On the role of reversible denaturation (unfolding) in the irreversible thermal inactivation of enzymes

The contribution of the reversible thermal unfolding of an enzyme toward the overall irreversible thermoinactivation process has been examined both theoretically and experimentally. Using bovine pancreatic ribonuclease as a model, we have studied the effect of such variables as pH and salts both on the equilibrium constant of reversible denaturation and on the rate constant of the overall irreversible process. It has been demonstrated that at temperatures where a significant fraction of the enzyme molecules are in the native conformation, there is a correlation between the enzyme thermostabilities with respect to the reversible and irreversible inactivations: greater stability against the former is accompanied by greater stability against the latter. On the other hand, at very high temperatures (where essentially all of the enzyme molecules are unfolded), such a correlation does not exist. These findings are considered in terms of a kinetic model for irreversible enzyme thermoinactivation, and the implications of the derived relationship are discussed.     

The thermal unfolding of ribonuclease A A 13C NMR study

The ¹³C nuclear magnetic resonance (NMR) spectra of ribonuclease A over the pH range 1–7 and between 6 and 70°C reveal many of the details of its reversible unfolding. Although the unfolding may loosely be described as ‘two-state’, evidence is presented for intermediate unfolding stages at least 10°C on either side of the main unfolding transition, particularly at low pH. The first residues to unfold are 17–24, in agreement with other results. The C-terminal region shows a steeper temperature dependence of its unfolding than does the main transition, which itself is shown to lead at all pH values to a semi-structured but internally flexible state which is far from being truly random-coil. This is confirmed by measurements of T₁ and of nuclear Overhauser enhancement. Indeed, even at pH 1.1 and 70°C there is evidence for considerable motional restriction of cysteine and proline residues, amongst others.The native protein has more variability of structure at low pH than at neutral pH, and also interchanges more rapidly with the semi-structured, denatured state.     

Pressure-induced unfolding/refolding of ribonuclease A: static and kinetic Fourier transform infrared spectroscopy study

In this paper, we illustrate the use of high-pressure Fourier transform infrared (FT-IR) spectroscopy to study the reversible presssure-induced unfolding and refolding of ribonuclease A (RNase A) and compare it with the results obtained for the temperature-induced transition. FT-IR spectroscopy monitors changes in the secondary structural properties (amide I' band) or tertiary contacts (tyrosine band) of the protein upon pressurization or depressurization. Analysis of the amide I' spectral components reveals that the pressure-induced denaturation process sets in at 5. 5 kbar at 20 degrees C and pH 2.5. It is accompanied by an increase in disordered structures while the content of beta-sheets and alpha-helices drastically decreases. The denatured state above 7 kbar retains nonetheless some degree of beta-like secondary structure and the molecule cannot be described as an extended random coil. Increase of pH from 2.5 to 5.5 has no influence on the structure of the pressure-denatured state; it slightly changes the stability of the protein only. All experimental evidence indicates that the pressure-denatured states of monomeric proteins have more secondary structure than the temperature-denatured states. Different modes of denaturation, including pressure, may correlate differently with the roughness of the energy scale and slope of the folding funnel. For these reasons we have also carried out pressure-jump kinetic studies of the secondary structural evolution in the unfolding/refolding reaction of RNase A. In agreement with the theoretical model presented by Hummer et al. [(1998) Proc. Natl. Acad. Sci. U.S.A. 95, 1552-1555], the experimental data show that pressure slows down folding and unfolding kinetics (here 1-2 orders of magnitude), corresponding to an increasingly rough landscape. The kinetics remains non-two-state under pressure. Assuming a two-step folding scenario, the calculated relaxation times for unfolding of RNase A at 20 degrees C and pH 2.5 can be estimated to be tau(1) approximately 0.7 min and tau(2) approximately 17 min. The refolding process is considerably faster (tau(1) approximately 0.3 min, tau(2) approximately 4 min). Our data show that the pressure stability and pressure-induced unfolding/refolding kinetics of monomeric proteins, such as wild-type staphylococcal nuclease (WT SNase) and RNase A, may be significantly different. The differences are largely due to the four disulfide bonds in RNase A, which stabilize adjacent structures. They probably lead to the much higher denaturation pressure compared to SNase, and this might also explain why the volume change of WT SNase upon unfolding is about twice as large.     

