Hydrophobic effect on the stability and folding of a hyperthermophilic protein

Ribonuclease HII from hyperthermophile Thermococcus kodakaraensis (Tk-RNase HII) is a kinetically robust monomeric protein. The conformational stability and folding kinetics of Tk-RNase HII were measured for nine mutant proteins in which a buried larger hydrophobic side chain is replaced by a smaller one (Leu/Ile to Ala). The mutant proteins were destabilized by 8.9 to 22.0 kJ mol^− 1 as compared with the wild-type protein. The removal of each -CH₂- group burial decreased the stability by 5.1 kJ mol^− 1 on average in the mutant proteins of Tk-RNase HII examined. This is comparable with the value of 5.3 kJ mol^− 1 obtained from experiments for proteins from organisms growing at moderate temperature. We conclude that the hydrophobic residues buried inside protein molecules contribute to the stabilization of hyperthermophilic proteins to a similar extent as proteins at normal temperature. In the folding experiments, the mutant proteins of Tk-RNase HII examined exhibited faster unfolding compared with the wild-type protein. These results indicate that the buried hydrophobic residues strongly contribute to the kinetic robustness of Tk-RNase HII. This is the first report that provides a practical cause of slow unfolding of hyperthermostable proteins.     

The effect of the osmolyte trimethylamine N-oxide on the stability of the prion protein at low pH

A study of the effect of trimethylamine N-oxide on the stability of two recombinant forms of the prion protein PrP, an ovine full-length and a human truncated form, is here reported. Both thermal denaturation and denaturation at room temperature were analyzed at pH values above and below the pKa of trimethylamine N-oxide, which is close to 4.7. Surprisingly, results showed that not only is trimethylamine N-oxide able to decrease PrP thermal stability at low pH but it also acts as a strong denaturant at room temperature. Likely, this destabilization is due to the capability of the cationic form of trimethylamine N-oxide to interact with the protein backbone as well as to weaken electrostatic interactions which are important for PrP fold. These results constitute the first experimental measurement of the effect of trimethylamine N-oxide on PrP stability and provide an unambiguous proof of the destabilizing effect of this osmolyte on PrP at low pH.     

Circular dichroism study on the early folding events of beta-lactoglobulin entrapped in wet silica gels

beta-Lactoglobulin is a predominantly beta-sheet protein that folds by forming excess alpha-helices within milliseconds. In this study, the refolding of beta-lactoglobulin was dramatically decelerated by entrapping in wet nanoporous silica gel matrices, and monitored on a time scale of minutes or hours by far-UV circular dichroism spectroscopy. Analysis of kinetics and transient spectra allowed to define the sequence of folding events that consist of alpha-helical formation, beta-sheet core formation, and alpha-to-beta transition. The results suggest that the initially formed alpha-helices, presumably including the native alpha-helix, help to guide the formation of the adjacent beta-sheet core.     

Secondary structure in ribonuclease. II. Relations between folding kinetics and secondary structure elements

The kinetics of regain of 2′-CMP binding are monitored during renaturation of RNAase S. Experiments were performed by mixing equimolar amounts of S-peptide with S-protein. The S-protein fragment was incubated initially (i.e. before mixing with S-peptide) at pH 6.2 or 1.7 and various guanidine hydrochloride (GuHCl) concentrations. Three well-resolved phases are observed. The fastest phase is second-order. The reciprocal half-time increases linearly with fragment concentration and is independent of initial conditions for the S-protein fragment. An apparent on rate of k_on = 2 × 10⁵m^?1s^?1 is measured in 0.5 m-GuHCl (pH 6.2) and 20 ° C. Identical association kinetics are observed by changes in tyrosine absorbance. The fraction of native RNAase S formed in this second-order reaction strictly equals the fraction of S-protein molecules with intact β-sheet in initial conditions. The relation holds for different pH values, GuHCl concentrations and temperatures. The fraction of apparent helical content of S-protein in initial conditions may also vary but this is not reflected by the association reaction. We interpret this to mean that the β-sheet but not the α-helices must be preformed in initial conditions in order to generate the high-affinity peptide binding site of S-protein. Furthermore, it is concluded that the S-protein moiety β-sheet forms or unfolds in a single one-step reaction. 2′-CMP binding reports, additionally, two slower phases of renaturation. These are produced by S-protein molecules that have their β-sheet unfolded in initial conditions. It is observed that a unique dependence of these two folding rates exists for RNAase A, RNAase S and S-protein as function of t_m, the temperature of half-completion of thermal denaturation as measured by unfolding of the β-sheet in the respective compound in final conditions. The t_m value varies with changing pH, with GuHCl concentration and (for RNAase S) with changing fragment concentration. The findings are interpreted to argue in favor of a sequential mechanism of folding, where the stability of a structural precursor determines the rate of folding.     

