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)

The dipole relaxational dynamics in the environment of a single tryptophan residue Trp-140 in staphylococcal nuclease was studied by time-resolved (multi-frequency phase-modulation) spectroscopy and selective red-edge excitation. The long-wavelength position of the fluorescence spectrum (at 343 nm) and the absence of red-edge excitation effects at 0 and 20 degrees C indicate that this residue is surrounded by very mobile protein groups which relax on the subnanosecond time scale. For these temperatures (0-20 degrees C) the steady-state emission spectra did not show the excitation-wavelength dependent shifts (red-edge effects) for excitation wavelengths from 295 to 308 nm; however, the anisotropy decay rate is slow (tens of nanoseconds). This suggests that the spectral relaxation is due to mobility of the surrounding groups rather than the motion of the tryptophan itself. The motions of the tryptophan surrounding are substantially retarded at reduced temperatures in viscous solvent (60% glycerol). The temperature dependence of the difference in position of fluorescence spectra at excitation wavelengths 295 and 305 nm demonstrate the existence of red-edge effect at sub-zero temperatures, reaching a maximum value at -50 degrees C, where the steady-state emission spectrum is shifted to 332 nm. The excitation and emission wavelength dependence of multi-frequency phase-modulation data at the half-transition point (-40 degrees C) demonstrates the existence of the nanosecond dipolar relaxations. At -40 degrees C the time-dependent spectral shift is close to monoexponential with the relaxation time of 1.4 ns.
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PMID:Intramolecular dynamics in the environment of the single tryptophan residue in staphylococcal nuclease. 825 66

Thermal denaturation of a staphylococcal nuclease mutant K78C, where lysine 78 is replaced by cysteine, was studied by circular dichroism (CD) and resonance energy transfer. CD spectra suggest that residual structures remain in the denatured state. Steady-state energy transfer from intrinsic tyrosines to a single and intrinsic tryptophan was measured at different temperatures. In the thermally-denatured state of K78C, there is still a substantial degree of energy transfer from tyrosine(s) to tryptophan, indicating residual structures in the denatured state. The cysteine residue in mutant K78C was labeled with a cysteine specific probe IAEDANS. Fluorescence decays of the tryptophan were measured to estimate distance distributions between Trp 140 and IAEDANS at position 78. Measurements were done as a function of temperature from 4 degrees C (native) to 65 degrees C (denatured) both with and without Ca2+ and inhibitor pdTp. Below 30 degrees C, the apparent distance distribution of both the ligand-free nuclease and the enzyme with bound pdTp can be adequately described by a Gaussian model. Above 40 degrees C, where the ligand-free nuclease but not the ternary complex begins to denature, two different populations are required to fit the data both with and without pdTp. One population has a compact structure and the other has an expanded structure. As temperature rises, the population of the expanded structure increases. At the highest temperature, the non-native compact structure is still the major form (60 to 70%). The overall thermally-denatured states of staphylococcal nuclease mutant K78C in the absence and presence of ligands are thus compact and heterogeneous.
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PMID:Compact thermally-denatured state of a staphylococcal nuclease mutant from resonance energy transfer measurements. 829 51

Three mutants of staphylococcal nuclease containing a tryptophan substitution have been examined in the full length (149 residues) protein and in a large fragment (residues 1 to 136). The large fragments are not in the native state and are a good model of the denatured state. However, these large fragments do show signs of residual structure that breaks down upon titration with guanidine hydrochloride. They share some similarities with what has become known as the molten globule state. The thermal unfolding of these mutant fragments was followed by tryptophan fluorescence. Tryptophan fluorescence was treated as an order parameter and analyzed to determine the order of the observed transition. The critical exponent of the order parameter as the transition temperature is approached is significantly higher than the value of 1/2 predicted by mean field theory for a second-order transition and is similar to that observed for the transition of the full length, wild-type, protein. This is strong evidence that the breakdown of this intermediate compact denatured state is a cooperative, first-order phenomenon.
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PMID:The phase transition between a compact denatured state and a random coil state in staphylococcal nuclease is first-order. 835 68

The role of proline in the stability and kinetics of folding of wild-type staphylococcal nuclease and its P117G, P117T, and P31A mutants was examined as a function of guanidinium thiocyanate (Gdn-SCN) concentration. Replacement of Pro-117 with Gly or Thr caused small increases in stability, whereas substitution of Pro-31 by Ala led to a small decrease in stability. The slopes of the plots of delta G against denaturant concentration (m) for the mutant proteins are significantly smaller than for the wild-type, suggesting a decrease in the solvent-accessible surface area of the denatured state relative to that of the wild-type. The rates of unfolding and refolding were monitored using tryptophan fluorescence. The kinetic traces for refolding in the presence of Gdn-SCN were triphasic for the wild-type protein and P31A but biphasic for P117G and P117T mutants. The slower phases were typically 10% of the total amplitude except in the transition region. The rates of the fastest and medium phases of the wild-type were essentially unaffected by the mutations. Double-jump experiments in which the protein was unfolded in a high concentration of denaturant for a short time period and then refolded to final Gdn-SCN concentrations near the Cm revealed a fast increase in fluorescence emission corresponding to formation of the native state, followed by a slower decrease with an amplitude that varied with the guanidine concentration and time of unfolding.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effect of proline mutations on the stability and kinetics of folding of staphylococcal nuclease. 844 12

