Recombination of S-peptide with S-protein during folding of ribonuclease S. I. Folding pathways of the slow-folding and fast-folding classes of unfolded S-protein.

The refolding kinetics of ribonuclease S have been measured by tyrosine absorbance, by tyrosine fluorescence emission, and by rapid binding of the specific inhibitor 2′CMP ² to folded RNAase S. The S-protein is first unfolded at pH 1.7 and then either mixed with S-peptide as refolding is initiated by a stopped-flow pH jump to pH 6.8, or the same results are obtained if S-protein and S-peptide are present together before refolding is initiated. The refolding kinetics of RNAase S have been measured as a function of temperature (10 to 40 °C) and of protein concentration (10 to 120 μm). The results are compared to the folding kinetics of S-protein alone and to earlier studies of RNAase A. A thermal folding transition of S-protein has been found below 30 °C at pH 1.7; its effects on the refolding kinetics are described in the following paper (Labhardt &; Baldwin, 1979).In this paper we characterize the refolding kinetics of unfolded S-protein, as it is found above 30 °C at pH 1.7, together with the kinetics of combination between S-peptide and S-protein during folding at pH 6.8. Two classes of unfolded S-protein molecules are found, fast-folding and slow-folding molecules, in a 20: 80 ratio. This is the same result as that found earlier for RNAase A; it is expected if the slow-folding molecules are produced by the slow cis-trans isomerization of proline residues after unfolding, since S-protein contains all four proline residues of RNAase A.The refolding kinetics of the fast-folding molecules show clearly that combination between S-peptide and S-protein occurs before folding of S-protein is complete. If combination occurred only after complete folding, then the kinetics of formation of RNAase S should be rather slow (5 s and 100 s at 30 °C) and nearly independent of protein concentration, as shown by separate measurements of the folding kinetics of S-protein, and of the combination between S-peptide and folded S-protein. The observed folding kinetics are faster than predicted by this model and also the folding rate increases strongly with protein concentration (apparent 1.6 order kinetics). The fact that RNAase S is formed more rapidly than S-protein alone is sufficient by itself to show that combination with S-peptide precedes complete folding of S-protein. Computer simulation of a simple, parallel-pathway scheme is able to reproduce the folding kinetics of the fast-folding molecules. All three probes give the same folding kinetics.These results exclude the model for protein folding in which the rate-limiting step is an initial diffusion of the polypeptide chain into a restricted range of three-dimensional configurations (“nueleation”) followed by rapid folding (“propagation”). If this model were valid, one would expect comparable rates of folding for RNAase A and for S-protein and one would also expect to find no populated folding intermediates, so that combination between S-peptide and S-protein should occur after folding is complete. Instead, RNAase A folds 60 times more rapidly than S-protein and also combination with S-peptide occurs before folding of S-protein is complete. The results demonstrate that the folding rate of S-protein increases after the formation, or stabilization, of an intermediate which results from combination with S-peptide. They support a sequential model for protein folding in which the rates of successive steps in folding depend on the stabilities of preceding intermediates.The refolding kinetics of the slow-folding molecules are complex. Two results demonstrate the presence of folding intermediates: (1) the three probes show different kinetic progress curves, and (2) the folding kinetics are concentration-dependent, in contrast to the results expected if complete folding of S-protein precedes combination with S-peptide. A faster phase of the slow-refolding reaction is detected both by tyrosine absorbance and fluorescence emission but not by 2′CMP binding, indicating that native RNAase S is not formed in this phase. Comparison of the kinetic progress curves measured by different probes is made with the use of the kinetic ratio test, which is defined here.     

15N backbone dynamics of the S-peptide from ribonuclease A in its free and S-protein bound forms: toward a site-specific analysis of entropy changes upon folding.