Salt-induced folding of a rabbit muscle pyruvate kinase intermediate at alkaline pH

The effect of alkaline denaturation on the structural and functional characteristics of rabbit muscle pyruvate kinase (PK) was investigated using enzymatic activity measurements and a combination of optical methods such as circular dichroism, fluorescence, and ANS binding. At a critical pH, 10.5, PK exists in an intermediate state (alkaline unfolded state) with predominant secondary structure along with some of the tertiary interactions and a strong binding to the hydrophobic dye ANS. This intermediate retains the enzymatic activity and corresponds to a dimeric state of the molecule. Above pH 10.5, a sudden fall in the spectral properties and enzymatic activity occurs suggesting the dissociation of the molecule followed by unfolding at very high pH. Addition of salts such as NaCl, KCl, and Na₂SO₄ to the alkali-induced state induces both secondary and tertiary structure to a level equivalent to that of native tetramer (salt-induced state). Chemical- and temperature-induced unfolding of the alkali-induced state as well as the salt-induced refolded state of PK reveal the presence of intermediate conformations in the unfolding pathway. The unfolding transition curves are noncoinciding and noncooperative along with ANS binding at intermediate concentrations of denaturants during unfolding. The observations presented in this paper suggest that the native pyruvate kinase tetramer dissociates to an active dimer around pH 10.5 and further to inactive monomer before attaining a completely unfolded monomeric conformation.     

Comparison of the stability of fish light meromyosins by guanidine hydrochloride denaturation

The guanidine hydrochloride denaturation of light meromyosins (LMMs) of fish (carp, sardine and greenling) and rabbit was investigated to determine their structural stability quantitatively. The circular dichroism (CD) and fluorescence spectroscopies were applied to monitor denaturation. The CD results indicate that the LMM α-helix undergoes a two-step unfolding. The free energy of denaturation was calculated based on the linear extrapolation method and the denaturant binding model. Total free energies of the two-step unfolding of the α-helix are related to the water temperatures in which the fish live and the body temperature of rabbit. The stability of α-helical structure of LMM was in the following descending order: rabbit>carp>sardine>greenling. The free energies of denaturation obtained by tryptophan fluorescence differ from the free energies of the unfolding α-helix. The data from the two spectroscopic measurements are discussed along with the conformational changes of LMMs.     

Metal ion substitution in phospholipase C (Bacillus cereus) and its effect on enzyme stability

The effect of guanidinium chloride solutions on the circular dichroism of native (ZnZn-) and apophospholipase C (Bacillus cereus) indicated marked protein unfolding at denaturant concentrations of 1.4–1.8 M and 0.1–0.6 M, respectively. With the apoenzyme near u.V. region circular dichroism bands remained even after all ordered structure appeared to have been lost. Apophospholipase C bound two equivalents of Ni²⁺, Cd²⁺, Co²⁺, Mn², Pb²⁺ or Cu²⁻, with only the latter metal causing marked changes either in circular dichroism or protein fluorescence relative to the native enzyme. Stability in guanidinium chloride for the metalloforms of phospholipase C decreased in the order: ZnZn->ZnCo->NiNi->CoCo->PbPb->CdCd->MnMn-apoenzyme.     