The reversible unfolding of two dissimilar proteins, phosphoglycerate kinase from Bacillus stearothermophilus (PGK) and Staphylococcus aureus nuclease (SAN), was induced with two denaturants, urea and guanidinium chloride (GuHCl). For each protein, structural transitions were monitored by intrinsic fluorescence intensity changes arising from a unique tryptophan residue. In the case of SAN the single, native tryptophan residue was used, whereas for PGK two versions, one with a tryptophan at position 315 and one at 379, were constructed genetically. The resultant folding curves were analyzed by considering the change in the solvation free energy of internal amino acid residues as the denaturant concentration was varied. We derive the following simple relationship: -RT ln K = delta Gw + n delta Gs,m[D]/Kden. + [D]) where K is the equilibrium constant describing the distribution of folded and unfolded forms at a given denaturant concentration [D], delta Gw is the free energy change for the transition in the absence of denaturant, and n is the number of internal side chains becoming exposed. delta Gs,m and Kden. are constants derived empirically from the solvation energies of model compounds and represent the behavior of an average internal side chain between 0 and 6 M GuHCl and 0 and 8 M urea. For proteins of known structure these values can easily be derived, and for others, average values in guanidinium chloride (delta Gs,m = 0.775 kcal/mol and Kden. = 5.4 M) or urea (delta Gs,m = 1.198 kcal/mol and Kden. = 25.25 M) can be used in the analysis. Results show that the parameters n and delta Gw are independent of the denaturant used for all 12 transitions studied. This supports the hypothesis that the unfolding activity of urea and GuHCl can be accounted for by their effect on the solvation energy of amino acid side chains which are buried in the folded but exposed in the unfolded protein. This simple analytical treatment allows the "cooperativity" of protein folding to be interpreted in terms of the number of side chains becoming exposed to the solvent in a given step and allows accurate estimation of the free energy irrespective of the denaturant concentration needed to induce the transition.
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PMID:The energetics and cooperativity of protein folding: a simple experimental analysis based upon the solvation of internal residues. 847 98

To quantitate the contributions of the ionizable amino acids to the stability of the native state of staphylococcal nuclease, each of the 23 lysines, 5 arginines, 4 histidines, 12 glutamic acids, and 8 aspartic acids was substituted with both alanine and glycine. This collection of 104 mutant proteins was analyzed by guanidine hydrochloride (GuHCl) denaturation, using intrinsic tryptophan fluorescence to quantitate the equilibrium between native and denatured states. From the analysis of these data, each mutant protein's stability in the absence of denaturant (delta GH2O) and sensitivity to changes in denaturant concentration [mGuHCl = d(delta G)/d[GuHCl]] were obtained. Several general trends in these values suggest that electrostatic interactions make only a minor contribution to the net stability of this protein. For the residue pairs that form ten salt bridges and ten charged hydrogen bonds between side chains, no correlation was observed between the stability losses (delta delta G) accompanying alanine substitution of each member of the pair. Little or no significant correlation was found between the magnitude of the loss in stability and the local electrostatic potential calculated from the three-dimensional structure by numerical and model dependent solutions of the linearized Poisson-Boltzmann equation. The structural parameters which correlated most strongly with stability loss are measures of the extent of burial of the residue in the native structure, as was previously observed for alanine and glycine substitutions of large hydrophobic residues [Shortle et al. (1990) Biochemistry 29, 8033] and of the polar, uncharged residues [Green et al. (1992) Biochemistry 31, 5717]. These results suggest that the ionizable amino acids contribute to stability predominantly through packing and bonding interactions that do not depend on their electrostatic charge.
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PMID:Contributions of the ionizable amino acids to the stability of staphylococcal nuclease. 863 91

