Hydrogen exchange and proteolytic degradation of ribonuclease A. Similarities and distinctions of the kinetic mechanisms

The studies by IR spectroscopy of the temperature dependence of the H-D exchange rate of the RNase A peptide NH atoms permit one to characterize two types of conformation fluctuations, local and global. A comparison with the temperature dependence of the proteolytic degradation rate of RNase A shows that similar in nature fluctuations allow for the H-D exchange of NH atoms and the splitting of peptide bonds of the native protein. In the low temperature region, both processes occur through local fluctuations, by way of the EX2 mechanism, and in the high temperature region, they occur through global fluctuations with the overall denaturation desorganization of the native structure, by way of the EX1 mechanism. The biphasic dependence of the rate of H-D exchange and proteolytic degradation of RNase A on urea concentration is also explained by the combination of local and global fluctuations.     

Hydrogen exchange and proteolytic degradation of ribonuclease A. The local splitting of the native structure and the conformation of loop segmentes

The limited proteolytic sites or nicksites are present only in one of the five loops of the RNase A molecule. The splitted loop 15-23 connects two structural domains in the hinge region of the interdomain contacts of the V-shaped molecule. The other four loops are inside two domains, 64-71 and 112-115 in the domain I (1-19, 47-81, 102-106) and 36-42 and 88-95 in the domain II (20-46, 82-101). Because of enhanced chain flexibility of the splitted loop in the pH-dependent conformational isomerization, deformation of its structure is slighter under the influence of the intermolecular contacts in the crystal lattice and more significant changes occur in loop conformation at the formation of the 3D swapped dimer of the RNase A molecule. The proteolytic splitting of the 15-23 loop proceeds due to the local fluctuation of the native protein structure.     

Hydrogen exchange and proteolytic degradation of ribonuclease A. The local splitting of the native structure and the conformation of loop segments

The limited proteolytic sites or nicksites are present only in one of the five loops of the RNase A molecule. The splitted loop 15–23 connects two structural domains in the hinge region of the interdomain contacts of the V-shaped molecule. The other four loops are inside two domains, 64–71 and 112–115 in the domain I (1–19, 47–81, 102–106) and 36–42 and 88–95 in the domain II (20–46, 82–101). Because of enhanced chain flexibility of the splitted loop in the pH-dependent conformational isomerization, deformation of its structure is slighter under the influence of the intermolecular contacts in the crystal lattice and more significant changes occur in loop conformation at the formation of the 3D swapped dimer of the RNase A molecule. The proteolytic splitting of the 15–23 loop proceeds due to the local fluctuation of the native protein structure.     

Dynamics of ribonuclease A and ribonuclease S: computational and experimental studies.

RNase S is a complex consisting of two proteolytic fragments of RNase A: the S peptide (residues 1-20) and S protein (residues 21-124). RNase S and RNase A have very similar X-ray structures and enzymatic activities. Previous experiments have shown increased rates of hydrogen exchange and greater sensitivity to tryptic cleavage for RNase S relative to RNase A. It has therefore been asserted that the RNase S complex is considerably more dynamically flexible than RNase A. In the present study we examine the differences in the dynamics of RNase S and RNase A computationally, by MD simulations, and experimentally, using trypsin cleavage as a probe of dynamics. The fluctuations around the average solution structure during the simulation were analyzed by measuring the RMS deviation in coordinates. No significant differences between RNase S and RNase A dynamics were observed in the simulations. We were able to account for the apparent discrepancy between simulation and experiment by a simple model. According to this model, the experimentally observed differences in dynamics can be quantitatively explained by the small amounts of free S peptide and S protein that are present in equilibrium with the RNase S complex. Thus, folded RNase A and the RNase S complex have identical dynamic behavior, despite the presence of a break in polypeptide chain between residues 20 and 21 in the latter molecule. This is in contrast to what has been widely believed for over 30 years about this important fragment complementation system.     