Backbone 15N relaxation parameters (R1, R2, 1H-15N NOE) have been measured for a 22-residue recombinant variant of the S-peptide in its free and S-protein bound forms. NMR relaxation data were analyzed using the "model-free" approach (Lipari & Szabo, 1982). Order parameters obtained from "model-free" simulations were used to calculate 1H-15N bond vector entropies using a recently described method (Yang & Kay, 1996), in which the form of the probability density function for bond vector fluctuations is derived from a diffusion-in-a-cone motional model. The average change in 1H-15N bond vector entropies for residues T3-S15, which become ordered upon binding of the S-peptide to the S-protein, is -12.6+/-1.4 J/mol.residue.K. 15N relaxation data suggest a gradient of decreasing entropy values moving from the termini toward the center of the free peptide. The difference between the entropies of the terminal and central residues is about -12 J/mol residue K, a value comparable to that of the average entropy change per residue upon complex formation. Similar entropy gradients are evident in NMR relaxation studies of other denatured proteins. Taken together, these observations suggest denatured proteins may contain entropic contributions from non-local interactions. Consequently, calculations that model the entropy of a residue in a denatured protein as that of a residue in a di- or tri-peptide, might over-estimate the magnitude of entropy changes upon folding.     

Hydrogen exchange from identified regions of the S-protein component of ribonuclease as a function of temperature,pH, and the binding of S-peptide

A medium resolution hydrogen exchange method (Rosa & Richards, 1979) has been used to measure the average rates of amide hydrogen exchange for known segments of the S-protein portion of ribonuclease-S. The analytical procedure permitted exchange rates to be monitored for seven S-protein fragments distributed throughout the structure, including regions of α-helix and β-sheet. Kinetics were measured as a function of pH, temperature and S-peptide binding.The pH dependence of exchange from isolated S-protein between pH 2·8 and pH 7·0 was found to deviate significantly from a first-order dependence on hydroxide ion concentration. The protection against exchange with increasing pH appeared to be closely related to the electrostatic stabilization of S-protein. It is suggested that such favorable electrostatic interactions result in increased energy barriers to the conformational fluctuations that provide solvent access to the time-average crystallographic structure. This explanation of the observed correlation between stability and exchange kinetics is also consistent with the calculated apparent activation energies for exchange from S-protein between 5·5 and 20 °C.S-peptide binding dramatically slows exchange from many S-protein sites, even those distant from the area of S-peptide contact. Interestingly, the effects of complex formation are not evenly propagated throughout S-protein. The most significantly perturbed sites (≥10³-fold reduction in exchange rate constants) lie within fragments derived from regions of secondary structure. Exchange from several other fragments is not significantly affected. The S-peptide—S-protein dissociation constant at neutral pH is so small that the measured exchange must have occurred from the complex and not from the dissociated parts.     

Characterization of a folding intermediate from HIV-1 ribonuclease H.

The RNase H domain from HIV-1 (HIV RNase H) encodes an essential retroviral activity. Refolding of the isolated HIV RNase H domain shows a kinetic intermediate detectable by stopped-flow far UV circular dichroism and pulse-labeling H/D exchange. In this intermediate, strands 1, 4, and 5 as well as helices A and D appear to be structured. Compared to its homolog from Escherichia coli, the rate limiting step in refolding of HIV RNase H appears closer to the native state. We have modeled this kinetic intermediate using a C-terminal deletion fragment lacking helix E. Like the kinetic intermediate, this variant folds rapidly and shows a decrease in stability. We propose that inhibition of the docking of helix E to this folding intermediate may present a novel strategy for anti HIV-1 therapy.     

Kinetic characterization of ribonuclease S mutants containing photoisomerizable phenylazophenylalanine residues.

Incorporation of the photoisomerizable amino acid phenylazophenylalanine (PAP) into enzyme structures has been proposed as a strategy for photoswitching enzyme activity. To evaluate the strengths and limitations of this approach to enzyme photo-control, we performed a kinetic analysis of RNase S analogues containing PAP in positions 4, 7, 8, 10, 11 or 13. For an enzyme containing a single PAP group, the maximum extent of photoconversion (between approximately 96% trans/4% cis and 10% trans/90% cis under standard conditions) sets a limit on the maximum fold change in the initial rate of approximately 25-fold, if the cis form is the more active isomer, and approximately 10-fold if the trans form is more active. This extent of photoswitching was not realized in the present case because the effects of photoisomerization on kinetic constants were small and distributed among effects on S-peptide binding, substrate binding and the rate of the chemical step. These results suggest that photoisomerization could substantially alter enzyme kinetic constants but that a directed combinatorial approach might be required for realizing maximal photo-control in such systems. The limit set by the extent of photoconversion might be overcome by coupling multiple PAP groups to one enzyme or by altering the behaviour of a system that required oligomerization for activity.     

