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PubMed1984

Characterization of nuclear estradiol receptors released by micrococcal nuclease and deoxyribonuclease I.

Interaction of estradiol receptor with chromatin was probed by nuclease digestion of receptor-chromatin complex. The complex was formed by incubating partially purified receptor with chromatin. Micrococcal nuclease digestion of the complex released a 7S form of receptor which could be converted to 2.8S form by DNAase I. Digestion of the complex with DNAase I yielded different forms of receptors ranging from 7S to 2.8S depending on the digestion time. Receptor distribution was also examined by isolating nuclei form tissue pre-incubated with radioactive estradiol. Micrococcal nuclease digestion of receptor-filled nuclei released 7S, 5.5S and 3.5S forms of receptors. Collectively, these results indicate that the 7S nuclear receptor may have an associated chromatin fragment which is sensitive to DNAase I activity. The desirable features of receptor-chromatin method over conventional methods for studies relating to receptor interaction at the gene site are discussed.

Animals↗
PubMed1985

Characterization of the 4-hydroxytamoxifen (4-OHTAM) bound estrogen receptor of MCF-7 cells solubilized by micrococcal nuclease.

In order to get an insight into the molecular mechanism of antiestrogen action at the chromatin level, we characterized the physical-chemical properties of the chromatin fragments released by micrococcal nuclease digestion of nuclei isolated from MCF-7 cells previously exposed to [3H]4-OHTAM. The [3H]4-OHTAM bound solubilized fragments were characterized in a low ionic strength buffer and in a high ionic strength buffer without and with urea. The following parameters were determined: sedimentation coefficients (S) on a sucrose gradient, Stokes radii (Rs) by gel filtration on a Sephadex G-200 column and the binding ability to a DNA-cellulose column. The molecular weights (Mr) and frictional ratios (f/fo) were calculated from the S and Rs values. Following mild nuclease digestion, the solubilized [3H]4-OHTAM bound receptor sedimented as an abundant 6-7 S form and a less abundant approximately 12 S species. Increasing the extensiveness of digestion resulted in one receptor form sedimenting at 5.2 S, Rs = 7.25 nm and Mr = 155,000. About 45% of the applied receptor bound to a DNA-cellulose column could be eluted by a high salt concentrated buffer. Dissociation of the micrococcal nuclease solubilized receptor in 0.4 M KCl resulted in a smaller receptor form with a 4.9 S, Rs = 5.87 nm and Mr = 119,000. Further dissociation in the presence of 3 M urea resulted in a receptor with a 3.5 S, Rs = 5.78 nm and Mr = 83,000. These results suggested that the antiestrogen bound estrogen receptor in chromatin, is associated with a tightly bound protein component and with an additional less tightly bound protein, complexes with DNA.

Breast Neoplasms↗
PubMed1981

Micrococcal nuclease as a probe of DNA sequence organization and chromatin structure.

We have investigated micrococcal nuclease digestion of chromatin and purified DNA at the heat-shock locus 67B in Drosophila melanogaster. At early stages of the reaction a distinct set of fragments is generated, indicating the presence of preferential cleavage sites. These sites are also observed when purified recombinant plasmid DNA is used as the substrate, demonstrating that the sites are specified by the DNA sequence. At Drosophila locus 67B, prominent sites occur frequently, spaced approximately 200 bp apart, within the nontranscribed portions of the locus, but are generally not observed within the regions that are transcribed. In contrast, such sites are randomly distributed along the procaryotic plasmid pBR322. The results indicate that specific patterns of digestion of eucaryotic chromatin by micrococcal nuclease cannot be simply interpreted as the consequence of the nucleosome array. However, it is possible that the organization of eucaryotic DNA sequences detected by micrococcal nuclease bears a functional relationship to the organization of DNA by nucleosomes and, in fact, was so selected through evolution.

Animals↗
PubMed1982

Fractionation of chromatin, released by nuclease digestion, on ECTHAM-cellulose. Separation of active and inactive chromatin.

