The Pro117 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.     

Distinct unfolding and refolding pathways of ribonuclease a revealed by heating and cooling temperature jumps

Heating and cooling temperature jumps (T-jumps) were performed using a newly developed technique to trigger unfolding and refolding of wild-type ribonuclease A and a tryptophan-containing variant (Y115W). From the linear Arrhenius plots of the microscopic folding and unfolding rate constants, activation enthalpy (ΔH^#), and activation entropy (ΔS^#) were determined to characterize the kinetic transition states (TS) for the unfolding and refolding reactions. The single TS of the wild-type protein was split into three for the Y115W variant. Two of these transition states, TS1 and TS2, characterize a slow kinetic phase, and one, TS3, a fast phase. Heating T-jumps induced protein unfolding via TS2 and TS3; cooling T-jumps induced refolding via TS1 and TS3. The observed speed of the fast phase increased at lower temperature, due to a strongly negative ΔH^# of the folding-rate constant. The results are consistent with a path-dependent protein folding/unfolding mechanism. TS1 and TS2 are likely to reflect X-Pro¹¹⁴ isomerization in the folded and unfolded protein, respectively, and TS3 the local conformational change of the β-hairpin comprising Trp¹¹⁵. A very fast protein folding/unfolding phase appears to precede both processes. The path dependence of the observed kinetics is suggestive of a rugged energy protein folding funnel.     

A kinetic study of the folding of staphylococcal nuclease using size-exclusion chromatography.

The kinetics of the hydrodynamic volume change accompanying the reversible unfolding of staphylococcal nuclease have been observed by size-exclusion chromatography at 4 degrees C and pH 7.0 using the denaturant guanidine hydrochloride. The observed chromatographic profiles have been simulated by a six-component unfolding/refolding mechanism using a consistent set of equilibrium and kinetic parameters. The native protein is an equilibrium mixture of the cis and trans isomers of the peptide bond preceding proline-117. The native conformation containing the cis isomer dominates the equilibrium mixture, is more stable, and unfolds more slowly at its transition midpoint. The denatured protein is an equilibrium mixture of at least four components, the cis/trans isomers of proline-117 and one of the five remaining prolines. The dominant refolding pathway is initiated from the denatured component containing the trans isomer of proline-117. The six-component mechanism is consistent with tryptophan fluorescence kinetic measurements of the wild-type protein and with chromatographic measurements of a mutant P117G protein.     

Kinetic coupling of folding and prolyl isomerization of beta2-microglobulin studied by mutational analysis

β₂-Microglobulin (β2-m), a protein responsible for dialysis-related amyloidosis, adopts a typical immunoglobulin domain fold with the N-terminal peptide bond of Pro32 in a cis isomer. The refolding of β2-m is limited by the slow trans-to-cis isomerization of Pro32, implying that intermediates with a non-native trans-Pro32 isomer are precursors for the formation of amyloid fibrils. To obtain further insight into the Pro-limited folding of β2-m, we studied the Gdn-HCl-dependent unfolding/refolding kinetics using two mutants (W39 and P32V β2-ms) as well as the wild-type β2-m. W39 β2-m is a triple mutant in which both of the authentic Trp residues (Trp60 and Trp95) are replaced by Phe and a buried Trp common to other immunoglobulin domains is introduced at the position of Leu39 (i.e., L39W/W60F/W95F). W39 β2-m exhibits a dramatic quenching of fluorescence upon folding, enabling a detailed analysis of Pro-limited unfolding/refolding. On the other hand, P32V β2-m is a mutant in which Pro32 is replaced by Val, useful for probing the kinetic role of the trans-to-cis isomerization of Pro32. A comparative analysis of the unfolding/refolding kinetics of these mutants including three types of double-jump experiments revealed the prolyl isomerization to be coupled with the conformational transitions, leading to apparently unusual kinetics, particularly for the unfolding. We suggest that careful consideration of the kinetic coupling of unfolding/refolding and prolyl isomerization, which has tended to be neglected in recent studies, is essential for clarifying the mechanism of protein folding and, moreover, its biological significance.     