Effect of pH and denaturants on the folding and stability of murine interleukin-6.

The conformation and stability of a recombinant mouse interleukin-6 (mIL-6) has been investigated by analytical ultracentrifugation, fluorescence spectroscopy, urea-gradient gel electrophoresis, and near- and far-ultraviolet circular dichroism. On decreasing the pH from 8.0 to 4.0, the tryptophan fluorescence of mIL-6 was quenched 40%, the midpoint of the transition occurring at pH 6.9. The change in fluorescence quantum yield was not due to unfolding of the molecule because the conformation of mIL-6, as judged by both urea-gradient gel electrophoresis and CD spectroscopy, was stable over the pH range 2.0-10.0. Sedimentation equilibrium experiments indicated that mIL-6 was monomeric, with a molecular mass of 22,500 Da over the pH range used in these physicochemical studies. Quenching of tryptophan fluorescence (20%) also occurred in the presence of 6 M guanidine hydrochloride upon going from pH 7.4 to 4.0 suggesting that an amino acid residue vicinal in the primary structure to one or both of the two tryptophan residues, Trp-36 and Trp-160, may be partially involved in the quenching of endogenous fluorescence. In this regard, similar results were obtained for a 17-residue synthetic peptide, peptide H1, which corresponds to an N-terminal region of mIL-6 (residues Val-27-Lys-43). The pH-dependent acid quenching of endogenous tryptophan fluorescence of peptide H1 was 30% in the random coil conformation and 60% in the presence of alpha-helix-promoting solvents. Replacement of His-33 with Ala-33 in peptide H1 alleviated a significant portion of the pH-dependent quenching of fluorescence suggesting that the interaction of the imidazole ring of His-33 with the indole ring of Trp-36 is a major determinant responsible for the quenching of the endogenous protein fluorescence of mIL-6.     

Thermodynamics of melittin tetramerization determined by circular dichroism and implications for protein folding.

The tetramerization of melittin, a 26-amino acid peptide from Apis mellifera bee venom, has been studied as a model for protein folding. Melittin converts from a monomeric random coil to an alpha-helical tetramer as the pH is raised from 4.0 to 9.5, as ionic strength is increased, as temperature is raised or lowered from about 37 degrees C, or as phosphate is added. The thermodynamics of this tetramerization (termed "folding") are explored using circular dichroism. The melittin tetramer has two pKa values of 7.5 and 8.5 corresponding to protonation of the N-terminus and Lys 23, respectively. pKa values calculated with the program DelPhi (Gilson, M.K., Sharp, K.A., & Honig, B.H., 1987, J. Comp. Chem. 9, 327-335; Gilson, M.K. & Honig, B.H., 1988a, Proteins 3, 32-52; Gilson, M.K. & Honig, B.H., 1988b, Proteins 4, 7-18) agree with experimental titration data. Greater electrostatic repulsion of these protonated groups destabilizes the tetramer by 3.6 kcal/mol at pH 4.0 compared to pH 9.5. Increasing the concentration of NaCl in the solution from 0 to 0.5 M stabilizes the tetramer by 5-6 kcal/mol at pH 4.0. The effect of NaCl is modeled with a ligand-binding approach. The melittin tetramer is found to have a temperature of maximum stability ranging from 35.5 to 43 degrees C depending on the pH, unfolding above and below that temperature. delta Cp0 for folding ranges from -0.085 to -0.102 cal g-1 K-1, comparable to that of other small globular proteins (Privalov, P.L., 1979, Adv. Protein Chem. 33, 167-241). delta H0 and delta S0 are found to decrease with temperature, presumably due to the hydrophobic effect (Kauzmann, W., 1959, Adv. Protein Chem. 14, 1-63). Phosphate is found to perturb the equilibrium substantially with a maximal effect at 150 mM, stabilizing the tetramer at pH 7.4 and 25 degrees C by 4.6 kcal/mol. The enthalpy change due to addition of phosphate (-7.5 kcal/mol at 25 degrees C) can be accounted for by simple dielectric screening. Both circular dichroism and crystallographic results suggest that phosphate may bind Lys 23 at the ends of the elongated tetramer. These detailed measurements give insight into the relative importance of various forces for the stability of melittin in the folded form and may provide an experimental standard for future tests of computational energetics on this simple protein system.     

