The process in which nucleotide is buried into the active site of heavy meromyosin

The process in which nucleotide is buried into the active site of heavy meromyosin was studied with stopped-flow apparatus by monitoring the time-course of the large fluorescence increase of 1,N6-ethenoadenosine triphosphate (epsilon-ATP) when it binds from acrylamide-containing solutions. We have recently reported that free epsilon-ATP fluorescence is effectively quenched by acrylamide while bound epsilon-ATP is resistant to quenching by acrylamide. In the present study it was found that in the first step the phosphate moiety binds at a high rate, while the adenine moiety is still on the rim of the active site; the adenine moiety is then pulled into a crevice, and finally epsilon-ATP hydrolysis occurs.     

A catalytic site of protein disulfide isomerase probed with adenosine-5'-triphosphate analogs

Anthraniloyl adenosine-5'-triphosphate (Ant-ATP) and etheno-adenosine-5'-triphosphate (epsilon-ATP) complexed to Mg(2+) ions are substrates of protein disulfide isomerase (PDI). epsilon-ATP, coordinated to Tb(3+) ions, was used as a probe of the ATPase binding site. Sensitized luminescence arising from resonance energy transfer from epsilon-adenine to Tb(3+) is quenched by PDI. The luminescence results are discussed in reference to a model in which the distance of separation between epsilon-adenine (donor) and Tb(3+) (acceptor) is increased upon binding of PDI. The interaction of a small peptide of 14 amino acid residues with the b/b' domain of the protein does not influence the ATPase activity. The phosphorescence, fluorescence and fluorescence anisotropy of bound epsilon-ATP are not perturbed by the binding of the small molecular weight peptide to PDI. It is suggested that the peptide and ATP do not share a common binding site on the b/b' domain.     

Fluorescence energy transfer between points in G-actin: the nucleotide-binding site, the metal-binding site and Cys-373 residue

Fluorescence energy transfers were studied in order to investigate the spatial relationships between the nucleotide-binding site, the metal-binding site and the Cys-373 residue in the G-actin molecule. When 1-N6-ethenoadenosine-5'-triphosphate (epsilon-ATP) in the nucleotide-binding site and Co2+ or Ni2+ in the metal-binding site were used as fluorescence donor and acceptor, respectively, the fluorescence intensity of epsilon-ATP was perfectly quenched by Ni2+ or Co2+. This indicated that the nucleotide-binding site is very close to the metal-binding site; the distance should be less than 10 A. When N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS) bound to Cys-373 residue and Co2+ in the metal-binding site were used as a fluorescence donor and an acceptor, respectively, the transfer efficiency was equal to 5 +/- 1%. The corresponding distance was calculated to be 23-32 A, assuming a random orientation factor K2 = 2/3.     

Biotin binding changes the conformation and decreases tryptophan accessibility of streptavidin

Biotin binding reduces the tryptophan fluorescence emissions of streptavidin by 39%, blue shifts the emission peak from 333 to 329 nm, and reduces the bandwidth at half height from 53 to 46 nm. The biotin-induced emission difference spectrum resembles that of a moderately polar tryptophan. Streptavidin fluorescence can be described by two lifetime classes: 2.6 nsec (34%) and 1.3 nsec (66%). With biotin bound, lifetimes are 1.3 nsec (26%) and 0.8 nsec (74%). Biotin binding reduces the average fluorescence lifetime from 1.54 to 0.88 nsec. Biotin does not quench the fluorescence of indoles. The fluorescence changes are consistent with biotin binding causing a conformational change which moves tryptophans into proximity to portions of streptavidin which reduce the quantum yield and lifetimes. Fluorescence quenching by acrylamide revealed two classes of fluorophores. Analysis indicated a shielded component comprising 20–28% of the initial fluorescence with (K_SV+V)0.55 M^–1. The more accessible component has a predominance of static quenching. Measurements of fluorescence lifetimes at different acrylamide concentrations confirmed the strong static quenching. Since static quenching could be due to acrylamide binding to streptavidin, a dye displacement assay for acrylamide binding was constructed. Acrylamide does bind to streptavidin (Ka=5 M^–1), and probably binds within the biotin-binding site. In the absence of biotin, none of streptavidin's fluorescence is particularly accessible to iodide. In the presence of biotin, iodide neither quenches fluorescence nor alters emission spectra, and acrylamide access is dramatically reduced. We propose that the three tryptophans which always line the biotin site are sufficiently close to the surface of the binding site to be quenched by bound acrylamide. These tryptophans are shielded from iodide, most probably due to steric or ionic hindrances against diffusion into the binding site. Most of the shielding conferred by biotin binding can be attributed to the direct shielding of these residues and of a fourth tryptophan which moves into the binding site when biotin binds, as shown by X-ray studies (Weberet al., 1989).     