Hydrogen exchange of the tryptophan residues in bovine alpha-lactalbumin studied by UV spectroscopy

The effect of Ca²⁺ ion on structural fluctuation of a milk Ca²⁺-binding protein, α-lactalbumin, under native conditions was investigated by comparing hydrogen-exchange reactions of tryptophan residues in the apo-form without Ca²⁺ and in the holo-form at 1 mM CaCl₂ at pH 7.0 in the presence of 0.1M Na⁺. The reactions were followed by measuring time-dependent absorption changes at 298–300 nm due to the ²H-¹H exchange of the tryptophan imino protons and were found to be biphasic under all the conditions examined. Two of the four tryptophan protons are insensitive to Ca²⁺ concentration and show a relatively fast exchange rate. The other two protons are much more extensively protected (a protection degree of 10³–10⁵) and are markedly affected by the presence of Ca²⁺. Examinations of the temperature dependence and pH dependence of the individual exchange rates have been utilized for elucidating the exchange mechanism. The fast protons show a low activation energy reaction with so-called EX₂ kinetics. The exchange reaction of the slow protons is accompanied by a high activation energy, and the exchange mechanism of the protons depended on the presence or absence of stabilizing Ca²⁺ ions—the EX₁ kinetics for the apo-protein and the EX₂ kinetics for the holo-protein at 1 mM Ca²⁺. The exchange reaction in the thermally unfolded state was also found to be biphasic, but the fast phase, which has an exchange rate in the fully exposed state, becomes predominant with decreasing temperature. By taking this fact and using a structural unfolding model of hydrogen exchange, the present results are fully consistent with thermodynamic parameters of the thermal transition and kinetic parameters of refolding reactions induced by concentration jumps of guanidine hydrochloride obtained in previous studies. It is demonstrated that the reaction of the slow protons in the native state is mediated by a transient global unfolding equivalent to the “thermal” unfolding under a native condition and that switching of the exchange mechanism from the EX₁ to EX₂ kinetics results from acceleration of the refolding rate with an increase in Ca²⁺ concentration. The transient global unfolding takes place even under a strongly native condition, e.g., at a temperature 20° below the beginning of the thermal transition.     

Replacement of proline with valine does not remove an apparent proline isomerization-dependent folding event in CRABP I

Site-directed mutagenesis has frequently been used to replace proline with other amino acids in order to determine if proline isomerization is responsible for a slow phase during refolding. Replacement of Pro 85 with alanine in cellular retinoic acid binding protein I (CRABP-I) abolished the slowest refolding phase, suggesting that this phase is due to proline isomerization in the unfolded state. To further test this assumption, we mutated Pro 85 to valine, which is the conservative replacement in the two most closely related proteins in the family (cellular retinoic acid binding protein II and cellular retinol binding protein I). The mutant protein was about 1 kcal/mole more stable than wild type. Retinoic acid bound equally well to wild type and P85V-CRABP I, confirming the functional integrity of this mutation. The refolding and unfolding kinetics of the wild-type and mutant proteins were characterized by stopped flow fluorescence and circular dichroism. The mutant P85V protein refolded with three kinetic transitions, the same number as wild-type protein. This result conflicts with the P85A mutant, which lost the slowest refolding rate. The P85V mutation also lacked a kinetic unfolding intermediate found for wild-type protein. These data suggest that proline isomerization may not be responsible for the slowest folding phase of CRABP I. As such, the loss of a slow refolding phase upon mutation of a proline residue may not be diagnostic for proline isomerization effects on protein folding.     