The mechanisms of the proteolytic degradation of native globular proteins. The role of local and global fluctuations of the native structure

Analysis of the proteolytic degradation of the native protein structure carried out by the comparison of the temperature dependence of the hydrogen exchange and proteolytic splitting rates of the hen egg-white lysozyme and human Hb and apoHb. Acceleration of the burst-like (all or none) proteolytic degradation in the high temperature range is provided by the intensification of the global fluctuations with overall unfolding revealed by hydrogen exchange. For Hb and apoHb the rate of burst-like proteolytic degradation and hydrogen exchange weakly depends on temperature in the range, where hydrogen exchange reveals only local fluctuations of the native protein structure. The splitting of the two proteins proceeds by the selfaccelerated burst-like mechanism with the initial rate-limiting single cleavage owing to the local fluctuation of the native structure. The local fluctuations play important role also upon the intracellular burst-like degradation of native proteins.     

Local fluctuations vs. global unfolding of proteins investigated by limited proteolysis

The structure of a protein molecule consists of both rigid and flexible sections to satisfy the demands for stability and catalysis. Because the flexibility of a protein segment is indispensable for a proteolytic attack, limited proteolysis is a superb tool to analyse both confined local fluctuations and global unfolding events in proteins. While the identification of the primary cleavage products allows the assignment of the flexible regions to the primary structure, the kinetics of proteolytic degradation enables differentiation between local fluctuations in the native protein molecule and the global unfolding process during denaturation. Modifications of the amino acid sequence in the concerned regions can tune proteolytic susceptibility and alter protein stability. In the present paper, we summarise our results on native-state and unfolded-state proteolysis of ribonuclease A (RNase A) and the effect of mutations in the detected flexible regions on the stability and unfolding of the RNase A molecule.     

Local fluctuations vs. global unfolding of proteins investigated by limited proteolysis

The structure of a protein molecule consists of both rigid and flexible sections to satisfy the demands for stability and catalysis. Because the flexibility of a protein segment is indispensable for a proteolytic attack, limited proteolysis is a superb tool to analyse both confined local fluctuations and global unfolding events in proteins. While the identification of the primary cleavage products allows the assignment of the flexible regions to the primary structure, the kinetics of proteolytic degradation enables differentiation between local fluctuations in the native protein molecule and the global unfolding process during denaturation. Modifications of the amino acid sequence in the concerned regions can tune proteolytic susceptibility and alter protein stability. In the present paper, we summarise our results on native-state and unfolded-state proteolysis of ribonuclease A (RNase A) and the effect of mutations in the detected flexible regions on the stability and unfolding of the RNase A molecule.     

Limited proteolysis of ribonuclease A with thermolysin in trifluoroethanol.

We have examined the proteolysis of bovine pancreatic ribonuclease A (RNase) by thermolysin when dissolved in aqueous buffer, pH 7.0, in the presence of 50% (v/v) trifluoroethanol (TFE). Under these solvent conditions, RNase acquires a conformational state characterized by an enhanced content of secondary structure (helix) and reduced tertiary structure, as given by CD measurements. It was found that the TFE-resistant thermolysin, despite its broad substrate specificity, selectively cleaves the 124-residue chain of RNase in its TFE state (20-42 degrees C, 6-24 h) at peptide bond Asn 34-Leu 35, followed by a slower cleavage at peptide bond Thr 45-Phe 46. In the absence of TFE, native RNase is resistant to proteolysis by thermolysin. Two nicked RNase species, resulting from cleavages at one or two peptide bonds and thus constituted by two (1-34 and 35-124) (RNase Th1) or three (1-34, 35-45 and 46-124) (RNase Th2) fragments linked covalently by the four disulfide bonds of the protein, were isolated to homogeneity by chromatography and characterized. CD measurements provided evidence that RNase Th1 maintains the overall conformational features of the native protein, but shows a reduced thermal stability with respect to that of the intact species (-delta Tm 16 degrees C); RNase Th2 instead is fully unfolded at room temperature. That the structure of RNase Th1 is closely similar to that of the intact protein was confirmed unambiguously by two-dimensional NMR measurements. Structural differences between the two protein species are located only at the level of the chain segment 30-41, i.e., at residues nearby the cleaved Asn 34-Leu 35 peptide bond. RNase Th1 retained about 20% of the catalytic activity of the native enzyme, whereas RNase Th2 was inactive. The 31-39 segment of the polypeptide chain in native RNase forms an exposed and highly flexible loop, whereas the 41-48 region forms a beta-strand secondary structure containing active site residues. Thus, the conformational, stability, and functional properties of nicked RNase Th1 and Th2 are in line with the concept that proteins appear to tolerate extensive structural variations only at their flexible or loose parts exposed to solvent. We discuss the conformational features of RNase in its TFE-state that likely dictate the selective proteolysis phenomenon by thermolysin.     