Nanosecond temperature jump and time-resolved Raman study of thermal unfolding of ribonuclease A

A nanosecond temperature jump (T-jump) apparatus was constructed and combined with time-resolved Raman measurements to investigate thermal unfolding of a protein for the first time. The 1.56-microm heat pulse with 9 ns width at 10 Hz was obtained through the two-step stimulated Raman scattering in D(2) gas involving seeding and amplification. To achieve uniform temperature rise, the counter-propagation geometry was adopted for the heat pulse. The temperature rise was determined by anti-Stokes to Stokes intensity ratios of the 317 and 897 cm(-1) bands of MoO(4)(2-) ions in an aqueous solution. The T-jump as large as 9 degrees C in 10 ns was attained. The unfolding of bovine pancreatic ribonuclease A was monitored with time-resolved Raman spectra excited at 532 nm. The C-S stretching band of Met residues exhibited 10% change of that expected from the stationary state temperature-difference spectra in the initial 200 ns following T-jump and another 10% in 5 ms. The Raman intensity of SO(4)(2-) ions around 980 cm(-1) increased at 100 micros, presumably due to some conformational changes of the protein around the active site. The S-S stretches and tyrosine doublet displayed little changes within 5 ms. Thus, the conformational changes in the initial step of unfolding are not always concerted.     

Laser Raman spectroscopic studies of the thermal unfolding of ribonuclease A.

The reversible thermal denaturation of bovine pancreatic ribonuclease A at pH 5 in 0.1 M NaCl over the range 32-70 degrees C as studied by Raman spectroscopy proceeds in a gradual manner consistent with a stepwise unfolding process rather than as a transition between two states. Conversion of residues from helical or pleated-sheet geometry to some intermediate geometry, as followed by means of the amide I and III lines, reveals that substantial amounts of the helical and pleated-sheet conformations remain at 70 degrees C. Changes in the strength of hydrogen bonding by the tyrosyl residues are indicated by the intensity ratio of the doublet at 830-850 cm(-1) and changes in the geometry of the disulfide bridges by the frequency and half-width of the Raman line near 510 cm(-1) due to the S-S vibration. Vibrations of C-S bonds in the methionines and cystines are used to monitor conformational changes in these residues. While there are small quantitative differences in temperature dependence among these probes, all agree in placing the malting temperature at or near 62 degrees C. The Raman data are quantitatively consistent with the six-stage scheme of unfolding of A.W. Burgess and H.A. Scheraga [(1975), J. Theor, Biol. 53, 403], except that no change in the environment of the tyrosines is seen until 45 degrees C.     

Zinc induces unfolding and aggregation of dimeric arginine kinase by trapping reversible unfolding intermediate

Arginine kinase plays an important role in the cellular energy metabolism of invertebrates. Dimeric arginine kinase (dAK) is unique in some marine invertebrates. The effects of Zn2(+) on the unfolding and aggregation of dAK from the sea cucumber Stichopus japonicus were investigated. Our results indicated that Zn2(+) caused dAK inactivation accompanied by conformational unfolding, the exposure of hydrophobic surface, and aggregation. Kinetic studies showed the inactivation and unfolding of dAK followed biphasic kinetic courses. Zn2(+) can affect unfolding and refolding of dAK by trapping the reversible intermediate. Our study provides important information regarding the effect of Zn2(+) on metabolic enzymes in marine invertebrates.     

The thermal unfolding of ribonuclease A. A 13C NMR study.

The 13C nuclear magnetic resonance (NMR) spectra of ribonuclease A over the pH range 1-7 and between 6 and 70 degrees C reveal many of the details of its reversible unfolding. Although the unfolding may loosely be described as 'two-state', evidence is presented for intermediate unfolding stages at least 10 degrees C on either side of the main unfolding transition, particularly at low pH. The first residues to unfold are 17-24, in agreement with other results. The C-terminal region shows a steeper temperature dependence of its unfolding than does the main transition, which itself is shown to lead at all pH values to a semi-structured but internally flexible state which is far from being truly random-coil. This is confirmed by measurements of T1 and of nuclear Overhauser enhancement. Indeed, even at pH 1.1 and 70 degrees C there is evidence for considerable motional restriction of cysteine and proline residues, amongst others. The native protein has more variability of structure at low pH than at neutral pH, and also interchanges more rapidly with the semi-structured, denatured state.     

