Native RNase Research Articles

Proteolytic degradation of ribonuclease A in the pretransition region of thermally and urea-induced unfolding.

The method of limited proteolysis has proven to be appropriate for the determination of unfolding rate constants (k(U)) of ribonuclease A in the transition region of thermal denaturation [Arnold, U. & Ulbrich-Hofmann, R. (1997) Biochemistry 36, 2166-2172]. The aim of the present paper was to extend this procedure to the pretransition region of thermally and urea-induced denaturation where spectroscopic methods do not allow direct measurement of k(U). The results show that the approach can be applied successfully to denaturing (free energy of unfolding Delta G < 10 kJ.mol(-1)) and to marginally native conditions (Delta G = 10-25 kJ.mol(-1)). Under moderately (Delta G = 25-30 kJ.mol(-1)) and strongly native conditions (Delta G > 30 kJ.mol(-1)), however, the determination of kU was not possible in this way as the proteolytic degradation of ribonuclease A by thermolysin or trypsin was no longer determined by global unfolding. Here, proteolysis proceeds via the native RNase A. In the presence of low concentrations of urea, the rate constants of proteolysis were, surprisingly, smaller than in the absence of urea. As the protease activity has been taken into account, this result points to a local stabilization of the RNase A molecule.

Conformational Studies of the Man8 Oligosaccharide on Native Ribonuclease B and on the Reduced and Denatured Protein

Site-specific presentation of oligosaccharides in the context of carrier proteins can influence markedly their recognition by carbohydrate-binding proteins. On RNaseB, the Man5–9N-glycans at Asn-34 are bound by the serum lectin conglutinin when the glycoprotein is reduced and denatured, but there is no binding to the N-glycans on the native form of RNaseB. The RNaseB Man8, which is a glycoform preferentially bound by conglutinin, is the subject of the present study. The conformational behavior of the protein-linked oligosaccharide Man8 is investigated on the native and on the reduced and denatured RNaseB, using a combination of NMR and theoretical calculations. Quantitative data on the NOESY crosspeaks have been obtained, thereby allowing the comparison of mobilities of homologous linkages within the glycan chain. Oligosaccharide conformations compatible with the NMR data have been explored by molecular modeling of the free oligosaccharide, using two different force fields (AMBER and SYBYL). There are some differences between the results produced by the two force fields, the AMBER simulations providing a better agreement with the experimental data. The results indicate that both on the native and on the reduced heat-denatured glycoprotein, the RNase Man8 oligosaccharide exhibits a conformational behavior very similar to that of the free oligosaccharide. However, this conformational freedom of the N-glcyan does not amount to full availability for carbohydrate-recognition proteins and enzymes.

Divalent metal cofactor binding in the kinetic folding trajectory of Escherichia coli ribonuclease HI.

Proteins often require cofactors to perform their biological functions and must fold in the presence of their cognate ligands. Using circular dichroism spectroscopy. we investigated the effects of divalent metal binding upon the folding pathway of Escherichia coli RNase HI. This enzyme binds divalent metal in its active site, which is proximal to the folding core of RNase HI as defined by hydrogen/deuterium exchange studies. Metal binding increases the apparent stability of native RNase HI chiefly by reducing the unfolding rate. As with the apo-form of the protein, refolding from high denaturant concentrations in the presence of Mg2+ follows three-state kinetics: formation of a rapid burst phase followed by measurable single exponential kinetics. Therefore, the overall folding pathway of RNase HI is minimally perturbed by the presence of metal ions. Our results indicate that the metal cofactor enters the active site pocket only after the enzyme reaches its native fold, and therefore, divalent metal binding stabilizes the protein by decreasing its unfolding rate. Furthermore, the binding of the cofactor is dependent upon a carboxylate critical for activity (Asp10). A mutation in this residue (D10A) alters the folding kinetics in the absence of metal ions such that they are similar to those observed for the unaltered enzyme in the presence of metal.

