The mechanism of folding of pancreatic ribonucleases is independent of the presence of covalently linked carbohydrate

The role of asparagine-linked oligosaccharides for the mechanism of protein folding was investigated. We compared the stability and folding kinetics for two sets of pancreatic ribonucleases (RNases) with identical amino acid sequences and differences in glycosylation. First the folding of RNases A (carbohydrate free) and B (a single N-linked oligosaccharide) from bovine pancreas was investigated. The kinetics of refolding were identical under a wide range of conditions. The rate of unfolding by guanidinium chloride was decreased in RNase B. In further experiments the folding of porcine RNase (three carbohydrate chains at Asn-21, -34, and -76) was compared with the corresponding data for the deglycosylated protein. Even for this RNase with almost 40% carbohydrate content the mechanism of refolding is independent of glycosylation. Although the folding mechanism is conserved, the rates of individual steps in folding are decreased about 2-fold upon deglycosylation. We interpret this to originate from a slight destabilization of folding intermediates by carbohydrate depletion. In control experiments with nonglycosylated bovine RNase A it was ascertained that treatment with HF (as used for deglycosylation) did not affect the folding kinetics. The in vitro folding mechanism of glycosylated RNases apparently does not depend on the presence of N-linked oligosaccharide chains. The information for the folding of glycoproteins is contained exclusively in the protein moiety, i.e. in the amino acid sequence. Carbohydrate chains are attached at chain positions which remain solvent exposed. This ensures that the presence of oligosaccharides does not interfere with correct folding of the polypeptide chain.     

Analysis of the differences in the folding kinetics of structurally homologous proteins based on predictions of the gross features of residue contacts

It is a general notion that proteins with very similar three-dimensional structures would show very similar folding kinetics. However, recent studies reveal that the folding kinetic properties of some proteins contradict this thought (i.e., the members in a same protein family fold through different pathways). For example, it has been reported that some beta-proteins in the intracellular lipid-binding protein family fold through quite different pathways (Burns et al., Proteins 1998;33:107-118). Similar differences in folding kinetics are also observed in the members of the globin family (Nishimura et al., Nat Struct Biol 2000;7:679-686). In our study, we examine the possibility of predicting qualitative differences in folding kinetics of the intracellular lipid-binding proteins and two globin proteins (i.e., myoglobin and leghemoglobin). The problem is tackled by means of a contact map based on the average distance statistics between residues, the Average Distance Map (ADM), as constructed from sequence. The ADMs for the three proteins show overall similarity, but some local differences among maps are also observed. Our results demonstrate that some properties of the protein folding kinetics are consistent with local differences in the ADMs. We also discuss the general possibility of predicting folding kinetics from sequence information.     

Matching simulation and experiment: a new simplified model for simulating protein folding.

Simulations of simplified protein folding models have provided much insight into solving the protein folding problem. We propose here a new off-lattice bead model, capable of simulating several different fold classes of small proteins. We present the sequence for an alpha/beta protein resembling the IgG-binding proteins L and G. The thermodynamics of the folding process for this model are characterized using the multiple multihistogram method combined with constant-temperature Langevin simulations. The folding is shown to be highly cooperative, with chain collapse nearly accompanying folding. Two parallel folding pathways are shown to exist on the folding free energy landscape. One pathway contains an intermediate--similar to experiments on protein G, and one pathway contains no intermediates-similar to experiments on protein L. The folding kinetics are characterized by tabulating mean-first passage times, and we show that the onset of glasslike kinetics occurs at much lower temperatures than the folding temperature. This model is expected to be useful in many future contexts: investigating questions of the role of local versus nonlocal interactions in various fold classes, addressing the effect of sequence mutations affecting secondary structure propensities, and providing a computationally feasible model for studying the role of solvation forces in protein folding.     