Effect of an engineered disulfide bond on the folding of T4 lysozyme at low temperatures

Equilibrium and kinetic effects on the folding of T4 lysozyme were investigated by fluorescence emission spectroscopy in cryosolvent. To study the role of disulfide cross-links in stability and folding, a comparison was made with a mutant containing an engineered disulfide bond between Cys-3 (Ile-3 in the wild type) and Cys-97, which links the C-terminal domain to the N terminus of the protein [Perry & Wetzel (1984) Science 226, 555]. In our experimental system, stability toward thermal and denaturant unfolding was increased slightly as a result of the cross-link. The corresponding reduced protein was significantly less stable than the wild type. Unfolding and refolding kinetics were carried out in 35% methanol, pH 6.8 at -15 degrees C, with guanidine hydrochloride as the denaturant. Unfolding/refolding of the wild-type and reduced enzyme showed biphasic kinetics both within and outside the denaturant-induced transition region and were consistent with the presence of a populated intermediate in folding. Double-jump refolding experiments eliminated proline isomerization as a possible cause for the biphasicity. The disulfide mutant protein, however, showed monophasic kinetics in all guanidine concentrations studied.     

Stability and folding of a mutant ribose-binding protein ofEscherichia coli

A mature mutant ribose-binding protein (RBP) ofEscherichia coli was obtained by site-directed mutagenesis, replacing Thr-3 in the N-domain of wild-type mature RBP (WT-mRBP) with a Trp residue (N-Trp-mRBP). The equilibrium unfolding properties and the refolding kinetics of this protein were monitored by fluorescence and circular dichroism (CD). The stability of N-Trp-mRBP appears to be the same as that of C-Trp-mRBP, another mutant obtained by replacing Phe-187 with a Trp, and lower than that of WT-mRBP. The overall refolding rate of N-Trp-mRBP is much smaller than that of C-Trp-mRBP, which, in turn, is similar to that of WT-mRBP. For the case of WT-mRBP, the rate constant obtained by Tyr fluorescence is identical to the value obtained by CD. But with C-Trp-mRBP, the rate constant from CD is smaller than the value from the Trp fluorescence and this difference in the rate constants is much greater with the N-TrpmRBP.     

Unfolding study of a trimeric membrane protein AcrB

The folding of a multi‐domain trimeric α‐helical membrane protein, Escherichia coli inner membrane protein AcrB, was investigated. AcrB contains both a transmembrane domain and a large periplasmic domain. Protein unfolding in sodium dodecyl sulfate (SDS) and urea was monitored using the intrinsic fluorescence and circular dichroism spectroscopy. The SDS denaturation curve displayed a sigmoidal profile, which could be fitted with a two‐state unfolding model. To investigate the unfolding of separate domains, a triple mutant was created, in which all three Trp residues in the transmembrane domain were replaced with Phe. The SDS unfolding profile of the mutant was comparable to that of the wild type AcrB, suggesting that the observed signal change was largely originated from the unfolding of the soluble domain. Strengthening of trimer association through the introduction of an inter‐subunit disulfide bond had little effect on the unfolding profile, suggesting that trimer dissociation was not the rate‐limiting step in unfolding monitored by fluorescence emission. Under our experimental condition, AcrB unfolding was not reversible. Furthermore, we experimented with the refolding of a monomeric mutant, AcrB_Δloop, from the SDS unfolded state. The CD spectrum of the refolded AcrB_Δloop superimposed well onto the spectra of the original folded protein, while the fluorescence spectrum was not fully recovered. In summary, our results suggested that the unfolding of the trimeric AcrB started with a local structural rearrangement. While the refolding of secondary structure in individual monomers could be achieved, the re‐association of the trimer might be the limiting factor to obtain folded wild‐type AcrB.     