A specific transition state for S-peptide combining with folded S-protein and then refolding

We measured the folding and unfolding kinetics of mutants for a simple protein folding reaction to characterize the structure of the transition state. Fluorescently labeled S-peptide analogues combine with S-protein to form ribonuclease S analogues: initially, S-peptide is disordered whereas S-protein is folded. The fluorescent probe provides a convenient spectroscopic probe for the reaction. The association rate constant, kon, and the dissociation rate constant, koff, were both determined for two sets of mutants. The dissociation rate constant is measured by adding an excess of unlabeled S-peptide analogue to a labeled complex (RNaseS*). This strategy allows kon and koff to be measured under identical conditions so that microscopic reversibility applies and the transition state is the same for unfolding and refolding. The first set of mutants tests the role of the alpha-helix in the transition state. Solvent-exposed residues Ala-6 and Gln-11 in the alpha-helix of native RNaseS were replaced by the helix destabilizing residues glycine or proline. A plot of log kon vs. log Kd for this series of mutants is linear over a very wide range, with a slope of -0.3, indicating that almost all of the molecules fold via a transition state involving the helix. A second set of mutants tests the role of side chains in the transition state. Three side chains were investigated: Phe-8, His-12, and Met-13, which are known to be important for binding S-peptide to S-protein and which also contribute strongly to the stability of RNaseS*. Only the side chain of Phe-8 contributes significantly, however, to the stability of the transition state. The results provide a remarkably clear description of a folding transition state.

Protein disulphide isomerase mediates platelet aggregation and secretion.

Platelet surface thiols and disulphides play an important role in platelet responses. Agents that reduce disulphide bonds expose the fibrinogen receptor in platelets and activate the purified glycoprotein (GP) IIbIIIa receptor. Protein disulphide isomerase (PDI), an enzyme that rearranges disulphides bonds, is found on the platelet surface where it is catalytically active. We investigated the role of PDI in platelet responses using (1) rabbit anti-PDI IgG specific for PDI, (2) a competing substrate (scrambled ribonuclease A), and (3) the PDI inhibitor, bacitracin. Fab fragments of the rabbit anti-PDI IgG inhibited platelet responses to the agonists tested (ADP and collagen), whereas Fab fragments prepared identically from normal rabbit IgG had no inhibitory effect. Scrambled ribonuclease A blocked platelet aggregation and secretion, but native ribonuclease A did not. When biphasic platelet aggregation was examined using platelets in citrated plasma, the principle effect of bacitracin was on second phase or irreversible aggregation responses and the accompanying secretion. Using flow cytometry and an antibody specific for activated GPIIbIIIa (PAC-1), the rabbit anti-PDI Fab fragments substantially inhibited activation of GPIIbIIIa when added before, but not after, platelet activation. In summary, we have demonstrated that protein disulphide isomerase mediates platelet aggregation and secretion, and that it activates GPIIbIIIa, suggesting this receptor as the target of the enzyme.

Quantitative Analysis of the Composition of the Native and Scrambled Ribonuclease A

Reversible conversion between the native and scrambled proteins can be applied to analyze the denaturation curve of a disulfide-containing protein. In the case of RNase A, scrambled species could not be well separated from the native species by HPLC to permit precise quantitative analysis of the extent of denaturation. Methods are developed here to overcome this problem. The methods exploit the difference of conformational stability between the native and scrambled RNase A. When a sample of partially denatured RNase A was placed under mild reducing conditions (0.2–1 mM dithiothreitol for 10 min), the disulfide bonds of the native RNase A remain intact, whereas those of scrambled isomers become fully reduced. The native and fully reduced species of RNase A can be completely separated by HPLC. Alternatively, a mixture of partially denatured RNase A can be treated with mild concentration of proteolytic enzymes (trypsin or thermolysin). In this approach, scrambled isomers of RNase A were totally fragmented and readily separated from the native RNase A. These methods allow analysis and construction of the denaturation curves of RNase A in the presence of urea, GdmCl and GdmSCN.

Protein variant separations by cation-exchange chromatography on tentacle-type polymeric stationary phases

We developed a set of prototype cation-exchange column packings that are based on a hydrophilic coated, pellicular polymeric support with a grafted tentacular surface chemistry that is highly suited to resolving closely related protein variants. These column packings (1) afford minimal band spreading in conjunction with extremely high selectivity, (2) exhibit a very hydrophilic character and (3) have moderate loading capacity. Cytochrome c variants (bovine, horse, rabbit) were baseline-separated, as was native ribonuclease A and its two deamidation products, the Asp 67 and isoAsp 67 forms. Humanized monoclonal antibody variants differing in the presence of lysine at the C terminus of the heavy chains were baseline-resolved. Finally, the separation of hemoglobin variants found in a sample containing elevated levels of glycated hemoglobin was also demonstrated.