The effect of additional disulfide bonds on the stability and folding of ribonuclease A

The significant contribution of disulfide bonds to the conformational stability of proteins is generally considered to result from an entropic destabilization of the unfolded state causing a faster escape of the molecules to the native state. However, the introduction of extra disulfide bonds into proteins as a general approach to protein stabilization yields rather inconsistent results. By modeling studies, we selected positions to introduce additional disulfide bonds into ribonuclease A at regions that had proven to be crucial for the initiation of the folding or unfolding process, respectively. However, only two out of the six variants proved to be more stable than unmodified ribonuclease A. The comparison of the thermodynamic and kinetic data disclosed a more pronounced effect on the unfolding reaction for all variants regardless of the position of the extra disulfide bond. Native-state proteolysis indicated a perturbation of the native state of the destabilized variants that obviously counterbalances the stability gain by the extra disulfide bond.     

The failure of simple empirical relationships to predict the viscosity of mixed aqueous solutions of guanidine hydrochloride and glucose has important implications for the study of protein folding

Viscosities of aqueous solutions of guanidine hydrochloride (GuHCl) were measured in the presence of varying amounts of glucose. At high concentrations of glucose or GuHCl, the measured viscosities showed significant deviation from the values computed using a method proposed by Tanford (1966, J Biol Chem 241:3228-3232). This method was originally derived to allow the calculation of the effects of buffer or low concentrations of salts and other additives on the density and viscosity of aqueous solutions of urea or GuHCl. Recently it has been used to estimate the viscosity of denaturant solutions that contain high concentrations of viscogens. Our results show that the extrapolation of this approach to solutions of highly concentrated viscous co-solutes leads to significant errors. The implications for experimental studies of the viscosity dependence of conformational transitions in proteins is discussed.     

Antigen Retrieval Causes Protein Unfolding: Evidence for a Linear Epitope Model of Recovered Immunoreactivity

Antigen retrieval (AR), in which formalin-fixed paraffin-embedded tissue sections are briefly heated in buffers at high temperature, often greatly improves immunohistochemical staining. An important unresolved question regarding AR is how formalin treatment affects the conformation of protein epitopes and how heating unmasks these epitopes for subsequent antibody binding. The objective of the current study was to use model proteins to determine the effect of formalin treatment on protein conformation and thermal stability in relation to the mechanism of AR. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to identify the presence of protein formaldehyde cross-links, and circular dichroism spectropolarimetry was used to determine the effect of formalin treatment and high-temperature incubation on the secondary and tertiary structure of the model proteins. Results revealed that for some proteins, formalin treatment left the native protein conformation unaltered, whereas for others, formalin denatured tertiary structure, yielding a molten globule protein. In either case, heating to temperatures used in AR methods led to irreversible protein unfolding, which supports a linear epitope model of recovered protein immunoreactivity. Consequently, the core mechanism of AR likely centers on the restoration of normal protein chemical composition coupled with improved accessibility to linear epitopes through protein unfolding.     