Trehalose favors a cutinase compact intermediate off-folding pathway

The folding of cutinase, an enzyme displaying lipolytic activity, has been studied in the presence of trehalose. Equilibrium unfolding data show that trehalose increases the free energy change between folded and unfolded states. Unfolding kinetics reveal the presence of an intermediate which is ca. 60% folded in terms of solvent exposure. Trehalose stabilizes this intermediate relative to the folded state. In contrast, the intermediate revealed by folding kinetics is more compact than the transition state, as shown by the positive slope observed at low denaturant concentration in the chevron plot, as well as the decrease in the observable rate constant for folding with the increase in trehalose concentration. This intermediate displays more than 50% of area buried from the solvent (relative to the native state) compared to around 40% for the transition state for folding and therefore appears to be off the folding pathway. Trehalose stabilizes and guanidine hydrochloride destabilizes this compact intermediate. Both unfolding and folding kinetics show that compact conformational states are stabilized by trehalose, in agreement with current models on the effect of compatible solutes. This effect occurs even for compact states that decelerate the folding as in the case of the intermediate revealed by folding kinetics.     

Relationship between protein folding kinetics and amino acid properties

The successful prediction of protein-folding rates based on the sequence-predicted secondary structure suggests that the folding rates might be predicted from sequence alone. To pursue this question, we directly predict the folding rates from amino acid sequences, which do not require any information on secondary or tertiary structure. Our work achieves 88% correlation with folding rates determined experimentally for proteins of all folding types and peptide, suggesting that almost all of the information needed to specify a protein's folding kinetics and mechanism is comprised within its amino acid sequence. The influence of residue on folding rate is related to amino acid properties. Hydrophobic character of amino acids may be an important determinant of folding kinetics, whereas other properties, size, flexibility, polarity and isoelectric point, of amino acids have contributed little to the folding rate constant.     

Multiple parallel-pathway folding of proline-free Staphylococcal nuclease

When a protein exhibits complex kinetics of refolding, we often ascribe the complexity to slow isomerization events in the denatured protein, such as cis/trans isomerization of peptidyl prolyl bonds. Does the complex folding kinetics arise only from this well-known reason? Here, we have investigated the refolding of a proline-free variant of staphylococcal nuclease by stopped-flow, double-jump techniques, to examine the folding reactions without the slow prolyl isomerizations. As a result, the protein folds into the native state along at least two accessible parallel pathways, starting from a macroscopically single denatured-state ensemble. The presence of intermediates on the individual folding pathways has revealed the existence of multiple parallel pathways, and is characterized by multi-exponential folding kinetics with a lag phase. Therefore, a "single" amino acid sequence can fold along the multiple parallel pathways. This observation in staphylococcal nuclease suggests that the multiple folding may be more general than we have expected, because the multiple parallel-pathway folding cannot be excluded from proteins that show simpler kinetics.     

Protein folding: simple models for a complex process

Twenty-eight years after its original publication, the diffusion-collision model has successfully been applied to describe the folding kinetics of two proteins with the same native structure but different sequences (Islam et al., this issue of Structure). The calculations show the relative importance of the primary and tertiary structure on the sequence of events and folding. For both proteins, the model suggests parallel folding pathways, a finding which has wide implications for the interpretations of experiments.     

Native-like folding intermediates of homologous ribonucleases

The mechanism of the slow refolding reactions of four different pancreatic ribonucleases from ox, sheep, red deer, and roe deer has been investigated. Refolding kinetics of these proteins were very similar. In particular, a native-like intermediate, IN, was shown to be populated on the slow refolding pathway of all ribonucleases. We conclude that, similar to the stability of the folded proteins, the pathway of slow refolding has been conserved despite the differences in amino acid sequence and the varying number of proline residues.     

Switching two-state to three-state kinetics in the helical protein Im9 via the optimisation of stabilising non-native interactions by design

The four-helix protein Im7 folds through an on-pathway intermediate at pH 7.0 and 10 degrees C. By contrast, under these conditions there is no evidence for a populated intermediate in the folding of its more stable homologue, Im9, even in the presence of 0.4 M sodium sulphate. Previous studies using phi-value analysis have shown that the Im7 intermediate is misfolded, in that three of its four native helices are formed, but are docked in a non-native manner. Using knowledge of the structure of the intermediate of Im7, we have used rational design to stabilise an intermediate formed during the folding of Im9 by the introduction of specific stabilising interactions at positions known to stabilise the Im7 folding intermediate through non-native interactions. We show that the redesigned Im9 sequence folds with three-state kinetics at pH 7.0 and have used phi-value analysis to demonstrate that this species resembles the misfolded intermediate populated during Im7 folding. The redesigned Im9 sequence folds 20-fold faster than the wild-type protein under conditions in which folding is two-state. The data show that intermediate formation is an important feature of folding, even for small proteins such as Im9 for which these partially folded states do not become significantly populated. In addition, they show that the introduction of stabilising interactions can lead to rapid refolding, even when the contacts introduced are non-native.     