The Subsites Structure of Bovine Pancreatic Ribonuclease A Accounts for the Abnormal Kinetic Behavior with Cytidine 2′,3′-Cyclic Phosphate

The kinetics of the hydrolysis of cytidine 2',3'-cyclic phosphate (C>p) to 3'-CMP by ribonuclease A are multiphasic at high substrate concentrations. We have investigated these kinetics by determining 3'-CMP formation both spectrophotometrically and by a highly specific and quantitative chemical sampling method. With the use of RNase A derivatives that lack a functional p2 binding subsite, evidence is presented that the abnormal kinetics with the native enzyme are caused by the sequential binding of the substrate to the several subsites that make up the active site of ribonuclease. The evidence is based on the following points. 1) Some of the unusual features found with native RNase A and C>p as substrate disappear when the derivatives lacking a functional p2 binding subsite are used. 2) The kcat/Km values with oligocytidylic acids of increasing lengths (ending in C>p) show a behavior that parallels the specific velocity with C>p at high concentrations: increase in going from the monomer to the trimer, a decrease from tetramer to hexamer, and then an increase in going to poly(C). 3) Adenosine increases the kcat obtained with a fixed concentration of C>p as substrate. 4) High concentrations of C>p protect the enzyme from digestion with subtilisin, which results in a more compact molecule, implying large substrate concentration-induced conformational changes. The data for the hydrolysis of C>p by RNase A can be fitted to a fifth order polynomial that has been derived from a kinetic scheme based on the sequential binding of several monomeric substrate molecules.

Thermal Denaturation of Ribonuclease A Characterized by Water 17O and 2H Magnetic Relaxation Dispersion

Water oxygen-17 and deuteron nuclear magnetic relaxation dispersion (NMRD) measurements were used to characterize ribonuclease A (RNase A) in the course of thermal denaturation at pH 2 and 4. The structure and dynamics of the protein were probed by specific long-lived water molecules, by the short-lived surface hydration, and by labile side-chain hydrogens. The NMRD data show that native RNase A contains at least three water molecules with a mean residence time of 8 ns at 27 degreesC and an activation enthalpy of ca. 40 kJ mol-1. These water molecules are identified with some or all of six ordered water molecules partly buried in surface pockets in the crystal structure of RNase A. The loss of the 17O dispersion at higher temperatures demonstrates that, in the thermally denatured protein, these surface pockets are either not present or undergoing large structural fluctuations on a subnanosecond time scale. The relaxation dispersion step vanishes monotonically and essentially in concert with the CD denaturation curves, thus ruling out the existence of equilibrium intermediates with a substantial amount of non-native and long-lived hydration water. The NMRD data show that thermally denatured RNase A has a relatively compact but highly flexible structure. The global solvent exposure and the hydrodynamic volume of the denatured protein are much less than for maximally unfolded disulfide-intact RNase A. The NMRD data show that thermal denaturation is accompanied by a large reduction of the mean-square orientational order parameter of side-chain O-H bonds, implying that, in the denatured state, these side chains sample a wide distribution of conformational states on a subnanosecond time scale.

Room Temperature Phosphorescence Study of Refolding of Disulfide Reduced RNase T1

Refolding kinetics of disulfide reduced ribonuclease T 1 (RNase T 1 ) was studied by the recovery of room temperature phosphorescence emitted from a tryptophan residue (Trp 59) of the protein. When unfolded in a deoxygenated aqueous solution, the enzyme does not emit phosphorescence at room temperature, but as soon as the protein was transferred into an anoxic refolding buffer solution, it began emitting phosphorescence within the mixing dead time (∼30 s) of our apparatus. The initial quick recovery of RNase T 1 phosphorescence was followed by a gradual increase in the phosphorescence lifetime lasting over several tens of minutes and then slight decrease to a final value comparable to that of native RNase T 1 . The initial quick recovery of phosphorescence suggests that the tryptophan residue, being exposed to surrounding water molecules when the protein is unfolded, is quickly isolated from the external water when the enzyme starts refolding, and the gradual change in the phosphorescence lifetime implies...