Investigation of the folding pathway of the TEM-1 β-lactamase

The TEM-1 β-lactamase is a globular protein containing 12 proline residues. The folding mechanism of this enzyme was investigated by kinetic and equilibrium experiments with the help of fluorescence spectroscopy and circular dichroism. The equilibrium denaturation of the protein induced by guanidine hydrochloride occurs in two discrete steps, indicating the existence of a thermodynamically stable intermediate state. Thisstate is 5.2 ± 0.4 kcal/mol less stable than the native conformation and 5.7 ± 0.2 kcal/mol more stable than the fully denaturedprotein. This intermediate state exhibits a high content of native secondary structure elements but is devoid of specific tertiary organization; its relation to the “molten globule” is discussed. Refolding kinetic experimentsrevealed the existence of a transient intermediate conformation between thethermodynamically stable intermediate and the native protein. This transient intermediate appears rapidly during the folding reaction. It exhibits a secondary structure content very similar to that of the native protein and has also recovered a significant amount of tertiary organisation. The final refolding step of the TEM-1 β-lactamase, leading to the native enzyme, is dominated by two major slow kinetic phases which probablyreflect a very complex process kinetically limited by proline cis/transisomerization. © 1995 Wiley-Liss, Inc.     

Transient intermediary states with high and low folding probabilities in the apparent two-state folding equilibrium of ACBP at low pH

Measurements of the stability as a function of pH for the acyl-coenzyme A binding protein (ACBP) has shown a significant difference in the pH transition midpoint measured by NMR spectroscopy at pH 3.12 and the transition midpoint measured at pH 2.92 and 2.97 by circular dichroism and by fluorescence spectroscopy, respectively. A similar behavior has not been observed in other proteins. It is suggested that these differences arise because the population of the unfolded molecules still contains significant amounts of native like secondary and tertiary structure. NMR spectroscopy measures the concentration of the two components of the folding unfolding equilibrium individually, whereas circular dichroism and fluorescence measure the concentration of the conformations of the light-absorbing chromophores present in both the folded and the unfolded molecules. In the narrow pH range, nascent structure can be detected as the average amount of secondary structure per unfolded molecule and hydrophobic interactions in the population of unfolded molecules. These structures are not observable immediately by NMR spectroscopy; however, a chemical shift analysis of the peptide backbone (13)C chemical shift indicates strongly the existence of short-lived and transient helical structures at pH 2.3. Magnetization transfer studies have been applied to study the equilibrium between folded and unfolded ACBP near the pH transition point measured by NMR. This study has shown that there are two categories of subpopulations in the population of unfolded ACBP. One for which magnetization can be transferred to the folded form during the folding process, and one for which transfer is not observed. The molecules of the latter population of unfolded protein apparently, do not fold within the time-frame of the magnetization transfer experiment. This result suggests the existence of a subpopulation of the acid-unfolded protein molecules with a high propensity for folding. It is suggested that in this subpopulation, a particular set of native like interactions in the peptide backbone and between side-chains in the peptide chain have to be formed.     

Linear correlation between thermal stability and folding kinetics of lysozyme

We have studied the refolding and thermal denaturation of hen egg white lysozyme in a wide range of pH values (from 1.5 to 9.4) using stopped-flow circular dichroism (CD) and differential scanning calorimetry (DSC). A linear correlation was found between the thermal denaturation temperature (T(m)) and the logarithm of the refolding rate of the slow folding phase of hen egg white lysozyme (lnk(2)).     

Sequential conformation change and activation of chicken liver dihydrofolate reductase in low concentration of guanidine hydrochloride

The conformation changes of dihydrofolate reductase (DHFR) from chicken liver in guanidine hy-drochloride were monitored by protein intrinsic fluorescence, hydrophobic fluorescence probe TNS and limited proteol-ysis by proteinase K. The kinetics of the enzyme denaturation were also studied and compared with its activity changes. It was indicated by the enhanced fluorescence of 2-p-toluidinylnaphthalene (TNS) that a subtle conforma-tional change of the enzyme in dilute GuHCl parallels GuHCl-induced activation. At GuHCl concentration higher than 0.75 mol/L, the conformational change can be detected by increased susceptibility of the enzyme to proteinase K, but no significant gross conformational change of the enzyme molecule is observed by intrinsic fluorescence up to a GuHCl concentration of 1.2 mol/L. The results suggest that the denaturation of DHFR by GuHCl does not follow strictly the two-state model. The enzyme seems to open up sequentially with increasing concentrations of denaturants, mainly at th     

