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.     

Recombination of S-peptide with S-protein during folding of ribonuclease S. II. Kinetic characterization of a stable folding intermediate shown by S-protein at pH 1.7.

At pH 1.7 S-peptide dissociates from S-protein but S-protein remains partly folded below 30 °C. A folded form of S-protein, labeled I₃, is detected and measured by its ability to combine rapidly with S-peptide at pH 6.8 and then to form native ribonuclease S. The second-order combination reaction (k = 0.7 × 10⁶m^?1s^?1 at 20 °C) can be monitored either by tyrosine absorbance or fluorescence emission; the subsequent first-order folding reaction (half-time, 68 ms; 20 °C) is monitored by 2′CMP ² binding. Combination with S-peptide and folding to form native RNAase S is considerably slower for both classes of unfolded S-protein (see preceding paper).I₃ shows a thermal folding transition at pH 1.7: it is completely unfolded above 32 °C and reaches a limiting low-temperature value of 65% below 10 °C. The 35% S-protein remaining at 10 °C is unfolded as judged by its refolding behavior in forming native RNAase S at pH 6.8. The folding transition of S-protein at pH 1.7 is a broad, multi-state transition. This is shown both by the large fraction of unfolded S-protein remaining at low temperatures and by the large differences between the folding transition curves monitored by I₃ and by tyrosine absorbance.The fact that S-protein remains partly folded after dissociation of S-peptide at pH 1.7 but not at pH 6.8 may be explained by two earlier observations. (1) Native RNAase A is stable in the temperature range of the S-protein folding transition at pH 1.7, and (2) the binding constant of S-protein for S-peptide falls steadily as the pH is lowered, by more than four orders of magnitude between pH 8.3 and pH 2.7, at 0 °C. The following explanation is suggested for why folding intermediates are observed easily in the transition of S-protein but not of RNAase A. The S-protein transition is shifted to lower temperatures, where folding intermediates should be more stable: consequently, intermediates in the folding of RNAase A which do not involve the S-peptide moiety and which are populated to almost detectable levels can be observed at the lower temperatures of the S-protein transition.     

Recombination of S-peptide with S-protein during folding of ribonuclease S. I. Folding pathways of the slow-folding and fast-folding classes of unfolded S-protein.

The refolding kinetics of ribonuclease S have been measured by tyrosine absorbance, by tyrosine fluorescence emission, and by rapid binding of the specific inhibitor 2′CMP ² to folded RNAase S. The S-protein is first unfolded at pH 1.7 and then either mixed with S-peptide as refolding is initiated by a stopped-flow pH jump to pH 6.8, or the same results are obtained if S-protein and S-peptide are present together before refolding is initiated. The refolding kinetics of RNAase S have been measured as a function of temperature (10 to 40 °C) and of protein concentration (10 to 120 μm). The results are compared to the folding kinetics of S-protein alone and to earlier studies of RNAase A. A thermal folding transition of S-protein has been found below 30 °C at pH 1.7; its effects on the refolding kinetics are described in the following paper (Labhardt &; Baldwin, 1979).In this paper we characterize the refolding kinetics of unfolded S-protein, as it is found above 30 °C at pH 1.7, together with the kinetics of combination between S-peptide and S-protein during folding at pH 6.8. Two classes of unfolded S-protein molecules are found, fast-folding and slow-folding molecules, in a 20: 80 ratio. This is the same result as that found earlier for RNAase A; it is expected if the slow-folding molecules are produced by the slow cis-trans isomerization of proline residues after unfolding, since S-protein contains all four proline residues of RNAase A.The refolding kinetics of the fast-folding molecules show clearly that combination between S-peptide and S-protein occurs before folding of S-protein is complete. If combination occurred only after complete folding, then the kinetics of formation of RNAase S should be rather slow (5 s and 100 s at 30 °C) and nearly independent of protein concentration, as shown by separate measurements of the folding kinetics of S-protein, and of the combination between S-peptide and folded S-protein. The observed folding kinetics are faster than predicted by this model and also the folding rate increases strongly with protein concentration (apparent 1.6 order kinetics). The fact that RNAase S is formed more rapidly than S-protein alone is sufficient by itself to show that combination with S-peptide precedes complete folding of S-protein. Computer simulation of a simple, parallel-pathway scheme is able to reproduce the folding kinetics of the fast-folding molecules. All three probes give the same folding kinetics.These results exclude the model for protein folding in which the rate-limiting step is an initial diffusion of the polypeptide chain into a restricted range of three-dimensional configurations (“nueleation”) followed by rapid folding (“propagation”). If this model were valid, one would expect comparable rates of folding for RNAase A and for S-protein and one would also expect to find no populated folding intermediates, so that combination between S-peptide and S-protein should occur after folding is complete. Instead, RNAase A folds 60 times more rapidly than S-protein and also combination with S-peptide occurs before folding of S-protein is complete. The results demonstrate that the folding rate of S-protein increases after the formation, or stabilization, of an intermediate which results from combination with S-peptide. They support a sequential model for protein folding in which the rates of successive steps in folding depend on the stabilities of preceding intermediates.The refolding kinetics of the slow-folding molecules are complex. Two results demonstrate the presence of folding intermediates: (1) the three probes show different kinetic progress curves, and (2) the folding kinetics are concentration-dependent, in contrast to the results expected if complete folding of S-protein precedes combination with S-peptide. A faster phase of the slow-refolding reaction is detected both by tyrosine absorbance and fluorescence emission but not by 2′CMP binding, indicating that native RNAase S is not formed in this phase. Comparison of the kinetic progress curves measured by different probes is made with the use of the kinetic ratio test, which is defined here.     