Topology, stability, sequence, and length: defining the determinants of two-state protein folding kinetics

The fastest simple, single domain proteins fold a million times more rapidly than the slowest. Ultimately this broad kinetic spectrum is determined by the amino acid sequences that define these proteins, suggesting that the mechanisms that underlie folding may be almost as complex as the sequences that encode them. Here, however, we summarize recent experimental results which suggest that (1) despite a vast diversity of structures and functions, there are fundamental similarities in the folding mechanisms of single domain proteins and (2) rather than being highly sensitive to the finest details of sequence, their folding kinetics are determined primarily by the large-scale, redundant features of sequence that determine a protein's gross structural properties. That folding kinetics can be predicted using simple, empirical, structure-based rules suggests that the fundamental physics underlying folding may be quite straightforward and that a general and quantitative theory of protein folding rates and mechanisms (as opposed to unfolding rates and thus protein stability) may be near on the horizon.     

Molecular cloning of four novel murine ribonuclease genes: unusual expansion within the ribonuclease A gene family.

We have characterized four novel murine ribonuclease genes that, together with the murine eosinophil-associated ribonucleases 1 and 2, form a distinct and unusual cluster within the RNase A gene superfamily. Three of these genes (mR-3, mR-4, mR-5) include complete open reading frames, encoding ribonucleases with eight cysteines and appropriately spaced histidines (His11 and His124) and lysine (Lys35) that are characteristic of this enlarging protein family; the fourth sequence encodes a non-functional pseudogene (mR-6P). Although the amino acid sequence similarities among these murine ribonucleases varies from 60 to 94%, they form a unique cluster, as each sequence is found to be more closely related to another of this group than to either murine angiogenin or to murine pancreatic ribonuclease. Interestingly, the relationship between the six genes in this 'mR cluster' and the defined lineages of the RNase A gene family could not be determined by amino acid sequence homology, suggesting the possibility that there are one or more additional ribonuclease lineages that have yet to be defined. Although the nature of the evolutionary constraints promoting this unusual expansion and diversification remain unclear, the implications with respect to function are intriguing.     

Simulating RNA folding kinetics on approximated energy landscapes

We present a general computational approach to simulate RNA folding kinetics that can be used to extract population kinetics, folding rates and the formation of particular substructures that might be intermediates in the folding process. Simulating RNA folding kinetics can provide unique insight into RNA whose functions are dictated by folding kinetics and not always by nucleotide sequence or the structure of the lowest free-energy state. The method first builds an approximate map (or model) of the folding energy landscape from which the population kinetics are analyzed by solving the master equation on the map. We present results obtained using an analysis technique, map-based Monte Carlo simulation, which stochastically extracts folding pathways from the map. Our method compares favorably with other computational methods that begin with a comprehensive free-energy landscape, illustrating that the smaller, approximate map captures the major features of the complete energy landscape. As a result, our method scales to larger RNAs. For example, here we validate kinetics of RNA of more than 200 nucleotides. Our method accurately computes the kinetics-based functional rates of wild-type and mutant ColE1 RNAII and MS2 phage RNAs showing excellent agreement with experiment.     