Extremely fast hydrogen exchange of ribonuclease-(1-118) as compared with native RNase A and its implication for the conformational energy state

RNase-(1-118) containing native disulfide bonds is similar in fold to native RNase A but not of lowest Gibbs energy as compared with the isomers containing non-native disulfide bonds. The present n.m.r. studies have indicated a dramatic increase in the exchange rate of all of the 'protected' amide protons of RNase-(1-118) over RNase A. A calculation shows a large increase in the rate of 'opening' of the structure. The exchange rate of the protected amide protons of RNase-(1-120) is slower than RNase-(1-118) but much faster than RNase A. Binding with a synthetic complementing fragment (114-124) markedly reduces the exchange rate of 20 to 25 amide protons of RNase-(1-118). It has previously been shown that binding with a complementing fragment of RNase-(1-118) generates a lowest Gibbs energy state. Thus, using available thermodynamic information for interpretation, we suggest that a) removal of six carboxy terminal residues of RNase A would disrupt coupling between these residues and those distant in the structure (loss of extra stabilizing energy), b) this would, in turn, alter the enthalpy-entropy compensation in such a way that the magnitude of Gibbs energy change favoring folding is significantly reduced without a large change of fold and c) in this activated state the molecule would be highly motile.     

Differences in the solution structures of the parallel beta-helical pectate lyases as determined by limited proteolysis

The pectate lyase family of proteins has been shown to fold into a novel domain motif, the right-handed parallel beta-helix. As a means of gaining insight to the solution structure of the pectate lyases, the enzymes were subjected to limited proteolytic digestion by the endoproteases AspN, GluC and trypsin. The effects of proteolytic cleavage on enzymatic activity were determined, and the early products of proteolysis were identified by capillary electrophoresis, MALDI-TOF mass spectrometry and HPLC. A single peptide bond between Lys158 and Asp159 in pectate lyase B (PLb) was cleaved by both AspN and trypsin, with no detectable hydrolysis of PLb by GluC. Pectate lyase E (PLe) was hydrolyzed by trypsin between Lys164 and Asp165, a bond on an analogous loop structure found to be susceptible to proteolytic attack in PLb. AspN and GluC preferentially hydrolyzed peptide bonds (at Asp127 and Glu124, respectively) on another loop extending from the central beta-helical core of PLe. A single beta-strand of the central cylinder of the pectate lyase C (PLc) molecule was susceptible to all three proteases used. These data demonstrate that the most susceptible peptide bonds to proteolytic scission within the native enzymes lie on or near one of the three parallel beta-sheets that compose the core domain motif Despite the proximity of the proteolytic cleavages to the catalytic sites of the enzymes, significant retention of lyase activity was observed after partial proteolysis, indicating preservation of functional tertiary structure in the proteolytic products.     

Hydrogen bond integrity between MHC class II molecules and bound peptide determines the intracellular fate of MHC class II molecules.

