Labelling of Histone H5 and Its Interaction with DNA. 1. Histone H5 Labelling with Fluorescein Isothiocyanate

Abstract

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 α-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.

FTTC-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 UTC was only slightly lowered; this is a consequence of the much lower content in positively-charged amino-acid side chains in this region.     

Labelling of histone H5 and its interaction with DNA. 2. Cooperative binding of histone H5 to DNA as probed by steady-state fluorescence and diffusion-enhanced energy transfer

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.     

A lysine residue involved in the inhibition of vacuolar H(+)-pyrophosphatase by fluorescein 5'-isothiocyanate

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.     

Specific labeling of the bovine heart mitochondrial phosphate carrier with fluorescein 5-isothiocyanate: roles of Lys185 and putative adenine nucleotide recognition site in phosphate transport

The amine/SH-modifying fluorescein 5-isothiocyanate (FITC) specifically labeled Lys(185) in the putative membrane-spanning region of the phosphate carrier from both the cytosolic and matrix sides of bovine heart mitochondria at 0 degrees C and pH 7.2, and the labeling inhibited the phosphate transport. Nonmodifying fluorescein derivatives having similar structural features to those of ADP and ATP (Majima, E., Yamaguchi, N., Chuman, H., Shinohara, Y., Ishida, M., Goto, S., and Terada, H. (1998) Biochemistry 37, 424-432) inhibited the specific FITC labeling and phosphate transport, but the nonfluorescein phenylisothiocyanate did not inhibit FITC labeling, suggesting that there is a region recognizing the adenine nucleotides in the phosphate carrier and that this region is closely associated with the transport activity. The phosphate transport inhibitor pyridoxal 5'-phosphate inhibited the specific FITC labeling, possibly due to competitive modification of Lys(185). In addition, FITC inhibited the ADP transport and specific labeling of the ADP/ATP carrier with the fluorescein SH reagent eosin 5-maleimide. Based on these results, we discuss the structural features of the phosphate carrier in relation to its transport activity.     

Modulation of histone H5-nucleosome interactions by H5 phosphorylation

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.     

Modification of actin with fluorescein isothiocyanate

Reaction of rabbit skeletal muscle G-actin at pH 8.5 with fluorescein isothiocyanate (FITC) resulted in incorporation of up to 1.20 mol FITC/mol actin. At pH 8.8, the level of incorporation was raised to 1.98 mol FITC/mol actin. When excited with ultraviolet light, the FITC-actin samples fluoresced strongly with an emission maximum near 517 nm. Tryptic digests of FITC-actin containing about 1.0 mol FITC/mol actin could be separated into a nonfluorescent 33.5 kDa trypsin-resistant core protein and a fluorescent pool of small peptides. Chromatography on DEAE-Bio-Gel or two-dimensional separation on cellulose TLC plates of the peptide pool revealed that FITC was highly selective in the site of its reaction with actin, resulting in a single highly fluorescent peptide after tryptic digestion. NH2-terminal and amino acid analyses demonstrated this peptide to be derived from residues 51 to 62, with Lys-61 proposed as the major FITC-sensitive site on actin. FITC-actin is similar to G-actin in gross conformation; circular dichroism spectra of actin before and after labelling are identical. FITC-actin is also able to interact strongly with deoxyribonuclease I. However, FITC-actin solution viscosities and fluorescence properties are not altered by the addition of KCl or MgCl2. Therefore, either a localized conformational change near Lys-61 or steric hindrance due to the FITC attached to Lys-61 blocks the polymerization of actin.     

The amino acid sequence of an active site peptide from the H,K-ATPase of gastric mucosa

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.     

Index to Subjects,Volume 5

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.     

Effect of H+ on the K+ activation of adenosine-5'-monophosphate aminohydrolase.

The activation of adenosine-5'-monophosphate aminohydrolase from rabbit skeletal muscle by H+ has been demonstrated. Evidence is presented which indicates that the binding of H+ and K+ is linked, in that the dissociation constant (KA) for K+ activation is reduced as the pH is lowered. Concomitantly, the pK of several enzyme functional groups is changed when K+ is added to a solution of enzyme. This change is pK results in an uptake or release of H+, depending on the pH, and shows that K+ interacts with the enzyme to achieve its effect. The uptake or release of H+ provides a simple method of following conformational changes in the enzyme following interaction of K+. The KD for K+ interaction monitored by following pH changes is the same within experimental error as that measured from kinetic data.     

