Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.31.1 (micrococcal nuclease)
2,818 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Effects of proline isomerizations on the equilibrium unfolding and kinetic refolding of staphylococcal nuclease were studied by circular dichroism in the peptide region (225 nm) and fluorescence spectra of a tryptophan residue. For this purpose, four single mutants (P11A, P31A, P42A, and P56A) and four multiple mutants (P11A/P47T/P117G, P11A/P31A/P47T/P117G, P11A/P31A/P42A/P47T/P117G, and P11A/P31A/P42A/P47T/P56A/P117G) were constructed. These mutants, together with the single and double mutants for Pro47 and Pro117 constructed in our previous study, cover all six proline sites of the nuclease. The P11A, P31A, and P42A mutations did not change the stability of the protein remarkably, while the P56A mutation increased protein stability to a small extent by 0.5 kcal/mol. The refolding kinetics of the protein were, however, affected remarkably by three of the mutations, namely, P11A, P31A, and P56A. Most notably, the amplitude of the slow phase of the triphasic refolding kinetics of the nuclease observed by stopped-flow circular dichroism decreased by increasing the number of the proline mutations; the slow phase disappeared completely in the proline-free mutant (P11A/P31A/P42A/P47T/P56A/P117G). The kinetic refolding reactions of the wild-type protein assessed in the presence of Escherichia coli cyclophilin A showed that the slow phase was accelerated by cyclophilin, indicating that the slow phase was rate-limited by cis-trans isomerization of the proline residues. Although the fast and middle phases of the refolding kinetics were not affected by cyclophilin, the amplitude of the middle phase decreased when the number of the proline mutations increased; the percent amplitudes for the wild-type protein and the proline-free mutants were 43 and 13%, respectively. In addition to these three phases detected with stopped-flow circular dichroism, a very fast phase of refolding was observed with stopped-flow fluorescence, which had a shorter dead time (3.6 ms) than the stopped-flow circular dichroism. The following conclusions were drawn. (1) The effects of the P11A, P31A, and P56A mutations on the refolding kinetics indicate that the isomerizations of the three proline residues are rate-limiting, suggesting that the structures around these residues (Pro11, Pro31, and Pro56) may be organized at an early stage of refolding. (2) The fast phase corresponds to the refolding of the native proline isomer, and the middle phase whose amplitude has decreased when the number of proline mutations was increased may correspond to the slow refolding of non-native proline isomers. The occurrence of the fast- and slow-refolding reactions together with the slow phase rate-limited by the proline isomerization suggests that there are parallel folding pathways for the native and non-native proline isomers. (3) The middle phase did not completely disappear in the proline-free mutant. This suggests that the slow-folding isomer is produced not only by the proline isomerizations but also by another conformational event that is not related to the prolines. (4) The very fast phase detected with the fluorescent measurements suggests that there is an intermediate at a very early stage of kinetic refolding.
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PMID:Effects of proline mutations on the folding of staphylococcal nuclease. 1002 6

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.
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PMID:Exploring the temperature-pressure phase diagram of staphylococcal nuclease. 1019 32

Fluorescence resonance energy transfer (FRET) is one of the few methods available to measure the rate at which a folding protein collapses. Using staphylococcal nuclease in which a cysteine residue was engineered in place of Lys64, permitted FRET measurements of the distance between the donor tryptophan 140 and 5-[[2-[(iodoacetyl)-amino]ethyl]amino]naphthalene-1-sulfonic acid-labeled Cys64. These measurements were undertaken on both equilibrium partially folded intermediates at low pH (A states), as well as transient intermediates during stopped-flow refolding. The results indicate that there is an initial collapse of the protein in the deadtime of the stopped-flow instrument, corresponding to a regain of approximately 60% of the native signal, followed by three slower transients. This is in contrast to circular dichroism measurements which show only 20-25% regain of the native secondary structure in the burst phase. Thus hydrophobic collapse precedes the formation of substantial secondary structure. The first two detected transient intermediate species have FRET properties essentially identical with those of the previously characterized equilibrium A state intermediates, suggesting similar structures between the equilibrium and transient intermediates. The effects of anions on the folding of acid-unfolded staphylococcal nuclease, and urea on the unfolding of the resulting A states, indicates that in folding the protein becomes compact prior to formation of major secondary structure, whereas in unfolding the protein expands prior to major loss of secondary structure. Comparison of the kinetics of refolding of staphylococcal nuclease, monitored by FRET, and for a proline-free variant, indicate that folding occurs via two partially folded intermediates leading to a native-like species with one (or more) proline residues in a non-native conformation. For the A states an excellent correlation between compactness measured by FRET, and compactness determined from small-angle X-ray scattering, was observed. Further, a linear relationship between compactness and free energy of unfolding was noted. Formation of soluble aggregates of the A states led to dramatic enhancement of the FRET, consistent with intermolecular fluorescence energy transfer.
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PMID:Fluorescence energy transfer indicates similar transient and equilibrium intermediates in staphylococcal nuclease folding. 1084 64