MHC class II molecules associate with peptides through pocket interactions and the formation of hydrogen bonds. The current paradigm suggests that the interaction of side chains of the peptide with pockets in the class II molecule is responsible for the formation of stable class II-peptide complexes. However, recent evidence has shown that the formation of hydrogen bonds between genetically conserved residues of the class II molecule and the main chain of the peptide contributes profoundly to peptide stability. In this study, we have used I-A(k), a class II molecule known to form strong pocket interactions with bound peptides, to probe the general importance of hydrogen bond integrity in peptide acquisition. Our studies have revealed that abolishing hydrogen bonds contributed by positions 81 or 82 in the beta-chain of I-A(k) results in class II molecules that are internally degraded when trafficked through proteolytic endosomal compartments. The presence of high-affinity peptides derived from either endogenous or exogenous sources protects the hydrogen bond-deficient variant from intracellular degradation. Together, these data indicate that disruption of the potential to form a complete hydrogen bond network between MHC class II molecules and bound peptides greatly diminishes the ability of class II molecules to bind peptides. The subsequent failure to stably acquire peptides leads to protease sensitivity of empty class II molecules, and thus to proteolytic degradation before export to the surface of APCs.     

The solvent dependence of hydrogen exchange kinetics of folded proteins.

The effects of ethanol, ethylene glycol, dioxane, and other organic co-solvents upon the hydrogen exchange rates of randomly coiled oxidized RNase, native RNase, and native trypsin have been measured. The exchange rate of oxidized RNase, the model compound for the proton transfer step in hydrogen exchange, is decreased by all of the co-solvents studied at temperatures in the range 3-20 degrees. This has been ascribed to the combined effects of the disruption of peptide bond solvation due to a reduction in the concentration of water, and of changes in [OH-] ion concentration due to changes in the acid dissociation constant of water, Kw. The solvent dependence for both native RNase and native trypsin is similar in all of the solvents studied. At a low temperature (3-20 degrees), the exchange rates go through a minimum as the solvent concentration is increased. At higher temperatures (20-35 degrees) the exchange rates are increased at all concentrations of the co-solvent. The apparent rate minimum at lower temperatures is due to two opposing effects. Co-solvents decrease the rate of exchange that occurs directly from the folded molecule. At higher concentrations and higer temperature. The decrease in rates for exchange directly from folded protein is primarily due to the effects on the proton transfer step, and not to binding or the solvent effects on protein structure. The solvents used in this study have no apparent effect on conformational processes contributing to the hydrogen exchange process in folded proteins.     

Disulfide bond formation during the folding of influenza virus hemagglutinin

To study the importance of individual sulfhydryl residues during the folding and assembly in vivo of influenza virus hemagglutinin (HA), we have constructed and expressed a series of mutant HA proteins in which cysteines involved in three disulfide bonds have been substituted by serine residues. Investigations of the structure and intracellular transport of the mutant proteins indicate that (a) cysteine residues in the ectodomain are essential both for efficient folding of HA and for stabilization of the folded molecule; (b) cysteine residues in the globular portion of the ectodomain are likely to form native disulfide bonds rapidly and directly, without involvement of intermediate, nonnative linkages; and (c) cysteine residues in the stalk portion of the ectodomain also appear not to form intermediate disulfide bonds, even though they have the opportunity to do so, being separated from their correct partners by hundreds of amino acids including two or more other sulfhydryl residues. We propose a role for the cellular protein BiP in shielding the cysteine residues of the stalk domain during the folding process, thus preventing them from forming intermediate, nonnative disulfide bonds.     

Implementation of a k/k(0) method to identify long-range structure in transition states during conformational folding/unfolding of proteins

A previously introduced kinetic-rate constant (k/k(0)) method, where k and k(0) are the folding (unfolding) rate constants in the mutant and the wild-type forms, respectively, of a protein, has been applied to obtain qualitative information about structure in the transition state ensemble (TSE) of bovine pancreatic ribonuclease A (RNase A), which contains four native disulfide bonds. The method compares the folding (unfolding) kinetics of RNase A, with and without a covalent crosslink and tests whether the crosslinked residues are associated in the folding (unfolding) transition state (TS) of the noncrosslinked version. To confirm that the fifth disulfide bond has not introduced a significant structural perturbation, we solved the crystal structure of the V43C-R85C mutant to 1.6 A resolution. Our findings suggest that residues Val43 and Arg85 are not associated, and that residues Ala4 and Val118 may form nonnative contacts, in the folding (unfolding) TSE of RNase A.     