An equilibrium folding intermediate detected in the thermal unfolding transition of ribonuclease S by circular dichroism

The thermal-denaturation transition of ribonuclease S (RNAase S) is measured by circular dichroism at 225 nm. Only conformational transitions involving the S-peptide–S-protein complex are detected at this wavelength. Different pathways of thermal unfolding at high and low concentrations are apparent: at low concentrations the temperature of half-completion of denaturation (T_m) varies with concentration. Above a total enzyme concentration of 50 μM, T_m remains constant. The observed data can be explained on the basis of a model where the association–dissociation step occurs between S-peptide and thermally (at least partly) unfolded S-protein. The complex as a whole undergoes a major folding–unfolding transition in the course of which the S-peptide μ-helix appears to be formed. The unfolded complex is well populated in the unfolding transition region for enzyme concentrations of 100 μM or more. The model succeeds in deducing thermodynamic parameters from the thermal denaturation curves in various different ways. The values thus obtained are fully self-consistent and, moreover, consistent with the values for the apparent association constant and apparent association enthalpy as measured in enzyme-dilution experiments and by batch calorimetry.     

Hydrogen exchange from identified regions of the S-protein component of ribonuclease as a function of temperature,pH, and the binding of S-peptide

A medium resolution hydrogen exchange method (Rosa & Richards, 1979) has been used to measure the average rates of amide hydrogen exchange for known segments of the S-protein portion of ribonuclease-S. The analytical procedure permitted exchange rates to be monitored for seven S-protein fragments distributed throughout the structure, including regions of α-helix and β-sheet. Kinetics were measured as a function of pH, temperature and S-peptide binding.The pH dependence of exchange from isolated S-protein between pH 2·8 and pH 7·0 was found to deviate significantly from a first-order dependence on hydroxide ion concentration. The protection against exchange with increasing pH appeared to be closely related to the electrostatic stabilization of S-protein. It is suggested that such favorable electrostatic interactions result in increased energy barriers to the conformational fluctuations that provide solvent access to the time-average crystallographic structure. This explanation of the observed correlation between stability and exchange kinetics is also consistent with the calculated apparent activation energies for exchange from S-protein between 5·5 and 20 °C.S-peptide binding dramatically slows exchange from many S-protein sites, even those distant from the area of S-peptide contact. Interestingly, the effects of complex formation are not evenly propagated throughout S-protein. The most significantly perturbed sites (≥10³-fold reduction in exchange rate constants) lie within fragments derived from regions of secondary structure. Exchange from several other fragments is not significantly affected. The S-peptide—S-protein dissociation constant at neutral pH is so small that the measured exchange must have occurred from the complex and not from the dissociated parts.     

