Protein fluorescence of nicotinamide nucleotide-dependent dehydrogenases

1. The decrease in the protein fluorescence (F) of Neurospora crassa glutamate dehydrogenase is linearly related to the increase in the fraction of the coenzyme sites occupied by NADPH (alpha) at pH6.35. Under these conditions NADPH causes this enzyme to dissociate to monomers. 2. There is a non-linear relationship of F to alpha for NADH binding to give the alcohol dehydrogenase-NADH-isobutyramide complex, the l-glycerol 3-phosphate dehydrogenase-NADH complex and the bovine glutamate dehydrogenase-NADH-glutamate complex. The non-linearity is accurately represented by F=[1-alpha(1-x)](n) where n is the number of NADH-binding sites per protein molecule. 3. The co-operative binding of GTP to bovine glutamate dehydrogenase in the presence of NADH gives a linear relationship between F and alpha. 4. The prediction from the equation F=[1-alpha(1-x)](n) that initial tangents to non-linear protein-fluorescence-quenching curves will intercept the fluorescence when alpha=1 at a value of total ligand concentration less than the sum of the concentration of binding sites in the solution plus the dissociation constant of ligand is quantitatively fulfilled. 5. Non-linear protein-fluorescence titrations may be used to obtain information about the distribution of ligand among the protein molecules in solution.     

Interaction of prostaglandins and fatty acids with native and acetaldehyde-alkylated proteins

Using intrinsic and probe fluorescence, microcalorimetry and isotopic methods, the interactions of prostaglandins (PG) E2 and F2 alpha and some fatty acids with native and alkylated proteins (human serum albumin (HSA) and rat liver plasma membrane PG receptors), were studied. The fatty acid and PG interactions with human serum albumin (HSA) resulted in effective quenching of fluorescence of the probe, 1.8-anilinonaphthalene sulfonate (ANS), bound to the protein. Fatty acids competed with ANS for the binding sites; the efficiency of this process increased with an increase in the number of double bonds in the fatty acid molecule. PG induced a weaker fluorescence quenching of HSA-bound ANS and stabilized the protein molecule in a lesser degree compared to fatty acids. The sites of PG E2 and F2 alpha binding did not overlap with the sites of fatty acid binding on the HSA molecule. Nonenzymatic alkylation of HSA by acetaldehyde resulted in the abnormalities of binding sites for fatty acids and PG. Modification of the plasma membrane proteins with acetaldehyde sharply diminished the density of PG E2 binding sites without changing the association constants. Alkylation did not interfere with the parameters of PG F2 alpha binding to liver membrane proteins.     

Malate dehydrogenase, anticooperative NADH, and L-malate binding in ternary complexes with Supernatant pig heart enzyme.

Supernatant malate dehydrogenase from pig heart, a dimeric protein containing two very similar or identical subunits, shows negatively cooperative (anticooperative) interactions between NADH binding sites in the presence, but not in the absence, of 0.1 M L-malate. This behavior is observed consitently whether the technique used employs protein fluorescence quenching, NADH fluorescence enhancement, or ultrafiltration dialysis. Fluorescence titration shows that L-malate is also anticooperatively bound in the presence of saturating concentrations of NADH. The data are consistent with an "induced asymmetry" model in which conformational change accompanies the formation of the ternary complex. Two of the three chromatographically resolvable forms of the enzyme have been tested and found to have anticooperative behavior.     

Coenzyme interaction with horse liver alcohol dehydrogenase. Evidence for allosteric coenzyme binding sites from thermodynamic equilibrium studies.

The techniques of fluorescence enhancement, fluorescence quenching, fluorescence polarization, and equilibrium dialysis are utilized to study the binding properties of coenzyme to horse liver alcohol dehydrogenase. Polarization of fluorescence and equilibrium dialysis show that NADH binds to alcohol dehydrogenase with a stoichiometry of 6 mol per mol of enzyme, in contrast to the value of 2 determined from fluorescence enhancement measurements. NAD+ also binds with a stoichiometry of six as was determined by equilibrium dialysis. The two NADH sites which bind coenzyme more tightly and which are revealed by fluorescence enhancement measurements are designated the catalytic sites. Binding of coenzyme to the four ancillary sites does not alter the quantum yield of NADH but results in a 20% contribution to quenching of enzyme's tryptophan fluorescence. From the emission anisotropy of bound NADH of 24.0% for the additional sites and 28.1% for the catalytic sites and their relative fluorescence lifetimes at the same wavelengths of excitation and emmision, we conclude that the nicotinamide ring of NADH bound to the additional sites exhibits a freedom of motion independent of the macromolecule, while that bound to the catalytic sites is more rigidly held. Polarization of fluorescence yields negative intrinsic free energies of 9.2 and 7.5 Cal M-1 for NADH interaction with the catalytic and additional sites, respectively. Although these values are 1.3 to 2.0 Cal higher than those determined by fluorescence quenching and equilibrium dialysis, the mean Hill coefficient of 1.76 plus or minus 0.06, the titration span of 2.4 logarithmic units and coupling free energies (in magnitude and sign) are the same for all these techniques. The above difference in the intrinsic free energies are attributed largely to the different modes of interaction of excited and unexcited NADH molecules with alcohol dehydrogenase.     