Dodine as a transparent protein denaturant for circular dichroism and infrared studies

The fungicide dodine combines the cooperative denaturation properties of guanidine with the mM denaturation activity of SDS. It was previously tested only on two small model proteins. Here we show that it can be used as a chemical denaturant for phosphoglycerate kinase (PGK), a much larger two‐domain enzyme. In addition to its properties as a chemical denaturant, dodine facilitates thermal denaturation of PGK, and we show for the first time that it also facilitates pressure denaturation of a protein. Much higher quality circular dichroism and amide I′ infrared spectra of PGK can be obtained in dodine than in guanidine, opening the possibility for use of dodine as a denaturant when UV or IR detection is desirable. One caution is that dodine denaturation, like other detergent‐based denaturants, is less reversible than guanidine denaturation.     

Equilibrium heat-induced denaturation of chitinase 40 from Streptomyces thermoviolaceus

High-precision differential scanning calorimetry (DSC) and circular dichroism (CD) have been employed to study the thermal unfolding of chitinase 40 (Chi40) from Streptomyces thermoviolaceus. Chi40 belongs to family 18 of glycosyl hydrolase superfamily bearing a catalytic domain with a "TIM barrel"-like fold, which exhibits deviations from the (beta/alpha)8 fold. The thermal unfolding is reversible at pH = 8.0 and 9.0. The denatured state is characterized by extensive structural changes with respect to the native. The process is characterized by slow relaxation kinetics. Even slower refolding rates are recorded upon cooling. It is shown that the denaturation calorimetric data obtained at slow heating rate (0.17 K/min) are in excellent agreement with equilibrium data obtained by extrapolation of the experimental results to zero scanning rate. Analysis of the DSC results reveals that the experimental data can be successfully fitted using either a non-two-state sequential model involving one equilibrium intermediate, or an independent transitions model involving the unfolding of two Chi40 energetic domains to intermediate states. The stability of the native state with respect to the final denatured state is estimated, deltaG = 24.0 kcal/mol at 25 degrees C. The thermal results are in agreement with previous findings from chemical denaturation studies of a wide variety of (beta/alpha)8 barrel proteins, that their unfolding is a non-two-state process, always involving at least one unfolding intermediate.     

RNA-dependent folding and stabilization of C5 protein during assembly of the E. coli RNase P holoenzyme

The pre-tRNA processing enzyme ribonuclease P is a ribonucleoprotein. In Escherichia coli assembly of the holoenzyme involves binding of the small (119 amino acid residue) C5 protein to the much larger (377 nucleotide) P RNA subunit. The RNA subunit makes the majority of contacts to the pre-tRNA substrate and contains the active site; however, binding of C5 stabilizes P RNA folding and contributes to high affinity substrate binding. Here, we show that RNase P ribonucleoprotein assembly also influences the folding of C5 protein. Thermal melting studies demonstrate that the free protein population is a mixture of folded and unfolded conformations under conditions where it assembles quantitatively with the RNA subunit. Changes in the intrinsic fluorescence of a unique tryptophan residue located in the folded core of C5 provide further evidence for an RNA-dependent conformational change during RNase P assembly. Comparisons of the CD spectra of the free RNA and protein subunits with that of the holoenzyme provide evidence for changes in P RNA structure in the presence of C5 as indicated by previous studies. Importantly, monitoring the temperature dependence of the CD signal in regions of the holoenzyme spectra that are dominated by protein or RNA structure permitted analysis of the thermal melting of the individual subunits within the ribonucleoprotein. These analyses reveal a significantly higher Tm for C5 when bound to P RNA and show that unfolding of the protein and RNA are coupled. These data provide evidence for a general mechanism in which the favorable free energy for formation of the RNA-protein complex offsets the unfavorable free energy of structural rearrangements in the RNA and protein subunits.