Mutations can cause large changes in the conformation of a denatured protein
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Deletion of 13 amino acids from the carboxyl terminus of staphylococcal nuclease (WTSNase delta) results in a denatured, partially unfolded molecule that lacks significant persistent secondary structure but is relatively compact and monomeric under physiological conditions [Shortle & Meeker (1989) Biochemistry 28, 936-944; Flanagan et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 748-752]. Because of these and other properties of the SNase delta polypeptide, it is a useful model system for investigating the conformation of the denatured state of a protein without using extreme temperature or solvent conditions. Moreover, since the modification is a carboxyl-terminal deletion, SNase delta may also resemble a transient state of the polypeptide chain as it emerges from a ribosome prior to its folding. In the present study, we have examined the sizes and conformations of mutated forms of SNase delta, using small-angle X-ray scattering and circular dichroism spectroscopy. Seven mutated forms were studied: four with single substitutions, two with double substitutions, and one triple substitution. When present in the full-length SNase, each of these mutated forms exhibited unusual behavior upon solvent or thermal denaturation. In the case of the truncated form (SNase delta), the small-angle scattering curves of the mutated forms fall into two classes: one resembling the scattering curve of compact native nuclease and the other having features consistent with those expected for an expanded coil-like polymer. In contrast, the scattering curve of WT SNase delta exhibits features intermediate between those observed for globular proteins and random polymers. The amino acid substitutions that gave rise to compact, native-like versions of SNase delta were all of the m--type (m-substitutions are predicted to decrease the size of the denatured state). Those which gave rise to versions of SNase delta that were more extended and coil-like than WT SNase delta were of the m+ type (m+ substitutions are predicted to increase the size of the denatured state). Estimates of the residual secondary structure present in WT SNase delta, as well as both the m+ and m-substituted versions of SNase delta, as determined by CD, suggest that the formation of secondary structure and compaction of the polypeptide chain occur concurrently. Our results show that single amino acid substitutions can radically alter the conformational distribution of a partially condensed polypeptide chain.(ABSTRACT TRUNCATED AT 400 WORDS)Keywords:
Micrococcal nuclease
Random coil
Denaturation (fissile materials)
Folding (DSP implementation)
Small-angle X-ray scattering
Actively transcribed regions of chromatin are more susceptible than bulk chromatin to digestion by nucleases, and useful information about the composition and structure of active chromatin may be obtained by studying the chromatin fragments released from nuclei by limited nuclease digestion. In the present study, we have used micrococcal nuclease to investigate the effects of TSH on protein phosphorylation in nucleasesensitive fractions of calf thyroid chromatin. Batches of calf thyroid slices were incubated for 2 h with 32Pi, with or without 50 mU/ml TSH. Nuclei were then prepared and the distribution of 32P-labeled histones, high mobility group(HMG) proteins, and other acid-soluble phosphoproteins between micrococcal nuclease-sensitive and resistant fractions of chromatin was examined. TSH increased the amount of 32P incorporated into HMG 14 and the histones H1 and H3. Hormone-dependent increases in the 32P-labeling of H1 and H3 were not selectively associated with micrococcal nuclease-sensitive chromatin. In contrast, [32P] HMG-14 was preferentially solubilized from nuclei by micrococcal nuclease. This lends support to the view that TSH-induced effects on the structure and function of transcriptionally active chromatin may be mediated in part by phosphorylation of HMG 14.
Micrococcal nuclease
Nuclease
Non-histone protein
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Small-angle X-ray scattering
Characterization
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Small-angle X-ray scattering
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Drying and denaturation kinetics of aqueous droplets of α-lactalbumin (α-lac), β-lactoglobulin (β-lg), and bovine serum albumin (BSA) were measured in a convective drying environment. Single droplets having an initial droplet diameter of 2 ± 0.1 mm and containing 10% (w/v) protein concentration were dried using conditioned air (65 and 80 °C, 2-3% RH, 0.5 m/s velocity) for 600 s. The denaturation of these proteins was measured by using reversed-phase HPLC. At the end of 600 s of drying 13.3 and 19.4% α-lac was found to be lost due to denaturation at 65 and 80 °C, respectively. Up to 31.0% of β-lg was found to be denatured, whereas BSA was not found to be significantly (p > 0.05) denatured in these drying conditions. The formation and strength of skin and the associated morphological features were found to be linked with the degree of denaturation of these proteins. The secondary structure of these proteins was significantly (p < 0.05) affected and altered by the drying stresses. The β-sheet and random coil contents were increased in α-lac by 6.5 and 4.0%, respectively, whereas the α-helix and β-turn contents decreased by 5.5 and 5.0%, respectively. The β-sheet and random coil contents in β-lg were increased by 7.5 and 2.0%, respectively, whereas the α-helix and β-turn contents decreased by 3.5 and 6.0%, respectively. In the case of BSA the β-sheet, α-helix, and random coil contents were found to increase, whereas the β-turn content decreased.
