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1.
Histone H5 has been labelled with fluorescein isothiocyanate (FITC) with particular attention to the reaction conditions (pH, reaction time and input FITC/H5 molar ratio) and to the complete elimination of non-covalently bound dye. We preferred to use reaction conditions which yielded non-specific uniform labelling rather than specific alpha-NH2 terminal labelling, in order to obtain higher sensitivity in further studies dealing with the detection of perturbation at the binding sites of H5 on DNA. FITC-labelled H5 was further characterized by absorption and circular dichroism spectroscopy, and the fluorescein probe titrated in the 4-8 pH range. The structural integrity of H5 was found to be preserved after labelling. The positive electrostatic potential of the environment in which the FITC probe is embedded in the arginine/lysine-rich tails of H5 is believed to be responsible for the drop of pK of 1 unit found for H5-FITC as compared to free FITC. For the globular part of H5, the pK of covalently-bound FITC was only slightly lowered; this is a consequence of the much lower content in positively-charged amino-acid side chains in this region. 相似文献
2.
Abstract The interaction of histone H5 labelled with fluorescein isothiocyanate (FITC) with DNA has been studied by fluorescence titration, and diffusion-enhanced fluorescence energy transfer (DEFET) measurements with Tb(III) lanthanide chelates as donors. Analysis of the binding data by the model of Schwarz and Watanabe (J. Mol. Biol. 163, 467-484 (1983)) yielded a mean stoichiometry of 60 nucleotides per H5 molecule, independently of ionic strength, in the range of 3 to 300 mM NaCl, at very low DNA concentration (6 μM in mononucleotide). It ensues an approximate electroneutrality of the saturated complexes. Histone H5 molecules appeared to be clustered along the DNA lattice in clusters containing on average 3 to 4 H5 molecules separated by about 79 base pairs, at mid-saturation of the binding sites. The interaction process was found highly cooperative but the cooperativity parameter was also insensitive to ionic strength in the above range. DEFET experiments indicated an important decrease of accessibility of the FITC label to the TbHED3A° and TbEDTA? chelates with ionic strength in the 0 to 100 mM NaCl range. In the presence of DNA, H5 appears already folded at low ionic strength so that the FITC probe is also not accessible to the donor chelate. The present study constitutes an indispensable preliminary step to further studies on the localization of histone H5 in condensed chromatin structures. 相似文献
3.
The interaction of histone H5 labelled with fluorescein isothiocyanate (FITC) with DNA has been studied by fluorescence titration, and diffusion-enhanced fluorescence energy transfer (DEFET) measurements with Tb(III) lanthanide chelates as donors. Analysis of the binding data by the model of Schwarz and Watanabe (J.Mol.Biol. 163, 467-484 (1983)) yielded a mean stoichiometry of 60 nucleotides per H5 molecule, independently of ionic strength, in the range of 3 to 300 mM NaCl, at very low DNA concentration (6 microM in mononucleotide). It ensues an approximate electroneutrality of the saturated complexes. Histone H5 molecules appeared to be clustered along the DNA lattice in clusters containing on average 3 to 4 H5 molecules separated by about 79 base pairs, at mid-saturation of the binding sites. The interaction process was found highly cooperative but the cooperativity parameter was also insensitive to ionic strength in the above range. DEFET experiments indicated an important decrease of accessibility of the FITC label to the TbHED3A and TbEDTA- chelates with ionic strength in the 0 to 100 mM NaCl range. In the presence of DNA, H5 appears already folded at low ionic strength so that the FITC probe is also not accessible to the donor chelate. The present study constitutes an indispensable preliminary step to further studies on the localization of histone H5 in condensed chromatin structures. 相似文献
4.