Two-dimensional mid-IR and near-IR correlation spectra of ribonuclease A: using overtones and combination modes to monitor changes in secondary structure.

We introduce near-IR spectroscopy as an ancillary tool for monitoring structural changes of proteins in aqueous solution using ribonuclease A (RNase A) as a model protein. The thermal unfolding of RNase A results in clear spectral changes in the near-IR and the mid-IR regions. In the near-IR the most pronounced changes are observed in the spectral region between 4820 and 4940 cm-1. The strong N-H combination band found at 4867 cm-1 in the spectrum of native RNase A shifts to 4878 cm-1 upon thermal unfolding. Hydrogen-deuterium exchange experiments that validate the N-H character of this mode can also be used to estimate the number of unexchanged amide protons after exposure to D2O. The transition profiles and temperatures derived from the temperature dependence of the N-H combination mode were found to be practically identical with those derived from the temperature dependence of the C = O amide I band in the mid-IR region, demonstrating that the near-IR region can be used as a conformation-sensitive monitor for the thermally induced unfolding of proteins in H2O solution. A 2-dimensional correlation analysis was applied to the mid-IR and near-IR spectra of RNase A to establish correlations between IR bands in both regions. The correlation analysis demonstrates that the thermal unfolding of RNase A is not a completely cooperative process; rather it begins with some changes in beta-sheet structure, followed by the loss of alpha-helical structures, and then ending with the unfolding of the remaining beta-sheets.

Effects of substrate binding on the dynamics of RNase A: Molecular dynamics simulations of UpA bound and native RNase A

RNase A has been extensively used as a model protein in several biophysical and biochemical studies. Using the available structural and biochemical results, RNase A-UpA interaction has been computationally modeled at an atomic level. In this study, the molecular dynamics (MD) simulations of native and UpA bound RNase A have been carried out. The gross dynamical behavior and atomic fluctuations of the free and UpA bound RNase A have been characterized. Principal component analysis is carried out to identify the important modes of collective motion and to analyze the changes brought out in these modes of RNase A upon UpA binding. The hydrogen bonds are monitored to study the atomic details of RNase A-UpA interactions and RNase A-water interactions. Based on these analysis, the stability of the free and UpA bound RNase A are discussed. © 1997 John Wiley & Sons, Inc. Biopoly 42: 505–520, 1997

Cross-linked trimers of bovine ribonuclease A: activity on double-stranded RNA and antitumor action

Trimers of bovine pancreatic RNase A were obtained by cross-linking native RNase A with dimethyl suberimidate. They degrade double-stranded RNA more efficiently than dimers and monomers of RNase A, and display significant cytotoxic and/or cytostatic actions against C4-I cells (a human cell line derived from squamous carcinoma of the uterus cervix). On the same cell line cross-linked dimers of RNase A appear to be ineffective.

Effect of chemical glycosylation of RNase A on the protein stability and surface histidines accessibility in immobilized metal ion affinity electrophoresis (IMAGE) system

Immobilized metal ion affinity gel electrophoresis (IMAGE) has been applied to study the change of the surface histidines topography of RNase A when chemically glycosylated on exposed carboxylic groups with glucosamine using carbodiimide as cross-linker, under mild conditions. Two populations of glycosylated RNase A, one with a single glucosamine and another with two glucosamine attached, were obtained. These chemically glycosylated RNase A conserved about 80% of native enzymatic activity and their p Is were increased in comparison to native RNase A. The chemically glycosylated RNase A showed dramatic enhancement for thermal stability, while proteolytic resistance was similar to that of native RNase A. Chemically glycosylated RNase A showed a slightly increased affinity to IDA-Cu(II) as compared to the native enzyme, which indicates that a conformational change and/or a decrease in steric hindrance around accessible surface histidines (His 12 or His 119 and His 105) has occured. IMAGE is a useful method to analyse subtle conformational changes in proteins which result in subtle changes in histidine accessibilities.