Toward minimalist models of larger proteins: a ubiquitin-like protein

Our recently developed off-lattice bead model capable of simulating protein structures with mixed alpha/beta content has been extended to model the folding of a ubiquitin-like protein and provides a means for examining the more complex kinetics involved in the folding of larger proteins. Using trajectories generated from constant-temperature Langevin dynamics simulations and sampling with the multiple multi-histogram method over five-order parameters, we are able to characterize the free energy landscape for folding and find evidence for folding through compact intermediates. Our model reproduces the observation that the C-terminus loop structure in ubiquitin is the last to fold in the folding process and most likely plays a spectator role in the folding kinetics. The possibility of a productive metastable intermediate along the folding pathway consisting of collapsed states with no secondary structure, and of intermediates or transition structures involving secondary structural elements occurring early in the sequence, is also supported by our model. The kinetics of folding remain multi-exponential below the folding temperature, with glass-like kinetics appearing at T/T(f) approximately 0.86. This new physicochemical model, designed to be predictive, helps validate the value of modeling protein folding at this level of detail for genomic-scale studies, and motivates further studies of other protein topologies and the impact of more complex energy functions, such as the addition of solvation forces.     

Oxidative folding of conotoxins sharing an identical disulfide bridging framework

Conotoxins are short, disulfide-rich peptide neurotoxins produced in the venom of predatory marine cone snails. It is generally accepted that an estimated 100,000 unique conotoxins fall into only a handful of structural groups, based on their disulfide bridging frameworks. This unique molecular diversity poses a protein folding problem of relationships between hypervariability of amino acid sequences and mechanism(s) of oxidative folding. In this study, we present a comparative analysis of the folding properties of four conotoxins sharing an identical pattern of cysteine residues forming three disulfide bridges, but otherwise differing significantly in their primary amino acid sequence. Oxidative folding properties of M-superfamily conotoxins GIIIA, PIIIA, SmIIIA and RIIIK varied with respect to kinetics and thermodynamics. Based on rates for establishing the steady-state distribution of the folding species, two distinct folding mechanisms could be distinguished: first, rapid-collapse folding characterized by very fast, but low-yield accumulation of the correctly folded form; and second, slow-rearrangement folding resulting in higher accumulation of the properly folded form via the reshuffling of disulfide bonds within folding intermediates. Effects of changing the folding conditions indicated that the rapid-collapse and the slow-rearrangement mechanisms were mainly determined by either repulsive electrostatic or productive noncovalent interactions, respectively. The differences in folding kinetics for these two mechanisms were minimized in the presence of protein disulfide isomerase. Taken together, folding properties of conotoxins from the M-superfamily presented in this work and from the O-superfamily published previously suggest that conotoxin sequence diversity is also reflected in their folding properties, and that sequence information rather than a cysteine pattern determines the in vitro folding mechanisms of conotoxins.     

Residues participating in the protein folding nucleus do not exhibit preferential evolutionary conservation

To what extent does natural selection act to optimize the details of protein folding kinetics? In an effort to address this question, the relationship between an amino acid's evolutionary conservation and its role in protein folding kinetics has been investigated intensively. Despite this effort, no consensus has been reached regarding the degree to which residues involved in native-like transition state structure (the folding nucleus) are conserved. Here we report the results of an exhaustive, systematic study of sequence conservation among residues known to participate in the experimentally (Phi-value) defined folding nuclei of all of the appropriately characterized proteins reported to date. We observe no significant evidence that these residues exhibit any anomalous sequence conservation. We do observe, however, a significant bias in the existing kinetic data: the mean sequence conservation of the residues that have been the subject of kinetic characterization is greater than the mean sequence conservation of all residues in 13 of 14 proteins studied. This systematic experimental bias gives rise to the previous observation that the median conservation of residues reported to participate in the folding nucleus is greater than the median conservation of all of the residues in a protein. When this bias is corrected (by comparing, for example, the conservation of residues known to participate in the folding nucleus with that of other, kinetically characterized residues) the previously reported preferential conservation is effectively eliminated. In contrast to well-established theoretical expectations, both poorly and highly conserved residues are apparently equally likely to participate in the protein-folding nucleus.     

Structural parameters affecting the kinetics of RNA hairpin formation

There is little experimental knowledge on the sequence dependent rate of hairpin formation in RNA. We have therefore designed RNA sequences that can fold into either of two mutually exclusive hairpins and have determined the ratio of folding of the two conformations, using structure probing. This folding ratio reflects their respective folding rates. Changing one of the two loop sequences from a purine- to a pyrimidine-rich loop did increase its folding rate, which corresponds well with similar observations in DNA hairpins. However, neither changing one of the loops from a regular non-GNRA tetra-loop into a stable GNRA tetra-loop, nor increasing the loop size from 4 to 6 nt did affect the folding rate. The folding kinetics of these RNAs have also been simulated with the program ‘Kinfold’. These simulations were in agreement with the experimental results if the additional stabilization energies for stable tetra-loops were not taken into account. Despite the high stability of the stable tetra-loops, they apparently do not affect folding kinetics of these RNA hairpins. These results show that it is possible to experimentally determine relative folding rates of hairpins and to use these data to improve the computer-assisted simulation of the folding kinetics of stem–loop structures.     