《Journal of receptor and signal transduction research》2013,33(1-4):195-221
AbstractTwo complementary approaches, covalent labelling and solubilization, have been used to study the biochemical properties of the central 5-HT1A receptor binding site. We have first designed a photoaffinity ligand containing the structure of 8-OH-DPAT, a potent and specific agonist of 5-HT1A sites. Thus, 8-methoxy-2[N-n-propyl,N-3-(2-nitro-4-azido-phenyl)- aminopropyl]aminotetralin or 8-methoxy-3'-NAP-amino-PAT, was found to displace, in the dark, [3H]8-OH-DPAT from 5-HT1A sites in rat hippocampal membranes with an IC50 of 6.6 nM. Under two cumulative UV irradiations (366 nm, for 20 min at 4°C), 8-methoxy-3-'-NAP-amino-PAT (30 nM) blocked irreversibly 55-60% of 5-HT1A binding sites. This blockade was specific of 5-HT1A sites since the other serotoninergic sites, 5-HT1B, 5-HT2 and also the presynaptic 5-HT3 sites were not affected by the treatment. In addition, the binding of [3H]Spiperone and [3H]7-OH-DPAT to striatal dopamine sites remained unchanged under similar photolysis conditions. The tritiated derivative of the photoaffinity ligand (92 Ci/mmol) was then synthesized for the identification of the covalently bound protein(s). SDS-PAGE of solubilized membranes irradiated in the presence of 20 nM 3H-8-methoxy-3'-NAP-amino-PAT allowed the detection of a 63 kD protein whose labelling appeared specific. Thus, 3H-incorporation into the 63 kD band could be prevented by uM concentrations of 5-HT, 8-OH-DPAT and other selective 5-HT1A ligands such as isapirone. In contrast, the 5-HT2 antagonist ketanserin, norepinephrine and dopamine-related ligands (including 7-OH-DPAT) were ineffective. Direct solubilization of 5-HT1A receptor binding sites was also attempted from rat hippocampal membranes. The best results were obtained using CHAPS (10 mM) plus NaCl (0.2 M), which led to 50 % recovery of 5-HT1A sites in the 100,000 g supernatant. The pharmacological properties and sensitivity to N-ethyl-maleimide and GppNHp of soluble sites appeared near identical to those of membrane-bound 5-HT1A sites. 相似文献
5.
Vacuolar proton pumping pyrophosphatase (H(+)-PPase; EC 3.6.1.1) plays a central role in the electrogenic translocation of protons from cytosol to the vacuole lumen at the expense of PP(i) hydrolysis. A fluorescent probe, fluorescein 5'-isothiocyanate (FITC), was used to modify a lysine residue of vacuolar H(+)-PPase. The enzymatic activity and its associated H(+) translocation of vacuolar H(+)-PPase were markedly decreased by FITC in a concentration-dependent manner. The inhibition of enzymatic activity followed pseudo-first-order rate kinetics. A double-logarithmic plot of the apparent reaction rate constant against FITC concentration yielded a straight line with a slope of 0.89, suggesting that the alteration of a single lysine residue on the enzyme is sufficient to inhibit vacuolar H(+)-PPase. Changes in K(m) but not V(max) values of vacuolar H(+)-PPase as inhibited by FITC were obtained, indicating that the labeling caused a modification in affinity of the enzyme to its substrate. FITC inhibition of vacuolar H(+)-PPase could be protected by its physiological substrate, Mg(2+)-PP(i). These results indicate that FITC might specifically compete with the substrate at the active site and the FITC-labeled lysine residue locates probably in or near the catalytic domain of the enzyme. The enhancement of fluorescence intensity and the blue shift of the emission maximum of FITC after modification of vacuolar H(+)-PPase suggest that the FITC-labeled lysine residue is located in a relatively hydrophobic region. 相似文献
6.
《Journal of receptor and signal transduction research》2013,33(2):159-169
AbstractHomogenates from dog cerebellum were fractionated using sucrose gradient centrifugation. The [3H]inositol 1, 4, 5-trisphosphate binding and the glucose 6-phosphatase activities were found to co-purify. The binding was saturable and had high affinity (Bmax=44 pmol/mg protein, Kd=116 nM). Selective chemical modification was used to examine amino acid residues of the microsomal receptor that might be critical for the binding of inositol trisphophate. Sulfhydryl reagents, p-chloromercuric-phenyl sulfonic acid, eosin 5-maleimide, N-ethyl maleimide and fluorescein 5-maleimide were found to be highly potent inhibitors of the binding with half-maximal inhibition occurring at about 20 µM, 70 µM, 1 mM, and 0.1 mM, respectively. The inhibition was specific since the presence of 10 µM of inositol trisphosphate during the reaction completely protected against the inhibition by these reagents. These results suggest that sulfhydryl group is essential for inositol trisphosphate binding to its receptor. 相似文献
7.