Renaturation of reduced ribonuclease A with a microsphere-induced refolding system

We intended to refold reduced ribonuclease A (RNase A) using polymeric microspheres. Polymeric microspheres were allowed to react with dithiothreitol (DTT) to immobilize the disulfide and thiol moieties on their surface. The fully reduced RNase A was added to the dispersion of the modified microspheres. Protein refolding and renaturation were estimated by the change in the number of disulfide bonds of RNase A and the recovery of the enzymatic activity, respectively. Without microspheres, the activity gradually recovered with the increase in the number of disulfide bonds. However, the formation of disulfide bonds of reduced RNase A was accelerated by adding the modified microspheres, and the rate of renaturation was increased depending on the amount of charged DTT and the reaction time of the immobilization. These results indicate that modified microspheres significantly catalyze the recovery of active RNase A from the reduced form. The protein adsorption data demonstrated that the disulfide moieties of the modified microspheres react with the thiol moieties of the reduced RNase A to form a mixed disulfide. The thiol/disulfide exchange reaction can possibly proceed at the microsphere/protein interface, resulting in the formation of a correct three-dimensional structure.     

Refolding simulations of an isolated fragment of barnase into a native-like β hairpin: Evidence for compactness and hydrogen bonding as concurrent stabilizing factors

Experimental evidence and theoretical models both suggest that protein folding is initiated within specific fragments intermittently adopting conformations close to that found in the protein native structure. These folding initiation sites encompassing short portions of the protein are ideally suited for study in isolation by computational methods aimed at peering into the very early events of folding. We have used Molecular Dynamics (MD) technique to investigate the behavior of an isolated protein fragment formed by residues 85 to 102 of barnase that folds into a β hairpin in the protein native structure. Three independent MD simulations of 1.3 to 1.8 ns starting from unfolded conformations of the peptide portrayed with an all-atom model in water were carried out at gradually decreasing temperature. A detailed analysis of the conformational preferences adopted by this peptide in the course of the simulations is presented. Two of the unfolded peptide conformations fold into a hairpin characterized by native and a larger bulk of nonnative interactions. Both refolding simulations substantiate the close relationship between interstrand compactness and hydrogen bonding network involving backbone atoms. Persistent compactness witnessed by side-chain interactions always occurs concomitantly with the formation of backbone hydrogen bonds. No highly populated conformations generated in a third simulation starting from the remotest unfolded conformer relative to the native structure are observed. However, nonnative long-range and medium-range contacts with the aromatic moiety of Trp94 are spotted, which are in fair agreement with a former nuclear magnetic resonance study of a denaturing solution of an isolated barnase fragment encompassing the β hairpin. All this lends reason to believe that the 85–102 barnase fragment is a strong initiation site for folding. Proteins 29:212–227, 1997. © 1997 Wiley-Liss, Inc.     

Dissecting the effect of trifluoroethanol on ribonuclease A. Subtle structural changes detected by nonspecific proteases.

With the aim to distinguish between local and global conformational changes induced by trifluoroethanol in RNase A, spectroscopic and activity measurements in combination with proteolysis by unspecific proteases have been exploited for probing structural transitions of RNase A as a function of trifluoroethanol concentration. At > 30% (v/v) trifluoroethanol (pH 8.0; 25 degrees C), circular dichroism and fluorescence spectroscopy indicate a cooperative collapse of the tertiary structure of RNase A coinciding with the loss of its enzymatic activity. In contrast to the denaturation by guanidine hydrochloride, urea or temperature, the breakdown of the tertiary structure in trifluoroethanol is accompanied by an induction of secondary structure as detected by far-UV circular dichroism spectroscopy. Proteolysis with the nonspecific proteases subtilisin Carlsberg or proteinase K, both of which attack native RNase A at the Ala20-Ser21 peptide bond, yields refined information on conformational changes, particularly in the pretransition region. While trifluoroethanol at concentrations > 40% results in a strong increase of the rate of proteolysis and new primary cleavage sites (Tyr76-Ser77, Met79-Ser80) were identified, the rate of proteolysis at trifluoroethanol concentrations < 40% (v/v) is much smaller (up to two orders of magnitude) than that of the native RNase A. The proteolysis data point to a decreased flexibility in the surrounding of the Ala20-Ser21 peptide bond, which we attribute to subtle conformational changes of the ribonuclease A molecule. These changes, however, are too marginal to alter the overall catalytic and spectroscopic properties of ribonuclease A.     