Amide proton exchange used to monitor the formation of a stable alpha-helix by residues 3 to 13 during folding of ribonuclease S

We make use of the known exchange rates of individual amide proton in the S-peptide moiety of ribonuclease S (RNAase S) to determine when during folding the alpha-helix formed by residues 3 to 13 becomes stable. The method is based on pulse-labeling with [3H]H2O during the folding followed by an exchange-out step after folding that removes 3H from all amide protons of the S-peptide except from residues 7 to 14, after which S-peptide is separated rapidly from S-protein by high performance liquid chromatography. The slow-folding species of unfolded RNAase S are studied. Folding takes place in strongly native conditions (pH 6.0, 10 degrees C). The seven H-bonded amide protons of the 3-13 helix become stable to exchange at a late stage in folding at the same time as the tertiary structure of RNAase S is formed, as monitored by tyrosine absorbance. At this stage in folding, the isomerization reaction that creates the major slow-folding species has not yet been reversed. Our result for the 3-13 helix is consistent with the finding of Labhardt (1984), who has studied the kinetics of folding of RNAase S at 32 degrees C by fast circular dichroism. He finds the dichroic change expected for formation of the 3-13 helix occurring when the tertiary structure is formed. Protected amide protons are found in the S-protein moiety earlier in folding. Formation or stabilization of this folding intermediate depends upon S-peptide: the intermediate is not observed when S-protein folds alone, and folding of S-protein is twice as slow in the absence of S-peptide. Although S-peptide combines with S-protein early in folding and is needed to stabilize an S-protein folding intermediate, the S-peptide helix does not itself become stable until the tertiary structure of RNAase S is formed.     

The influence of amino acid side-chains on α-helix stability: S-peptide analogues and related ribonucleases S′

In order to determine the influence of amino acid side-chains on α-helix stability, in relation to the protein folding process, the coil-helix transitions of some synthetic [Orn 10]-S-peptide analogues, containing, in position 8, Phe, Tyr, Ile, Ala, cpGly² and Gly, were investigated by the technique of circular dichroism under two different sets of conditions. First, the transitions of the Speptide analogues in water/trifluoroethanol mixtures were recorded. From the pattern of the transitions and from the ellipticity values in 97% trifluoroethanol, the following increasing order of amino acids as α-helix formers was found: Gly < Tyr ≤ Phe < cpGly < Ala < Ile. This finding indicates that the conformational parameters (Chou & Fasman, 1974) of the residues in position 8 play an important but not exclusive role in α-helix stability, since the hydrophobicity of the side-chain (Nozaki & Tanford, 1971) of residue 8 exerts a strong influence. From the second approach, studying the capability of the S-peptide analogues to bind to S-protein, the following increasing order was found: (Gly, Ala) < Ile < cpGly < Tyr < Phe. This result reveals that the conformational parameters of the residues in position 8 play no role, whereas their hydrophobic character and side-chain interactions with surrounding residues in the S-protein portion are the determining binding factors. This finding explains the reason for the Phe8 invariance in RNAase A during evolution, and furnishes evidence for the relevant role of long-range interactions in the protein folding process.     

On the thermal unfolding character of globular proteins

A theoretical model is presented to study the stepwise thermal unfolding of globular proteins using the stabilizing/destabilizing characters of amino acid residues in protein crystals. A multiple regression relation connecting the melting temperature and the amounts of stabilizing and destabilizing groups of residues in a protein, when used for the thermal behavior of peptide segments, provides reliable results on the stepwise unfolding nature of the protein. In ribonuclease A, the shell residues 16–22 are predicted to unfold earlier in the temperature range 30–45°C; the -sheet structures undergo thermal denaturation as a single cooperative unit and there is evidence indicating the segment 106–118 as a nucleation site. In ribonuclease S, the S-peptide unfolds earlier than S-protein. The predicted average and the range of melting temperatures, and the folding pathways of a set of globular proteins, agree very well with the experimental results. The results obtained in the present study indicate that (i) most of the nucleation parts possess high relative thermal stability, (ii) the unfolded state retains some residual structure, and (iii) some segments undergo gradual and overlapping thermal denaturation.     

