Proline peptide isomerization and protein folding

The unfolding-refolding of proteins is a cooperative process and, as judged by equilibrium properties, occurs in one step involving the native,N, and the unfoldedU, conformational states. Kinetic studies have shown that the denatured protein exists as a mixture of slow-(U)_Sand fast-(U)_Frefolding forms produced by proline peptidecis-trans isomerization. Proline residues inU _Fare in the same configuration as in the native protein while they are in non-native configuration inU _S. For protein folding to occur quicklyU _Smust be converted intoU _F. The fact that the equilibrium and kinetic properties of are the same as those found for prolinecis-trans isomerization taken together with the absence of slow phase in the kinetics of refolding of a protein devoid of proline, support this view. However, the absence of a linear correlation between half-time of reactivation of denatured enzymes and their proline-contents, as well as the dissimilarities in the kinetic properties of in unfolding and refolding experiments are not consistent with the model. Conformational energy calculation and experimental results on refolding of proteins suggest that some proline residues are non-essential. They will not block protein folding even in wrong isomeric form. The native-like folded structure with incorrect proline isomers will serve as intermediate state(s) in which these prolines will more readily isomerize to the correct isomeric form. The picture becomes more complex when one considers the consequence ofcis-trans isomerism of non-proline residues on protein folding.     

Kinetics of unfolding and refolding of proteins. 3. Results for lysozyme

The kinetics of the reversible denaturation of lysozyme by guanidine hydrochloride have been studied over a wide range of pH and denaturant concentration. The results contrast sharply with those observed for cytochrome c. The interconversion of native (N) and denatured (D) states is strictly first-order in both directions under most conditions, showing that no intermediate forms of any kind accumulate to a significant extent during the reaction. At one pH (pH 2.6) experiments were extended to extreme denaturant concentrations, and under these conditions kinetic intermediates were observed. Analysis by the procedures of the first paper of this series Ikai &; Tanford 1973 showed that the principal intermediate observed at low denaturant concentration must be different from that observed at high denaturant concentration, and that both must be on the direct pathway from N to D. This suggests that the over-all reaction mechanism is of the form

and this mechanism is able to account for all of the observed results. The intermediate X₁ which accumulates transiently in the renaturation reaction at low concentrations of guanidine hydrochloride is spectrally very similar to the native state, i.e. it is probably a highly ordered state, but most of the interactions between surface acidic and basic groups, which are responsible for the anomalous titration behavior of native lysozyme, do not yet exist in this state. It is probable that the difference between the kinetics of refolding of lysozyme and cytochrome c may be ascribed to the existence of disulfide cross-links in lysozyme, which were intact in these experiments. These cross-links greatly limit the possible pathways for folding and thus make it much less likely that incorrectly folded states on dead-end pathways are readily accessible.     

Unfolding and refolding of the constant fragment of the immunoglobulin light chain

The kinetics of reversible unfolding and refolding by guanidine hydrochloride of the constant fragment of the immunoglobulin light chain are described. The kinetic measurements were made at pH 7.5 and 25 °C using tryptophyl fluorescence and farultraviolet circular dichroism.The kinetics of unfolding of the constant fragment showed two phases in the conformational transition zone and a single phase above the transition zone. A double-jump experiment confirmed the presence of two forms of the unfolded molecule. These results were thoroughly explained on the basis of the three-species mechanism, U₁

U₂

N, where U₁ and U₂ are the slow-folding and fast-folding species, respectively, of unfolded protein and N is native protein. The equilibrium constant for the process of U₂ to U₁ was estimated to be about 10 and was independent of the conditions of denaturation. These findings were consistent with the view that the U₁

U₂ reaction is proline isomerization. The rates of interconversion between N and U₂ changed greatly with the concentration of guanidine hydrochloride. On the other hand, the refolding kinetics below the transition zone showed behavior unexpected from the three-species mechanism. Whereas the apparent rate constant of the slow phase of refolding was independent of the refolding conditions, its amplitude decreased markedly with the decrease in the final concentration of guanidine hydrochloride. On the basis of this and other results, formation of an intermediate during refolding was ascertained and the refolding kinetics were consistently explained in terms of a more general mechanism involving a kinetic intermediate probably containing non-native proline isomers. The intermediate seemed to have a folded conformation similar to native protein. Comparison of the refolding kinetics of the constant fragment with those of other domains of the immunoglobulin molecule suggested that Pro143 is responsible for the appearance of the slow phase.     