Effect of glutaredoxin and protein disulfide isomerase on the glutathione-dependent folding of ribonuclease A.

Protein folding, associated with oxidation and isomerization of disulfide bonds, was studied using reduced and denatured RNase A (rd-RNase A) and mixed disulfide between glutathione and reduced RNase A derivative (GS-RNase A) as starting materials. Folding was initiated by addition of free glutathione (GSH + GSSG) and was monitored by electrospray mass spectrometry (ESMS) time-course analysis and recovery of the native catalytic activity. The ESMS analysis permitted both the identification and quantitation of the population of intermediates present during the refolding process. Refolding of rd-RNase A and GS-RNase A was also performed in the presence of glutaredoxin (Grx) and/or protein disulfide isomerase (PDI). All the analyses indicate a pathway of sequential reactions in the formation of native RNase A. First, the reduced protein reacts with a single glutathione molecule to form a mixed disulfide which then evolves to an intramolecular S-S bond via thiol-disulfide exchange. Only at this stage, the intermediate containing one intramolecular S-S reacts with a further glutathione molecule, reiterating the process. An analogous mechanism occurs in the refolding of GS-RNase A. The structural analysis of the intermediates formed during the refolding of RNase A showed for the first time that Grx is actually able to catalyze both formation and reduction of mixed disulfides involving glutatione. In both refolding processes, starting from either rd-RNase A or GS-RNase A, Grx displays a significant catalysis at the early stages of the process. Addition of PDI led to a net catalysis of the entire process without appearing to alter the refolding pathway. In the presence of both Grx and PDI, the two enzymes showed a synergistic activity either starting from rd-RNase A, as previously reported [Lundström, J., and Holmgren, A. (1995) J. Biol. Chem. 270, 7822-7828], or starting from GS-RNase A. Present data suggest that the synergistic effect can be explained assuming that Grx actually facilitates PDI action by catalyzing formation or reduction of mixed disulfides. The mixed disulfides are then rapidly converted into intramolecular disulfides in the presence of PDI. These steps are repeated sequentially throughout the whole refolding, resulting in an immediate formation of fully oxidized species even at the very beginning of the reaction. Finally, a Grx mutant, C14S Grx, in which one of the active site cysteine residues (Cys14) had been replaced by serine, had a similar effect on the distribution of folding intermediates, compared to the wild-type protein, thus demonstrating that Grx acts by a monothiol mechanism either in the reduction or in the oxidation step.

A Bipartite Model of 2-5A-dependent RNase L

The 2-5A-dependent RNase (RNase L) is a tightly regulated endoribonuclease of higher vertebrates that is catalytically active only after engaging unusual effector molecules consisting of the 2',5'-linked oligoadenylates, p1-3A(2'p5'A)>/=2 (2-5A). Progressive truncations from either terminus have provided insight into the structure, function, and regulation of RNase L. We determined that deletion of the N-terminal 335 amino acids of RNase L, about 45% of the enzyme, produced a constitutively active endoribonuclease, thus effectively eliminating the requirement for 2-5A. The truncated nuclease had 6-fold lower catalytic activity against an oligo(rU) substrate than wild type RNase L. However, the two enzymes showed identical RNA cleavage site preferences with an mRNA as substrate. The repressor function required only the last three of a series of nine ankyrin-like repeats present in the N-terminal part of RNase L. In contrast, the entire ankyrin repeat region was necessary and sufficient for 2-5A binding activity. Deletion of a 10-amino acid sequence near the C terminus of RNase L, between residues 710 and 720, eliminated both the catalytic and RNA substrate binding functions of the enzyme. The ability to bind native RNase L in response to 2-5A required amino acid sequences near both termini of the protein. A bipartite model for the structure of RNase L emerged in which the regulatory functions of the molecule are located in the N-terminal half, while the catalytic domain is present in the C-terminal half.