Kinetic Control in Protein Folding for Light Chain Amyloidosis and the Differential Effects of Somatic Mutations

Light chain amyloidosis is a devastating disease where immunoglobulin light chains form amyloid fibrils, resulting in organ dysfunction and death. Previous studies have shown a direct correlation between the protein thermodynamic stability and the propensity for amyloid formation for some proteins involved in light chain amyloidosis. Here we investigate the effect of somatic mutations on protein stability and in vitro fibril formation of single and double restorative mutants of the protein AL-103 compared to the wild-type germline control protein. A scan rate dependence and hysteresis in the thermal unfolding and refolding was observed for all proteins. This indicates that the unfolding/refolding reaction is kinetically determined with different kinetic constants for unfolding and refolding even though the process remains experimentally reversible. Our structural analysis of AL-103 and AL-103 delP95aIns suggests a kinetic coupling of the unfolding/refolding process with cis–trans prolyl isomerization. Our data reveal that the deletion of proline 95a (AL-103 delP95aIns), which removes the trans–cis di-proline motif present in the patient protein AL-103, results in a dramatic increment in the thermodynamic stability and a significant delay in fibril formation kinetics with respect to AL-103. Fibril formation is pH dependent; all proteins form fibrils at pH 2; reactions become slower and more stochastic as the pH increases up to pH 7. Based on these results, we propose that, in addition to thermodynamic stability, kinetic stability (possibly influenced by the presence of cis proline 95a) plays a major role in the AL-103 amyloid fibril formation process.     

Effects of proline mutations on the unfolding and refolding of human lysozyme: the slow refolding kinetic phase does not result from proline cis-trans isomerization

The unfolding and refolding kinetics of six proline mutants of the human lysozyme (h-lysozyme) were carried out and compared to that of the wild-type protein. Our results show that the slow refolding phase observed in the h-lysozyme refolding kinetics cannot be ascribed to proline isomerization reactions. The h-lysozyme contains two proline residues at positions 71 and 103, both in the trans conformation in the native state. The refolding kinetics of the P71G/P103G mutant, in which both prolines have been replaced by a glycine, were found to be similar to those of the wild-type protein. The same slow phase amplitude of about 10% was found for both proteins, and the slow phase rate constants were also identical within experimental error. Other mutants such as P103G or P71G, in which only one of the two prolines has been replaced by a glycine, and A47P with its three prolines, gave identical slow refolding phases. The X-ray structure analysis and scanning microcalorimetric study of each protein (Herning et al., unpublished experiments) have confirmed that none of the considered mutations affects significantly protein structure and that no major changes in protein stability were brought about by these mutations. Therefore, comparison of the properties of the mutant and wild-type proteins is legitimate. Interestingly, the refolding kinetics of the V110P mutant, in which a proline residue has been introduced at position 110 (N-terminus of an alpha-helix), were clearly triphasic. For this mutant an additional very slow phase with properties similar to those expected from the proline hypothesis was detected. Equilibrium denaturation studies were conducted for each protein, and the refolding pathway of h-lysozyme is partly presented. We also discuss the effect of proline mutations on the energetics of the folding pathway of the h-lysozyme in water.     

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.     

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.     

Preformed GroES Oligomers Are Not Required as Functional Cochaperonins

We have previously shown that the C-terminal sequence of GroES is required for oligomerization [Seale and Horowitz (1995), J. Biol. Chem. 270, 30268–30270]. In this report, we have generated a C-terminal deletion mutant of GroES with a significantly destabilized oligomer and have investigated its function in the chaperonin-assisted protein folding cycle. Removal of the two C-terminal residues of GroES results in a cochaperonin [GroESD(96–97)] that is monomeric at concentrations where GroES function is assessed. Using equilibrium ultracentrifugation, we measured the dissociation constant for the oligomer–monomer equilibrium to be 7.3×10^–34M⁶. The GroESD(96–97) is fully active as a cochaperonin. This mutant is able to inhibit the ATPase activity of GroEL to levels comparable to wild-type GroES. It is also able to assist the refolding of urea-denatured rhodanese by GroEL. While GroESD(96–97) can function at levels comparable to wild-type GroES, higher concentrations of mutant are required to produce the same effect. These results support the idea that the preformed GroES heptamer is not required for function, but they suggest that the oligomeric cochaperonin is most efficient.     