Chromatin released by two nucleases under various ionic conditions has been fractionated by chromatography on ECTHAM-cellulose. Mg2+ -soluble chromatin, which according to Gottesfeld and Partington is enriched in transcribed DNA sequences (Gottesfeld, J.M. and Partington, G.A., (1977) Cell 12, 953-962) and produced by DNAase II digestion at intermediate ionic strength, comprises material eluting from ECTHAM-cellulose at 80-100 mM Cl-, pH 6.8-7.0, whereas bulk, Mg2+ -insoluble chromatin comprises more tightly binding material. Free hnRNP particles elute at 30 mM Cl-, pH 6.8. Oligonucleosomes, which according to Dimitriadis and Tata are enriched in transcribed sequences (Dimitriadis, G.J. and Tata, J.R. (1980) Biochem. J. 187, 467-477) and produced by micrococcal nuclease digestion at physiological ionic strength, also elute predominantly at 80-100 mM Cl-, pH 6.8-7.0. When liver nuclei are digested with micrococcal nuclease at low ionic strength, the most rapidly released chromatin is enriched in nascent RNA and hnRNP particles, and binds weakly to ECTHAM-cellulose. More slowly solubilised chromatin, containing fewer hnRNP particles, binds much more strongly to ECTHAM-cellulose. In confirmation of results with mechanically sheared chromatin, the affinity of particular chromatin fractions is not dependent on the size of chromatin particles, rather it reflects the differing composition, and in particular the non-histone protein and hnRNP content, which, we propose, determines the conformation adopted by different chromatin fractions in the cation conditions used for elution from ECTHAM-cellulose.

Animals↗
PubMed1983

Effects of thyrotropin on thyroid chromatin. Enhanced sensitivity to micrococcal nuclease and increased nuclear protein phosphorylation.

Thyroid slices were incubated with or without TSH for 2 or 5 h. Nuclei were then prepared, subjected to mild digestion with micrococcal nuclease, and centrifuged at 1200 X g. The amount of DNA in 1200 X g supernatants was increased by TSH at 5 h, but not at 2 h. In parallel studies, thyroid slices were incubated with 32Pi and labeling of acid-soluble nuclear proteins was examined. TSH-dependent increases in labeling of histones H1 and H3, and of the high mobility group protein HMG 14, were observed at 2 h; however, there were no apparent changes in TSH-dependent labeling between 2 and 5 h, in nuclease-sensitive or in bulk chromatin. These results suggest that the observed TSH-dependent changes in the micrococcal nuclease-sensitivity of thyroid nuclear chromatin were not induced directly by changes in the phosphorylation of the histones or HMG 14.

Animals↗
PubMed1983

Ion competition and micrococcal nuclease digestion studies of spermidine-condensed calf thymus DNA. Evidence for torus organization by circumferential DNA wrapping.

Spermidine-condensed calf thymus DNA structures have been studied by ion competition using a sedimentation assay and by micrococcal nuclease digestion. Competitor ions Mg2+, Ca2+ and putrescine2+ show specific ion effects; but all three appear to affect the DNA condensation-decondensation equilibrium caused by spermidine3+ in a qualitatively similar manner, suggesting the spermidine3+-DNA interaction is largely electrostatic. Our data show a hysteresis in condensation and decondensation transition directions. We interpret this in terms of a kinetic block in the condensation direction with decondensation representing the equilibrium state of the system. These results agree with results obtained from related systems using different measurement techniques. Micrococcal nuclease digestion of spermidine-condensed calf thymus DNA produces broad but discrete bands in gel electrophoresis experiments. At least two bands determined to be 760 +/- 87 bp and 1355 +/- 135 bp, possess the size ratio 1:1.8 +/- 0.4 consistent with their forming the monomer and dimer fragments of an arithmetic band series. We rationalize this result in terms of a localized micrococcal nuclease cleavage model of circumferentially-wrapped DNA toruses proposed previously by Marx, K.A. and Reynolds, T.C. (Proc. Natl. Acad. Sci. (1982) 79, 6484-6488). The arithmetic series monomer band (760 +/- 87 bp), corresponding to wrapping B DNA once circumferentially about the torus, is in agreement with the electron microscopic measurements of hydrated calf thymus DNA torus circumferences presented by Marx, K.A. and Ruben, G.C. (Nucleic Acids Res. (1983) 11, 1839-1853).