Kinetic coupling between protein folding and prolyl isomerization. II. Folding of ribonuclease A and ribonuclease T1.

The folding and unfolding kinetics within the transition region were measured for RNase A and for RNase T1. The data were used to evaluate the theoretical models for the influence of prolyl isomerization on the observed folding kinetics. These two proteins were selected, since the folding reaction of RNase A is faster than prolyl isomerization, whereas in RNase T1, folding is slower than isomerization in the transition region. Folding of RNase T1 was investigated for three variants with different numbers of cis prolyl residues. The results indicate that in the transition region the folding rates are indeed strongly dependent on the number of prolyl residues. The variant of RNase T1 that contains only one cis prolyl residue folds about ten times faster than two variants that contain two cis prolyl residues. For both RNase A and RNase T1, the apparent rates of folding and unfolding as well as the corresponding amplitudes depend on the concentration of denaturant in a manner that was predicted by the model calculations. When refolding was started from the fast-folding species, additional kinetic phases could be observed in the transition region for both proteins. The obtained values could be used to calculate the microscopic rate constants of folding and isomerization on the basis of theoretical models.     

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.     

Amide proton exchange used to monitor the formation of a stable alpha-helix by residues 3 to 13 during folding of ribonuclease S

We make use of the known exchange rates of individual amide proton in the S-peptide moiety of ribonuclease S (RNAase S) to determine when during folding the alpha-helix formed by residues 3 to 13 becomes stable. The method is based on pulse-labeling with [3H]H2O during the folding followed by an exchange-out step after folding that removes 3H from all amide protons of the S-peptide except from residues 7 to 14, after which S-peptide is separated rapidly from S-protein by high performance liquid chromatography. The slow-folding species of unfolded RNAase S are studied. Folding takes place in strongly native conditions (pH 6.0, 10 degrees C). The seven H-bonded amide protons of the 3-13 helix become stable to exchange at a late stage in folding at the same time as the tertiary structure of RNAase S is formed, as monitored by tyrosine absorbance. At this stage in folding, the isomerization reaction that creates the major slow-folding species has not yet been reversed. Our result for the 3-13 helix is consistent with the finding of Labhardt (1984), who has studied the kinetics of folding of RNAase S at 32 degrees C by fast circular dichroism. He finds the dichroic change expected for formation of the 3-13 helix occurring when the tertiary structure is formed. Protected amide protons are found in the S-protein moiety earlier in folding. Formation or stabilization of this folding intermediate depends upon S-peptide: the intermediate is not observed when S-protein folds alone, and folding of S-protein is twice as slow in the absence of S-peptide. Although S-peptide combines with S-protein early in folding and is needed to stabilize an S-protein folding intermediate, the S-peptide helix does not itself become stable until the tertiary structure of RNAase S is formed.     

Salt-induced folding of a rabbit muscle pyruvate kinase intermediate at alkaline pH