Concentration-dependent hydrogen exchange kinetics of 3H-labeled S-peptide in ribonuclease S.

The hydrogen exchange kinetics of the S-peptide in ribonuclease S can be measured by first tritiating the S-peptide in the absence of S-protein and then allowing it to recombine rapidly with S-protein. Afterwards the exchange reactions of this specific segment of ribonuclease S can be studied. The exchange kinetics of bound S-peptide are complex, indicating that different protons exchange at markedly different rates. The terminal exchange reaction, involving at least five highly protected protons, has been studied as a function of pH.At low concentrations of ribonuclease S the exchange kinetics become concentration-dependent, owing to the dissociation of the S-peptide. Although the fraction of free S-peptide is always very small, its rate of exchange is several orders of magnitude faster than that of bound S-peptide, and the concentration dependence of the exchange kinetics is readily measurable. It provides a highly sensitive method for determining small dissociation constants (K_D). Values of K_D ranging from 10^?6m at pH 2.7, 0 °C, to 2 × 10^?10m at pH 7.0, 0 °C, are reported here. Our value for K_D at pH 7.0, 0 °C, confirms the data and extrapolation to 0 °C of Hearn et al. (1971).At high concentrations of ribonuclease S the terminal exchange reaction is independent of concentration. It probably results from a local unfolding reaction of the bound S-peptide. Above pH 4 the strong pH dependence of K_D closely resembles that of the apparent equilibrium constant for this local unfolding reaction. The latter may be one step in the dissociation process and we present such a model for ribonuclease S dissociation.Measurement of concentration-dependent exchange kinetics should provide a useful method of determining small dissociation constants in other systems: for example, in studies of protein-nucleic acid interactions.     

Molecular dynamics simulation of temperature induced unfolding of animal prion protein

To elucidate the structural stability and the unfolding dynamics of the animal prion protein, the temperature induced structural evolution of turtle prion protein (tPrPc) and bank vole prion protein (bvPrPc) have been performed with molecular dynamics (MD) simulation. The unfolding behaviors of secondary structures showed that the α-helix was more stable than β-sheet. Extension and disruption of β-sheet commonly appeared in the temperature induced unfolding process. The conversion of α-helix to π-helix occurred more readily at the elevating temperature. Furthermore, it was suggested in this work that the unfolding of prion protein could be regulated by the temperature.

Thermodynamics of the helix-coil transition: Binding of S15 and a hybrid sequence, disulfide stabilized peptide to the S-protein

Pancreatic ribonuclease A may be cleaved to produce two fragments: the S-peptide (residues 1-20) and the S-protein (residues 21-124). The S-peptide, or a truncated version designated as the S15 peptide (residues 1-15), combines with the S-protein to produce catalytically active complexes. The conformation of these peptides and many of their analogues is predominantly random coil at room temperature; however, they populate a significant fraction of helical form at low temperature under certain solution conditions. Moreover, they adopt a helical conformation when bound to the S-protein. A hybrid sequence, disulfide-stabilized peptide (ApaS-25), designed to stabilize the helical structure of the S-peptide in solution, also combines with the S-protein to yield a catalytically active complex. We have performed high-precision titration microcalorimetric measurements to determine the free energy, enthalpy, entropy, and heat capacity changes for the binding of ApaS-25 to S-protein within the temperature range 5-25 degrees C. The thermodynamic parameters for both the complex formation reactions and the helix-to-coil transition also were calculated, using a structure-based approach, by calculating changes in accessible surface area and using published empirical parameters. A simple thermodynamic model is presented in an attempt to account for the differences between the binding of ApaS-25 and the S-peptide. From this model, the thermodynamic parameters of the helix-to-coil transition of S15 can be calculated.     