Transient kinetics of the interaction of 1,N6-ethenoadenosine 5'-triphosphate with myosin subfragment 1 under normal and cryoenzymic conditions: a comparison with adenosine 5'-triphosphate

The kinetics of the interaction of the fluorescent analogue 1,N6-ethenoadenosine 5'-triphosphate (epsilon-ATP) with myosin subfragment 1 (S1) were studied at 15 and -7.5 degrees C with 40% ethylene glycol as cryosolvent. Two techniques were used: fluorescence stopped flow and rapid flow-quench. When S1 is mixed with epsilon-ATP in a stopped-flow apparatus, biphasic fluorescence transients are obtained which are difficult to assign. Chemical sampling by the rapid-flow-quench method led to the chemical identity and the kinetics of interconversion of key intermediates, and by this method the optical signals were assigned and information about the cleavage and release of products was obtained. The data were interpreted by a shortened form of the Bagshaw-Trentham scheme for myosin adenosinetriphosphatase: M + ATP K1 in equilibrium M.ATP k2----M*.ATP k3 in equilibrium k3 M**.ADP.Pi k4----M + ADP + Pi The constants obtained were compared with those for ATP under identical conditions. In agreement with Rosenfeld and Taylor [Rosenfeld, S. S., & Taylor, E. W. (1984) J. Biol. Chem. 259, 11920-11929] we find that epsilon-ATP is bound tightly to S1 and that the chemical step is slower than with ATP. We show that the fast fluorescence transient is due to the tight binding of epsilon-ATP with K1 = 32 microM and k2 = 58 s-1 at 15 degrees C. With ATP these values are 8 microM and 16 s-1, respectively. There is a large difference in the delta H for k2: 50 kJ.mol-1 for epsilon-ATP and 119 kJ.mol-1 for ATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism for nucleotide exchange in monomeric actin

Rabbit skeletal muscle G-actin has been treated to obtain ADP, 1,N6-ethenoadenosine diphosphate (epsilon-ADP), or 1,N6-ethenoadenosine triphosphate (epsilon-ATP) at the nucleotide binding site and either Mg2+ or Ca2+ at high- and moderate-affinity metal binding sites. Apparent rates or rate constants for the displacement of the actin-bound nucleotides by epsilon-ATP or ATP have been obtained by stopped-flow measurements at pH 8 and 20 degrees C of the fluorescence difference between bound and free epsilon-ATP or epsilon-ADP. In the presence of Ca2+, displacement of ADP by epsilon-ATP or epsilon-ADP by ATP is a biphasic process, but in the presence of low (less than 10 microM) Mg2+ concentrations, it is a slow first-order process. At high levels of Mg2+ (greater than 50 microM), low ADP concentrations displace epsilon-ATP from G-actin as a consequence of Mg2+ binding to moderate-affinity sites on the actin. Displacement of epsilon-ATP by ATP in the presence of either Ca2+ or Mg2+ is slow at low ATP concentrations, but the rate is increased by high ATP concentrations. Using ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, we find that nucleotide exchange is affected differently by the removal of Ca2+ from the high-affinity site compared to Ca2+ removal from moderate-affinity sites. A mechanism for the displacement reaction is proposed in which there are two forms of an actin-ADP complex and metal binding influences the ratio of these forms as well as the binding of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescence lifetime reference standards for the range 0.189 to 115 nanoseconds