Fluorescein isothiocyanate (FITC) reactivity with the (Na+ + K+)-ATPase was studied at pH 6.5 and 9.0. Reaction with FITC is nearly complete in 30 min and is irreversible at both pH values. Differential inhibition of enzyme activity is observed at the two pH values as follows: at pH 6.5 the maximal inhibition reached is only 35–45% of the ATPase or p-nitrophenylphosphatase activities, whereas at pH 9.0 ATPase activity can be completely inhibited while maximal phosphatase inhibition is ca. 50%. At all concentrations of FITC tested, more FITC is incorporated into the enzyme at pH 9.0 than at 6.5. At both pH values NaCl increases the inhibition due to FITC while KCl protects against the inhibition. ATP protects the enzyme at both pH values with a K0.5 in the range of 8–20 μm. Enzyme that is partially inactivated at either pH shows no significant change in the K0.5 values for Na+ or K+ or in the Km app for ATP or p-nitrophenylphosphate for the remaining activity. The binding of 48VO4 is not changed by reaction with FITC at either pH, while [3H]ouabain binding is inhibited after reaction at pH 9.0 only in the presence of Mg+2 + Na+ + ATP. [3H]Ouabain binding in the presence of Mg+2 + inorganic phosphate is not inhibited by FITC reaction. Enzyme reacted at both pH values exhibits the expected fluorescein fluorescence (λex = 490, λem = 520) but only with enzyme reacted at pH 9.0 is fluorescence quenching by K+ or reversal by Na+ observed. These results suggest that different classes of amino groups react with FITC at the two pH values tested, and that these groups have distinct roles in the different activities of the enzyme. 相似文献
8.
《Biochimica et Biophysica Acta (BBA)/General Subjects》2020,1864(2):129313
BackgroundHemagglutinin (HA) of influenza A is one of the key virulence factors that mediates the release of viral components in host cells. HA is initially synthesized as a trimeric precursor (HA0) and then it is cleaved by proteases to become a functional HA. Low pH induces irreversible conformational changes in both HA0 and HA but only HA is fusion compatible. Here, we used high-speed atomic force microscopy (HS-AFM) to record conformational changes in HA0 trimers (H5N1) from neutral to acidic conditions at a millisecond scale.MethodsPurified HA0 protein was diluted with either neutral Tris-HCl (pH 7.4) or acetic acid-titrated Tris-HCl (pH 5.0) and then loaded onto bare mica. Neutral or acidic Tris-HCl was used as the scanning buffer. HS-AFM movies were recorded and processed using Image J software.ResultsThe conformation of HA0neutral visualized using HS-AFM was comparable to the HA trimer structures depicted in the PDB data and the AFM simulator. HA0 underwent rapid conformational changes under low pH condition. The circularity and area of HA0acid were significantly higher than in HA0neutral. In contrast, the height of HA0acid was significantly lower than in HA0neutral.ConclusionsWe have captured real-time images of the native HA0 trimer structure under physiological conditions using HS-AFM. By analyzing the images, we confirm that HA0 trimer is sensitive to acidic conditions.General significanceThe dynamic nature of the HA structure, particularly in the host endosome, is essential for H5N1 infectivity. Understanding this acidic behavior is imperative for designing therapeutic strategies against H5N1. This article reports a sophisticated new tool for studying the spatiotemporal dynamics of the HA precursor protein. 相似文献
9.
目的 阐明纳米二氧化锆暴露对人永生化角质形成细胞Ha Ca T组蛋白H3常见修饰位点的影响,探讨组蛋白H3修饰变化的潜在机制,为纳米材料的进一步安全应用提供理论基础。方法 在利用扫描电子显微镜、激光粒度仪、X射线衍射仪等技术对纳米二氧化锆进行详细表征的基础上,通过蛋白质免疫印迹及流式细胞术等方法评价纳米二氧化锆暴露对细胞生存率、细胞内蓄积量以及组蛋白H3修饰等的影响。结果 在分散介质中纳米二氧化锆明显团聚,比表面积减少,二次粒径增大,其短时间内(1 h)即诱导了组蛋白H3第10位丝氨酸的磷酸化、第9及14位赖氨酸的乙酰化、第4及27位赖氨酸的三甲基化修饰水平的升高。进一步分析发现,纳米二氧化锆的细胞内蓄积量及其引起的DNA损伤水平,与纳米二氧化锆诱导的组蛋白H3修饰水平均呈线性相关。结论 纳米二氧化锆暴露后诱导了Ha Ca T细胞组蛋白H3常见修饰位点的变化,其细胞内的蓄积是诱导组蛋白H3修饰变化的关键因素之一,且组蛋白H3修饰的调控机制可能涉及DNA损伤修复途径。 相似文献
10.