Tryptophan luminescence from liver alcohol dehydrogenase in its complexes with coenzyme. A comparative study of protein conformation in solution

The extent of fluorescence quenching and that of phosphorescence quenching of Trp-15 and Trp-314 in alcohol dehydrogenase from horse liver as well as the intrinsic phosphorescence lifetime of Trp-314 in fluid solution have been utilized as structural probes of the macromolecule in binary and ternary complexes formed with coenzyme, analogous, and various substrate/inhibitors. Luminescence quenching by the coenzyme reveals that (1) while the reduced form quenches Trp emission exclusively from the fluorescent state, the oxidized form is very effective on the phosphorescent state as well and that (2) among the series of NADH binary and ternary complexes known by crystallographic studies to attain the closed form, distinct nicotinamide/indole geometrical arrangements are inferred from a variable degree of fluorescence quenching. Information of the dynamic structure of the coenzyme-binding domain derived from the phosphorescence lifetime of Trp-314 points out that within the series of closed NADH complexes there is considerable conformational heterogeneity. In solution, the variability in dynamical structure among the various protein complexes emphasizes that the closed/open forms identified by crystallographic studies are not two well-defined macrostates of the enzyme.     

Subunit interactions in rabbit-muscle glyceraldehyde-phosphate dehydrogenase, as measured by NAD+ and NADH binding.

1. The binding parameters for NADH and NAD+ to rabbit-muscle glyceraldehyde-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) have been measured by quenching of the flourescence of the protein and the NADH. 2. The fact that the degree of protein fluorescence quenching by bound NAD+ or NADH, excited at 285 nm and measured at 340 nm ('blue' tryptophans), is not linearly related to the saturation functions of these nucleotides, leads to a slight overestimation of the interaction energy and an underestimation of the concentration of sites, if linearity is assumed. 3. This is also the case for NADH, but not for NAD+, when the protein fluorescence is excited at 305 nm and measured at 390 nm ('red' tryptophans). 4. The binding of NAD+ can be described by a model in which the binding of NAD+, via negative interactions within the dimer, induces weaker binding sites, with the result that the microscopic dissociation constant is 0.08 microM at low saturation and 0.18 microM for the holoenzyme. 5. The binding of NADH can be described on the basis of the same model, the dissociation constant at low saturation being 0.5 microM and of the holoenzyme 1.0 microM. 6. The fluorescence of bound NADH is not sensitive to the conformational changes that cause the decrease in affinity of bound NAD+ or NADH. 7. The binding of NAD+ to the 3-phosphoglyceroyl enzyme can be described by a dissociation constant that is at least two orders of magnitude greater than the dissociation constants of the unacylated enzyme. The affinity of NAD+ to this form of the enzyme is in agreement with the Ki calculated from product inhibition by NAD+ of the reductive dephosphorylation of 1,3-diphosphoglycerate.     

Sturgeon glyceraldehyde-3-phosphate dehydrogenase.