A review of the modern literature reveals that the values for quinine fluorescence lifetime are in good agreement, the mean value being 18.91±0.56 nsec. By virtue of some very unusual properties, quinine appears suitable for use as a lifetime reference standard for any value from 0.189 to 18.9 nsec, with an expected accuracy of ±3% throughout this range. Cl^?, not normally considered a quenching agent, quenches quinine emission at diffusion-controlled rates. The Stern-Volmer plot was unique in that the strict lincarity, indicating pure collisional quenching, was maintained even when fluorescence was > 99% quenched. Thus, solutions of quinine-NaCl can be made up having lifetimes known to great accuracy. Similarly, γ-pyrenebutyrate solutions containing KI are suitable standards for the range up to 115 nsec. The compositions of such solutions have been calculated and tabulated. It is argued that the lifetimes of these solutions are at least as reliable as any of the hundreds of lifetimes which have been reported in the literature. Several important applications of such lifetime standards are discussed.     

Dehydration of the lipid-protein microinterface on binding of phospholipase A2 to lipid bilayers

A novel method is described to demonstrate inaccessibility to the bulk aqueous phase of the microinterface between pig pancreatic phospholipase A₂ and lipid bilayers to which this protein is bound. The method is based on the fact that the fluorescence emission quantum yields of the tryptophan residue of the protein and of a 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) chromophore attached to a lipid are lower in water as compared to that in deuterated water. The fluorescence emission quantum yield of these chromophores is measured in water and in deuterated water under conditions where the protein is either bound or not bound to the surface of a lipid bilayer containing the dansyl chromophore. Under conditions where the protein is tightly bound to the surface of the bilayer, desolvation of both fluorophores abolishes the observed effect of deuterated water. The tryptophan residue in the bound phospholipase A₂ also becomes inaccessible to fluorescence quenching by acrylamide or succinimide. Desolvation of the microinterface is observed only under conditions that are significant for the catalytic action of phospholipase A₂ in the scooting mode and not in the hopping mode. Also, under similar conditions, binding of pro-phospholipase A₂ to anionic vesicles does not cause dehydration of the microinterface. The mechanistic significance of these observations for lipid-protein interactions, in general, and for interfacial catalysis and interfacial activation, in particular, is discussed.     

Fluorescence of human liver alanine aminopeptidase.

Fluorescence of human liver alanine aminopeptidase has been attributed to tryptophan fluorescence. The fluorescence maximum is at 330 nm, 20 nm lower than that for free tryptophan, suggesting that most of the enzyme tryptophans are in a nonpolar environment and are shielded from solvent. Quenching of enzyme fluorescence by iodide, pyridine, and N-methyl nicotinamide also demonstrates that enzyme tryptophan residues are largely buried and inaccessible to solvent. Those accessible are in negatively charged environments. 8-(1'-dimethylaminonaphthalene-5'-sulfonylamido-octanoic acid (8-DNS-octanoic acid) and epsilon-DNS-L-Lys inhibit aminopeptidase. One molecule of inhibitor when bound to the enzyme quenched 57% and 63% of enzyme fluorescence, respectively. Such efficient quenching may indicate a degree of segregation of tryptophan toward the active center.     

Characterization of the binding domain of the beta-adrenergic receptor with the fluorescent antagonist carazolol. Evidence for a buried ligand binding site

The antagonist carazolol has been used as a fluorescent probe for the binding site of the beta-adrenergic receptor (beta AR). The fluorescence properties of carazolol are dominated by the emission of the carbazole group, with the fine structure of the spectrum, but not the quantum yield, sensitive to the environment of the probe. The fluorescence emission spectrum of the bound probe is consistent with an extremely hydrophobic environment in the binding site of the receptor. Binding of carazolol to the purified beta AR increases the polarization of the fluorophore. Exposure to collisional quenchers has demonstrated the bound carazolol to be completely inaccessible to the solvent. Furthermore, the fluorescence of bound carazolol is not quenched by exposure to sodium nitrite, a F?rster energy acceptor which has an R0 value of 11.7 A with carazolol. Thus, physical analysis of the binding site of the beta AR by carazolol fluorescence indicates that the antagonist binds to the beta AR in a rigid hydrophobic environment which is buried deep within the core of the protein.     