The stability and folding kinetics of wild-type and a mutant staphylococcal nuclease (SNase) at neutral pH are significantly perturbed by the presence of moderate to high concentrations of salts. Very substantial increases in stability toward thermal and urea denaturation were observed; for example, 0.4 M sodium sulfate increased the free energy of wild-type SNase by more than 2 kcal/mol. For the NCA SNase mutant, the presence of the salts abolished the cold denaturation observed at neutral pH with this variant, and substantially increased its stability. Significant effects of salts on the kinetics of refolding were also observed. For NCA SNase, the presence of the salts markedly increased the folding rates (up to 5-fold). On the other hand, chloride, in particular, substantially decreased the rate of folding of the wild-type protein. Since the rates of the slow phases due to proline isomerization were increased by salt, these steps must be coupled to conformational processes. Fluorescence energy transfer between the lone tryptophan (Trp140) and an engineered fluorescent acceptor at residue 64 revealed that the addition of a high concentration of KCl led to the formation of a transient folding intermediate not observed at lower salt concentrations, and in which residues 140 and 64 were much closer than in the native state. The salt-induced effects on the kinetics of folding are attributed to the enhanced stability of the transient folding intermediates. It is likely that the combination of the high net charge, due to the high isoelectric point, and the relatively low intrinsic hydrophobicity, leads to staphylococcal nuclease having only marginal stability at neutral pH. The salt-induced effects on the structure, stability, and kinetics of staphylococcal nuclease are attributed to the binding of counterions, namely, anions, resulting in minimization of intramolecular electrostatic repulsion. This leads to increased stability, more structure, and greater compactness, as observed. Consequently, localized electrostatic repulsion is present at neutral pH in SNase, probably contributing to its marginal stability. The results suggest that, in general, marginally stable globular proteins will be significantly stabilized by salts under conditions where they have a substantial net charge.
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PMID:Effect of salts on the stability and folding of staphylococcal nuclease. 1132 80

The dipolar relaxation process induced by the excitation of the single tryptophan residue of four proteins (staphylococcal nuclease, ribonuclease-T1, phosphofructokinase, and superoxide dismutase) has been studied by dynamic fluorescence measurements. A new algorithm taking into account the relaxation effect has been applied to the fluorescence decay function obtained by phase-shift and demodulation data. This approach only requires that fluorescence be collected through the whole emission spectrum, avoiding the time-consuming determination of the data at different emission wavelengths, as usual with time-resolved emission spectroscopy. The results nicely match those reported in the literature for staphylococcal nuclease and ribonuclease-T1, demonstrating the validity of the model. Furthermore, this new methodology provides an alternative explanation for the complex decay of phosphofructokinase and human superoxide dismutase suggesting the presence of a relaxation process even in proteins that lack a lifetime-dependent spectral shift. These findings may have important implications on the analysis of small-scale protein dynamics, since dielectric relaxation directly probes a local structural change around the excited state of tryptophan.
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PMID:The recovery of dipolar relaxation times from fluorescence decays as a tool to probe local dynamics in single tryptophan proteins. 1294 Dec 97

Sugars are known to stabilize proteins. This study addresses questions of the nature of sugar and proteins incorporated in solid sugar films. Infrared (IR) and Raman spectroscopy was used to examine trehalose and sucrose films and glycerol/water solvent. Proteins and indole-containing compounds that are imbedded in the sugar films were studied by IR and optical (absorption, fluorescence, and phosphorescence) spectroscopy. Water is able to move in the sugar films in the temperature range of 20-300 K as suggested by IR absorption bands of HOH bending and OH stretching modes that shift continuously with temperature. In glycerol/water these bands reflect the glass transition at approximately 160 K. The fluorescence of N-acetyl-L-tryptophanamide and tryptophan of melittin, Ca-free parvalbumin, and staphylococcal nuclease in dry trehalose/sucrose films remains broad and red-shifted over a temperature excursion of 20-300 K. In contrast, the fluorescence of these compounds in glycerol/water solvent shift to the blue as temperature decreases. The fluorescence of the buried tryptophan in Ca-bound parvalbumin in either sugar film or glycerol/water remains blue-shifted and has vibronic resolution over the entire temperature range. The red shift for fluorescence of indole groups exposed to solvent in the sugars is consistent with the motion of water molecules around the excited-state molecule that occurs even at low temperature, although the possibility of static complex formation between the excited-state molecule and water or other factors is discussed. The phosphorescence yield for protein and model indole compounds is sensitive to the matrix glass transition. Phosphorescence emission spectra are resolved and shift little in different solvents or temperature, as predicted by the small dipole moment of the excited triplet state molecule. The conclusion is that the sugar film maintains the environment present at the glass formation temperature for surface Trp and amide groups over a wide temperature excursion. In glycerol/water these groups reflect local changes in the environment as temperature changes.
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PMID:Protein in sugar films and in glycerol/water as examined by infrared spectroscopy and by the fluorescence and phosphorescence of tryptophan. 1294 11