Rapid folding with and without populated intermediates in the homologous four-helix proteins Im7 and Im9

The kinetics and thermodynamics of the folding of the homologous four-helix proteins Im7 and Im9 have been characterised at pH 7.0 and 10 degrees C. These proteins are 60 % identical in sequence and have the same three-dimensional structure, yet appear to fold by different kinetic mechanisms. The logarithm of the folding and unfolding rates of Im9 change linearly as a function of urea concentration and fit well to an equation describing a two-state mechanism (with a folding rate of 1500 s-1, an unfolding rate of 0. 01 s-1, and a highly compact transition state that has approximately 95 % of the native surface area buried). By contrast, there is clear evidence for the population of an intermediate during the refolding of Im7, as indicated by a change in the urea dependence of the folding rate and the presence of a significant burst phase amplitude in the refolding kinetics. Under stabilising conditions (0.25 M Na2SO4, pH 7.0 and 10 degrees C) the folding of Im9 remains two-state, whilst under similar conditions (0.4 M Na2SO4, pH 7.0 and 10 degrees C) the intermediate populated during Im7 refolding is significantly stabilised (KUI=125). Equilibrium denaturation experiments, under the conditions used in the kinetic measurements, show that Im7 is significantly less stable than Im9 (DeltaDeltaG 9.3 kJ/mol) and the DeltaG and m values determined accord with those obtained from the fit to the kinetic data. The results show, therefore, that the population of an intermediate in the refolding of the immunity protein structure is defined by the precise amino acid sequence rather than the global stability of the protein. We discuss the possibility that the intermediate of Im7 is populated due to differences in helix propensity in Im7 and Im9 and the relevance of these data to the folding of helical proteins in general.     

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.     

A comparison of the GroE chaperonin requirements for sequentially and structurally homologous malate dehydrogenases: the importance of folding kinetics and solution environment

Escherichia coli malate dehydrogenase (EcMDH) and its eukaryotic counterpart, porcine mitochondrial malate dehydrogenase (PmMDH), are highly homologous proteins with significant sequence identity (60%) and virtually identical native structural folds. Despite this homology, EcMDH folds rapidly and efficiently in vitro and does not seem to interact with GroE chaperonins at physiological temperatures (37 degrees C), whereas PmMDH folds much slower than EcMDH and requires these chaperonins to fold to the native state at 37 degrees C. Double jump experiments indicate that the slow folding behavior of PmMDH is not limited by proline isomerization. Although the folding enhancer glycerol (<5 m) does not alter the renaturation kinetics of EcMDH, it dramatically accelerates the spontaneous renaturation of PmMDH at all temperatures tested. Kinetic analysis of PmMDH renaturation with increasing glycerol concentrations suggests that this osmolyte increases the on-pathway kinetics of the monomer folding to assembly-competent forms. Other osmolytes such as trimethylamine N-oxide, sucrose, and betaine also reactivate PmMDH at nonpermissive temperatures (37 degrees C). Glycerol jump experiments with preformed GroEL.PmMDH complexes indicate that the shift between stringent (requires ATP and GroES) and relaxed (only requires ATP) complex conformations is rapid (<3-5 s). The similarity in irreversible misfolding kinetics of PmMDH measured with glycerol or the activated chaperonin complex (GroEL.GroES.ATP) suggests that these folding aids may influence the same step in the PmMDH folding reaction. Moreover, the interactions between glycerol-induced PmMDH folding intermediates and GroEL.GroES.ATP are diminished. Our results support the notion that the protein folding kinetics of sequentially and structurally homologous proteins, rather than the structural fold, dictates the GroE chaperonin requirement.