Structural and functional characterization of a novel lipolytic enzyme from a Brazilian Cerrado soil metagenomic library

Objective

To isolate putative lipase enzymes by screening a Cerrado soil metagenomic library with novel features.

Results

Of 6720 clones evaluated, Clone W (10,000 bp) presented lipolytic activity and four predicted coding sequences, one of them LipW. Characterization of a predicted esterase/lipase, LipW, showed 28% sequence identity with an arylesterase from Pseudomonas fluorescens (pdb|3HEA) from protein database (PDB). Phylogenetic analysis showed LipW clustered with family V lipases; however, LipW was clustered in different subclade belonged to family V, suggesting a different subgroup of family V. In addition, LipW presented a difference in family V GH motif, a glycine replaced by a serine in GH motif. Estimated molecular weight and stokes radius values of LipW were 29,338.67–29,411.98 Da and 2.58–2.83 nm, respectively. Optimal enzyme activity was observed at pH 9.0–9.5 and at 40 °C. Circular dichroism analysis estimated secondary structures percentages as approximately 45% α-helix and 15% β-sheet, consistent with the 3D structure predicted by homology.

Conclusion

Our results demonstrate the isolation of novel family V lipolytic enzyme with biotechnological applications from a metagenomic library.

     

Secondary structure perturbations in salt-induced protein precipitates

The secondary structure implications of precipitation induced by a chaotropic salt, KSCN, and a structure stabilizing salt, Na₂SO₄, were studied for twelve different proteins. α-helix and β-sheet content of precipitate and native structures were estimated from the analysis of amide I band Raman spectra. A statistical analysis of the estimated perturbations in the secondary structure contents indicated that the most significant event is the formation of β-sheet structures with a concomitant loss of α-helix on precipitation with KSCN. The conformational changes for each protein were also analyzed with respect to elements of primary, secondary and tertiary structure existing in the native protein; primary structure was quantified by the fractions of hydrophobic and charged amino acids, secondary structure by x-ray estimates of α-helix and β-sheet contents of native proteins and tertiary structure by the dipole moment and solvent-accessible surface area. For the KSCN precipitates, factors affecting β-sheet content included the fraction of charged amino acids in the primary sequence and the surface area. Changes in α-helix content were influenced by the initial helical content and the dipole moment. The enhanced β-sheet contents of precipitates observed in this work parallel protein structural changes occurring in other aggregative phenomena.     

Effects of <Emphasis Type="Italic">GSH1</Emphasis> and <Emphasis Type="Italic">GSH2</Emphasis> Gene Mutation on Glutathione Synthetases Activity of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

In this paper, three mutants from wild Saccharomyces cerevisiae HBU2.558, called U2.558, UN2.558, and UNA2.558, were screened by UV, sodium nitrite, Atmospheric and room temperature plasma, respectively. Glutathione production of the three mutants increased by 41.86, 72.09 and 56.76%, respectively. We detected the activity of glutathione synthetases and found that its activity was improved. Amino acid sequences of three mutant colonies were compared with HBU2.558. Four mutants: Leu51→Pro51 (L51P), Glu62→Val62 (E62V), Ala332→Glu332 (A332E) and Ser653→Gly653 (S653G) were found in the analysis of γ-glutamylcysteine ligase. L51 is located adjacently to the two active sites of GCL/E/Mg²⁺/ADP complex in the overall GCL structure. L51P mutant spread distortion on the β-sheet due to the fact that the φ was changed from ?50.4° to ?40.2°. A mutant Leu54→Pro54 (L54P) was found in the analysis of glutathione synthetase, and L54 was an amino acid located between an α-helix and a β-sheet. The results confirm that introduction of proline located at the middle of the β-sheet or at the N- or C-terminal between α-helix and β-sheet or, i.e., L51P and L54P, changed the φ, rigidity, hydrophobicity and conformational entropy, thus increased protein stability and improved the enzyme activity.