used to monitor the Mg2+-induced tertiary folding of pyrene-labeled P4-P6. At 35 degrees C with [Mg2+] approximately 10 mM, P4-P6 folds on the tens of milliseconds timescale with k(obs) = 15-31 s(-1). From these values, an activation free energy deltaG(double dagger) of approximately 8-16 kcal/mol is calculated, where the large range for deltaG(double dagger) arises from uncertainty in the pre-exponential factor relating k(obs) and delta G(double dagger). The folding rates of six mutant P4-P6 RNAs were measured and found to be similar to that of the wild-type RNA, in spite of significant thermodynamic destabilization or stabilization. The ratios of the kinetic and thermodynamic free energy changes phi = delta deltaG(double dagger)/delta deltaG(o') are approximately 0, implying a folding transition state in which most of the native-state tertiary contacts are not yet formed (an early folding transition state). The k(obs) depends on the Mg2+ concentration, and the initial slope of k(obs) versus [Mg2+] suggests that only approximately 1 Mg2+ ion is bound in the rate-limiting folding step. This is consistent with an early folding transition state, because folded P4-P6 binds many Mg2+ ions. The observation of a substantial deltaG(double dagger) despite an early folding transition state suggests that a simple two-state folding diagram for Mg2+-induced P4-P6 folding is incomplete. Our kinetic data are some of the first to provide quantitative values for an activation barrier and location of a transition state for tertiary folding of an RNA domain.

Nonglassy kinetics in the folding of a simple single-domain protein

Theory suggests that the otherwise rapid folding of simple heteropolymer models becomes "glassy"-dominated by multiple kinetically trapped misfolded states-at low temperatures or when the overall bias toward the native state is reduced relative to the depth of local minima. Experimental observations of nonsingle-exponential protein-folding kinetics have been taken as evidence that the protein-folding free energy landscape is similarly rough. No equivalent analysis, however, has been reported for a simple single-domain protein lacking prolines, disulfide bonds, prosthetic groups, or other gross structural features that might complicate folding. In an effort to characterize the glassiness of a folding free energy landscape in the absence of these potentially complicating factors, we have monitored the folding of a kinetically simple protein, peptostreptococcal protein L (protein L). We observe no statistically significant deviation from homogeneous single-exponential relaxation kinetics across temperatures ranging from near the protein's melting temperature to as low as -15 degrees C. On the basis of these observations, we estimate that, if there is a glass transition in the folding of protein L, it occurs at least 45 degrees C and possibly more than 145 degrees C below the freezing point of water. Apparently the folding free energy landscape of protein L is extremely smooth, which may be indicative of a rate-limiting step in folding that is, effectively, a nonglassy process.

Evidence concerning rate-limiting steps in protein folding from the effects of trifluoroethanol.

The refolding kinetics of 13 proteins have been studied in the presence of 2,2,2-trifluoroethanol (TFE). Low concentrations of TFE increased the folding rates of all the proteins, whereas higher concentrations have the opposite effect. The extent of deceleration of folding correlates closely with similar effects of guanidine hydrochloride and can be related to the burial of accessible surface area during folding. For those proteins folding in a two-state manner, the extent of acceleration of folding correlates closely with the number of local backbone hydrogen bonds in the native structure. For those proteins that fold in a multistate manner, however, the extent of acceleration is much smaller than that predicted from the data for two-state proteins. These results support the concept that for two-state proteins the search for native-like contacts is a key aspect of the folding reaction, whereas the rate-determining steps for folding of multistate proteins are associated with the reorganization of stable structure within a collapsed state or with the search for native-like interactions within less structured regions.

Methionine and alanine substitutions show that the formation of wild-type-like structure in the carboxy-terminal domain of T4 lysozyme is a rate-limiting step in folding.

In an attempt to identify a systematic relation between the structure of a protein and its folding kinetics, the rate of folding was determined for 20 mutants of T4 lysozyme in which a bulky, buried, nonpolar wild-type residue (Leu, Ile, Phe, Val, or Met) was substituted with alanine. Methionine, which approximated the size of the original side chain but which is of different shape and flexibility, was also substituted at most of the same sites. Mutations that substantially destabilize the protein and are located in the carboxy-terminal domain generally slow the rate of folding. Destabilizing mutations in the amino-terminal domain, however, have little effect on the rate of folding. Mutations that have little effect on stability tend to have little effect on the rate, no matter where they are located. These results suggest that, at the rate-limiting step, elements of structure in the C-terminal domain are formed and have a structure similar to that of the fully folded protein. Consistent with this, two variants that somewhat increase the rate of folding (Phe104 --> Met and Val149 --> Met) are located within the carboxy-terminal domain and maintain or improve packing with very little perturbation of the wild-type structure.