Unfolding and refolding of the reduced constant fragment of the immunoglobulin light chain: Kinetic role of the intrachain disulfide bond

The constant fragment of the immunoglobulin light chain whose intrachain disulfide bond is reduced (reduced C_L fragment) assumes a conformation very similar to the intact C_L fragment (Goto & Hamaguchi, 1979). The kinetics of reversible unfolding and refolding of the reduced C_L fragment by guanidine hydrochloride at pH 7.5 and 25 °C were studied and were compared with those of the intact C_L fragment described in the accompanying paper (Goto & Hamaguchi, 1982). Tryptophyl fluorescence, far-ultraviolet circular dichroism, and reactivity of the SH groups toward 5,5′-dithiobis-(2-nitrobenzoic acid) were used to follow the kinetics. The results obtained were thoroughly explained on the basis of the three-species mechanism, U₁

U₂

N, where U₁ and U₂ are slow-folding and fast-folding species, respectively, of unfolded protein and N is native protein. The rate constants of interconversion between U₁ and U₂ and the rate constant for the process from N to U₂ were very similar to the respective values for the intact C_L fragment. Only the rate constant for the process from U₂ to N was greatly different between the intact and reduced C_L fragments; the rate constant for the reduced C_L fragment was about 100 times smaller than that for the intact C_L fragment. These results indicated that the slow isomerization of the unfolded molecule is independent of the presence of the disulfide bond and that the kinetic role of the intrachain disulfide bond is to accelerate the folding process. This kinetic role in the folding of the C_L fragment was explainable only in terms of the decreased entropy in the unfolded state of the intact C_L fragment due to the presence of the disulfide bond.     

The nature of protein folding pathways: The classical versus the new view

Summary Pulsed hydrogen exchange and other studies of the kinetic refolding pathways of several small proteins have established that folding intermediates with native-like secondary structures are well populated, but these studies have also shown that the folding kinetics are not well synchronized. Older studies of the kinetics of formation of the native protein, monitored by optical probes, indicate that the folding kinetics should be synchronized. The model commonly used in these studies is the simple sequential model, which postulates a unique folding pathway with defined and sequential intermediates. Theories of the folding process and Monte Carlo simulations of folding suggest that neither the folding pathway nor the set of folding intermediates is unique, and that folding intermediates accumulate because of kinetic traps caused by partial misfolding. Recent experiments with cytochrome c lend support to this new view of folding pathways. These different views of the folding process are discussed. Misfolding and consequent slowing down of the folding process as a result of cis-trans isomerization about prolyl peptide bonds in the unfolded protein are well known; isomerization occurs before refolding is initiated. The occurrence of equilibrium intermediates on the kinetic folding pathways of some proteins, such as -lactalbumin and apomyoglobin, argues that these intermediates are not caused by kinetic traps but rather are stable intermediates under certain conditions, and this conclusion is consistent with a sequential model of folding. Folding reactions with successive kinetic intermediates, in which late intermediates are more highly folded than early intermediates, indicate that folding is hierarchical. New experiments that test the predictions of the classical and the new views are needed.     

Test of the extended two-state model for the kinetic intermediates observed in the folding transition of ribonuclease A

A test has been made of the proposal that: (a) the extended two-state model describes the kinetic intermediates seen in the folding transition of RNAase A, i.e. that the only species present in folding experiments are the native protein and multiple forms of the completely unfolded protein; and (b) that the interconversion between the two known unfolded forms of RNAase A (the U₁

U₂ reaction) is described solely by the cis-trans isomerization of the proline residues. The test is to measure the rate of the U₁

U₂ reaction in a wide range of refolding conditions and to compare these data with the kinetic properties of proline isomerization.The main results are as follows. (1) The activation enthalpy of the U₁

U₂ reaction in refolding conditions (pH 6, 20 ° to 40 °C) is less than 5 kcal/mol. This is much too small to be explained as proline isomerization. (2) Both the rate and the activation enthalpy change sharply at guanidine hydrochloride concentrations below 2 m. There appear to be two pathways for the U₁

U₂ reaction in refolding conditions, and the slower pathway is favored by adding guanidine hydrochloride. (3) The rate and activation enthalpy for proline isomerization in l-alanyl-l-proline are unaffected by 2 m-guanidine hydrochloride.The results show that the proline isomerization hypothesis and the extended two-state model cannot both be correct for RNAase A. They suggest that partial folding occurs rapidly in refolding conditions and that the extended two-state model is invalid. They leave open the question of whether or not proline isomerization is the rate-limiting step in the U₁

U₂ reaction.Another possible source of slow configurational reactions in the unfolded state is mentioned. The three major, overlapping, disulfide-bonded loops of RNAase A can exist in two isomeric configurations. Interconversion of these isomers requires pulling one loop, or one end of the polypeptide chain, through a second loop and this is likely to be a slow process.In some conditions, heat-unfolded but not guanidine-unfolded RNAase A shows a second slow-refolding process. It may result from aggregates of the heatunfolded protein which are formed and broken up slowly. Conditions are given for eliminating this reaction.     

