Slow folding of a three-helix protein via a compact intermediate

The KIX domain of CREB binding protein (CBP) forms a small three-helix bundle which folds autonomously. Previous equilibrium unfolding experiments led to the suggestion that folding may not be strictly two-state. To investigate the folding mechanism in more detail, the folding kinetics of KIX have been studied by urea jump fluorescence-detected stopped-flow experiments. Clear evidence for an intermediate is obtained from the plot of the natural log of the observed rate constant versus denaturant concentration, the chevron plot, and from analysis of the initial fluorescence amplitudes of the stopped-flow experiments. The chevron plot exhibits a change in shape, rollover, at low denaturant concentrations, characteristic of the formation of an intermediate. The kinetic data can be fit to a three-state model involving a compact intermediate. An on-pathway model predicts that the position of the intermediate lies close to the native state. The folding rate in the absence of denaturant is 260 s(-)(1) at pH 7.5 and 25 degrees C. This is significantly slower than the rates of other helical proteins similar in size. The slow folding may be due to the necessity of forming a buried polar interaction in the native state. The potential functional significance of the folding intermediate is discussed.     

More compact protein globules exhibit slower folding rates

We have demonstrated that, among proteins of the same size, alpha/beta proteins have on the average a greater number of contacts per residue due to their more compact (more "spherical") structure, rather than due to tighter packing. We have examined the relationship between the average number of contacts per residue and folding rates in globular proteins according to general protein structural class (all-alpha, all-beta, alpha/beta, alpha+beta). Our analysis demonstrates that alpha/beta proteins have both the greatest number of contacts and the slowest folding rates in comparison to proteins from the other structural classes. Because alpha/beta proteins are also known to be the oldest proteins, it can be suggested that proteins have evolved to pack more quickly and into looser structures.     

Conformational equilibration time of unfolded protein chains and the folding speed limit

The speed with which the conformers of unfolded protein chains interconvert is a fundamental question in the study of protein folding. Kinetic evidence is presented here for the time constant for interconversion of disparate unfolded chain conformations of a small globular protein, cytochrome c, in the presence of guanidine hydrochloride denaturant. The axial binding reactions of histidine and methionine residues with the Fe(II) heme cofactor were monitored with time-resolved magnetic circular dichroism spectroscopy after photodissociation of the CO complexes of unfolded protein obtained from horse and tuna and from several histidine mutants of the horse protein. A kinetic model fitting both the reaction rate constants and spectra of the intermediates was used to obtain a quantitative estimate of the conformational diffusion time. The latter parameter was approximated as a first-order time constant for exchange between conformational subensembles presenting either a methionine or a histidine residue to the heme iron for facile binding. The mean diffusional time constant of the wild type and variants was 3 +/- 2 mus, close to the folding "speed limit". The implications of the relatively rapid conformational equilibration time observed are discussed in terms of the energy landscape and classical pathway time regimes of folding, for which the conformational diffusion time can be considered a pivot point.     

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.     

Function of trigger factor and DnaK in multidomain protein folding: increase in yield at the expense of folding speed

Trigger factor and DnaK protect nascent protein chains from misfolding and aggregation in the E. coli cytosol, but how these chaperones affect the mechanism of de novo protein folding is not yet understood. Upon expression under chaperone-depleted conditions, multidomain proteins such as bacterial beta-galactosidase (beta-gal) and eukaryotic luciferase fold by a rapid but inefficient default pathway, tightly coupled to translation. Trigger factor and DnaK improve the folding yield of these proteins but markedly delay the folding process both in vivo and in vitro. This effect requires the dynamic recruitment of additional trigger factor molecules to translating ribosomes. While beta-galactosidase uses this chaperone mechanism effectively, luciferase folding in E. coli remains inefficient. The efficient cotranslational domain folding of luciferase observed in the eukaryotic system is not compatible with the bacterial chaperone system. These findings suggest important differences in the coupling of translation and folding between bacterial and eukaryotic cells.     

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.     

Predicting the protein folding nucleus from sequences [correction of a sequence]

Understanding the mechanism of protein folding would allow prediction of the three-dimensional structure from sequence data alone. It has been shown that small proteins fold in a small number of kinetic steps and that significantly populated intermediate states exist for some of them. Studies of these intermediates have demonstrated the existence of specific interactions established during the initial stages of folding. Comparison of the amino acids participating in these specific and essential interactions and constituting the folding nucleus with conserved hydrophobic positions of a given fold shows a striking correspondence. This finding opens the perspective of predicting the folding nucleus knowing only a set of divergent sequences of a protein family.     