The effect of alkaline denaturation on the structural and functional characteristics of rabbit muscle pyruvate kinase (PK) was investigated using enzymatic activity measurements and a combination of optical methods such as circular dichroism, fluorescence, and ANS binding. At a critical pH, 10.5, PK exists in an intermediate state (alkaline unfolded state) with predominant secondary structure along with some of the tertiary interactions and a strong binding to the hydrophobic dye ANS. This intermediate retains the enzymatic activity and corresponds to a dimeric state of the molecule. Above pH 10.5, a sudden fall in the spectral properties and enzymatic activity occurs suggesting the dissociation of the molecule followed by unfolding at very high pH. Addition of salts such as NaCl, KCl, and Na₂SO₄ to the alkali-induced state induces both secondary and tertiary structure to a level equivalent to that of native tetramer (salt-induced state). Chemical- and temperature-induced unfolding of the alkali-induced state as well as the salt-induced refolded state of PK reveal the presence of intermediate conformations in the unfolding pathway. The unfolding transition curves are noncoinciding and noncooperative along with ANS binding at intermediate concentrations of denaturants during unfolding. The observations presented in this paper suggest that the native pyruvate kinase tetramer dissociates to an active dimer around pH 10.5 and further to inactive monomer before attaining a completely unfolded monomeric conformation.     

The equilibrium unfolding of Azotobacter vinelandii apoflavodoxin II occurs via a relatively stable folding intermediate.

A flavodoxin from Azotobacter vinelandii is chosen as a model system to study the folding of alpha/beta doubly wound proteins. The guanidinium hydrochloride induced unfolding of apoflavodoxin is demonstrated to be reversible. Apoflavodoxin thus can fold in the absence of the FMN cofactor. The unfolding curves obtained for wild-type, C69A and C69S apoflavodoxin as monitored by circular dichroism and fluorescence spectroscopy do not coincide. Apoflavodoxin unfolding occurs therefore not via a simple two-state mechanism. The experimental data can be described by a three-state mechanism of apoflavodoxin equilibrium unfolding in which a relatively stable intermediate is involved. The intermediate species lacks the characteristic tertiary structure of native apoflavodoxin as deduced from fluorescence spectroscopy, but has significant secondary structure as inferred from circular dichroism spectroscopy. Both spectroscopic techniques show that thermally-induced unfolding of apoflavodoxin also proceeds through formation of a similar molten globule-like species. Thermal unfolding of apoflavodoxin is accompanied by anomalous circular dichroism characteristics: the negative ellipticity at 222 nM increases in the transition zone of unfolding. This effect is most likely attributable to changes in tertiary interactions of aromatic side chains upon protein unfolding. From the presented results and hydrogen/deuterium exchange data, a model for the equilibrium unfolding of apoflavodoxin is presented.     

An equilibrium folding intermediate detected in the thermal unfolding transition of ribonuclease S by circular dichroism

The thermal-denaturation transition of ribonuclease S (RNAase S) is measured by circular dichroism at 225 nm. Only conformational transitions involving the S-peptide–S-protein complex are detected at this wavelength. Different pathways of thermal unfolding at high and low concentrations are apparent: at low concentrations the temperature of half-completion of denaturation (T_m) varies with concentration. Above a total enzyme concentration of 50 μM, T_m remains constant. The observed data can be explained on the basis of a model where the association–dissociation step occurs between S-peptide and thermally (at least partly) unfolded S-protein. The complex as a whole undergoes a major folding–unfolding transition in the course of which the S-peptide μ-helix appears to be formed. The unfolded complex is well populated in the unfolding transition region for enzyme concentrations of 100 μM or more. The model succeeds in deducing thermodynamic parameters from the thermal denaturation curves in various different ways. The values thus obtained are fully self-consistent and, moreover, consistent with the values for the apparent association constant and apparent association enthalpy as measured in enzyme-dilution experiments and by batch calorimetry.     

pH-induced insertion of pHLIP into a lipid bilayer: In-situ SEIRAS characterization of a folding intermediate at neutral pH