Effects of solvent viscosity and different guanidine salts on the kinetics of ribonuclease A chain folding

The kinetics of folding of the two forms of unfolded ribonuclease A have been measured as a function of solvent viscosity by adding either glycerol or sucrose. The aim is to find out if either reaction is rate limited by segmental motion whose rate depends on external friction. The fast folding reaction (U₂ ? N) is known to be the direct folding process, and the slow folding reaction (U₁ ? N) is known to be rate limited by an interconversion between two forms (U₁ ? U₂) which are present after unfolding in strongly denaturing conditions. No dependence on solvent viscosity is found, either for the direct folding reaction or for the interconversion reaction. Each folding reaction has also been tested to see if its rate depends on the concentration of one or more partly folded intermediates, by adding denaturants destabilize any partly folded structures. Different guanidine salts are used as denaturants to vary the denaturing effectiveness of the salt while holding the guanidinium ion concentration constant. The rates both of the direct folding reaction and of the interconversion reaction decrease in relation to the denaturing effectiveness of the salt. However, there is a basic difference between the responses of the fast and slow folding reactions to low concentrations of denaturants. Although each folding reaction produces native protein, there is an 800-fold decrease in the rate of the fast folding reaction in 1M guanidine thiocyanate and only a 13-fold decrease in the rate of the slow folding reaction. This is consistent with the fast reaction being the direct folding process and the slow reaction being rate limited by the initial conversion of the slowrefolding to the fast-refolding form. Both the lack of viscosity dependence and the effects of denaturants indicate that the formation of structure is rate limiting in the direct folding reaction, U₂ ? N. The failure to find a viscosity dependence for the interconversion reaction, U₁ ? U₂, indicates that in this reaction also friction-limited segmental motion is not the rate-limiting process. Since the U₁ ? U₂ interconversion still occurs when the polypeptide chain is completely unfolded, the surprising result is that its rate in refolding conditions depends significantly on a reaction intermediate which is “denatured” by guanidine salts.     

Molecular dynamics characterisations of the Trp-cage folding mechanisms: in the absence and presence of water solvents

Protein folding is an important and yet challenging topic in current molecular biology. In this work, the folding dynamics and mechanisms of the Trp-cage mini-protein were studied with molecular dynamics simulations, in the absence and presence of water solvents. The important intermediates during the Trp-cage folding were determined by gradually decreasing the simulation temperature. The folding transition temperature was identified to be approximately 400 K, and the folding pathway was decomposed into six steps: U ? I ₁ ? I ₂ ? I ₃ ? I ₄ ? F ₁ ? F ₂, where U, I and F represent the unfolded, intermediate and folded states, respectively. The finding that the two helical subunits are successively formed is consistent with the experimental observations, and the Asp9/Arg16 salt bridge forms at the final stage and does not play a significant role during folding kinetics. The presence of water solvents induces hydrophobic collapse as the whole cage comparatively closes. Within aqueous solutions, the Trp-cage folding begins to contract into the meta-stable state, and by traversing the transition state it arrives at the native-like structure, which resembles the experimental structure closely.     

Characterization of the transition state of lysozyme unfolding. II. Effects of the intrachain crosslinking and the inhibitor binding on the transition state

The reversible refolding of a lysozyme derivative containing an extra crosslink between Glu 35 and Trp 108 was observed at pH 3.7 in solutions of 4.5M LiBr and 4.SM 1-PrOH. The rate constants of unfolding and folding for crosslinked lysozyme were compared with those for intact lysozyme. In LiBr solutions, the crosslinking greatly increases k_f but only slightly decreases k_uf. This means that the crosslinking restricts possible conformations of the U state to some conformations on the folding pathway, whereas the possible conformations of the transition state are little restricted. Although the crosslinking produces an apparently different effect on the rate constants in a PrOH solution, this could be explained by assuming that the crosslinking counteracts the effect of PrOH on the transition state. These observations suggest that the Glu 35 and Trp 108 of intact lysozyme already come into contact with each other in the course of refolding to the transition state. Kinetics of the unfolding and folding reactions of intact lysozyme were measured in the presence of 8mAf (NAG)₃. The apparent folding rate (k) was nearly equal to k_f, while the apparent unfolding rate (k) decreased sixfold. This means that the inhibitor binding stabilizes the native conformation but makes no contribution to the stabilization of the transition state. Specific interactions between (NAG)₃ and the cleft of lysozyme become available only at the last stage of folding.     