Kinetic and thermodynamic analysis of thermal unfolding of recombinant erythropoietin

Thermal stress was used to assess the stability of recombinant human erythropoietin (EPO) derived from Chinese hamster ovary cells. In 20 mm phosphate at pH 7.0, this protein had a highly reversible thermal unfolding as observed by far UV circular dichroism (CD) and native gel analysis, with no indication of protein aggregation. It had a relatively low melting temperature at 53 degrees C. Assuming a two-state transition, the observed reversibility permits thermodynamic analysis of the unfolding of EPO, which shows that the free energy of unfolding at 25 degrees C is only 6-7 kcal/mol. Upon heating to 79 degrees C over 30 min, however, this protein does undergo aggregation as assessed by native gel. In 20 mm phosphate and citrate at pH 7.0, the results are similar, i.e., EPO suffered a substantial aggregation, while it showed little aggregation in 20 mm Tris or histidine at pH 7.0 and 20 mm glycine at pH 6.3 under identical heat treatment.     

Local and long-range interactions in the thermal unfolding transition of bovine pancreatic ribonuclease A

This research was undertaken to distinguish between local and global unfolding in the reversible thermal denaturation of bovine pancreatic ribonclease A (RNase A). Local unfolding was monitored by steady-state and time-resolved fluorescence of nine mutants in each of which a single tryptophan was substituted for a wild-type residue. Global unfolding was monitored by far-UV circular dichroism and UV absorbance. All the mutants (except F8W and D38W) exhibited high specific enzymatic activity, and their far-UV CD spectra were very close to that of wild-type RNase A, indicating that the tryptophan substitutions did not affect the structure of any of the mutants (excluding K1W and Y92W) under folding conditions at 20 degrees C. Like wild-type RNase A, the various mutants exhibited reversible cooperative thermal unfolding transitions at pH 5, with transition temperatures 2.5-11 degrees C lower than that of the wild-type transition, as detected by far-UV CD or UV absorbance. Even at 80 degrees C, well above the cooperative transition of all the RNase A mutants, a considerable amount of secondary and tertiary structure was maintained. These studies suggest the following two-stage mechanism for the thermal unfolding transition of RNase A as the temperature is increased. First, at temperatures lower than those of the main cooperative transition, long-range interactions within the major hydrophobic core are weakened, e.g., those involving residues Phe-8 (in the N-terminal helix) and Lys-104 and Tyr-115 (in the C-terminal beta-hairpin motif). The structure of the chain-reversal loop (residues 91-95) relaxes in the same temperature range. Second, the subsequent higher-temperature cooperative unfolding transition is associated with a loss of secondary structure and additional changes in the tertiary contacts of the major hydrophobic core, e.g., those involving residues Tyr-73, Tyr-76, and Asp-38 on the other side of the molecule. The hydrophobic interactions of the C-terminal loop of the protein are enhanced by high temperature, and perhaps are responsible for the preservation of the local structural environment of Trp-124 at temperatures slightly above the major cooperative transition. The results shed new light on the thermal unfolding transitions, generally supporting the thermal unfolding hypothesis of Burgess and Scheraga, as modified by Matheson and Scheraga.     

Equilibrium and kinetic studies on reversible and irreversible denaturation of micrococcal nuclease

The effect of pH and temperature on the thermal denaturation of micrococcal nuclease wer4e investigated. The ranges employed were between pH3.30 and pH9.70 and between 10 degrees C and 85 degrees C, respectively. The reversible denaturation involved in the whole process was clearly discriminated from the irreversible one. The former took place with a large enthalpy change of 384 kJ mol(-1) at pH 9.70, where the enzyme exhibited it s maximum activity. The latter probably led to aggregation because the successive long incubation after complete deactivation caused precipitation. A reasonable scheme explaining the process involving both denaturations was proposed and the kinetic on the irreversible deactivation was performed. It was revealed that the irreversible deactivation involved two types of reactions whose activation energies were relatively small: 22.2 kJ mol(-1) and 18.8kJ mol(-1). The presence of sucrose suppressed the reversible denaturation without significant influence on enthalpy change, whereas it affected little the irreversible deactivation kinetically. The effects of pH change and addition of sucrose on the denaturation were discussed thermodynamically, especially in terms of the entropy change. (c) 1994 John Wiley & Sons, Inc.     