The environment of the high-affinity cation binding site on actin and the separation between cation and ATP sites as revealed by proton NMR and fluorescence spectroscopy

The lanthanide ions Lu3+ (diamagnetic) and Gd3+ (paramagnetic broadening probe) were used to displace Ca2+ from the high-affinity cation binding site on G-actin. The effects of these higher-affinity ions on the proton nuclear magnetic resonance spectrum of actin were recorded. The aliphatic proton envelope in the Gd-actin sample exhibited a complex array of changes due to the proximity of Gd to several aliphatic residues. No such changes were observed in the diamagnetic Lu-actin control spectrum. By contrast, the aromatic proton envelope remained largely unaffected in both Gd-actin and Lu-actin samples. However, the adenosine moiety on the actin-bound ATP became increasingly mobilized without the triphosphate chain being released from the ATP binding site. Maximum adenosine mobilization occurred with approximately 1 mol of lanthanide ion bound per mol of actin. The absence of changes in the aromatic proton envelope suggests that the high-affinity cation binding site is in a region well removed from the adenosine moiety of bound ATP as well as any aromatic side-chains. The separation of the ATP and cation sites was further explored using the fluorescent ATP analogues FTP and epsilon-ATP. Tb3+ bound to the high-affinity cation site was found to be separated by 16 A from the FTP chromophore bound to the nucleotide binding site on actin. Since this distance is greater than can be accommodated on a model of the Tb-ATP complex, we conclude that the sites are physically separate. This conclusion was further reinforced by experiments involving the quenching of epsilon-ATP fluorescence by Mn2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Susceptibilities of phospholipid membranes containing cholesterol or ergosterol to gramicidin and its derivative incorporated in lysophospholipid micelles

Complex formation of gramicidin (GA) and desformylgramicidin (des-GA) with sterols was investigated by measuring the intrinsic Trp fluorescence. In organic solvents, the Trp fluorescence of momeric GA was quenched upon binding either cholesterol or ergosterol, but that of monomeric des-GA was not quenched by adding cholesterol. Both dimeric GA and des-GA bound highly to ergosterol, but not to cholesterol, determined by quenching of Trp fluorescence. Furthermore, GA- and des-GA-loaded lysophosphatidylcholine micelles were incubated with phosphatidylcholine vesicles containing cholesterol or ergosterol. The results showed that both monomeric and dimeric peptides hardly bound to cholesterol incorporated into phospholipid vesicles, but markedly bound to ergosterol incorporated into the bilayer membranes. Interestingly, des-GA bound more specifically to the two sterols than GA. In addition, fluorescence resonance energy transfer analysis showed that des-GA bound more specifically to the two sterol than GA.     

Sequence-selective DNA binding to the regulatory subunit of cAMP-dependent protein kinase

The fluorescence of Trp-226 in the regulatory subunit of bovine type II cAMP-dependent protein kinase is unaffected by the binding of cAMP, but is quenched by the binding of 2'-dansyl-cAMP (DNS-cAMP). Up to 67% of the fluorescence of Trp-226 can be quenched by resonant energy transfer to the DNS-cAMP bound to the first site, and 96% of the fluorescence can be quenched by saturating both sites with DNS-cAMP. The observed efficiencies of energy transfer gave a distance of 16 A between Trp-226 and the DNS-cAMP bound at the first site and a distance of 12.7 A between Trp-226 and the DNS-cAMP bound at second site. The fluorescence of Trp-226 was suppressed by incubation of RII with the self-complementary octanucleotide TGACGTCA (CRE) due to binding of the oligonucleotide to RII. A detailed study of the binding equilibrium showed that each RII(cAMP)2 molecule binds 1 molecule of CRE with Kd = 80 nM. The corresponding Kd value for cAMP-depleted RII was found to be 25-fold higher. RII was also found to bind randomly selected DNA fragments with an average Kd value much higher than that of CRE. These observations show for the first time that the binding of oligonucleotide to RII is cAMP-enhanced and sequence-selective.     