Animals↗
PubMed1987

The use of micrococcal nuclease as a probe for drug-binding sites on DNA.

The cutting pattern produced by micrococcal nuclease on three DNA fragments has been determined in the absence and presence of various DNA-binding drugs. The enzyme itself cuts almost exclusively at pA and pT bonds, showing a greater activity at (A-T)n than in homopolymeric runs of A and T. Each drug produces distinct changes in the cleavage pattern. The protected regions can not be pinpointed with sufficient precision to assess the exact drug-binding sites on account of the sequence selectivity of the enzyme, although where a direct comparison is possible these include most of those seen as DNAase I footprints. The enzyme is most useful for assessing the selectivity of drugs which bind to AT-rich regions. Several drugs protect the DNA from micrococcal nuclease attack in regions which do not contain their acknowledged best binding sites. It appears that micrococcal nuclease is sensitive to the existence of secondary drug-binding sites which are not evident with other footprinting techniques.

Base Sequence↗
PubMed1995

Random mutagenesis of staphylococcal nuclease and phage display selection.

Multiple cycles of mutagenesis and phage display selection have been investigated as a method for obtaining enzymes with altered catalytic properties. A library of staphylococcal nuclease mutants displayed on phage was created by error-prone PCR mutagenesis and selected for binding to thymidine- or guanosine-containing substrate analogs. After discarding non-binders, the binding mutants were then subjected to further mutagenesis and selection rounds. After four mutagenesis and selection cycles, the catalytic properties of some of the resulting nucleases were studied and one nuclease with nine accumulated mutations was found to have a two-fold reduction in kcat for DNA hydrolysis, but a two-fold increase in kcat/Km for hydrolysis of a thymidine containing small molecule substrate. The possibility of this technique for in vitro evolution of enzyme properties is discussed.

Amino Acid Sequence↗
PubMed1993

NMR analysis of the residual structure in the denatured state of an unusual mutant of staphylococcal nuclease.

BACKGROUND: Staphylococcal nuclease is a well-developed model system for analyzing the effects of mutations on protein folding and stability. Substitution of glycine 88 with valine (Gly88Val) destabilizes staphylococcal nuclease by 1.0 kcal mole-1 and reduces its sensitivity to the denaturant guanidine hydrochloride, a phenomenon which may indicate an increase in residual structure in the denatured state. To assess its effects on denatured state structure, the Gly88Val mutation was incorporated into a 136 residue nonsense fragment which has been developed as a model of the wild type denatured state. RESULTS: Application of two- and three-dimensional NMR spectroscopy to the Gly88Val fragment uniformly labeled with 15N and 13C has led to the assignment of 93 of the 136 residues. Comparison of chemical shifts of backbone resonances to those of wild type native nuclease, analysis of the secondary shifts of the assigned resonances and nuclear Overhauser effects involving backbone protons indicate that, unlike the wild type fragment, most if not all of the five-stranded beta-barrel structure persists in this denatured state. CONCLUSION: One major effect of the Gly88Val mutation is to perturb the cooperative breakdown of the folded conformation, leading to a denatured state which is both more ordered and more stable than that formed by the wild type sequence. Since the equilibrium between the native and denatured states depends on the free energy difference between them, stabilization of the denatured state by the Gly88Val mutation indirectly destabilizes the native state.

Amino Acid Sequence↗
PubMed2004

Replacement of staphylococcal nuclease hydrophobic core residues with those from thermophilic homologues indicates packing is improved in some thermostable proteins.

The importance of tight hydrophobic core packing in stabilizing proteins found in thermophilic organisms has been vigorously disputed. Here, portions of the cores found in three thermophilic homologues were transplanted into the core of staphylococcal nuclease, a protein of modest stability. Packing of the core was evaluated by comparing interaction energy of the three mutants to the comprehensive mutant library built up previously at these same sites in staphylococcal nuclease. It was found that the interaction energy of one thermophilic sequence is extraordinarily favorable and the interaction energies of other two transplanted thermophilic sequences are good, comparable to the interaction energies of mutant cores based on cores found in mesophilic homologues. As expected when transferring just a portion of the core sequence, the mutant proteins were destabilized overall relative to wild-type staphylococcal nuclease. The overall conclusion is that improvement of packing interactions is a mechanism to confer stability employed in some proteins from thermophiles, but not all.