A kinetic molecular model of the reversible unfolding and refolding of titin under force extension.

Molecular elasticity is a physicomechanical property that is associated with a select number of polypeptides and proteins, such as the giant muscle protein, titin, and the extracellular matrix protein, tenascin. Both proteins have been the subject of atomic force microscopy (AFM), laser tweezer, and other in vitro methods for examining the effects of force extension on the globular (FNIII/Ig-like) domains that comprise each protein. In this report we present a time-dependent method for simulating AFM force extension and its effect on FNIII/Ig domain unfolding and refolding. This method treats the unfolding and refolding process as a standard three-state protein folding model (U right arrow over left arrow T right arrow over left arrow F, where U is the unfolded state, T is the transition or intermediate state, and F is the fully folded state), and integrates this approach within the wormlike chain (WLC) concept. We simulated the effect of AFM tip extension on a hypothetical titin molecule comprised of 30 globular domains (Ig or FNIII) and 25% Pro-Glu-Val-Lys (PEVK) content, and analyzed the unfolding and refolding processes as a function of AFM tip extension, extension rate, and variation in PEVK content. In general, we find that the use of a three-state protein-folding kinetic-based model and the implicit inclusion of PEVK domains can accurately reproduce the experimental force-extension curves observed for both titin and tenascin proteins. Furthermore, our simulation data indicate that PEVK domains exhibit extensibility behavior, assist in the unfolding and refolding of FNIII/Ig domains in the titin molecule, and act as a force "buffer" for the FNIII/Ig domains, particularly at low and moderate extension forces.     

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

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

A measure of conformational entropy change during thermal protein unfolding using neutron spectroscopy

Thermal unfolding of proteins at high temperatures is caused by a strong increase of the entropy change which lowers Gibbs free energy change of the unfolding transition (DeltaG(unf) = DeltaH - TDeltaS). The main contributions to entropy are the conformational entropy of the polypeptide chain itself and ordering of water molecules around hydrophobic side chains of the protein. To elucidate the role of conformational entropy upon thermal unfolding in more detail, conformational dynamics in the time regime of picoseconds was investigated with neutron spectroscopy. Confined internal structural fluctuations were analyzed for alpha-amylase in the folded and the unfolded state as a function of temperature. A strong difference in structural fluctuations between the folded and the unfolded state was observed at 30 degrees C, which increased even more with rising temperatures. A simple analytical model was used to quantify the differences of the conformational space explored by the observed protein dynamics for the folded and unfolded state. Conformational entropy changes, calculated on the basis of the applied model, show a significant increase upon heating. In contrast to indirect estimates, which proposed a temperature independent conformational entropy change, the measurements presented here, demonstrated that the conformational entropy change increases with rising temperature and therefore contributes to thermal unfolding.     

Molecular, kinetic and thermodynamic characterization of Mycobacterium tuberculosis orotate phosphoribosyltransferase

Tuberculosis (TB) is a chronic infectious disease caused mainly by Mycobacterium tuberculosis. The worldwide emergence of drug-resistant strains, the increasing number of infected patients among immune compromised populations, and the large number of latent infected individuals that are reservoir to the disease have underscored the urgent need of new strategies to treat TB. The nucleotide metabolism pathways provide promising molecular targets for the development of novel drugs against active TB and may, hopefully, also be effective against latent forms of the pathogen. The orotate phosphoribosyltransferase (OPRT) enzyme of the de novo pyrimidine synthesis pathway catalyzes the reversible phosphoribosyl transfer from 5'-phospho-α-D-ribose 1'-diphosphate (PRPP) to orotic acid (OA), forming pyrophosphate and orotidine 5'-monophosphate (OMP). Here we describe cloning and characterization of pyrE-encoded protein of M. tuberculosis H37Rv strain as a homodimeric functional OPRT enzyme. The M. tuberculosis OPRT true kinetic constants for forward reaction and product inhibition results suggest a Mono-Iso Ordered Bi-Bi kinetic mechanism, which has not been previously described for this enzyme family. Absence of detection of half reaction and isothermal titration calorimetry (ITC) data support the proposed mechanism. ITC data also provided thermodynamic signatures of non-covalent interactions between substrate/product and M. tuberculosis OPRT. These data provide a solid foundation on which to base target-based rational design of anti-TB agents and should inform us how to better design inhibitors of M. tuberculosis OPRT.     