A. M. Cesura R. Imhof B. Takacs M. D. Galva G. B. Picotti M. Da Prada 《Journal of neurochemistry》1988,50(4):1037-1043
Abstract: [3H]Ro 16–6491 [N-(2-aminoethyl)-p-chloroben-zamide HCl], a reversible “mechanism-based” inhibitor of monoamine oxidase (MAO) type B, binds selectively and with high affinity to the active site of MAO-B in brain and platelet membranes. Under normal conditions, the binding of [3H]Ro 16–6491 is fully reversible. However, [3H]Ro 16–6491 could be irreversibly bound (covalently) to membranes by the addition of the reducing agent NaBH3CN to the sample and adjusting to pH 4.5 with acetic acid. No irreversible labelling occurred in the absence of NaBH3CN and at neutral pH. The presence of the irreversible MAO-B inhibitor /-deprenyl completely abolished the irreversible labelling of the membranes by [3H]Ro 16–6491. The selective inactivation of MAO-B, e.g., by /-deprenyl prevented the covalent incorporation of [3H]Ro 16–6491 whereas selective inhibition of the MAO-A by clorgyline was without effect. The covalent linkage to membranes of unlabelled Ro 16–6491 and Ro 19–6327 (a selective and reversible MAO-B inhibitor closely related to Ro 16–6491) after the addition of NaBH3CN at pH 4.5 irreversibly inactivated MAO-B activity whereas MAO-A activity was unaffected. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of labelled membranes showed that [3H]Ro 16–6491 was incorporated into a single polypeptide with a molecular mass identical to the one labelled by [3H]pargyline (58 kilodaltons). Our results indicate that the polypeptide that is covalently labelled by [3H]Ro 16–6491 corresponds to one of the two MAO-B subunits. Therefore, [3H]Ro 16–6491 represents a selective probe for affinity labelling of MAO-B and for the investigation of the structural composition of the active site of the enzyme. Whether the reduction with NaBH3CN at pH 4.5 of the [3H]Ro 16–6491-MAO-B complex results in the formation of a stable adduct with the amino acid chain of the MAO-B or with its prosthetic group, FAD, remains to be elucidated. 相似文献
11.
Emmanuelle Trévisiol Annabelle Renard Eric Defrancq Jean Lhomme 《Nucleosides, nucleotides & nucleic acids》2013,32(9):1427-1439
Abstract We describe the reaction of oligonucleotides containing an aldehydic group at the 5′-end or inside the sequence with an oxyamino label. The reaction was found to be highly selective and represents an efficient method for derivatization of oligonucleotides. 相似文献
12.
D. Yu. Lando S. G. Haroutiunian A. M. Kul′ba E. B. Dalian P. Orioli S. Mangani 《Journal of biomolecular structure & dynamics》2013,31(2):355-366
Abstract The theoretical approach to the calculation of the influence of selective binding of small ligands on DNA helix-coil transition has been described in the previous paper (Lando D.Yu., J. Biomol. Struct. Dyrt., (1994)). In the present paper that method is used for the study of DNA protonation and deprotonation in acidic and alkaline medium by theoretical analysis of pH effect on DNA heat denaturation. The mechanism of DNA protonation in acidic medium and pK values of nucleotides are well known. It gave us an opportunity to check the theory without any fitting of pK values. A good agreement between experimental and calculated functions Tm(pH) and ΔT(pH) (melting temperature and melting range width) obtained for acidic medium proved the validity of the theory. However, for alkaline medium there was not even qualitative agreement when the agreed-upon mechanism of deprotonation was considered. Looking into the cause of the discrepancy, we have studied the DNA melting for different mechanisms of deprotonation by calculation of Tm(pH) and ΔT(pH). As a result, it has been established that the discrepancy is due to deprotonation of bonded GC base pairs of helical DNA regions (pK= 11). It was shown that the early known protonation and newly found deprotonation of helical DNA essentially stabilised double helix in alkaline and acidic medium. 相似文献
13.
The amino acid sequence of an active site peptide from the H,K-ATPase of gastric mucosa 总被引:1,自引:0,他引:1
The gastric H,K-ATPase is an active transport protein that is responsible for the maintenance of a large pH gradient across the secretory canaliculus of the mammalian parietal cell. Acid secretion across these epithelial cell membranes is coupled to the potassium-stimulated hydrolysis of ATP catalyzed by H,K-ATPase, but the mechanism of coupling between ion transport and ATP hydrolysis is unknown. In order to investigate the enzymatic mechanism of this coupling, a peptide derived from the ATP binding site of H,K-ATPase has been purified and its amino acid sequence has been determined. The peptide was identified by the incorporation of a fluorescent probe, fluorescein 5'-isothiocyanate (FITC), into the active site before trypsin digestion of the protein. The labeling of the enzyme by FITC was associated with the irreversible inhibition of enzymatic activity, and both the labeling of the tryptic peptide and inhibition of activity were prevented when the reaction was performed in the presence of ATP. At 100% inhibition of activity, 3.5 +/- 1.6 nmol of FITC were incorporated per mg of protein. The amino acid sequence of the active site peptide is His-Val-Leu-Val-Met-Lys-Gly-Ala-Pro-Glu-Gln-Leu-Ser-Ile-Arg, and FITC reacts with the lysine. This sequence is very similar to sequences of fluorescein-labeled peptides from the ATP binding sites of Na,K-ATPase and Ca2+-ATPase, and suggests that the active site structures of these ion transport ATPases are similar. 相似文献
14.