Control of 5-aminolaevulinate synthetase activity in Rhodopseudomonas spheroides. Binding of pyridoxal phosphate to 5-aminolaevulinate synthetase.

1. Pyridoxal 5'-phosphate is a cofactor essential for the enzymic activity of aminolaevulinate synthetase from Rhodopseudomonas spheroides. It also aids activation of the low-activity enzyme by trisulphides such as cystine trisulphide, whereas inactivation of enzyme is facilitated by its absence. 2. The fluorescence spectrum of purified high-activity enzyme is that expected for a pyridoxal phosphate--Schiff base, but the firmly bound cofactor does not appear to be at the active centre. In dilute solutions of enzyme this grouping is inaccessible to nucleophiles such as glycine, hydroxylamine, borohydride and cyanide, at pH 7.4. 3. An active-centre Schiff base is formed between enzyne and added pyridoxal phosphate, which is accessible to nucleophiles. Concentrated solutions of this enzyme--Schiff base on treatment with glycine yield apo- and semi-apoenzyme, which can re-bind pyridoxal phosphate. 4. Two types of binding of pyridoxal phosphate are distinguishable in dilute solution of enzyme, but these become indistinguishable when concentrated solutions are treated with cofactor. A change occurs in the susceptibility towards borohydride of the fluorescence of the "structural" pyridoxal phosphate. 5. One or two molecules of cofactor are bound per subunit of mol. wt. 50 000 in semiapo- or holo-enzyme. The fluorescence of pyridoxamine phosphate covalently bound to enzyme also indicates one to two nmol of reducible Schiff base per 7000 units of activity in purified and partially purified samples of enzyme. 6. Cyanide does not convert high-activity into low-activity enzyme, but with the enzyme-pyridoxal phosphate complex it forms a yellow fluorescent derivative that is enzymically active.     

Fluorescence energy transfer measurements between the nucleotide binding site and Cys-373 in actin and their application to the kinetics of actin polymerization

Intramonomer fluorescence energy transfer between the donor epsilon-ATP bound to the nucleotide-binding site and the acceptor 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole bound to Cys-373 in G-actin was measured by steady-state fluorimetry. Assuming for the orientation factor its dynamic limit K2 = 2/3, the donor and acceptor distance in a G-actin molecule was calculated to be about 3 nm. The intermonomer energy transfer in F-actin occurring between the donor bound to an actin monomer and the acceptor bound to the nearest-neighbour actin monomer was also measured and the distance was calculated to be about 4 nm. The kinetics of the actin polymerization process was studied by following the decrease in fluorescence intensity upon addition of salts to G-actin solution. The initial velocity of the fluorescence intensity change was proportional to the square of the initial G-actin concentration. The temperature dependence of the velocity was proportional to the square of the initial G-actin concentration. The temperature dependence of the velocity was proportional to exp(-10/RT). These results indicated that the initial fluorescence intensity change corresponds to monomer-dimer transformation and its activation enthalpy was 10 kcal/mol.     

The conformation of the tRNAPhe anticodon loop monitored by fluorescence

The intrinsic fluorescence of the Wye base was used to study the conformational change of the anticodon loop of yeast tRNAPhe brought about by the addition of magnesium. The fluorescence emission and excitation spectra show dramatic changes as magnesium is added to the solution. The rotational relaxation time changes from 6 nsec without added magnesium to 33 nsec with 10 mM magnesium at an ionic strength of 0.1 M. Stern-Volmer quenching by iodide or iodoethanol shows greater access of the base to the quencher with no added magnesium. A plausible interpretation of this data is that the base stack of the anticodon loop is altered by tilting or twisting the Wye base with respect to the adjacent bases and the base becomes parallel to its neighbors upon the addition of magnesium.     