Methanol-stabilized intermediates in the thermal unfolding of ribonuclease A: Characterization by 1H nuclear magnetic resonance

The thermal unfolding of ribonuclease A has been studied in solutions of 25, 35 and 50% methanol ($\text{v}\text{v}$), using 360 MHz proton magnetic resonance spectroscopy. Several observations indicate that the native structure of the protein in methanol cryosolvents is very similar to that in aqueous solution. A detailed analysis of the unfolding process has been made using the C-2 protons of the imidazole side-chains of the four histidine residues. As denaturation proceeds new resonances appear, whose chemical shifts correspond to neither native nor fully unfolded species. These have been assigned to particular His residues by selective deuteration studies. The thermal denaturation transitions reveal a multiphasic process in each of the solvents, and become less co-operative with increasing concentrations of methanol. The denaturation is fully reversible with no evidence of hysteresis.The new resonances that appear during the unfolding process are attributed to partially folded species, which are stabilized by the presence of the relatively hydrophobic methanol. Based on the temperature dependence of the chemical shifts and the relative areas of the various resonances, a detailed sequence of events has been proposed to describe the unfolding process. Key features include the initial general loosening of the two domains, the subsequent movement of the upper S-peptide region (residues 13 to 25) away from the main body of the protein, followed by partial separation of the sheet structure and full exposure of the N-terminal helix, leading to complete separation of the “winged domains”, and ultimately the loss of the residual sheet and helix structure.     

Steady-state kinetic study of action of ribonuclease A, involving a conformational change between 30 and 40 degrees C

The steady-state kinetics of the reaction of ribonuclease A with cyclic cytidine 2',3'-phosphate as substrate are investigated as a function of temperature at pH 5 and ionic strength 0.1 M. The results suggest, but cannot prove, that a conformational change near 32 degrees C is involved in the rate-limiting step of the reaction mechanism. This conformational change is proposed to be the same one that was observed in studies of the free enzyme and of enzyme-inhibitor complexes near the same temperature.     

Purification, kinetic and thermodynamic studies of a new ribonuclease from a mutant of Aspergillus niger

Ribonuclease was purified from Aspergillus niger SA-13-20 to homogeneity level by using (NH(4))(2)SO(4) precipitation, DEAE-cellulose anion-exchange chromatography, ultrafiltration and Sephacryl HR-200 chromatography. The molecular weight and isoelectric point of the enzyme was 40.1kDa and 5.3, respectively. The pH- and temperature-dependent kinetic parameters were determined. The RNase showed the strongest affinity with RNA as the substrate, and the highest catalytic efficiency for hydrolysis of the substrate at pH 3.5 and 65 degrees C. It exhibited Michaelis-Menten Kinetics with k(cat) of 118.1s(-1) and K(m) of 57.0 microg ml(-1), respectively. Thermodynamic parameters for catalysis and thermal denaturation were also determined. Activation energy (E(a)) for catalysis of A. niger SA-13-20 RNase was 50.31 kJ mol(-1) and free energy (DeltaG(#)), enthalpy (DeltaH(#)) and entropy (DeltaS(#)) of activation for catalysis of the enzyme at 65 degrees C were 69.76, 47.50 and -65.83 Jmol(-1)K(-1), respectively. Activation energy (E(a,d)) for denaturation of the enzyme was 200.53 kJ mol(-1) and free energy (DeltaG(d)(#)), enthalpy (DeltaH(d)(#)) and entropy (DeltaS(d)(#)) of activation for denaturation of the enzyme at 45 degrees C were 79.18 kJ mol(-1), 197.88 and 373.09 Jmol(-1)K(-1), respectively.