A continuous-flow mixing device with a dead time of 100 micros coupled with intrinsic tryptophan and 1-anilinonaphthalene-8-sulfonate (ANS) fluorescence was used to monitor structure formation during early stages of the folding of staphylococcal nuclease (SNase). A variant with a unique tryptophan fluorophore in the N-terminal beta-barrel domain (Trp76 SNase) was obtained by replacing the single Trp140 in wild-type SNase with His in combination with Trp substitution of Phe76. A common background of P47G, P117G and H124L mutations was chosen in order to stabilize the protein and prevent accumulation of cis proline isomers under native conditions. In contrast to WT(*) SNase, which shows no changes in tryptophan fluorescence prior to the rate-limiting folding step ( approximately 100 ms), the F76W/W140H variant shows additional changes (enhancement) during an early folding phase with a time constant of 75 micros. Both proteins exhibit a major increase in ANS fluorescence and identical rates for this early folding event. These findings are consistent with the rapid accumulation of an ensemble of states containing a loosely packed hydrophobic core involving primarily the beta-barrel domain while the specific interactions in the alpha-helical domain involving Trp140 are formed only during the final stages of folding. The fact that both variants exhibit the same number of kinetic phases with very similar rates confirms that the folding mechanism is not perturbed by the F76W/W140H mutations. However, the Trp at position 76 reports on the rapid formation of a hydrophobic cluster in the N-terminal beta-sheet region while the wild-type Trp140 is silent during this early stage of folding. Quantitative modeling of the (un)folding kinetics and thermodynamics of these two proteins versus urea concentration revealed that the F76W/W140H mutation selectively destabilizes the native state relative to WT(*) SNase while the stability of transient intermediates remains unchanged, leading to accumulation of intermediates under equilibrium conditions at moderate denaturant concentrations.
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PMID:Early events during folding of wild-type staphylococcal nuclease and a single-tryptophan variant studied by ultrarapid mixing. 1506 39

It is unclear whether the thermal denaturation of staphylococcal nuclease is a two state, three state, or variable two state process. The thermal denaturation of wild-type staphylococcal nuclease was followed by tryptophan fluorescence and circular dichroism signal at 222 nm, forty-two and fourteen times, respectively. Analysis of this data using a simple two state model gave melting temperatures of 53.0+/-0.4 degrees C (fluorescence) and 52.7+/-0.6 degrees C (CD) and van't Hoff enthalpies of 82.4+/-2.6 kcal/mol and 88.6+/-4.2 kcal/mol. Ninety-seven mutants also had these parameters determined by both fluorescence and CD. The average difference between the melting temperatures was 1.05+/-0.75 degrees and the average difference between van't Hoff enthalpies was 1.6+/-4.8 kcal/mol. These very similar results for the two spectroscopic probes of structure are discussed in the context of the different models that have been proposed for nuclease denaturation. It is concluded, for most nuclease variants, that the errors introduced by a two state assumption are negligible and either virtually all helical structure is lost in any initial unfolding event or any intermediate must have low stability.
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PMID:Thermal denaturations of staphylococcal nuclease wild-type and mutants monitored by fluorescence and circular dichroism are similar: lack of evidence for other than a two state thermal denaturation. 1713 19

Folding stability and cooperativity of the three forms of 1-110 residues fragment of staphylococcal nuclease (SNase110) have been studied by various biophysical and NMR methods. Samples of G-88W- and V-66W-mutant SNase110, namely G-88W110 and V-66W110, in aqueous solution and SNase110 in 2.0 M TMAO are adopted in this study. The unfolding transitions and folded conformations of the three SNase fragments were detected by far- and near-ultraviolet circular dichroism and intrinsic tryptophan fluorescence measurements. The tertiary structures and internal motions of the fragments were determined by NMR spectroscopy. Both G-88W and V-66W single mutations as well as a small organic osmolyte (Trimethylamine N-oxide, TMAO) can fold the fragment into a native-like conformation. However, the tertiary structures of the three fragments exhibit different degrees of folding stability and compactness. G-88W110 adopts a relatively rigid structure representing a most stable native-like beta-subdomain conformation of the three fragments. V-66W110- and TMAO-stabilized SNase110 produce less compact structures having a less stable "beta-barrel" structural region. The different folding status accounts for the different backbone dynamic and urea-unfolding transition features of the three fragments. The G-20I/G-29I-mutant variants of the three fragments have provided the evidence that the folding status is correlated closely to the packing of the beta-strands in the beta-barrel of the fragments. The native-like beta-barrel structural region acts as a nonlocal nucleus for folding the fragment. The tertiary folding of the three fragments is initiated by formation of the local nucleation sites at two beta-turn regions, I-18-D-21 and Y-27-Q-30, and developed by the formation of a nonlocal nucleation site at the beta-barrel region. The formation of beta-barrel and overall structure is concerted, but the level of cooperativity is different for the three 1-110 residues SNase fragments.
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PMID:Folding stability and cooperativity of the three forms of 1-110 residues fragment of staphylococcal nuclease. 1717 96

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.
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PMID:Folding kinetics of staphylococcal nuclease studied by tryptophan engineering and rapid mixing methods. 1733 34


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