Confirmation of the hierarchical folding of RNase H: a protein engineering study.

The kinetic intermediate of RNase H is structured in a core region of the protein. To probe the role of this intermediate in the folding of RNase H, the folding kinetics of mutant proteins with altered native state stabilities were investigated. Mutations within the folding core destabilize the kinetic intermediate and slow refolding in a manner consistent with an obligatory intermediate model. Mutations outside of the folding core, however, do not affect the stability of the kinetic intermediate but do perturb the native state and transition state. These results indicate that interactions formed in the intermediate persist in the transition and native states and that RNase H folds through a hierarchical mechanism.     

Exploring the folding pathway of green fluorescent protein through disulfide engineering

We have introduced two disulfide crosslinks into the loop regions on opposite ends of the beta barrel in superfolder green fluorescent protein (GFP) in order to better understand the nature of its folding pathway. When the disulfide on the side opposite the N/C‐termini is formed, folding is 2× faster, unfolding is 2000× slower, and the protein is stabilized by 16 kJ/mol. But when the disulfide bond on the side of the termini is formed we see little change in the kinetics and stability. The stabilization upon combining the two crosslinks is approximately additive. When the kinetic effects are broken down into multiple phases, we observe Hammond behavior in the upward shift of the kinetic m‐value of unfolding. We use these results in conjunction with structural analysis to assign folding intermediates to two parallel folding pathways. The data are consistent with a view that the two fastest transition states of folding are "barrel closing" steps. The slower of the two phases passes through an intermediate with the barrel opening occurring between strands 7 and 8, while the faster phase opens between 9 and 4. We conclude that disulfide crosslink‐induced perturbations in kinetics are useful for mapping the protein folding pathway.     

Glycerol-induced formation of the molten globule from acid-denatured cytochrome c: implication for hierarchical folding

At high concentration (98% or higher, v/v), glycerol induces collapse of acid-denatured cytochrome c into a compact state, the G_U state, showing a molten globule character. The G_U state possesses a nativelike -helix structure but a tertiary conformation less packed with respect to the native state. The spectroscopic properties of the G_U state closely resemble those of the molten globule stabilized by the organic solvent from the native protein (called the G_N state), indicating that glycerol can stabilize the molten globule of cytochrome c either from the native or the acid-denatured protein. The G_U and the G_N states show spectroscopic (and, thus, structural) properties and stabilities comparable to those of molten globules stabilized by different effectors, despite the fact that the mechanisms involved in the molten globule formation may significantly differ. This implies in cytochrome c a hierarchy for the rupture (native-to-molten globule) or the formation (unfolded-to-molten globule) of intramolecular interactions leading to the stabilization of the molten globule state of the protein, independently from the effector responsible for the structural transition, in accord with the sequential model proposed by Englander and collaborators.     

Equilibrium amide hydrogen exchange and protein folding kinetics

The classical Linderstrøm-Lang hydrogen exchange (HX) model is extended to describe the relationship between the HX behaviors (EX1 and EX2) and protein folding kinetics for the amide protons that can only exchange by global unfolding in a three-state system including native (N), intermediate (I), and unfolded (U) states. For these slowly exchanging amide protons, it is shown that the existence of an intermediate (I) has no effect on the HX behavior in an off-pathway three-state system (IUN). On the other hand, in an on-pathway three-state system (UIN), the existence of a stable folding intermediate has profound effect on the HX behavior. It is shown that fast refolding from the unfolded state to the stable intermediate state alone does not guarantee EX2 behavior. The rate of refolding from the intermediate state to the native state also plays a crucial role in determining whether EX1 or EX2 behavior should occur. This is mainly due to the fact that only amide protons in the native state are observed in the hydrogen exchange experiment. These new concepts suggest that caution needs to be taken if one tries to derive the kinetic events of protein folding from equilibrium hydrogen exchange experiments.     