Peptide Binding to a PDZ Domain by Electrostatic Steering via Nonnative Salt Bridges

We have captured the binding of a peptide to a PDZ domain by unbiased molecular dynamics simulations. Analysis of the trajectories reveals on-pathway encounter complex formation, which is driven by electrostatic interactions between negatively charged carboxylate groups in the peptide and positively charged side chains surrounding the binding site. In contrast, the final stereospecific complex, which matches the crystal structure, features completely different interactions, namely the burial of the hydrophobic side chain of the peptide C-terminal residue and backbone hydrogen bonds. The simulations show that nonnative salt bridges stabilize kinetically the encounter complex during binding. Unbinding follows the inverse sequence of events with the same nonnative salt bridges in the encounter complex. Thus, in contrast to protein folding, which is driven by native interactions, the binding of charged peptides can be steered by nonnative interactions, which might be a general mechanism, e.g., in the recognition of histone tails by bromodomains.     

Mapping the lifetimes of local opening events in a native state protein.

The rate constants for the processes that lead to local opening and closing of the structures around hydrogen bonds in native proteins have been determined for most of the secondary structure hydrogen bonds in the four-helix protein acyl coenzyme A binding protein. In an analysis that combines these results with the energies of activation of the opening processes and the stability of the local structures, three groups of residues in the protein structure have been identified. In one group, the structures around the hydrogen bonds have frequent openings, every 600 to 1,500 s, and long lifetimes in the open state, around 1 s. In another group of local structures, the local opening is a very rare event that takes place only every 15 to 60 h. For these the lifetime in the open state is also around 1 s. The majority of local structures have lifetimes between 2,000 and 20,000 s and relatively short lifetimes of the open state in the range between 30 and 400 ms. Mapping of these groups of amides to the tertiary structure shows that the openings of the local structures are not cooperative at native conditions, and they rarely if ever lead to global unfolding. The results suggest a mechanism of hydrogen exchange by progressive local openings.     

The native state conformational ensemble of the SH3 domain from alpha-spectrin.

The folding/unfolding equilibrium of the alpha-spectrin SH3 domain has been measured by NMR-detected hydrogen/deuterium exchange and by differential scanning calorimetry. Protection factors against exchange have been obtained under native conditions for more than half of the residues in the domain. Most protected residues are located at the beta-strands, the short 3(10) helix, and part of the long RT loop, whereas the loops connecting secondary structure elements show no measurable protection. Apparent stability constants per residue and their corresponding Gibbs energies have been calculated from the exchange experiments. The most stable region of the SH3 domain is defined by the central portions of the beta-strands. The peptide binding region, on the other hand, is composed of a highly stable region (residues 53-57) and a highly unstable region, the loop between residues 34-41 (n-Src loop). All residues in the domain have apparent Gibbs energies lower than the global unfolding Gibbs energy measured by differential scanning calorimetry, indicating that under our experimental conditions the amide exchange of all residues in the SH3 domain occurs primarily via local unfolding reactions. A structure-based thermodynamic analysis has allowed us to predict correctly the thermodynamics of the global unfolding of the domain and to define the ensemble of conformational states that quantitatively accounts for the observed pattern of hydrogen exchange protection. These results demonstrate that under native conditions the SH3 domain needs to be considered as an ensemble of conformations and that the hydrogen exchange data obtained under those conditions cannot be interpreted by a two-state equilibrium. The observation that specific regions of a protein are able to undergo independent local folding/unfolding reactions indicates that under native conditions the scale of cooperative interactions is regional rather than global.