Folding dynamics of the B1 domain of protein G explored by ultrarapid mixing.

For many proteins, compact conformations are known to accumulate in advance of the rate-limiting step in folding. To understand the nature and significance of these early conformational events, we employed ultrarapid mixing methods to fully characterize the kinetics of folding of the 57-residue B1 domain of protein G. Continuous-flow fluorescence measurements exhibit a major exponential phase on the submillisecond time scale (600-700 micros), which is followed by a slower phase with a denaturant-dependent time constant (2-30 ms) observable by conventional stopped-flow measurements. The combined kinetic traces quantitatively account for the total change in Trp 43 fluorescence upon folding, including the previously unresolved 'burst phase' signal. The denaturant dependence of the two rate constants and their relative amplitudes are fully consistent with a three-state mechanism, U right harpoon over left harpoon I right harpoon over left harpoon N, where I is a productive intermediate with native-like fluorescence properties. The relatively slow rate and exponential time course of the initial folding phase indicates that a substantial free energy barrier is encountered during chain condensation, resulting in a partially organized ensemble of states distinct from the initial unfolded conformations.

Chain collapse can occur concomitantly with the rate-limiting step in protein folding.

We have directly characterized the extent of chain collapse early in the folding of protein L using time-resolved small angle X-ray scattering. We find that, immediately after the initiation of refolding, the protein exhibits dimensions indistinguishable from those observed under highly denaturing, equilibrium conditions and that this expanded initial state collapses with the same rate as that of the overall folding reaction. The observation that chain compaction need not significantly precede the rate-limiting step of folding demonstrates that rapid chain collapse is not an obligatory feature of protein folding reactions.

Effects of varying the local propensity to form secondary structure on the stability and folding kinetics of a rapid folding mixed α/β protein: characterization of a truncation mutant of the N-terminal domain of the ribosomal protein L9

The N-terminal domain of the ribosomal protein L9 forms a split βαβ structure with a long C-terminal helix. The folding transitions of a 56 residue version of this protein have previously been characterized, here we report the results of a study of a truncation mutant corresponding to residues 1–51. The 51 residue protein adopts the same fold as the 56 residue protein as judged by CD and two-dimensional NMR, but it is less stable as judged by chemical and thermal denaturation experiments. Studies with synthetic peptides demonstrate that the C-terminal helix of the 51 residue version has very little propensity to fold in isolation in contrast to the C-terminal helix of the 56 residue variant. The folding rates of the two proteins, as measured by stopped-flow fluorescence, are essentially identical, indicating that formation of local structure in the C-terminal helix is not involved in the rate-limiting step of folding.

Peptidyl-prolyl cis-trans isomerases, a superfamily of ubiquitous folding catalysts.

Cyclosporine A therapy for prophylaxis against graft rejection revolutionized human organ transplantation. The immunosuppressant drugs cyclosporin A (CsA), FK506 and rapamycin block T-cell activation by interfering with the signal transduction pathway. The target proteins for CsA and FK506 were found to be cyclophilins and FK506-binding proteins, (FKBPs), respectively. They are unrelated in primary sequence, although both are peptidyl-prolyl cis-trans isomerases catalyzing the interconversion of peptidylprolyl imide bonds in peptide and protein substrates. However, the prolyl isomerase activity of these proteins is not essential for their immunosuppressive effects. Instead, the specific surfaces of the cyclophilin-CsA and FKBP-FK506 complexes mediate the immunosuppressive action. Moreover, the natural cellular functions of all but a few remain elusive. In some cases it could be demonstrated that prolyl isomerization is the rate-limiting step in protein folding in vitro, but many knockout mutants of single and multiple prolyl isomerases were viable with no detectable phenotype. Even though a direct requirement for in vivo protein folding could not be demonstrated, some important natural substrates of the prolyl isomerases are now known, and they demonstrate the great variety of prolyl isomerization functions in the living cell: (i) A human cyclophilin binds to the Gag polyprotein of the human immunodeficiency virus-1 (HIV-1) virion and was found to be essential for infection with HIV to occur, probably by removal of the virion coat. (ii) Together with heat shock protein (HSP) 90, a member of the chaperone family, high molecular weight cyclophilins and FKBPs bind and activate steroid receptors. This example also demonstrates that prolyl isomerases act together with other folding enzymes, for example the chaperones, and protein disulfide isomerases. (iii) An FKBP was found to act as a modulator of an intracellular calcium release channel. (iv) Along with the cyclophilins and FKBPs, a third class of prolyl isomerases exist, the parvulins. The human parvulin homologue Pin1 is a mitotic regulator essential for the G2/M transition of the eukaryotic cell cycle. These findings place proline isomerases at the intersection of protein folding, signal transduction, trafficking, assembly and cell cycle regulation.