The formation of binary complexes between sturgeon apoglyceralddhyde-3-phosphate dehydrogenase, coenzymes (NAD+ and NADH) and substrates (phosphate, glyceraldehyde 3-phosphate and 1,3-bisphosphoglycerate) has been studied spectrophotometrically and spectrofluorometrica-ly. Coenzyme binding to the apoenzyme can be characterized by several distinct spectroscopic properties: (a) the low intensity absorption band centered at 360 nm which is specific of NAD+ binding (Racker band); (b) the quenching of the enzyme fluorescence upon coenzyme binding; (c) the quenching of the fluorescence of the dihydronicotinamide moiety of the reduced coenzyme (NADH); (D) the hypochromicity and the red shift of the absorption band of NADH centered at 338 nm; (e) the coenzyme-induced difference spectra in the enzyme absorbance region. The analysis of these spectroscopic properties shows that up to four molecules of coenzyme are bound per molecule of enzyme tetramer. In every case, each successively bound coenzyme molecule contributes identically to the total observed change. Two classes of binding sites are apparent at lower temperatures for NAD+ Binding. Similarly, the binding of NADH seems to involve two distinct classes of binding sites. The excitation fluorescence spectra of NADH in the binary complex shows a component centered at 260 nm as in aqueous solution. This is consistent with a "folded" conformation of the reduced coenzyme in the binary complex, contradictory to crystallographic results. Possible reasons for this discrepancy are discussed. Binding of phosphorylated substrates and orthophosphate induce similar difference spectra in the enzyme absorbance region. No anticooperativity is detectable in the binding of glyceraldehyde 3-phosphate. These results are discussed in light of recent crystallographic studies on glyceraldehyde-3-phosphate dehydrogenases.     

Detection of ligand-induced conformation changes in lactate dehydrogenase by using fluorescent probes

The binding of ANS to apolactate dehydrogenase (apo-LDH) is accompanied by a 300-fold increase in dye fluorescence with a shift of the emission maximum from 515 to 479 nm, as well as by quenching of intrinsic protein fluorescence. A tetrameric LDH molecule has 6.4 +/- 1.6 non-interacting dye-binding sites with an association constant equal to (4.3 +/- 1.6) X 10(3) M-1. NAD+ added at saturating concentrations does not alter the number of ANS binding sites or the association constant value. The formation of binary LDH.NAD+, LDH.NADH, LDH.AMP and LDH.pyruvate complexes causes the quenching of fluorescence of the enzyme-bound ANS. The extent of quenching observed at ligand saturating concentrations differs for each ligand. Pyruvate added to the binary LDH.AMP complex exerts no effect on the fluorescence of protein-bound ANS; this indicates that the binding of AMP causes some alterations in the microenvironment of the substrate-binding site. Nicotinamide mononucleotide (NMN) can act as a coenzyme in the LDH-catalyzed reaction. AMP added together with NMN displays an inhibitory effect. The cationic (auramine O) and anionic (ANS) fluorescent probes bound to LDH exhibit different responses to conformational changes accompanying the transition from the apoenzyme to the LDH X NAD-pyruvate complex.     

The fluorescence spectrum of the introduced tryptophans in the alpha 3(beta F155W)3gamma subcomplex of the F1-ATPase from the thermophilic Bacillus PS3 cannot be used to distinguish between the number of nucleoside di- and triphosphates bound to catalytic sites.

It has been reported that shifts in the fluorescence emission spectrum of the introduced tryptophans in the betaF155W mutant of Escherichia coli F(1) (bovine heart mitochondria F(1) residue number) can quantitatively distinguish between the number of catalytic sites occupied with ADP and ATP during steady-state ATP hydrolysis (Weber, J., Bowman, C., and Senior, A. E. (1996) J. Biol. Chem. 271, 18711--18718). In contrast, addition of MgADP, Mg-5'-adenylyl beta,gamma-imidophosphate (MgAMP-PNP), and MgATP in 1:1 ratios to the alpha(3)(betaF155W)(3)gamma subcomplex of thermophilic Bacillus PS3 F(1) (TF(1)) induced nearly identical blue shifts in the fluorescence emission maximum that was accompanied by quenching. Addition of 2 mm MgADP induced a slightly greater blue shift and a slight increase in intensity over those observed with 1:1 MgADP. However, addition of 2 mm MgAMP-PNP or MgATP induced a much greater blue shift and substantially enhanced fluorescence intensity over those observed in the presence of stoichiometric MgADP or MgAMP-PNP. It is clear from these results that the fluorescence spectrum of the introduced tryptophans in the betaF155W mutant of TF(1) does not respond in regular increments at any wavelength as catalytic sites are filled with nucleotides. The fluorescence spectrum observed after entrapping MgADP-fluoroaluminate complexes in two catalytic sites of the betaF155W subcomplex indicates that the fluorescence emission spectrum of the enzyme is maximally perturbed when nucleotides are bound to two catalytic sites. This finding is consistent with accumulating evidence suggesting that only two beta subunits in the alpha(3)beta(3)gamma subcomplex of TF(1) can simultaneously exist in the completely closed conformation.     