Contact versus energy transfer fluorescence quenching in the sulfur substituted form of the enzyme rhodanese: a study using cesium ion resolved emission spectra.

The intrinsic fluorescence of the enzyme rhodanese (EC 2.8.1.1) can be resolved into separate contributions from solvent accessible and solvent inaccessible tryptophan residues by comparing spectra run in 2 $\text{M}$ NaCl with those run in the quenching solution, 2 $\text{M}$ CsCl. Both these classes of tryptophan residues are quenched when sulfur is transferred to rhodanese forming a sulfur substituted enzyme which is an intermediate in the catalytic cycle. This observation is consistent with a non-radiative energy transfer mechanism for quenching as opposed to a mechanism requiring direct contact between the bound sulfur and an active site tryptophan. Therefore, the data supports the hypothesis that the primary stabilizing influence in forming the substituted enzyme intermediate is a persulfide bond between an active site sulfhydryl group and the transferred sulfur.     

Binding of 1,N6-ethanoadenosine triphosphate to actin

G-actin is known to bind one molecule of ATP. Its polymerization to F-actin is accompanied by the splitting off of the terminal phosphate of the bound nucleotide. We have found that the fluorescent 1,N⁶-ethanoadenosine triphosphate (?ATP) can substitute for ATP in G-actin and that G-actin containing bound ?ATP possesses essentially full polymerizability. The binding of this ATP analog has been studied by following the inactivation of the ?ATP·G-actin complex. The binding constant (4?5.7 × 10⁶ M^?1) obtained in the absence of EDTA is about 50% of that for ATP, while the binding constant obtained in the presence of EDTA (0.9?3.0 × 10⁵ M^?1) is comparable to those for ATP and ADP. These findings suggest that ?ATP can be used as a structural probe for actin. The fluorescence lifetime of ?ATP bound to G·actin is 36 nsec. The rotational relaxation time of ?ATP·G-actin is near 60 nsec. at 20°C.     

Fluorescence resonance energy transfer between the nucleotide binding site and Cys-10 in G-actin and F-actin

Intramonomer fluorescence resonance energy transfer between the donor epsilon-ATP bound to the nucleotide site and the acceptor N-(4-dimethylamino-3,5-dinitrophenyl)maleimide (DDPM) or 4-dimethylaminophenyl-azophenyl-4'-maleimide bound to Cys-10 in G-actin was measured. The donor-acceptor distance was calculated to be about 40 A. The intermonomer energy transfer in F-actin occurring between epsilon-ADP and DABMI was also measured. The radial coordinate of Cys-10 was calculated to be 25 A based on the helical symmetry of F-actin and the recently calculated radial coordinate of the nucleotide binding site in F-actin i.e. 25 A (Miki, M., Hambly, B. and dos Remedios, C.G. (1986) Biochim. Biophys. Acta 871, 137-141). (The assumption has been made in calculating these distances that the energy donor and acceptor rotate rapidly relative to the fluorescence lifetime.) Corresponding distances separating the donor nucleotide in one monomer from acceptors on Cys-10 in the first and second nearest neighbours in F-actin are 39-40 A and 41-43 A.     

Is Tb3+ fluorescence enhancement only due to binding to single stranded polynucleotides.

Enhancement of Tb3+ fluorescence upon binding to double-stranded ribo- and deoxyribo-duplexes was investigated. It was observed that certain double stranded ribopolynucleotides completely quenched the Tb3+ fluorescence and others did not. It is concluded that the nature of the base in the duplex is critical for this enhancement. - Polydeoxyduplexes also showed enhancement of Tb3+ fluorescence, but much higher terbium concentrations were necessary to obtain similar fluorescence signals, indicative of unspecific effects. CD spectra evidence considerable conformational changes of these duplexes, in particular poly(dG-C) . poly(dG-C( which assumes the Z-form in 0.1 nM Tb3+.