Hmo1p, a high mobility group 1/2 homolog, genetically and physically interacts with the yeast FKBP12 prolyl isomerase.

The immunosuppressive drugs FK506 and rapamycin bind to the cellular protein FKBP12, and the resulting FKBP12-drug complexes inhibit signal transduction. FKBP12 is a ubiquitous, highly conserved, abundant enzyme that catalyzes a rate-limiting step in protein folding: peptidyl-prolyl cis-trans isomerization. However, FKBP12 is dispensible for viability in both yeast and mice, and therefore does not play an essential role in protein folding. The functions of FKBP12 may involve interactions with a number of partner proteins, and a few proteins that interact with FKBP12 in the absence of FK506 or rapamycin have been identified, including the ryanodine receptor, aspartokinase, and the type II TGF-beta receptor; however, none of these are conserved from yeast to humans. To identify other targets and functions of FKBP12, we have screened for mutations that are synthetically lethal with an FKBP12 mutation in yeast. We find that mutations in HMO1, which encodes a high mobility group 1/2 homolog, are synthetically lethal with mutations in the yeast FPR1 gene encoding FKBP12. Deltahmo1 and Deltafpr1 mutants share two phenotypes: an increased rate of plasmid loss and slow growth. In addition, Hmo1p and FKBP12 physically interact in FKBP12 affinity chromatography experiments, and two-hybrid experiments suggest that FKBP12 regulates Hmo1p-Hmo1p or Hmo1p-DNA interactions. Because HMG1/2 proteins are conserved from yeast to humans, our findings suggest that FKBP12-HMG1/2 interactions could represent the first conserved function of FKBP12 other than mediating FK506 and rapamycin actions.

Exploring the temperature-pressure phase diagram of staphylococcal nuclease.

The temperature dependence of the pressure-induced equilibrium unfolding of staphylococcal nuclease (Snase) was determined by fluorescence of the single tryptophan residue, FTIR absorption for the amide I' and tyrosine O-H bands, and small-angle X-ray scattering (SAXS). The results from these three techniques were similar, although the stability as measured by fluorescence was slightly lower than that measured by FTIR and SAXS. The resulting phase diagram exhibits the well-known curvature for heat and cold denaturation of proteins, due to the large decrease in heat capacity upon folding. The volume change for unfolding became less negative with increasing temperatures, consistent with a larger thermal expansivity for the unfolded state than for the folded state. Fluorescence-detected pressure-jump kinetics measurements revealed that the curvature in the phase diagram is due primarily to the rate constant for folding, indicating a loss in heat capacity for the transition state relative to the unfolded state. The similar temperature dependence of the equilibrium and activation volume changes for folding indicates that the thermal expansivities of the folded and transition states are similar. This, along with the fact that the activation volume for folding is positive over the temperature range examined, the nonlinear dependence of the folding rate constant upon temperature implicates significant dehydration in the rate-limiting step for folding of Snase.

Viscosity dependence of the folding kinetics of a dimeric and monomeric coiled coil.

We measured whether solvent viscosity, and hence chain diffusion, plays a role in the rate-limiting step of the folding reactions of GCN4-p2', a simple alpha-helical coiled coil derived from the leucine zipper region of bZIP transcriptional activator GCN4. To deconvolute the dual effects of viscosogenic solvents on both viscosity, eta, and stability, earlier attempts assumed that the cosolvent and denaturant interact to the same degree in the transition state. Applying this analysis to GCN4-p2' yielded a nearly 1/eta dependence between folding rates and viscosity for both the dimeric and the cross-linked, monomeric versions of the coiled coil, but it revealed no such coherent relationship for cytochrome c. We also developed a method to determine the relative viscosity dependence of the dimeric and monomeric forms of the coiled coil independent of the assumption concerning the transition state's relative interaction with cosolvents and denaturants. Application of this method indicated that the effect of viscosity on both the folding and the unfolding rates was the same for the dimeric and monomeric versions, further supporting the view that the folding of the dimeric version is folding-limited rather than encounter-limited. The finding that GCN4-p2' folding appears to exhibit a 1/eta viscosity dependence implies that the rate-limiting step in folding is opposed predominantly by solvent-derived rather than internal frictional forces. These results are interpreted in relation to various models for protein folding.