Kinetic analysis of the folding and unfolding of a mutant form of bovine pancreatic trypsin inhibitor lacking the cysteine-14 and -38 thiols

The kinetics of the disulfide-coupled unfolding-refolding transition of a mutant form of bovine pancreatic trypsin inhibitor (BPTI) lacking Cys-14 and -38 were measured and compared to previous results for the wild-type protein and other modified forms. The altered cysteines, which were changed to serine in the mutant protein, are normally paired in a disulfide in the native protein but from disulfides with Cys-5 in two-disulfide kinetic intermediates during folding. Although the mutant protein could fold efficiently, the kinetics of both folding and unfolding were altered, reflecting the roles of these cysteines in the two-disulfide intermediates with "wrong" disulfides. The intramolecular rate constant for the formation of the second disulfide of the native mutant protein was more than 10(3)-fold lower than that for the formation of a second disulfide during the refolding of the wild-type protein. The observed rate of unfolding of the mutant protein was also lower than that of the wild-type protein, demonstrating that the altered cysteines are involved in the intramolecular rearrangements that are the rate-determining step in the unfolding of the wild-type protein. These results confirm the previous conclusion [Creighton, T.E. (1977) J. Mol. Biol. 113, 275-293] that the energetically preferred pathway for folding and unfolding of BPTI includes intramolecular rearrangements of intermediates in which Cys-14 and -38 are paired in disulfides not present in the native protein. The present results are also consistent with other, less detailed, studies with similar mutants lacking Cys-14 and -38 [Marks, C.B., Naderi, H., Kosen, P.A., Kuntz, I.D., & Anderson, S. (1987) Science (Washington, D.C.) 235, 1370-1371].     

Folding mechanism of mutant human lysozyme C77/95A with increased secretion efficiency in yeast.

A mutant human lysozyme C77/95A, in which Cys77 and Cys95 are replaced with alanine, has been characterized by 8-fold greater secretion in yeast (Taniyama, Y., Yamamoto, Y., Nakao, M., Kikuchi, M., and Ikehara, M. (1988) Biochem. Biophys. Res. Commun. 152, 962-967) and almost the same three-dimensional structure as wild-type human lysozyme (Inaka, K., Taniyama, Y., Kikuchi, M., Morikawa, K., and Matsushima, M. (1991) J. Biol. Chem. 266, 12599-12603). To clarify the molecular features of C77/95A and the reason for its increased secretion in yeast, the stabilities of the mutant C77/95A and the wild-type proteins were examined by guanidine hydrochloride denaturation, and the unfolding-refolding kinetics were determined from circular dichroism and fluorescence stopped-flow measurements. Equilibrium experiments showed that the delta G of unfolding of C77/95A in water was 5.8 kcal/mol less stable than that of the wild-type protein at pH 4.0 and 10 degrees C. The unfolding rate of C77/95A was 4 orders of magnitude faster than that of the wild-type protein whereas the two proteins shared similar refolding rates. The slowly refolding phase of the wild-type protein disappeared in C77/95A, indicating that the disulfide bond affects this phase. These observations show that the disulfide bond Cys77-Cys95 contributes to the stabilization of the folded form of human lysozyme by suppressing the unfolding rate and that the increase in the unfolding rate, or the disappearance of the slowly refolding phase in vitro, could correlate with the increase in secretion efficiency in vivo.     

Energetics of protein structure and folding

The available experimental date on the kinetics of unfolding and refolding of small proteins are reviewed. Excluding slow transitions in the unfolded protein due to cis–trans isomerization of peptide bonds, the rate-limiting transition state in both unfolding and refolding is concluded to be a high-energy distortion of the fully folded state. Partially folded intermediates are undoubtedly important for folding, but their formation is normally not rate limiting. A simple model is used to illustrate some of the aspects of protein-folding energetics.     