Evaluation of fluorochromes for flow cytometric detection of Cryptosporidium parvum oocysts labelled by fluorescent in situ hybridization

Oligonucleotide probes specific to Cryptosporidium parvum (CRY1) were conjugated with a range of fluorochromes. The fluorescence after in situ hybridization (FISH) labelling of oocysts and controls was assessed. The objective was to determine the most suitable conjugate for FISH labelling, followed by analysis with a 488 nm laser flow cytometer. The most promising candidate was fluorescein isothiocyanate but only when linked to the CRY1 probe via an 18-carbon spacer arm consisting of six ethylene glycol moieties. The use of the spacer increased fluorescent signals fivefold compared with an equivalent probe in which the FITC was linked directly to the 5'-amino group of the DNA.     

The ATP/AMP binding site of pyruvate,phosphate dikinase: selective modification with fluorescein isothiocyanate

Pyruvate,phosphate dikinase from Propionibacterium shermanii is strongly inhibited by fluorescein 5'-isothiocyanate (FITC). The time course of inactivation is biphasic, but the dependence of the pseudo-first-order rate constants on the inhibitor concentration indicates the formation of a reversible complex with the enzyme prior to covalent modification. The substrate/product nucleotide pairs MgATP and MgAMP protected against inactivation, while in the absence of Mg2+, both the nucleotides were ineffective. Previously, an essential lysine at the ATP/AMP subsite of the enzyme from Bacteroides symbiosus had been implicated by use of the 2',3'-dialdehyde of AMP (oAMP) [Evans, C. T., Goss, N. H., & Wood, H. G. (1980) Biochemistry 19, 5809]. The inhibition by FITC was competitive with MgAMP, and a multiple inhibition analysis plot indicated that binding of oAMP and FITC was mutually exclusive. These observations suggest that FITC and oAMP bind at the nucleotide binding site and probably to the same reactive lysine that is modified by oAMP. With peptide mapping by high-performance liquid chromatography, FITC was found to be a suitable probe for isolating the peptide from the ATP/AMP subsite.     

pK changes of ionizable reporter groups as an index of conformational changes in proteins. A study of fluorescein-labelled ribonuclease A.

A chemical derivative of bovine pancreatic ribonuclease A (RNase A) has been prepared by reaction with fluorescein-isothiocyanate at pH 6. This derivative has a fluorescein group covalently attached to the alpha-amino group of the protein. The enzymic properties of the modified protein are similar to those of RNase A. It is shown that the pK of the fluorescein group can be used as an index of protein conformation to monitor structural changes in the protein. In this work, the binding of a specific inhibitor (cytidine 2'-monophosphate) to RNase A, the isomerization process occurring in RNase A around pH 6, and the thermal unfolding of RNase A, were studied by mean of the pK changes of the fluorescein group. The results obtained by this method are fully consistent with those obtained by other methods. It is proposed that using ionizable reporter groups and their changes in pK to monitor conformational changes in proteins may be a sensitive tool both in equilibrium and kinetic studies.     

Fluorescein conjugates as indicators of subcellular pH. A critical evaluation

Fluorescein and tetramethylrhodamine conjugates to protein or dextran were used to determine subcellular pH. The pH dependence of fluorescence of fluorescein isothiocyanate (FITC) conjugates could be described by a single proton dissociation (pK'a approximately 6.8). This allowed pH to be derived accurately from spectra using the simple Henderson-Hasslebach equation. FITC and TRITC conjugates were delivered to mouse macrophage lysosomes by pinocytosis. Lysosomal pH was then determined in several different ways. First, by direct matching of the subcellular fluorescence spectrum with calibration spectra obtained in free solution. Secondly, monensin was used to equilibrate internal and extracellular pH. Subcellular pH could then be determined by the relative increase in fluorescence of the FITC conjugate without loss of probe from the lysosomes. This allowed the calibration of pH dependence with the probe in situ. Finally, macrophages were permitted to pinocytose FITC and TRITC dextran conjugates simultaneously. pH could be determined from the ratio of emissions from the two dyes within the lysosomes. Each of these different methods gave a similar value for lysosomal pH (4.8 +/- 0.1).     

Effect of the labelling ratio on the photophysics of fluorescein isothiocyanate (FITC) conjugated to bovine serum albumin.