On the location and function of tyrosine beta 331 in the catalytic site of Escherichia coli F1-ATPase.

1) Using a combination of site-directed mutagenesis and fluorescence spectroscopy we have studied the location and function of residue beta Y331 in the catalytic site of Escherichia coli F1-ATPase. The fluorescent analog lin-benzo-ADP was used as a catalytic-site probe, and was found to bind to three sites in normal F1, with Kd1 = 0.20 microM and Kd2,3 = 5.5 microM. lin-Benzo-ATP was a good substrate for hydrolysis. 2) The mutants investigated were beta Y331F, L, A and E. kcat/KM for ATP hydrolysis in purified F1 was reduced according to the series Y greater than or equal to F greater than L greater than A greater than E, with E being severely impaired; concomitant decreases in binding affinity for lin-benzo-ADP were seen. 3) Fluorescence properties of lin-benzo-ADP bound to F1 differed widely, depending on the residue present at position beta 331. Red shifts of excitation and emission spectra occurred with F and L residues, but not with Y, A, or E. There was strong quenching of fluorescence with wild-type (Y), partial quenching with A, and no quenching with F, L, or E. 4) We conclude that (a) the environment around the bound adenine moiety in the catalytic site is nonpolar, (b) residue beta 331 is part of the adenine-binding subdomain and when tyrosine is the residue, the phenolic hydroxyl makes direct interaction with the fluorophore, (c) an aromatic residue is not absolutely required at position beta 331 for catalytic function, but an increase in polarity leads to functional impairment, and (d) in terms of fluorescence response of bound lin-benzo-ADP all three catalytic sites behaved the same. 5) F1 from mutant beta Y297F bound lin-benzo-ADP with the same fluorescence and binding characteristics as normal F1, and catalytic properties were similar to normal. Therefore, there was no reason to conclude that residue beta Y297 is involved in binding the adenine moiety of ATP.     

Fluorescence investigations of coenzyme and substrate interactions in mannitol-1-phosphate dehydrogenase ofEscherichia coli

Coenzyme and substrate interactions with mannitol-1-phosphate dehydrogenase fromEscherichia coli (a dimer of MW 45,000) have been studied by fluorescence spectroscopy. NAD⁺ quenches the fluorescence emission of the protein tryptophan residues; shifting the excitation wavelength from 280 to 290 nm results in an increase in this quenching and a red shift in the emission maximum. NAD⁺ also quenches the fluorescence of covalently attached pyridoxyl phosphate, and this quenching is accompanied by a spectral broadening above 425 nm. Fructose-6-phosphate increases the binding of NAD⁺, but causes a slight reduction in the quenching of the tryptophan fluorescence observed at saturating levels of coenzyme, and reverses the NAD⁺-induced broadening in the pyridoxyl phosphate emission spectrum. NADH quenches the protein emission much less than NAD⁺; this quenching is not changed by shifting the excitation wavelength and is not affected by the presence of bound mannitol-1-phosphate. Titrations monitoring the quenching by NADH indicate a single class of NADH binding sites, while titrations monitoring NADH fluorescence suggest that coenzyme fluorescence is more enhanced when NADH is bound to less than half of the total enzyme subunits, with the emission per NADH molecule bound decreasing as the number of NADH molecules bound increases. In the absence of coenzyme, neither fructose-6-phosphate nor mannitol-1-phosphate have any effect on the protein tryptophan emission; however, both substrates induce specific changes in the emission spectrum of covalently attached pyridoxyl phosphate. These results suggest that the different coenzymes and substrates cause specific conformational changes in mannitol-1-phosphate dehydrogenase.     