We have studied the equilibrium unfolding staphylococcal nuclease and two of its variants, V66W and V66W', over two perturbation axes (acid-induced unfolding as a function of urea concentration and urea-induced unfolding as a function of pH). The transitions were monitored by simultaneous measurements of circular dichroism and fluorescence. With this multidimensional array of data (2 perturbation axes and 2 signals), we present a strategy of performing a global analysis, over as many as 12 individual data sets, to test various models for the unfolding process, to determine with greater confidence the pertinent thermodynamic parameters, and to characterize unfolding intermediates. For example, wildtype nuclease shows a cooperative two-state transition with either urea or pH as denaturant, but the global fits are improved when the model is expanded to include a pH dependence of the urea m value or when two distinct classes of protonic groups are considered. The best fit for wild-type nuclease is with delta G degree 0,UN = 6.4 kcal/mol at pH 7, with the acid-induced unfolding being triggered by protonation of three to five carboxylate groups (with possible contribution from His121), and with the urea m = 2.5 kcal mol-1 M-1. V66W' lacks the last 13 amino acids on the C-terminus, has a tryptophan at position 66, has a predominantly beta-sheet structure, and is less stable than the wild type. For V66W', delta G degree 0,UN = 1.6 kcal/mol, m = 1.2 kcal mol-1 M-1, and there are two or three groups responsible for acid unfolding. V66W, a full-length mutant with two tryptophan residues, unfolds via a three-state mechanism: native reversible intermediate reversible unfolded. It appears that its beta-barrel subdomain retains structure in the intermediate state. Assuming that the unfolding of V66W' and the beta-barrel subdomain of V66W can be described by the same thermodynamic parameters, a global analysis enabled a description of the alpha subdomain of V66W with delta G degree 0,IN = 2.7 kcal/mol, mIN = 1.1 kcal mol-1 M-1, and with the acid unfolding being triggered by protonation of a single group. This group has a pKa around 6 in the unfolded state, suggesting that the state of protonation of a histidine residue may contribute significantly to the stability of V66W.
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PMID:Global analysis of the acid-induced and urea-induced unfolding of staphylococcal nuclease and two of its variants. 903 4

5-Hydroxytryptophan (5HW) and 7-azatryptophan (7AW) are analogue of tryptophan that potentially can be incorporated biosynthetically into proteins and used as spectroscopic probes for studying protein-DNA and protein-protein complexes. The utility of these probes will depend on the extent to which they can be incorporated and the demonstration that they cause minimal perturbation of a protein's structure and stability. To investigate these factors in a model protein, we have incorporated 5HW and 7AW biosynthetically into staphylococcal nuclease A, using a trp auxotroph Escherichia coli expression system containing the temperature-sensitive lambda cI repressor, Both tryptophan analogues are incorporated into the protein with good efficiency. From analysis of absorption spectra, we estimate approximately 95% incorporation of 5HW into position 140 of nuclease, and we estimate approximately 98% incorporation of 7AW, CD spectra of the nuclease variants are similar to that of the tryptophan-containing protein, indicating that the degree of secondary structure is not changed by the tryptophan analogues. Steady-state fluorescence data show emission maxima of 338 nm for 5HW-containing nuclease and 355 nm for 7AW-containing nuclease. Time-resolved fluorescence intensity and anisotropy measurements indicate that the incorporated 5HW residue, like tryptophan at position 140, has a dominant rotational correlation time that is approximately the value expected for global rotation of the protein. Guanidine-hydrochloride-induced unfolding studies show the unfolding transition to be two-state for 5HW-containing protein, with a free energy change for unfolding that is equal to that of the tryptophan-containing protein. In contrast, the guanidine-hydrochloride-induced unfolding of 7AW-containing nuclease appears to show a non-two-state transition, with the apparent stability of the protein being less than that of the tryptophan form.
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PMID:Biosynthetic incorporation of tryptophan analogues into staphylococcal nuclease: effect of 5-hydroxytryptophan and 7-azatryptophan on structure and stability. 907 Apr 51

In order to help determine the extent to which side chain interactions within the staphylococcal nuclease beta-barrel affect its global stability, a full set of point mutants was generated for residue 27. Intrinsic tryptophan fluorescence was monitored during solvent denaturation with guanidine hydrochloride (GuHCl) and was used to calculate DeltaGH2O unfolding and m values for each mutant. In the wild type protein, residue 27 is a tyrosine which is at the first position of a type I' beta-turn, and which participates in both hydrophobic interactions and side chain to side chain hydrogen bonding. The hydrophobicity of the mutant residue was found to be the dominant factor in determining global protein stability within this series of nuclease mutants.
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PMID:Stability studies of amino acid substitutions at tyrosine 27 of the staphylococcal nuclease beta-barrel. 931 53

We developed a recombinant DNA system to overexpress a fusion protein between the small, minimally soluble acute phase serum protein, serum amyloid A (SAA), and the bacterial enzyme staphylococcal nuclease (SN). This fusion protein is very soluble and is immunoreactive to polyclonal anti-SAA antibodies. Tryptophan fluorescence shows smooth denaturation curves for the fusion protein in guanidinium HCl or potassium thiocyanate. Fluorescence also indicates that only a single tryptophan residue (of the four present) is accessible to iodide quenching and, presumably, is exposed on the surface of the fusion protein. Circular dichroism (CD) shows a significant signal indicating alpha-helix, which can be attributed to the SAA portion of the molecule; these are the first CD spectral data available for SAA. pH titration shows persistence of helix domains for the fusion protein at pH 3.0, in contrast to the denaturation of SN under the same conditions. (The entire fusion protein shows a random coil pattern below pH 3.0.) By exploiting the structural and solubility properties of SN, this fusion protein has provided the first structural data about SAA-the precursor of the amyloid deposits in secondary amyloidosis. This fusion protein should be useful for further physical and physiologic studies of SAA.
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PMID:A fusion protein between serum amyloid A and staphylococcal nuclease--synthesis, purification, and structural studies. 953 22


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