Multiple tryptophan probes reveal that ubiquitin folds via a late misfolded intermediate

Much of our understanding of protein folding mechanisms is derived from experiments using intrinsic fluorescence of natural or genetically inserted tryptophan (Trp) residues to monitor protein refolding and site-directed mutagenesis to determine the energetic role of amino acids in the native (N), intermediate (I) or transition (T) states. However, this strategy has limited use to study complex folding reactions because a single fluorescence probe may not detect all low-energy folding intermediates. To overcome this limitation, we suggest that protein refolding should be monitored with different solvent-exposed Trp probes. Here, we demonstrate the utility of this approach by investigating the controversial folding mechanism of ubiquitin (Ub) using Trp probes located at residue positions 1, 28, 45, 57, and 66. We first show that these Trp are structurally sensitive and minimally perturbing fluorescent probes for monitoring folding/unfolding of the protein. Using a conventional stopped-flow instrument, we show that ANS and Trp fluorescence detect two distinct transitions during the refolding of all five Trp mutants at low concentrations of denaturant: T₁, a denaturant-dependent transition and T₂, a slower transition, largely denaturant-independent. Surprisingly, some Trp mutants (Ub^M1W, Ub^S57W) display Trp fluorescence changes during T₁ that are distinct from the expected U → N transition suggesting that the denaturant-dependent refolding transition of Ub is not a U → N transition but represents the formation of a structurally distinct I-state (U → I). Alternatively, this U → I transition could be also clearly distinguished by using a combination of two Trp mutations Ub^F45W-T66W for which the two Trp probes that display fluorescence changes of opposite sign during T₁ and T₂ (Ub^F45W-T66W). Global fitting of the folding/unfolding kinetic parameters and additional folding-unfolding double-jump experiments performed on Ub^M1W, a mutant with enhanced fluorescence in the I-state, demonstrate that the I-state is stable, compact, misfolded, and on-pathway. These results illustrate how transient low-energy I-states can be characterized efficiently in complex refolding reactions using multiple Trp probes.     

Nuclease A (Gbs0661), an extracellular nuclease of Streptococcus agalactiae,attacks the neutrophil extracellular traps and is needed for full virulence

Most bacteria of the genus Streptococcus are opportunistic pathogens, and some of them produce extracellular DNases, which may be important for virulence. Genome analyses of Streptococcus agalactiae (GBS) neonate isolate NEM316 revealed the presence of seven genes putatively encoding secreted DNases, although their functions, if any, are unknown. In this study, we observed that respiration growth of GBS led to the extracellular accumulation of a putative nuclease, identified as being encoded by the gbs0661 gene. When overproduced in Lactococcus lactis, the protein was found to be a divalent cation‐requiring, pH‐stable and heat‐stable nuclease that we named Nuclease A (NucA). Substitution of the histidine¹⁴⁸ by alanine reduced nuclease activity of the GBS wild‐type strain, indicating that NucA is the major nuclease ex vivo. We determined that GBS is able to degrade the DNA matrix comprising the neutrophil extracellular trap (NET). The nucA^H148A mutant was impaired for this function, implicating NucA in the virulence of GBS. In vivo infection studies confirmed that NucA is required for full infection, as the mutant strain allowed increased bacterial clearance from lung tissue and decreased mortality in infected mice. These results show that NucA is involved in NET escape and is needed for full virulence.     

Stability and folding of a mutant ribose-binding protein ofEscherichia coli

A mature mutant ribose-binding protein (RBP) ofEscherichia coli was obtained by site-directed mutagenesis, replacing Thr-3 in the N-domain of wild-type mature RBP (WT-mRBP) with a Trp residue (N-Trp-mRBP). The equilibrium unfolding properties and the refolding kinetics of this protein were monitored by fluorescence and circular dichroism (CD). The stability of N-Trp-mRBP appears to be the same as that of C-Trp-mRBP, another mutant obtained by replacing Phe-187 with a Trp, and lower than that of WT-mRBP. The overall refolding rate of N-Trp-mRBP is much smaller than that of C-Trp-mRBP, which, in turn, is similar to that of WT-mRBP. For the case of WT-mRBP, the rate constant obtained by Tyr fluorescence is identical to the value obtained by CD. But with C-Trp-mRBP, the rate constant from CD is smaller than the value from the Trp fluorescence and this difference in the rate constants is much greater with the N-TrpmRBP.