The pH low insertion peptide (pHLIP) is a pH-sensitive cell penetrating peptide that transforms from an unstructured coil on the membrane surface at pH > 7, to a transmembrane (TM) α-helix at pH < 5. By exploiting this unique property, pHLIP attracts interest as a potential tool for drug delivery and visualisation of acidic tissues produced by various maladies such as cancer, inflammation, hypoxia etc. Even though the structures of initial and end states of pHLIP insertion have been widely accepted, the intermediate structures in between these two states are less clear. Here, we have applied in situ Surface-Enhanced Infrared Absorption spectroscopy to examine the pH-induced insertion and folding processes of pHLIP into a solid-supported lipid bilayer. We show that formation of partially helical structure already takes place at pH only slightly below 7.0, but with the helical axis parallel to the membrane surface. The peptide starts to reorientate its helix from horizontal to vertical direction, accompanied by the insertion into the TM region at pH < 6.2. Further insertion into the TM region of the peptide results in an increase of inherent α-helical structure and complete secondary structure formation at pH 5.3. Analysis of the changes of the carboxylate vibrational bands upon pH titration shows two distinctive groups of aspartates and glutamates with pK_a values of 4.5 and 6.3, respectively. Comparison to the amide bands of the peptide backbone suggests that the latter Asp/Glu groups are directly involved in the conformational changes of pHLIP in the respective intermediate states.     

Structural studies of a folding intermediate of bovine pancreatic ribonuclease A by continuous recycled flow

A new technique, continuous recycled flow (CRF) spectroscopy, has been developed for observing intermediates of any thermally induced, reversible reaction with a half-life of 10 s or longer. The structure can be probed by any spectroscopic method which does not perturb the system. Prolonged signal acquisitions of 8 h for ribonuclease A are possible. CRF was used to investigate the structure of the slow-folding intermediates of chemically intact ribonuclease A (RNase A) during thermal unfolding/folding under acidic conditions. The following conclusions were reached on the basis of the proton nuclear magnetic resonance and far-ultraviolet circular dichroism spectra of a folding intermediate(s): (A) The conformation of the detected folding intermediate(s) is similar to that of the heat-denatured protein. There is only limited formation of new structures. (B) The N-terminal alpha-helix is partially stable under these conditions and is in rapid (less than 10 ms) equilibrium with the denatured conformation. (C) There are long-range interactions between the hydrophobic residues of the N-terminal alpha-helix and the rest of the protein. These interactions persist well above the melting point. (D) An aliphatic methyl group reports on the formation of a new structure(s) that lie(s) outside of the N-terminal region. (E) The structures detected in chemically modified, nonfolding forms of the RNase A are also present in the folding intermediate(s). There are, however, additional interactions that are unique to chemically intact RNase A.     

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.     

Observing a late folding intermediate of Ubiquitin at atomic resolution by NMR

The study of intermediates in the protein folding pathway provides a wealth of information about the energy landscape. The intermediates also frequently initiate pathogenic fibril formations. While observing the intermediates is difficult due to their transient nature, extreme conditions can partially unfold the proteins and provide a glimpse of the intermediate states. Here, we observe the high resolution structure of a hydrophobic core mutant of Ubiquitin at an extreme acidic pH by nuclear magnetic resonance (NMR) spectroscopy. In the structure, the native secondary and tertiary structure is conserved for a major part of the protein. However, a long loop between the beta strands β3 and β5 is partially unfolded. The altered structure is supported by fluorescence data and the difference in free energies between the native state and the intermediate is reflected in the denaturant induced melting curves. The unfolded region includes amino acids that are critical for interaction with cofactors as well as for assembly of poly‐Ubiquitin chains. The structure at acidic pH resembles a late folding intermediate of Ubiquitin and indicates that upon stabilization of the protein's core, the long loop converges on the core in the final step of the folding process.     

Refolding behavior of a kinetic intermediate observed in the low pH unfolding of ribonuclease A.

A transient intermediate (I3) observed previously in the unfolding of ribonuclease A has been studied by employing a sequential mixing instrument to populate selectively this species. This approach has made it possible both to determine the refolding behavior of this species and to characterize further the kinetics of its formation. (1) Formation of I3 represents the earliest detectable change in unfolding. (2) The loss of the 2'CMP binding site occurs in parallel with the exposure of the interior of the protein to solvent. (3) I3 is distinct from previously described intermediates in refolding. (4) Overall condensation of the protein to exclude solvent from the interior, as well as the formation of a substrate binding site, takes place in approximately 30 ms (pH 5.8, 47 degrees C), indicating that the formation of native structure can take place faster than had previously been supposed.