Denaturation (fissile materials)
Random coil
Bovine serum albumin
Alpha-lactalbumin
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Structural and functional organization of the 5' region of the SUP35 gene was analyzed in Saccharomyces cerevisiae yeast. Indirect DNA end labeling allowed two nuclease-hypersensitive sites and a region involved in nucleosomes to be revealed. DNase I and micrococcal nuclease hypersensitive sites were localized to almost the same regions: -461 ... -372 bp and -271 ... -91 bp for DNase I and -461 ... -356 bp and -231 ... -79 for micrococcal nuclease. Nucleosomes were localized to a region +22 ... +339 bp. Both the location of DNase I and micrococcal nuclease hypersensitive sites within the promoter region and the location of nucleosomes within the coding region remain the same at different cell culture growth phases. However, positioning nucleosomes were revealed within the SUP35 coding region only at the late logarithmic phase; the radioautographic pattern of them does not depend on the extent of nuclease digestion.
Micrococcal nuclease
Nuclease
Hypersensitive site
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Journal Article Sequence specific cleavage of DNA by micrococcal nuclease Get access Wolfram Hörz, Wolfram Hörz Institut für Physiologische Chemie, Physikalische Biochemie und Zellbiologie der Universität MünchenGoethestrasse 33, 8000 München 2, GFR Search for other works by this author on: Oxford Academic PubMed Google Scholar Werner Altenburger Werner Altenburger Institut für Physiologische Chemie, Physikalische Biochemie und Zellbiologie der Universität MünchenGoethestrasse 33, 8000 München 2, GFR Search for other works by this author on: Oxford Academic PubMed Google Scholar Nucleic Acids Research, Volume 9, Issue 12, 25 June 1981, Pages 2643–2658, https://doi.org/10.1093/nar/9.12.2643 Published: 25 June 1981 Article history Received: 23 April 1981 Published: 25 June 1981
Micrococcal nuclease
Nuclease
Cleave
Nucleoprotein
Cleavage (geology)
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Little is known about the mechanisms of action of polypeptide hormones on chromatin structure and nuclear function. We have employed micrococcal nuclease to examine the effect of TSH on the accessibility of DNA in thyroid nuclei. Brief digestion of nuclear suspensions with 0.05-0.2 U/ml micrococcal nuclease at 26–-28 C decreased their opacity at 600 nm. The decrease in opacity was linear with increasing nuclear concentration up to 0.2 mg/ml DNA. This response to nuclease was enhanced in nuclear suspensions prepared from thyroid slices that had been incubated with TSH (50 mU/ml) for 5 h (P < 0.001). To determine whether TSH also increased the digestion of DNA, we measured the amount of DNA released into 1200 × g supernatants by nuclease treatment of nuclei prepared from control and TSH-treated slices. When TSH-treated nuclei (110 μg/100μl) were digested with 0.2 U micrococcal nuclease/ml at 37 C for 30 sec, a mean of 12.6 μg ± 3.6 (SD) DNA appeared in the upernatant, as compared to 8.4 /×g± 1.9 DNA from control nuclei (P < 0.05). This increase in the sensitivity of nuclear DNA to microcpccal nuclease may reflect some conformational change in chromatin in response to TSH. Since micrococcal nuclease sensitivity may reflect transcriptional competence of DNA, we speculate that polypeptide hormones may enhance the accessibility of DNA to RNA polymerase or to endogenous stimulators of transcription.
Micrococcal nuclease
Nuclease
Nuclear DNA
Digestion
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Small-angle X-ray scattering
Divinylbenzene
Morphology
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Endogenous nuclease is present in the nuclear sap of chicken erythrocyte nuclei. This enzyme resembles the nuclease of mammalian nuclei in requirements for bivalent cations and in production of large chromatin fragments that gradually decrease in size, but differs in that the products do not go through the stage of discrete bands on gel electrophoresis. Endogenous nuclease and micrococcal nuclease are also detectable in mononucleosomes prepared from chicken erythrocytes with the aid of micrococcal nuclease. Both nucleases are extractable with 0.35 M NaCl, and both are inhibited by pTp. In the absence of Ca2+, the micrococcal nuclease is totally inactive, whereas the endogenous nuclease shows a low level of activity.
Micrococcal nuclease
Nuclease
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This chapter focuses on methodological developments aiming to interpret small-angle X-ray scattering (SAXS) data for topological structure characterization of large biomolecular assemblies. It also focuses on developments on modeling large protein complexes that can adopt a single conformation or exist in a mixture of multiple conformations. In particular, conformation generation from large-scale computations provides a solid theoretical foundation for SAXS data interpretation. In the midst of broadened SAXS applications, the emerging potential of a SAXS analysis for visualizing the protein topology of biomolecular complexes is apparent, especially when already known structures of individual components are productively used in theoretical and computational studies designed for SAXS data analysis. In fact, it is counter-intuitive that the sample preparation needed for a SAXS measurement could be more stringent when compared to crystallographic requirements given that crystallization itself is a highly efficient purification process.
Small-angle X-ray scattering
Characterization
Small-Angle Scattering
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