The Pro to glycine mutation of staphylococcal nuclease simplifies the unfolding—folding kinetics

Kinetics of unfolding and refolding of a staphylococcal nuclease mutant, in which Pro¹¹⁷ is replaced by glycine, have been investigated by stopped-flow circular dichroism, and the results are compared with those for the wild-type protein. In contrast to the biphasic unfolding of the wild-type nuclease, the unfolding of the mutant is represented by a single-phase reaction, indicating that the biphasic unfolding for the wild-type protein is caused by cis-trans isomerization about the prolyl peptide bond in the native state. The proline mutation also simplifies the kinetic refolding. Importance of the results in elucidating the folding mechanism is discussed.     

NMR analysis of staphylococcal nuclease thermal quench refolding kinetics.

Thermally unfolded staphylococcal nuclease has been rapidly quenched to temperatures near 0 degree C and the refolding behavior examined using an NMR kinetic experiment. Unfolded protein, exhibiting random coil chemical shifts, persists following the quench and refolds in two distinct kinetic phases. A protein folding intermediate with a trans Lys 116-Pro 117 peptide bond is transiently overpopulated and relaxes to the predominantly cis native cis-trans equilibrium. The rate of trans-->cis isomerization in the native-like nuclease intermediate is approximately 100-fold faster than that observed in a Lys-Pro model peptide. The activation enthalpy of 20 kcal/mol observed for the nuclease Lys 116-Pro 117 peptide bond is comparable to that observed for other X-Pro isomerizations.     

Pressure-jump small-angle x-ray scattering detected kinetics of staphylococcal nuclease folding

The kinetics of chain disruption and collapse of staphylococcal nuclease after positive or negative pressure jumps was monitored by real-time small-angle x-ray scattering under pressure. We used this method to probe the overall conformation of the protein by measuring its radius of gyration and pair-distance-distribution function p(r) which are sensitive to the spatial extent and shape of the particle. At all pressures and temperatures tested, the relaxation profiles were well described by a single exponential function. No fast collapse was observed, indicating that the rate limiting step for chain collapse is the same as that for secondary and tertiary structure formation. Whereas refolding at low pressures occurred in a few seconds, at high pressures the relaxation was quite slow, approximately 1 h, due to a large positive activation volume for the rate-limiting step for chain collapse. A large increase in the system volume upon folding implies significant dehydration of the transition state and a high degree of similarity in terms of the packing density between the native and transition states in this system. This study of the time-dependence of the tertiary structure in pressure-induced folding/unfolding reactions demonstrates that novel information about the nature of protein folding transitions and transition states can be obtained from a combination of small-angle x-ray scattering using high intensity synchrotron radiation with the high pressure perturbation technique.     

The foldon substructure of staphylococcal nuclease

To search for submolecular foldon units, the spontaneous reversible unfolding and refolding of staphylococcal nuclease under native conditions was studied by a kinetic native-state hydrogen exchange (HX) method. As for other proteins, it appears that staphylococcal nuclease is designed as an assembly of well-integrated foldon units that may define steps in its folding pathway and may regulate some other functional properties. The HX results identify 34 amide hydrogens that exchange with solvent hydrogens under native conditions by way of large transient unfolding reactions. The HX data for each hydrogen measure the equilibrium stability (ΔG_HX) and the kinetic unfolding and refolding rates (k_op and k_cl) of the unfolding reaction that exposes it to exchange. These parameters separate the 34 identified residues into three distinct HX groupings. Two correspond to clearly defined structural units in the native protein, termed the blue and red foldons. The remaining HX grouping contains residues, not well separated by their HX parameters alone, that represent two other distinct structural units in the native protein, termed the green and yellow foldons. Among these four sets, a last unfolding foldon (blue) unfolds with a rate constant of 6 × 10^− 6 s^− 1 and free energy equal to the protein's global stability (10.0 kcal/mol). It represents part of the β-barrel, including mutually H-bonding residues in the β4 and β5 strands, a part of the β3 strand that H-bonds to β5, and residues at the N-terminus of the α2 helix that is capped by β5. A second foldon (green), which unfolds and refolds more rapidly and at slightly lower free energy, includes residues that define the rest of the native α2 helix and its C-terminal cap. A third foldon (yellow) defines the mutually H-bonded β1-β2-β3 meander, completing the native β-barrel, plus an adjacent part of the α1 helix. A final foldon (red) includes residues on remaining segments that are distant in sequence but nearly adjacent in the native protein. Although the structure of the partially unfolded forms closely mimics the native organization, four residues indicate the presence of some nonnative misfolding interactions. Because the unfolding parameters of many other residues are not determined, it seems likely that the concerted foldon units are more extensive than is shown by the 34 residues actually observed.     