Human aldehyde dehydrogenase: coenzyme binding studies

The binding of NADH and NAD+ to the human liver cytoplasmic, E1, and mitochondrial, E2, isozymes at pH 7.0 and 25 degrees C was studied by the NADH fluorescence enhancement technique, the sedimentation technique, and steady-state kinetics. The binding of radiolabeled [14C]NADH and [14C]NAD+ to the E1 isozyme when measured by the sedimentation technique yielded linear Scatchard plots with a dissociation constant of 17.6 microM for NADH and 21.4 microM for NAD+ and a stoichiometry of ca. two coenzyme molecules bound per enzyme tetramer. The dissociation constant, 19.2 microM, for NADH as competitive inhibitor was found from steady-state kinetics. With the mitochondrial E2 isozyme, the NADH fluorescence enhancement technique showed only one, high-affinity binding site (KD = 0.5 microM). When the sedimentation technique and radiolabeled coenzymes were used, the binding studies showed nonlinear Scatchard plots. A minimum of two binding sites with lower affinity was indicated for NADH (KD = 3-6 microM and KD = 25-30 microM) and also for NAD+ (KD = 5-7 microM and KD = 15-30 microM). A fourth binding site with the lowest affinity (KD = 184 microM for NADH and KD = 102 microM for NAD+) was observed from the steady-state kinetics. The dissociation constant for NAD+, determined by the competition with NADH via fluorescence titration, was found to be 116 microM. The number of binding sites found by the fluorescence titration (n = 1 for NADH) differs from that found by the sedimentation technique (n = 1.8-2.2 for NADH and n = 1.2-1.6 for NAD+).(ABSTRACT TRUNCATED AT 250 WORDS)     

The binding of reduced nicotinamide adenine dinucleotide to citrate synthase of Escherichia coli K12.

Citrate synthase from Escherichia coli enhances the fluorescence of its allosteric inhibitor, NADH, and shifts the peak of emission of the coenzyme from 457 to 428 nm. These effects have been used to measure the binding of NADH to this enzyme under various conditions. The dissociation constant for the NADH-citrate synthase complex is about 0.28 muM at pH 6.2, but increases toward alkaline pH as if binding depends on protonation of a group with a pKa of about 7.05. Over the pH range 6.2-8.7, the number of binding sites decreases from about 0.65 to about 0.25 per citrate synthase subunit. The midpoint of this transition is at about pH 7.7, and it may be one reflection of the partial depolymerization of the enzyme which is known to occur in this pH range. A gel filtration method has been used to verify that the fluorescence enhancement technique accurately reveals all of the NADH molecules bound to the enzyme in the concentration range of interest. NAD+ and NADP+ were weak competitive inhibitors of NADH binding at pH 7.8 (Ki values greater than 1 mM), but stronger inhibition was shown by 5'-AMP and 3'-AMP, with Ki values of 83 +/- 5 and 65 +/- 4 muM, respectively. Acetyl-CoA, one of the substrates, and KCl, an activator, also inhibit the binding in a weakly cooperative manner. All of these effects are consistent with kinetic observations on this system. We interpret our results in terms of two types of binding site for nucleotides on citrate synthase: an active site which binds acetyl-CoA, the substrate, or its analogue 3'-AMP; and an allosteric site which binds NADH or its analogue 5'-AMP and has a lesser affinity for other nicotinamide adenine dinucloetides. When the active site is occupied, we propose that NADH cannot bind to the allosteric site, but 5'-AMP can; conversely, when NADH is the in the allosteric site, the active site cannot be occupied. In addition to these two classes of sites, there must be points for interaction with KCl and other salts. Oxaloacetate, the second substrate, and alpha-ketoglutarate, an inhibitor whose mode of action is believed to be allosteric, have no effect on NADH binding to citrate synthase at pH 7.8. When NADH is bound to citrate synthase, it quenches the intrinsic tryptophan fluorescence of the enzyme. The amount of quenching is proportional to the amount of NADH bound, at least up to a binding ratio of 0.50 NADH per enzyme subunit. This amount of binding leads to the quenching of 53 +/- 5% of the enzyme fluorescence, which means that one NADH molecule can quench all the intrinsic fluorescence of the subunit to which it binds.     

Location of lysine-beta 162 in mitochondrial F1-adenosinetriphosphatase

The quenching of the fluorescence of bovine heart F1-adenosinetriphosphatase labeled specifically at its essential Lys-beta 162 with 7-chloro-4-nitro-2,1,3-benzoxadiazole (N-NBD-F1) by 2',3'-O-(2,4,6-trinitrocyclohexadienylidene)adenosine 5'-triphosphate (TNP-ATP) has been studied. Analysis of the fluorescence data in the presence of 1 mM ATP shows that the dissociation constant of TNP-ATP from its first binding site in the covalently labeled enzyme is 250-fold lower than that of ATP, which it replaces in pH 7.0 buffer containing 25% glycerol, and that this binding causes a 54% quenching of the fluorescence of the N-NBD label due to energy transfer to the weakly fluorescent TNP-ATP molecule. Computation based on the observed quenching gives a distance of 25.6 +/- 0.4 A between the NBD label and the chromophore of the bound TNP-ATP molecule. Since the distance between the chromophore and the farthest O atom of the bound TNP-ATP is about 16 A, it seems quite likely that the epsilon-amino group of Lys-beta 162 is near the gamma-phosphate group of the TNP-ATP bound at the catalytic site. Similar measurements in the presence of 1 mM ADP show that the replacement of ADP at the catalytic site by TNP-ATP causes a 49% quenching of the fluorescence of the N-NBD label, which gives a distance of 26.5 +/- 0.4 A between the label and the chromophore of the bound TNP-ATP molecule.     