Amino Acids↗
PubMed1989

Fluorescence lifetime studies with staphylococcal nuclease and its site-directed mutant. Test of the hypothesis that proline isomerism is the basis for nonexponential decays.

Using frequency domain methods, the fluorescence decay of Trp-140 in staphylococcal nuclease and its site-directed mutant (Pro-117----Gly) has been examined. Based on nuclear magnetic resonance (NMR) studies (Evans, P. A., C. M. Dobson, R. A. Kautz, G. Hatfull, and R. O. Fox. 1987. Nature [Lond.]. 329:266-268), it is believed that nuclease exists in two macroscopic, native conformations and that the slow interconversion of these conformations is controlled by the cis----trans isomerization of Pro-117. The above mutant shows only one native conformation in NMR experiments. To test the hypothesis that the biexponential fluorescence decay of Trp-140 of nuclease can also be related to the existence of these conformational states of the protein, we have compared the decay patterns of the wild type and mutant. Essentially no difference was observed, which indicates that there is some other basis for the nonexponential decay of Trp-140. We have used global nonlinear least squares analysis to link the fit of data at several temperatures.

Binding Sites↗
PubMed2003

Development of a 5' nuclease assay to detect ciprofloxacin resistant isolates of the biowarfare agent Yersinia pestis.

The detection of antibiotic resistance in agents of biowarfare is extremely critical in order to start appropriate therapy in a timely manner. We have developed a 5' nuclease assay to detect ciprofloxacin resistance (Cip(r)) in Yersinia pestis. Two groups of fluorogenic probes were developed. The first group included a probe homologous to the wild type Y. pestis gyrA sequence with two corresponding probes that were homologous with two different mis-sense mutations in codon 81 of GyrA. The second group of probes included a wild type probe and two corresponding probes that recognized mis-sense mutations in codon 83 or gyrA. These probes specifically reacted only with the homologous DNA sequences. The 5' nuclease assay was sensitive to 1 pg (approximately 1 colony forming unit) of starting template and could be used on semi-purified DNA. We tested our optimized assay against a group of 38 Cip(r) Y. pestis isolates and demonstrated that it was accurate at determining the gyrA allele encoded by these strains. The 5' nuclease assay performed on the LightCycler proved to be sensitive, rapid and accurate at identification of Cip(r) Y. pestis and thus should be useful in characterization of this organism in the event of a future act of bioterrorism.

Animals↗
PubMed1995

The equilibrium folding pathway of staphylococcal nuclease: identification of the most stable chain-chain interactions by NMR and CD spectroscopy.

In a previous report [Alexandrescu, A. T., Abeygunawardana, C., & Shortle, D. (1994) Biochemistry 33, 1063-1072], NMR methods were used to characterize the residual structure in delta 131 delta, a large fragment of staphylococcal nuclease that serves as a model denatured state under nondenaturing conditions. On the basis of a large number of missing amide protons for the residues that form a three-strand antiparallel beta sheet in the native state, it was concluded that this beta meander may be highly populated in delta 131 delta, with severe line broadening due to relatively slow exchange between different conformational states. In the present report, results from circular dichroism spectroscopy and NMR spectroscopy indicate strands beta 2-beta 3 form a beta hairpin at urea concentrations below 6 M. Amide proton resonances from several hydrophobic residues adjacent to this beta hairpin disappear in concert with all of the beta 2-beta 3 residues, suggesting a local, non-native hydrophobic interaction may help stabilize the beta hairpin. At concentrations below 3 M, all amide resonances from strand beta 1 in delta 131 delta also disappear, suggesting that beta 1 may combine with the beta 2-beta 3 hairpin to form a native-like beta meander. In addition, the hydrophobic helix alpha 2 decreases from approximately 30% population in 0 M urea to approximately 10%-15% at 6 M urea, whereas helix alpha 1 goes from 10%-15% populated in 0 M urea to undetectable in 6 M urea. Characterization of a second, distinctly different denatured state, WT nuclease at pH 3.0 and low salt, reveals that this low-density acid-denatured state is structurally similar to delta 131 delta at low concentrations of urea. From these and previously published data, a tenative equilibrium folding pathway can be constructed for staphylococcal nuclease which describes the relative strengths and interdependencies of the chain-chain interactions involved in forming the native state.