The effects of amino acid replacements of glycine 20 on conformational stability and catalysis of staphylococcal nuclease

Staphylococcal nuclease (SNase) is a well-established model for protein folding studies. Its three-dimensional structure has been determined. The enzyme, Ca2+, and DNA or RNA substrate form a ternary complex. Glycine 20 is the second position of the first beta-turn of SNase, which may serve as the folding initiation site for the SNase polypeptide. To study the role of Gly20 in the conformational stability and catalysis of SNase, three mutants, in which Gly20 was replaced by alanine, valine, or isoleucine, were constructed and studied by using circular dichroism spectra, intrinsic and ANS-binding fluorescence spectra, stability and activity assays. The mutations have little effect on the conformational integrity of the mutants. However, the catalytic activity is reduced drastically by the mutations, and the stability of the protein is progressively decreased in the order G20A相似文献   

Genetic control of the immune response to staphylococcal nuclease

Summary Antibody responses of inbred strains of mice to staphylococcal nuclease were studied by isoelectric focusing in polyacrylamide gels followed by in situ labeling of focused antibodies with radioactive antigen. All A/J mice examined produced antinuclease antibodies of limited heterogeneity, and although there was individual variation in the focusing patterns observed, a characteristic spectrotype produced by all of the animals could be discerned. In order to determine the possible relationship between this characteristic spectrotype and the cross-reactive idiotypes of A/J antinuclease antibodies previously described (7), focused antibodies were also examined with a radioactively labeled pig anti-(A/J antinuclease) anti-idiotypic antibody preparation. Using this reagent, similar spectrotypes to those observed for antigen binding were seen in all of the individual A/J sera, suggesting that cross-reactive idiotype expression is a reflection of the characteristic spectrotypes observed. The same labeled anti-idiotypic reagent revealed characteristic but different spectrotypes when used to develop focused antinuclease antibodies from individual mice of other strains, suggesting that the use of similar variable region structures may be a common feature of the antinuclease response in mice of different allotypes. These studies thus provide a structural basis for the genetics of idiotype expression defined previously by serologic analysis.     

Export defect adjacent to the processing site of staphylococcal nuclease is suppressed by a prlA mutation.

Plasmids have been constructed in which the Escherichia coli alkaline phosphatase promoter and signal sequence have been fused to the staphylococcal nuclease gene to promote the high-level expression and secretion of this gene product in E. coli. We determined that the first amino acid residue after the signal sequence can determine whether this protein was processed and exported to the periplasmic space. Fractionation and protease accessibility studies were used to show that the export-defective, nuclease precursor is internal to the cytoplasmic membrane barrier of the cell. Furthermore, this export defect was suppressed in a strain containing a prlA mutation. These findings are novel in that this region of the polypeptide chain has been implicated in processing but not export and that prlA mutations have not been previously known to suppress such defects.     

Folding kinetics of staphylococcal nuclease studied by tryptophan engineering and rapid mixing methods

To monitor the development of tertiary structural contacts during folding, a unique tryptophan residue was introduced at seven partially buried locations (residues 15, 27, 61, 76, 91, 102 and 121) of a tryptophan-free variant of staphylococcal nuclease (P47G/P117G/H124L/W140H). Thermal unfolding measurements by circular dichroism indicate that the variants are destabilized, but maintain the ability to fold into a native-like structure. For the variants with Trp at positions 15, 27 and 61, the intrinsic fluorescence is significantly quenched in the native state due to close contact with polar side-chains that act as intramolecular quenchers. All other variants exhibit enhanced fluorescence under native conditions consistent with burial of the tryptophan residues in an apolar environment. The kinetics of folding was observed by continuous and stopped-flow fluorescence measurements over refolding times ranging from 100 micros to 10 s. The folding kinetics of all variants is quantitatively described by a mechanism involving a major pathway with a series of intermediate states and a minor parallel channel. The engineered tryptophan residues in the beta-barrel and the N-terminal part of the alpha-helical domain become partially shielded from the solvent at an early stage (<1 ms), indicating that this region undergoes a rapid collapse. For some variants, a major increase in fluorescence coincides with the rate-limiting step of folding on the 100 ms time scale, indicating that these tryptophan residues are buried only during the late stages of folding. Other variants exhibit a transient increase in fluorescence during the 10 ms phase followed by a decrease during the rate-limiting phase. These observations are consistent with burial of these probes in a collapsed, but loosely packed intermediate, followed by the rate-limiting formation of the densely packed native core, which brings the tryptophan residues into close contact with intramolecular quenchers.     

Cold denaturation of staphylococcal nuclease

It has been shown that the structure of staphylococcal nuclease breaks down reversibly both at a temperature increase above 20 degrees C and at its decrease. Both the heat and cold denaturations of protein are well approximated by a transition between two states differing in heat capacity, which means that the whole protein molecule represents a unique cooperative system with a well developed hydrophobic core. The transfer to a denatured state at a temperature decrease is accompanied by heat release and leads to a complete loss of the unique tertiary structure, decrease of the helicity and increase of the hydrodynamic volume of the molecule.     