Small angle X-ray scattering reveals a compact intermediate in RNA folding

We have used small angle X-ray scattering (SAXS) to monitor changes in the overall size and shape of the Tetrahymena ribozyme as it folds. The native ribozyme, formed in the presence of Mg2+, is much more compact and globular than the ensemble of unfolded conformations. Time-resolved measurements show that most of the compaction occurs at least 20-fold faster than the overall folding to the native state, suggesting that a compact intermediate or family of intermediates is formed early and then rearranges in the slow steps that limit the overall folding rate. These results lead to a kinetic folding model in which an initial 'electrostatic collapse' of the RNA is followed by slower rearrangements of elements that are initially mispositioned.     

Clustering of large hydrophobes in the hydrophobic core of two-stranded alpha-helical coiled-coils controls protein folding and stability

The de novo design and biophysical characterization of two 60-residue peptides that dimerize to fold as parallel coiled-coils with different hydrophobic core clustering is described. Our goal was to investigate whether designing coiled-coils with identical hydrophobicity but with different hydrophobic clustering of non-polar core residues (each contained 6 Leu, 3 Ile, and 7 Ala residues in the hydrophobic core) would affect helical content and protein stability. The disulfide-bridged P3 and P2 differed dramatically in alpha-helical structure in benign conditions. P3 with three hydrophobic clusters was 98% alpha-helical, whereas P2 was only 65% alpha-helical. The stability profiles of these two analogs were compared, and the enthalpy and heat capacity changes upon denaturation were determined by measuring the temperature dependence by circular dichroism spectroscopy and confirmed by differential scanning calorimetry. The results showed that P3 assembled into a stable alpha-helical two-stranded coiled-coil and exhibited a native protein-like cooperative two-state transition in thermal melting, chemical denaturation, and calorimetry experiments. Although both peptides have identical inherent hydrophobicity (the hydrophobic burial of identical non-polar residues in equivalent heptad coiled-coil positions), we found that the context dependence of an additional hydrophobic cluster dramatically increased stability of P3 (Delta Tm approximately equal to 18 degrees C and Delta[urea](1/2) approximately equal to 1.5 M) as compared with P2. These results suggested that hydrophobic clustering significantly stabilized the coiled-coil structure and may explain how long fibrous proteins like tropomyosin maintain chain integrity while accommodating polar or charged residues in regions of the protein hydrophobic core.     

Cooperative folding in a multi-domain protein

Most protein domains are found in multi-domain proteins, yet most studies of protein folding have concentrated on small, single-domain proteins or on isolated domains from larger proteins. Spectrin domains are small (106 amino acid residues), independently folding domains consisting of three long alpha-helices. They are found in multi-domain proteins with a number of spectrin domains in tandem array. Structural studies have shown that in these arrays the last helix of one domain forms a continuous helix with the first helix of the following domain. It has been demonstrated that a number of spectrin domains are stabilised by their neighbours. Here we investigate the molecular basis for cooperativity between adjacent spectrin domains 16 and 17 from chicken brain alpha-spectrin (R16 and R17). We show that whereas the proteins unfold as a single cooperative unit at 25 degrees C, cooperativity is lost at higher temperatures and in the presence of stabilising salts. Mutations in the linker region also cause the cooperativity to be lost. However, the cooperativity does not rely on specific interactions in the linker region alone. Most mutations in the R17 domain cause a decrease in cooperativity, whereas proteins with mutations in the R16 domain still fold cooperatively. We propose a mechanism for this behaviour.     

Predicting repeat protein folding kinetics from an experimentally determined folding energy landscape

The Notch ankyrin domain is a repeat protein whose folding has been characterized through equilibrium and kinetic measurements. In previous work, equilibrium folding free energies of truncated constructs were used to generate an experimentally determined folding energy landscape (Mello and Barrick, Proc Natl Acad Sci USA 2004;101:14102–14107). Here, this folding energy landscape is used to parameterize a kinetic model in which local transition probabilities between partly folded states are based on energy values from the landscape. The landscape‐based model correctly predicts highly diverse experimentally determined folding kinetics of the Notch ankyrin domain and sequence variants. These predictions include monophasic folding and biphasic unfolding, curvature in the unfolding limb of the chevron plot, population of a transient unfolding intermediate, relative folding rates of 19 variants spanning three orders of magnitude, and a change in the folding pathway that results from C‐terminal stabilization. These findings indicate that the folding pathway(s) of the Notch ankyrin domain are thermodynamically selected: the primary determinants of kinetic behavior can be simply deduced from the local stability of individual repeats.     