Fluorescence studies of the interactions of serum albumin and rat alpha1-fetoprotein with aflatoxin B1. Specificity and binding parameters.

The interaction of bovine serum albumin and rat alpha1-fetoprotein with aflatoxin B1 has been followed by the fluorescence quenching of the protein in presence of the ligand. The binding parameters (n, number of sites and Kd, dissociation constant) have been determined for the bovine serum albumin-alflatoxin B1 system: n = 3.5 and Kd = 3.1 +/- 0.5 . 10(-5) M and for the alpha-fetoprotein-aflatoxin system: n = 4 and Kd = 3.7 +/-0.5 . 10(-5) M. The competition of anilino-naphthalene-sulfonate and aflatoxin B1 for the same hydrophobic sites on bovine serum albumin has been demonstrated. The fluorescence quenching of various proteins (lysozymes, egg-albumin, gamma-globulin) by aflatoxin B1 have shown that there is not a strict specificity of aflatoxin towards proteins.     

Induced circular dichroism spectra reveal binding of the antiinflammatory curcumin to human alpha1-acid glycoprotein

This paper reports the first experimental evidence on binding of the plant derived curcumin molecule to human alpha1-acid glycoprotein (AGP), an acute phase protein in blood. Oppositely signed induced circular dichroism (CD) bands measured in the visible spectral region in pH7.4 phosphate buffer indicate that the protein binds this natural polyphenol molecule in a left-handed chiral conformation. Decreasing of the intrinsic fluorescence of AGP upon addition of curcumin confirmed the binding to take place. Fluorescence quenching titration curve of AGP allowed to calculate the association constant of the ligand (Ka = 4 x 10(4) M(-1)). Modification of near UV CD spectrum of the protein suggests that curcumin induces changes in the tertiary structure of AGP, which leads to the decrease of binding affinity. By using rac-warfarin and amitriptyline, selective high affinity ligands of F1-S and A genetic variants of AGP, CD displacement experiments showed that curcumin is able to bind to both variants. Molecular docking calculations performed on curcumin-AGP and warfarin-AGP complexes suggest the existence of two alternative binding sites for curcumin; either at the open end of the central hydrophobic cavity or in a surface cleft of the protein.     

Relation between the secondary structure of carbohydrate residues of alpha1-acid glycoprotein (orosomucoid) and the fluorescence of the protein

We studied in this work the relation that exists between the secondary structure of the glycans of alpha(1)-acid glycoprotein and the fluorescence of the Trp residues of the protein. We calculated for that the efficiency of quenching and the radiative and non-radiative constants. Our results indicate that the glycans display a spatial structure that is modified upon asialylation. The asialylated conformation is closer to the protein matrix than the sialylated form, inducing by that a decrease in the fluorescence parameters of the Trp residues. In fact, the mean quantum yield of Trp residues in sialylated and asialylated alpha(1)-acid glycoprotein are 0.0645 and 0.0385, respectively. Analysis of the fluorescence emission of alpha(1)-acid glycoprotein as the result of two contributions (surface and hydrophobic domains) indicates that quantum yields of both classes of Trp residues are lower when the protein is in the asialylated form. Also, the mean fluorescence lifetime of Trp residues decreases from 2.285 ns in the sialylated protein to 1.948 ns in the asialylated one. The radiative rate constant k(r) of the Trp residues in the sialylated alpha(1)-acid glycoprotein is higher than that in the asialylated protein. Thus, the carbohydrate residues are closer to the Trp residues in the absence of sialic acid. The modification of the spatial conformation of the glycans upon asialylation is confirmed by the decrease of the fluorescence lifetimes of Calcofluor, a fluorophore that binds to the carbohydrate residues. Finally, thermal intensity quenching of Calcofluor bound to alpha(1)-acid glycoprotein shows that the carbohydrate residues have slower residual motions in the absence of sialic acid residues.     