The role of tryptophan in staphylococcal nuclease stability

Staphylococcal nuclease (SNase) has a single Trp residue at position 140. Circular dichroism, intrinsic and ANS-binding fluorescence, chemical titrations and enzymatic assays were used to measure the changes of its structure, stability and activities as the Trp was mutated or replaced to other positions. The results show that W140 is critical to SNase structure, stability, and function. Mutants such as W140A, F61W/W140A, and Y93W/W140A have unfolding, corrupted secondary and tertiary structures, diminished structural stability and attenuated catalytic activity as compared to the wild type. The deleterious effects of W140 substitution cannot be compensated by concurrent changes at topographical locations of position 61 or 93. Local hydrophobicity defined as a sum of hydrophobicity around a given residue within a distance is found to be a relevant property to SNase folding and stability.     

Thermal denaturation of staphylococcal nuclease

The fully reversible thermal denaturation of staphylococcal nuclease in the absence and presence of Ca2+ and/or thymidine 3',5'-diphosphate (pdTp) from pH 4 to 8 has been studied by high-sensitivity differential scanning calorimetry. In the absence of ligands, the denaturation is accompanied by an enthalpy change of 4.25 cal g-1 and an increase in specific heat of 0.134 cal K-1 g-1, both of which are usual values for small globular proteins. The temperature (tm) of maximal excess specific heat is 53.4 degrees C. Each of the ligands, Ca2+ and pdTp, by itself has important effects on the unfolding of the protein which are enhanced when both ligands are present. Addition of saturating concentrations of these ligands raises the denaturational enthalpy to 5.74 cal g-1 in the case of Ca2+ and to 6.72 cal g-1 in the case of pdTp. The ligands raise the tm by as much as 11 degrees C depending on ligand concentration. From the variation of the denaturational enthalpies with ligand concentrations, binding constants at 53 degrees C equal to 950 M-1 and 1.4 X 10(4) M-1 are estimated for Ca2+ and pdTp, respectively, and from the enthalpies at ligand saturation, binding enthalpies at 53 degrees C of -15.0 and -19.3 kcal mol-1.     

An immunological approach to the conformational equilibrium of staphylococcal nuclease.

The conformational equilibrium constant, K_conf, of staphylococcal nuclease, describing the equilibrium between the native conformation and non-native or disordered conformations, has been estimated using an immunologic method and an interpretive model. Using goat antisera prepared toward a conformationally disordered nuclease fragment (99–149), antibodies specific for the disordered form of the helix-rich sequence 99 to 126, anti-(99–126)_R, were isolated by sequential immunoabsorption. Anti-(99–126)_R forms soluble 7 S complexes with fragment (99–149), but this interaction may be inhibited by a large excess of nuclease. By using fragment (99–149) preferentially carbamylated at the α-amino terminus with KN¹⁴CO and rabbit anti-goat immunoglobulin to distinguish between antibody-bound and free fragment (99–149), an assay for the quantitation of the degree of inhibition of anti-(99–126)_R. (99–149) complex formation by nuclease was developed.Using a formal analysis based on the hypothesis that nuclease is in a conformational equilibrium between a folded and unfolded form and that anti-(99–126)_R binds effectively only to the unfolded form, the K_conf of nuclease was estimated to be 2900. In the presence of the ligands Ca(II), or Ca(II) and thymidine-3′,5′-diphosphate, K_conf values of 6500 and 30,000 to 50,000 were estimated, respectively. The K_conf of nuclease at 4 °C and 39 °C was 3900 and 400, respectively.     

Exploring the temperature-pressure phase diagram of staphylococcal nuclease

The temperature dependence of the pressure-induced equilibrium unfolding of staphylococcal nuclease (Snase) was determined by fluorescence of the single tryptophan residue, FTIR absorption for the amide I' and tyrosine O-H bands, and small-angle X-ray scattering (SAXS). The results from these three techniques were similar, although the stability as measured by fluorescence was slightly lower than that measured by FTIR and SAXS. The resulting phase diagram exhibits the well-known curvature for heat and cold denaturation of proteins, due to the large decrease in heat capacity upon folding. The volume change for unfolding became less negative with increasing temperatures, consistent with a larger thermal expansivity for the unfolded state than for the folded state. Fluorescence-detected pressure-jump kinetics measurements revealed that the curvature in the phase diagram is due primarily to the rate constant for folding, indicating a loss in heat capacity for the transition state relative to the unfolded state. The similar temperature dependence of the equilibrium and activation volume changes for folding indicates that the thermal expansivities of the folded and transition states are similar. This, along with the fact that the activation volume for folding is positive over the temperature range examined, the nonlinear dependence of the folding rate constant upon temperature implicates significant dehydration in the rate-limiting step for folding of Snase.