The non-linearity of the fluorescence emission on increasing the probe to protein ratio has long been regarded as problematic and has lead to the development of dyes to overcome this effect. One of the causes of this non-linear response can be ascribed to the overlap of the label's own absorption and emission spectra. At higher labelling ratios, this affords the possibility of a reasonably efficient energy migration pathway, thus reducing the observed quantum yield of the dye. In this work we study the photophysics of fluorescein isothiocyanate (FITC) when conjugated to bovine serum albumin (BSA) at different labelling ratios (in the range FITC : BSA 1 : 17-15 : 1) using both steady state and time-resolved fluorescence techniques where on going from under labelled to over labelled samples a decrease in the initial (and steady state) anisotropy is observed, accompanied by an increase in the complexity of the decay kinetics and a decrease in the average lifetime. The band structure, elucidated by synchronous scan fluorescence spectroscopy, is also found to change on increased labelling. These results can be applied to the study of protein conformation and were confirmed by the analysis of denaturing BSA using urea.     

The fluorescein isothiocyanate-binding site of the plasma-membrane H+-ATPase of Neurospora crassa

The mammalian (Na+,K+), Ca2+-, and (H+,K+)-ATPases contain a well-characterized lysine residue that reacts with fluorescein 5'-isothiocyanate (FITC); enzymatic activity is protected by ATP, suggesting that the residue is located in or near the nucleotide-binding domain. In this study, the plasma-membrane H+-ATPase of Neurospora crassa is also shown to be sensitive to FITC. The reaction occurs with pseudo first-order kinetics, has a pKa of 8.0, and is stimulated by Mg2+. Enzymatic activity is protected by MgADP with a Kd of 0.2-0.3 mM, close to the Ki with which MgADP serves as a competitive inhibitor of ATP hydrolysis. A tryptic peptide labeled with FITC in the absence, but not in the presence, of MgADP has been isolated and sequenced. The FITC-sensitive residue is Lys474, located in a region that exhibits significant homology with the mammalian cation-transporting ATPases.     

Dopa decarboxylase exhibits low pH half-transaminase and high pH oxidative deaminase activities toward serotonin (5-hydroxytryptamine)

Dopa decarboxylase (DDC) catalyzes not only the decarboxylation of L-aromatic amino acids but also side reactions including half-transamination of D-aromatic amino acids and oxidative deamination of aromatic amines. The latter reaction produces, in equivalent amounts, an aromatic aldehyde or ketone (depending on the nature of the substrate), and ammonia, accompanied by O(2) consumption in a 1 : 2 molar ratio with respect to the products. The kinetic mechanism and the pH dependence of the kinetic parameters have been determined in order to obtain information on the chemical mechanism for this reaction toward 5-hydroxytryptamine (5-HT). The initial velocity studies indicate that 5-HT and O(2) bind to the enzyme sequentially, and that D-Dopa is a competitive inhibitor versus 5-HT and a noncompetitive inhibitor versus O(2). The results are consistent with a mechanism in which 5-HT binds to DDC before O(2). The pH dependency of log V for the oxidative deaminase reaction shows that the enzyme possesses a single ionizing group with a pK value of approximately 7.8 that must be unprotonated for catalysis. In addition to an ionizing residue with a pK value of 7.9 similar to that found in the V profile, the (V/K)(5-HT) profile exhibits a pK value of 9.8, identical to that of free substrate. This pK was therefore tentatively assigned to the alpha-amino group of 5-HT. No titratable ionizing residue was detected in the (V/K)(O2) profile, in the pH range examined. Surprisingly, at pH values lower than 7, where oxidative deamination does not occur to a significant extent, a half-transamination of 5-HT takes place. The rate constant of pyridoxamine 5'-phosphate formation increases below a single pK of approximately 6.7. This value mirrors the spectrophotometric pK(spec) of the shift 420-384 nm of the external aldimine between DDC and 5-HT. Nevertheless, the analysis of the reaction of DDC with 5-HT under anaerobic conditions indicates that only half-transamination occurs with a pH-independent rate constant over the pH range 6-8.5. A model accounting for these data is proposed that provides alternative pathways leading to oxidative deamination or half-transamination.     

Structural dynamics and oligomeric interactions of Na+,K(+)-ATPase as monitored using fluorescence energy transfer.