Tertiary structure of human alpha1-acid glycoprotein (orosomucoid). Straightforward fluorescence experiments revealing the presence of a binding pocket

Binding of hemin to alpha1-acid glycoprotein has been investigated. Hemin binds to the hydrophobic pocket of hemoproteins. The fluorescent probe 2-(p-toluidino)-6-naphthalenesulfonate (TNS) binds to a hydrophobic domain in alpha1-acid glycoprotein with a dissociation constant equal to 60 microM. Addition of hemin to an alpha1-acid glycoprotein-TNS complex induces the displacement of TNS from its binding site. At saturation (1 hemin for 1 protein) all the TNS has been displaced from its binding site. The dissociation constant of hemin-alpha1-acid glycoprotein was found equal to 2 microM. Thus, TNS and hemin bind to the same hydrophobic site: the pocket of alpha1-acid glycoprotein. Energy-transfer studies performed between the Trp residues of alpha1-acid glycoprotein and hemin indicated that efficiency (E) of Trp fluorescence quenching was equal to 80% and the F?rster distance, R0 at which the efficiency of energy transfer is 50% was calculated to be 26 A, revealing a very high energy transfer.     

Efficient reconstitution of mitochondrial energy-transfer reactions from depleted membranes and F1-ATPase as a function of the amount of bound oligomycin sensitivity-conferring protein (OSCP)

Pig heart mitochondrial membranes depleted of F1 and OSCP by various treatments were analyzed for their content in alpha and beta subunits of F1 and in OSCP using monoclonal antibodies. Membrane treatments and conditions of rebinding of F1 and OSCP were optimized to reconstitute efficient NADH- and ATP-dependent proton fluxes, ATP synthesis and oligomycin-sensitive ATPase activity. F1 and OSCP can be rebound independently to depleted membranes but to avoid unspecific binding of F1 to depleted membranes (ASUA) which is not efficient for ATP synthesis, F1 must be rebound before the addition of OSCP. The rebinding of OSCP to depleted membranes reconstituted with F1 inhibits the ATPase activity of rebound F1, while it restores the ATP-driven proton flux measured by the quenching of ACMA fluorescence. The rebinding of OSCP also renders the ATPase activity of bound F1 sensitive to uncouplers. The rebinding of OSCP alone or F1 alone, does not modify the NADH-dependent proton flux, while the rebinding of both F1 and OSCP controls this flux, inducing an inhibition of the rate of NADH oxidation. Similarly, oligomycin, which seals the F0 channel even in the absence of F1 and OSCP, inhibits the rate of NADH oxidation. OSCP is required to adjust the fitting of F1 to F0 for a correct channelling of protons efficient for ATP synthesis. All reconstituted energy-transfer reactions reach their optimal value for the same amount of OSCP. This amount is consistent with a stoichiometry of two OSCP per F1 in the F0-F1 complex.     

Investigation of the aurovertin binding site of Escherichia coli F1-ATPase by fluorescence spectroscopy and site-directed mutagenesis.

(1) Previous mutational analyses have shown that residue beta R398 of the beta-subunit is a key residue for binding of the inhibitory antibiotic aurovertin to Escherichia coli F1Fo-ATP synthase. Here, we studied purified F1 from the beta R398C and beta R398W mutants. ATPase activity in both cases was resistant to aurovertin inhibition. The fluorescence spectrum (lambda exc = 278 or 295 nm) of beta R398W F1 showed a significant red-shift as compared to wild-type and beta R398C enzymes, indicating that residue beta R398 lies in a polar environment. On the basis of this and previous evidence, we propose that aurovertin binding to F1-ATPase involves a specific charged donor-acceptor H-bond between residue beta R398 and the 7-hydroxyl group of aurovertin. (2) The fluorescent substrate analog lin-benzo-ADP was shown to bind to beta R398W F1 catalytic sites with the same Kd values as to wild-type F1, and with the same quenching of the fluorescence of the analog. Fluorescence energy transfer was seen between the beta R398W residue and bound lin-benzo-ADP. Analysis of transfer efficiency at varying stoichiometry of bound lin-benzo-ADP showed that interaction occurred between one beta R398W residue and one catalytic-site-bound analog molecule at a distance of approximately 23 A. The relationships of the aurovertin and catalytic sites in the primary and tertiary structure are discussed.