Qi Zhang Dongyang He Long Xu Shikun Ge Jinquan Wang 《Preparative biochemistry & biotechnology》2020,50(8):788-793
AbstractIn order to evaluate the possibility of using IgY as the secondary antibody in immunoassay, specific IgY (1: 128,000) was generated by immunizing hens with mouse serum IgG purified by protein A column. IgY was extracted from egg yolk by polyethylene glycol 6000 (PEG-6000), and further purified using protein M affinity chromatography column. The purified IgY was conjugated with horseradish peroxidase (HRP) and fluorescein?isothiocyanate (FITC), in that order. The reactivity of conjugated antibodies was evaluated by ELISA, Western blot and Immunofluorescence, demonstrating that the obtained IgY was able to conjugate with enzymes, react with mouse primary IgG antibody, and subsequently amplify the antigen-antibody signals in different immune reaction conditions, in a comparable secondary effect to conventional goat anti-mouse IgG antibody. The obtained conjugated antibodies showed high stability in broad pH ranges (4–10; >70%) and high thermostability at 37?°C for 84?h (>85%). Despite the need to further consider and evaluate the industrial standardization and production process, our data provided the primary evidence that conjugated IgY antibodies can be used as a secondary antibody for broad immunological analysis. 相似文献
15.
Tsann-Long Su Kazuho Harada Kyoichi A. Watanabe 《Nucleosides, nucleotides & nucleic acids》2013,32(5):513-524
Abstract 7-Amino-6-substituted-1-(β-D-ribofuranosyl)pyrido [2,3–d]pyrimidine-2,4 (1H, 3H)-diones were prepared in good yields from 5-cyanouridine by application of a novel ring transformation reaction recently developed in our laboratory. Treatment of 3-benzyloxymethyl-2', 3'-O-isopropylidene-5'-O-trityl-5-cyanouridine with malononitrile, cyanoacetamide or ethyl cyanoacetate in base gave directly the pyridopyrimidine nucleosides bearing a CN, CONH2 and CO2 Et at C-6, respectively. The benzyloxymethyl and trityl protecting groups were removed by hydrogenolysis and the isopropylidene group by acid hydrolysis. 相似文献
16.
I C Mackenzie 《The American journal of anatomy》1975,144(2):127-136
Epidermal Langerhans cells may be identified by light microscopy by their strongly positive reaction following incubation for ATPase activity. Intact sheets of epidermis from mice killed at various time intervals following a single pulse label of H3-thymidine were incubated to demonstrate ATPase activity and subsequently processed for autoradiography. In specimens taken one hour after labelling, many basal keratinocytes were labelled but very few ATPase-positive dendritic cells. At subsequent time periods a few pairs of labelled ATPase-positive cells were found but individually labelled cells were not observed. The findings suggest that epidermal Langerhans cells form a very stable (labelling index less than 0.01%) self-replicating population which divides to maintain cell spacing during growth. No evidence was found for migration and interchange of Langerhans cells with the connective tissue, or for an origin of Langerhans cells by transformation of another cell type. 相似文献
17.