Amino Acid Sequence↗
PubMed2001

Effect of salts on the stability and folding of staphylococcal nuclease.

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.

Amino Acid Substitution↗
PubMed1992

Contributions of the polar, uncharged amino acids to the stability of staphylococcal nuclease: evidence for mutational effects on the free energy of the denatured state.

In order to quantitate the contributions of the polar, uncharged amino acids to the stability of the native state of staphylococcal nuclease, each of the 13 alanines, 9 glycines, 9 threonines, 6 prolines, 6 glutamines, 6 asparagines, and 3 serines was substituted, either with both alanine and glycine or with 1 of these 2 amino acids plus valine. For each mutant, the stability to reversible denaturation (delta GH2O) was quantitated by determining the Kapp for this reaction as a function of guanidine hydrochloride concentration. In addition, the parameter mGuHCl (= d(delta G)/d[GuHCl]) was calculated from the data. To identify the local structural features responsible for the relatively large and variable changes in delta GH2O and mGuHCl observed for the same type of substitution at different locations in nuclease, statistical correlations were sought between delta GH2O, mGuHCl, and a number of descriptors of the local structure. As with substitutions of the large hydrophobic amino acids [Shortle, D., Stites, W. E., & Meeker, A. K. (1990) Biochemistry 29, 8033-8041], mutation of polar, uncharged residues to Gly leads to a change in stability that, on average, correlates well with the degree to which the wild-type residue is buried. This correlation is especially significant for threonine, an amino acid with both polar and hydrophobic character, but is not demonstrated for the more typically hydrophobic residue alanine. As reported in the previous study of alanine/glycine substitutions of hydrophobic residues, a significant correlation between changes in stability and changes in the value of mGuHCl is again observed, strengthening the conclusion that the putative structural changes in the denatured state which lead to increases or decreases in mGuHCl are responsible for a significant fraction of the stability loss for an average mutant. The existence of this correlation is consistent with the denatured state of wild-type staphylococcal nuclease having evolved to a relatively high free energy via optimization of a balance between a maximal exposure of hydrophobic surface and a minimal gain in chain entropy. On average, mutations are less stable in proportion to the extent of which they perturb this balance. A new and puzzling correlation is reported between the extent of buriedness of a residue in the wild-type native state versus the difference in mGuHCl between the Ala mutation and the Gly mutation at that position.

Alanine↗
PubMed1992

Compact denatured state of a staphylococcal nuclease mutant by guanidinium as determined by resonance energy transfer.

The protein from a mutant clone of staphylococcal nuclease with a cysteine substituting for a lysine at position 78 was prepared and labeled with a cysteine-specific fluorescent probe 5-[[2-[(iodoacetyl)-amino]ethyl]amino]naphthalene-1-sulfonic acid (IAEDANS). Time-resolved nonradiative energy-transfer studies were done using the single tryptophan at position 140 as the energy donor and the IAEDANS as the receptor. Changes in distance and distance distributions were observed as a function of increasing guanidinium (GuHCl) concentration (0-2 M) and in the presence or absence of Ca2+ and inhibitor 2'-deoxythymidine 3',5'-diphosphate (pdTp). In the native state, both the ternary complex and the noncomplexed protein are best fit with one population having an average donor-acceptor distance of approximately 23 A and an "apparent" full width at half-maximum (fwhm) of distance distribution of approximately 18 A. Besides the contribution of linker arm of the acceptor, it appears that there are some conformational heterogeneties either due to the disordering of the tryptophan region or due to the whole protein in the native state. During GuHCl unfolding, the average distance remains relatively constant up to GuHCl concentrations where both the ternary complex and the ligand-free protein are denatured (1-1.3 M). The compact denatured states persist up to 2 M GuHCl. At 2 M GuHCl, the heterogeneity of the denatured state in the ternary complex is much larger than that of the ligand-free nuclease. The results show that the denatured states of staphylococcal nuclease mutant K78C by GuHCl are compact and these compact denatured states are likely due to residual structures or incompletely disrupted hydrophobic cores under these conditions.