Equilibrium and kinetic folding of hen egg-white lysozyme under acidic conditions

The equilibrium and kinetic folding of hen egg-white lysozyme was studied by means of circular dichroism spectra in the far- and near-ultraviolet (UV) regions at 25 degrees C under the acidic pH conditions. In equilibrium condition at pH 2.2, hen lysozyme shows a single cooperative transition in the GdnCl-induced unfolding experiment. However, in the GdnCl-induced unfolding process at lower pH 0.9, a distinct intermediate state with molten globule characteristics was observed. The time-dependent unfolding and refolding of the protein were induced by concentration jumps of the denaturant and measured by using stopped-flow circular dichroism at pH 2.2. Immediately after the dilution of denaturant, the kinetics of refolding shows evidence of a major unresolved far-UV CD change during the dead time (<10 ms) of the stopped-flow experiment (burst phase). The observed refolding and unfolding curves were both fitted well to a single-exponential function, and the rate constants obtained in the far- and near-UV regions coincided with each other. The dependence on denaturant concentration of amplitudes of burst phase and both rate constants was modeled quantitatively by a sequential three-state mechanism, U<-->I<-->N, in which the burst-phase intermediate (I) in rapid equilibrium with the unfolded state (U) precedes the rate-determining formation of the native state (N). The role of folding intermediate state of hen lysozyme was discussed.     

Protein folding rates and thermodynamic stability are key determinants for interaction with the Hsp70 chaperone system

The Hsp70 family of molecular chaperones participates in vital cellular processes including the heat shock response and protein homeostasis. E. coli''s Hsp70, known as DnaK, works in concert with the DnaJ and GrpE co-chaperones (K/J/E chaperone system), and mediates cotranslational and post-translational protein folding in the cytoplasm. While the role of the K/J/E chaperones is well understood in the presence of large substrates unable to fold independently, it is not known if and how K/J/E modulates the folding of smaller proteins able to fold even in the absence of chaperones. Here, we combine experiments and computation to evaluate the significance of kinetic partitioning as a model to describe the interplay between protein folding and binding to the K/J/E chaperone system. First, we target three nonobligatory substrates, that is, proteins that do not require chaperones to fold. The experimentally observed chaperone association of these client proteins during folding is entirely consistent with predictions from kinetic partitioning. Next, we develop and validate a computational model (CHAMP70) that assumes kinetic partitioning of substrates between folding and interaction with K/J/E. CHAMP70 quantitatively predicts the experimentally measured interaction of RNase H^D as it refolds in the presence of various chaperones. CHAMP70 shows that substrates are posed to interact with K/J/E only if they are slow-folding proteins with a folding rate constant k_f <50 s⁻¹, and/or thermodynamically unstable proteins with a folding free energy ΔG⁰_UN ≥−2 kcal mol⁻¹. Hence, the K/J/E system is tuned to use specific protein folding rates and thermodynamic stabilities as substrate selection criteria.     

The folding process of apomyoglobin

Apomyoglobin (apoMb) folds through at least two partially folded forms that are detected both as transient intermediates during folding/unfolding kinetics or as stable intermediates at equilibrium. Here, I summarize the results of recent kinetic studies, which combined with detailed characterizations of equilibrium forms of the protein, provide a very detailed picture of apoMb folding process. The data are consistent with a linear U<->Ia<->Ib<->N model where compaction and structure are progressively acquired.     

Slow folding kinetics of RNase P RNA.

Understanding the folding mechanisms of large, highly structured RNAs is important for understanding how these molecules carry out their function. Although models for the three-dimensional architecture of several large RNAs have been constructed, the process by which these structures are formed is only now beginning to be explored. The kinetic folding pathway of the Tetrahymena ribozyme involves multiple intermediates and both Mg2+-dependent and Mg2+-independent steps. To determine whether this general mechanism is representative of folding of other large RNAs, a study of RNase P RNA folding was undertaken. We show, using a kinetic oligonucleotide hybridization assay, that there is at least one slow step on the folding pathway of RNase P RNA, resulting in conformational changes in the P7 helix region on the minute timescale. Although this folding event requires the presence of Mg2+, the slow step itself does not involve Mg2+ binding. The P7 and P2 helix regions exhibit distinctly different folding behavior and ion dependence, implying that RNase P folding is likely to be a complex process. Furthermore, there are distinct similarities in the folding of RNase P RNA from both Bacillus subtilis and Escherichia coli, indicating that the folding pathway may also be conserved along with the final structure. The slow folding kinetics, Mg2+-independence of the rate, and existence of intermediates are basic features of the folding mechanism of the Tetrahymena group I intron that are also found in RNase P RNA, suggesting these may be general features of the folding of large RNAs.