Zhang HX Jijakli H Courtois P Sener A Malaisse WJ 《Molecular and cellular biochemistry》2003,252(1-2):247-251
In pancreatic islets prepared from either normal or GK rats and incubated at either low (2.8 mM) or high (16.7 mM) D-glucose concentration, the labelling of both lipids and their glycerol moiety is higher in the presence of D-[1-14C]glucose than D-[6-14C]glucose. The rise in D-glucose concentration augments the labelling of lipids, the paired 14C/3H ratio found in islets exposed to both D-[1-14C]glucose or D-[6-14C]glucose and D-[3-3H]glucose being even slightly higher at 16.7 mM D-glucose than that found, under otherwise identical conditions, at 2.8 mM D-glucose. Such a paired ratio exceeds unity in islets exposed to D-[1-14C]glucose. The labelling of islet lipids by D-[6-14C]glucose is about 30 times lower than the generation of acidic metabolites from the same tracer. These findings indicate (i) that the labelling of islet lipids accounts for only a minor fraction of D-glucose catabolism in pancreatic islets, (ii) a greater escape to L-glycerol-3-phosphate of glycerone-3-phosphate generated from the C1-C2-C3 moiety of D-glucose than D-glyceraldehyde-3-phosphate produced from the C4-C5-C6 moiety of the hexose, (iii) that only a limited amount of [3-3H]glycerone 3-phosphate generated from D-[3-3H]glucose is detritiated at the triose phosphate isomerase level before being converted to L-glycerol-3-phosphate, and (iv) that a rise in D-glucose concentration results in an increased labelling of islet lipids, this phenomenon being somewhat more pronounced in the case of D-[1-14C]glucose or D-[6-14C]glucose rather than D-[3-3H]glucose. 相似文献
18.
K.-W. Hofmann H.-A. Arfmann J. Bode A. Arellano 《International journal of biological macromolecules》1980,2(1):25-32
In nucleosomal particles of 180 base pairs, part of the histone H5 binding site is preserved. After fluorescein labelling of H5 from chicken erythrocytes comparative equilibrium binding studies have been performed and on these particle as well as on core particles (140 base pairs) and on free DNA (180 base pairs). While nucleosomal particles can accommodate about the same number of H5 molecules as the free DNA derived from it, affinities are decreased by a factor of 3. A further decrease by factors of 3–4 is the consequence of phosphorylating three of the H5 serines: hence phosphorylation should facilitate thermodynamically controlled complexing of red cell chromatin during erythropoiesis. The most dramatic effect of an H5 phosphorylation is a reduction in the binding sites from 56 to 36 nucleotides (free DNA) and an even more pronounced effect upon interacting with nucleosomes which should make the H5-chromatin association sterically favourable. Related studies with protamines from herring are included for comparison. 相似文献
19.
We have synthesized a radioactive derivative of fluorescein isothiocyanate (PITC) by lactoperoxidase-catalyzed iodination of fluorescein amine using 125I. The iodinated amine was purified by thin-layer chromatography and converted to the isothiocyanate by reaction with thiophosgene. The product was inferred to be the diiodo derivative of FITC by comparing its absorbance and fluorescence emission spectra with those of known standards. This reagent, [125I]diI-FITC, shares many of the useful features of its congener, FITC. Specifically, it may be used to label under mild conditions of temperature and pH, and it is chemically stable. When erythrocytes were labeled with [125I]diI-FITC, radioactivity was found principally in a major exposed protein of the cell surface, and very little hemoglobin was labeled. [125I]diI-FITC may prove generally useful as a means of labeling proteins and cell surfaces to high specific radioactivity. 相似文献
20.
Daniel C.F. Chan Joelle Biard-Roche Claude Gorka Jean-Luc Girardet Jean-Jacques Lawrence Lawrence H. Piette 《Journal of biomolecular structure & dynamics》2013,31(2):319-332
Abstract Histone H5 contains three tryosines in the central, a polar region of the molecule. All three tryosines can be spin labeled at low ionic strength. When the central globular domain is folded at high ionic strength, only one tyrosine becomes accessible to the imidazole spin label. Spin labeling the buried tyrosines prevents the folding of the globular structure, which, in turn, affects the proper binding of the H5 molecule to stripped chromatin. Chromatin complexes reconstituted from such an extensively modified H5 molecule show a weaker protection of the 168 base pair chromatosome during nuclease digestion. However, when only the surface tyrosine of the H5 molecule is labeled, such a molecule can still bind correctly to stripped chromatin, yielding a complex very similar to that of native chromatin. Our data supports the idea that not just the presence of the linker histone H5, but the presence of an intact H5 molecule with a folded, globular central domain is essential in the recognition of its specific binding sites on the nucleosomes. Our data also show that during the chromatin condensation process, the tumbling environment of the spin label attached to the surface tyrosine in the H5 molecule is not greatly hindered but remains partially mobile. This suggests that either the labeled domain of the H5 molecule is not directly involved in the condensation process or the formation of the higher-order chromatin structure does not result in a more viscous or tighter environment around the spin label. The folded globular domain of H5 molecule serves in stabilizing the nucleosome structure, as well as the higherorder chromatin structure. 相似文献