Amino Acid Sequence↗
PubMed1994

Structure and dynamics of a denatured 131-residue fragment of staphylococcal nuclease: a heteronuclear NMR study.

A partially folded form of staphylococcal nuclease has been obtained by deleting residues 4-12 and 141-149 of the 149-residue wild-type protein. Sequence-specific NMR resonance assignments have been obtained for 106 of the 131 residues in this protein fragment by using multi-dimensional triple resonance NMR of samples enriched with 13C and 15N. Residues corresponding to helix 2 (residues 98-106) and helix 1 (residues 54-68) of the native state give chemical shifts and NOE effects characteristic of helical structure. These same residues, however, give coupling constants and NOE effects indicative of fast conformational averaging between helical and extended conformations. The residual helix structure observed in the nuclease fragment is thus considerably less persistent than the corresponding structure in the native state. Based on H alpha chemical shifts, we estimate the fractional population of helical conformers to be 30% for helix 2 and 10% for helix 1. Two segments, 83-86 and 94-97, show NOE effects, coupling constants, and lowered amide temperature coefficients consistent with a native-like reverse-turn structure. The C-terminal alpha-helix as well as the fourth and fifth strands of the 5-strand beta-barrel show little evidence for ordered structure. The first three strands of the beta-sheet, part of the catalytic loop, and the first turn of helix 3 give significantly poorer NMR data than the rest of the protein, possibly as a result of exchange broadening, and could not be characterized in detail. That the most persistent elements of structure in the fragment are native-like suggests that nuclease may fold by a hierarchical mechanism.

Amides↗
PubMed1994

Mapping staphylococcal nuclease conformation using an EDTA-Fe derivative attached to genetically engineered cysteine residues.

Six single cysteine variants of staphylococcal nuclease were reacted with the iron complex of (EDTA-2-aminoethyl) 2-pyridyl disulfide (EPD-Fe) [Ermácora, M. R., Delfino, J. M., Cuenoud, B., Schepartz, A., & Fox, R. O. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 6383-6387] and used to assess the ability of this cleavage reagent to faithfully report on the structure of nonnative protein states. The act of mutation and modification did not significantly alter the protein's global structure, as measured by CD and enzymatic activity, and only modestly affected its stability. The reaction was conformation dependent and generated specific cleavage products that mapped tertiary interactions present in the folded state. Several parameters relevant to the cleavage reaction and its use as a conformational probe were analyzed. Proximity and solvent accessibility are the most important parameters in determining the cleavage pattern and can be used to predict cleavage sites in the native protein. The cleavage reaction can be performed in the presence of high denaturant concentration, in the presence of SDS, and under a wide range of pH values; thus it can readily be applied to the study of equilibrium folding intermediates. Mass spectrometric analysis combined with N-terminal sequencing identified cleavage products consistent with a single cleavage event per protein molecule and revealed one cleavage mechanism which was not previously considered for protein oxidative degradation, although it was reported for hydroxyl radical induced cleavage of small peptides. Identification of the cleavage sites obtained from each variant allowed a nearest-neighbor mapping of the secondary structural elements of nuclease. Quantitation of specific cleavage products was used to monitor the disruption of the interaction between helices H2 and H3 in equilibrium unfolding experiments. The resulting unfolding curve revealed a local conformational heterogeneity at low denaturant concentration which was not observed when the same transition was monitored by the change in fluorescence of a single nuclease tryptophan. Interestingly, the midpoint of the transition and the second half of the unfolding curve were the same, as monitored by the two probes. This indicates that the lifetime of the reactive oxygen species generated by the cleavage reagent is short compared to the unfolding equilibrium rate constants and that the cleavage technique identifies a native-like folding intermediate not detected by fluorescence. The experiments presented herein demonstrate that EPD-Fe-mediated protein cleavage is an appropriate technique for the study of nonnative protein structure.(ABSTRACT TRUNCATED AT 400 WORDS)

Computer Graphics↗