Matrix Effects on Spin−Lattice Relaxation in the Triplet State of Tryptophan

Spin−lattice relaxation (SLR) of the photoexcited triplet state of tryptophan both as the free amino acid (Trp) and also incorporated at a buried site of ribonuclease T1 from Aspergillus oryzae (RNase T1) is investigated in the ethylene glycol (EG)−water mixed solvent system. Global analysis of microwave-induced delayed phosphorescence (MIDP) transients measured at 1.2 K is employed to obtain the triplet state kinetic parameters over a range of solvent composition between 10 and 90 vol % EG. These parameters are used to calculate the phosphorescence decay profile of the triplet states that contribute to the MIDP. The experimental phosphorescence profile is fitted at each solvent composition as the sum of the calculated nonexponential decay and a simple exponential decay with lifetime equal to the average tryptophan lifetime. The phosphorescence is modeled to originate from two distinct populations characterized by either slow SLR or rapid SLR relative to the triplet state lifetime. Only the former contributes to the MIDP signals while the latter is responsible for the exponential decay component. The fraction of tryptophan undergoing slow SLR, Φs, is at a maximum (ca. 0.7) for EG in the range 30−40% (v/v) for both Trp and RNase T1, diminishes sharply with increasing water content, and diminishes slightly with increasing EG content. The SLR of this population is independent of solvent composition. Since the tryptophan triplet state is not kinetically homogeneous in EG−water, the method of microwave-saturated phosphorescence decay analysis produces erroneous kinetic parameters for this system. The native RNase T1 structure becomes unstable above 60 vol % EG as revealed by changes in phosphorescence and ODMR.

Pyridoxamine-amino acid chimeras in semisynthetic aminotransferase mimics.

The transaminase activity of two new semisynthetic RNase-S proteins incorporating a pyridoxamine moiety at the active site has been evaluated. A chemically competent derivative of pyridoxamine phosphate was incorporated into the C-peptide fragments of these non-covalent protein complexes in the form of an unnatural coenzyme-amino acid chimera, 'Pam'. The chimeric Pam residue integrates the heterocyclic functionality of pyridoxamine phosphate into the side chain of an alpha-amino acid and was introduced instead of Phe8 into the C-peptide sequence via standard solid phase methodology. The two semisynthetic Pam-RNase constructs were designed to probe whether the native ribonuclease catalytic machinery could be enlisted to modulate a pyridoxamine-dependent transamination reaction. Both RNase complexes, H1SP and S1SP, exhibited modest rate enhancements in the Cu(II)-assisted transamination of pyruvate to alanine under single turnover conditions, relative to 5'-deoxypyridoxamine and the uncomplexed C-peptide fragments. Furthermore, multiple turnovers of substrates were achieved in the presence of added L-phenylalanine due to recycling of the pyridoxamine moiety. The modest chiral inductions observed in the catalytic production of alanine and the differences in reactivity between the two proteins could be rationalized by the participation of a general base (His12) in complex H1SP, and by the increased tolerance for large amino acid substrates by complex S1SP, which contains serine at this position. The pyridoxamine-amino acid chimera will be useful in the future for examining the coenzyme structure/ function relationships in a native-like peptidyl architecture.

Role of histidine 46 in the hydrolysis and the reverse transphosphorylation reaction of RNase Rh from Rhizopus niveus.

In order to study the reaction mechanism of RNase Rh from Rhizopus niveus, the rates of cleavage of four 2',3'-cyclic nucleotides by mutant enzymes of RNase Rh, H46F, H109F, E105Q, and K108L were measured. H46F is virtually inactive towards cyclic nucleotides, but H109F hydrolyzed these substrates at 0.7-4.5% of the rates of the native RNase Rh. The other mutants hydrolyzed 2',3'-cyclic nucleotides at 15-20% of the rates of the native enzyme. Relative enzymatic activities towards four cyclic nucleotides of H109F in the hydrolysis reaction (2nd step) were much higher than in the transphosphorylation reaction (the 1st step). In the presence of a 13-fold excess of uridine, H109F catalyzed the transphosphorylation reaction of 2',3'-cyclic AMP (A>p) to ApU. However, this reaction was not catalyzed by H46F mutant or native RNase Rh. These results showed that His46 is crucial to the hydrolysis reaction, and to the reversed reaction of the transphosphorylation reaction. We suggest that His46 in RNase Rh plays a major role in these reactions by acting as a base catalyst to activate water and the 5'-hydroxyl group of nucleosides, respectively.

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.