Monomer topology defines folding speed of heptamer

Small monomeric proteins often fold in apparent two-state processes with folding speeds dictated by their native-state topology. Here we test, for the first time, the influence of monomer topology on the folding speed of an oligomeric protein: the heptameric cochaperonin protein 10 (cpn10), which in the native state has seven beta-barrel subunits noncovalently assembled through beta-strand pairing. Cpn10 is a particularly useful model because equilibrium-unfolding experiments have revealed that the denatured state in urea is that of a nonnative heptamer. Surprisingly, refolding of the nonnative cpn10 heptamer is a simple two-state kinetic process with a folding-rate constant in water (2.1 sec(-1); pH 7.0, 20 degrees C) that is in excellent agreement with the prediction based on the native-state topology of the cpn10 monomer. Thus, the monomers appear to fold as independent units, with a speed that correlates with topology, although the C and N termini are trapped in beta-strand pairing with neighboring subunits. In contrast, refolding of unfolded cpn10 monomers is dominated by a slow association step.     

Changes of protein stiffness during folding detect protein folding intermediates

Single-molecule force-quench atomic force microscopy (FQ-AFM) is used to detect folding intermediates of a simple protein by detecting changes of molecular stiffness of the protein during its folding process. Those stiffness changes are obtained from shape and peaks of an autocorrelation of fluctuations in end-to-end length of the folding molecule. The results are supported by predictions of the equipartition theorem and agree with existing Langevin dynamics simulations of a simplified model of a protein folding. In the light of the Langevin simulations the experimental data probe an ensemble of random-coiled collapsed states of the protein, which are present both in the force-quench and thermal-quench folding pathways.     

Thermodynamics of a diffusional protein folding reaction

The folding reactions of several proteins are well described as diffusional barrier crossing processes, which suggests that they should be analyzed by Kramers' rate theory rather than by transition state theory. For the cold shock protein Bc-Csp from Bacillus caldolyticus, we measured stability and folding kinetics, as well as solvent viscosity as a function of temperature and denaturant concentration. Our analysis indicates that diffusional folding reactions can be treated by transition state theory, provided that the temperature and denaturant dependence of the solvent viscosity is properly accounted for, either at the level of the measured rate constants or of the calculated activation parameters. After viscosity correction the activation barriers for folding become less enthalpic and more entropic. The transition from an enthalpic to an entropic folding barrier with increasing temperature is, however, apparent in the data before and after this correction. It is a consequence of the negative activation heat capacity of refolding, which is independent of solvent viscosity. Bc-Csp and its mesophilic homolog Bs-CspB from Bacillus subtilis differ strongly in stability but show identical enthalpic and entropic barriers to refolding. The increased stability of Bc-Csp originates from additional enthalpic interactions that are established after passage through the activated state. As a consequence, the activation enthalpy of unfolding is increased relative to Bs-CspB.     

Design and folding of a multidomain protein

To test whether the folding process of a large protein can be understood on the basis of the folding behavior of the domains that constitute it, we coupled two well-studied small -helical proteins, the B-domain of protein A (60 amino acids) and Rd-apocytochrome b562 (Rd-apocyt b562, 106 amino acids), by fusing the C-terminal helix of the B-domain of protein A with the N-terminal helix of Rd-apocyt b562 without changing their hydrophobic core residues. The success of the design was confirmed by determining the structure of the engineered protein with multidimensional NMR methods. Kinetic studies showed that the logarithms of the folding/unfolding rate constants of the engineered protein are linearly dependent on concentrations of guanidinium chloride in the measurable range from 1.7 to 4 M. Their slopes (m-values) are close to those of Rd-apocyt b562. In addition, the 1H-15N HSQC spectrum taken at 1.5 M guanidinium chloride reveals that only the Rd-apocyt b562 domain in the designed protein remained folded. These results suggest that the two domains have weak energetic coupling. Interestingly, the redesigned protein folds faster than Rd-apocyt b562, suggesting that the fused helix stabilizes the rate-limiting transition state.     

Sequence determinants of a protein folding pathway

Local hydrophobic collapse of the polypeptide chain and transient long-range interactions in unfolded states of apomyoglobin appear to occur in regions of the amino acid sequence which, upon folding, bury an above-average area of hydrophobic surface. To explore the role of these interactions in protein folding, we prepared and characterized apomyoglobins with compensating point mutations designed to change the average buried surface area in local regions of the sequence, while conserving as much as possible the constitution of the hydrophobic core. The behavior of the mutants in quench-flow experiments to determine the folding pathway was exactly as predicted by the changes in the buried surface area parameter calculated from the amino acid sequence. In addition, spin label experiments with acid-unfolded mutant apomyoglobin showed that the transient long-range contacts that occur in the wild-type protein are abolished in the mutant, while new contacts are observed between areas that now have above-average buried surface area. We conclude that specific groupings of amino acid side-chains, which can be predicted from the sequence, are responsible for early hydrophobic interactions in the first phase of folding in apomyoglobin, and that these early interactions determine the subsequent course of the folding process.