The oligomeric nature of the purified lamb kidney Na+,K(+)-ATPase was investigated by measuring the fluorescence energy transfer between catalytic (alpha) subunits following sequential labeling with fluorescein 5'-isothiocyanate (FITC) and erythrosin 5'-isothiocyanate (ErITC). Although these two probes had different spectral responses upon reaction with the enzyme, our studies suggest that a sizeable proportion of their binding occurs at the same ATP protectable, active site domain of alpha. Fluorescence energy transfer (FET) from donor (FITC) to acceptor (ErITC) revealed an apparent 56 A distance between the putative ATP binding sites of alpha subunits, which is consistent with (alpha beta)2 dimers rather than randomly spaced alpha beta heteromonomers. In this work, methods were introduced to eliminate the contribution of nonspecific probe labeling to FET values and to determine the most probable orientation factor (K2) for these rigidly bound fluorophores. FET measurements between anthroylouabain/ErITC, 5'-iodoacetamide fluorescein (5'IAF)/ErITC, and TNP-ATP/FITC, donor/acceptor pairs were also made. Interestingly, none of these distances were affected by ligand-dependent changes in enzyme conformation. These results and those from electron microscopy imaging (Ting-Beall et al. 1990. FEBS Lett. 265:121) suggest a model in which ATP binding sites of (alpha beta)2 dimers are 56 A apart, and reside 30 A from the intracellular surface of the membrane contiguous with the phosphorylation domain.     

Fluorescence properties of free and protein bound fluorescein dyes. I. Macrospectrofluorometric measurements.

Excitation and emission properties of fluorescein derivatives were studied macrofluorometrically. Measurements were performed with solutions of various concentrations (0.07-100 microgram/ml) of free sodium fluorescein prepared from fluorescein diacetate (FDA), fluorescein isothiocyanate (FITC) and FITC bound to rabbit gamma-globulin. Both excitation and emission spectra as well as fluorescence intensities at constant excitation/emission wavelengths (496/515 nm) were recorded. The findings indicate that (1) FDA gives about twice the fluorescence intensity compared to equal concentrations of FITC. (2) The fluorescence properties of FITC upon excitation with blue light (lambda = 496 nm) are only slightly altered by the conjugation to rabbit gamma-globulin. (3) Considerable quenching due to conjugation could, however, be shown to occur upon UV excitation (lambda = 340 nm). (4) Fluorescence emission excited by visible blue light (496 nm) increases linearly to dye concentration in a range of 0.07-2.5 microgram/ml. Beginning at 5 microgram/ml (10-(5) M/1) all three compounds show a sharp decrease of fluorescence intensity with further increasing concentration. Practical aspects of these data for the immunofluorescence method are discussed.     

Electron acceptors of bacterial photosynthetic reaction centers. II. H+ binding coupled to secondary electron transfer in the quinone acceptor complex.

The photoreduction of ubiquinone in the electron acceptor complex (QIQII) of photosynthetic reaction centers from Rhodopseudomonas sphaeroides, R26, was studied in a series of short, saturating flashes. The specific involvement of H+ in the reduction was revealed by the pH dependence of the electron transfer events and by net H+ binding during the formation of ubiquinol, which requires two turnovers of the photochemical act. On the first flash QII receives an electron via QI to form a stable ubisemiquinone anion (QII-); the second flash generates QI-. At low pH the two semiquinones rapidly disproportionate with the uptake of 2 H+, to produce QIIH2. This yields out-of-phase binary oscillations for the formation of anionic semiquinone and for H+ uptake. Above pH 6 there is a progressive increase in H+ binding on the first flash and an equivalent decrease in binding on the second flash until, at about pH 9.5, the extent of H+ binding is the same on all flashes. The semiquinone oscillations, however, are undiminished up to pH 9. It is suggested that a non-chromophoric, acid-base group undergoes a pK shift in response to the appearance of the anionic semiquinone and that this group is the site of protonation on the first flash. The acid-base group, which may be in the reaction center protein, appears to be subsequently involved in the protonation events leading to fully reduced ubiquinol. The other proton in the two electron reduction of ubiquinone is always taken up on the second flash and is bound directly to QII-. At pH values above 8.0, it is rate limiting for the disproportionation and the kinetics, which are diffusion controlled, are properly responsive to the prevailing pH. Below pH 8, however, a further step in the reaction mechanism was shown to be rate limiting for both H+ binding electron transfer following the second flash.     

Site-specific fluorescent derivatization and liquid chromatographic-mass spectrometric characterization of long R(3) IGF-I for bioanalytical applications

Recombinant Long R(3) IGF-I was derivatized with fluorescein isothiocyanate (FITC) at a single location by careful selection of reaction conditions (i.e. pH, and FITC/protein amino group ratio). High-performance liquid chromatography (LC) and electrospray mass spectrometry (MS) were used to confirm the extent of fluorescein conjugation. The protein conjugate was isolated and subjected to cyanogen bromide (CNBr) cleavage, followed by LC-MS to determine the site of modification. The isolated species of Long R(3) IGF-I-FITC was labeled at the N-terminal Met residue. Recognition of this fluorescent analog by monoclonal anti-IGF-I was preserved, indicating its potential for immunodiagnostic applications.