Membrane-protein structural mapping of chloroplast coupling factor in asolectin vesicles

The spatial relationship of specific sites on chloroplast coupling factor, reconstituted in asolectin vesicles, to the bilayer surface has been studied with fluorescence methods. Fluorescence resonance energy transfer measurements have been used to map the distances of closest approach of the N,N'-dicyclohexylcarbodiimide-binding site and the disulfide on the gamma-polypeptide to the bilayer center. The dicyclohexylcarbodiimide site was labeled with N-cyclohexyl-N'-pyrenylcarbodiimide and the gamma-disulfide site with a coumarinyl derivative. The bilayer center was labeled with 25-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-N-methylamino]-27-norc holesterol. The distances obtained, 15 and 43 A, respectively, were combined with previous measurements of the distance of closest approach between these sites and the membrane surface to estimate the perpendicular distances of the sites from the membrane surface. The depth of the dicyclohexylcarbodiimide site was also determined by studying the quenching of fluorescence by 5-, 7-, 12-, and 16-doxylstearic acids. The model developed suggests that the dicyclohexylcarbodiimide site is 6-10 A below the membrane surface and the gamma-disulfide is 16 A above the membrane surface. The distances measured are subject to a considerable uncertainty, but the proposed model provides a useful starting point for further structural studies.     

Structural organization of chloroplast coupling factor

Fluorescence resonance energy transfer measurements have been used to construct spatial maps for the accessible sulfhydryl of the gamma subunit (dark site) and the essential tyrosine residue of the beta subunits relative to previously mapped sites on the H+-ATPase from chloroplasts. The extent of energy transfer was measured between a coumarinylmaleimide derivative reacted covalently at the dark site and acceptor species selectively bound at the gamma-disulfide and the three nucleotide binding sites of the solubilized coupling factor complex. The nucleotide energy acceptor was 2'(3')-(trinitrophenyl)adenosine triphosphate, and the gamma-disulfide site was labeled with fluoresceinylmaleimide. The dark-site sulfhydryl also was labeled with pyrenylmaleimide which served as an energy donor for 7-chloro-4-nitro-2,1,3-benzoxadiazole reacted at the beta-tyrosine sites. Similar measurements were also made with pyrenylmaleimide covalently attached to the gamma-sulfhydryl accessible only under energized conditions on the thylakoid membrane surface (light site). The observed transfer efficiencies indicate that the dark-site sulfhydryl is approximately 45 A from all three nucleotide sites and 41-46 A from the gamma-disulfide site. The average distances separating the essential beta-tyrosines and the light- and dark-site sulfhydryls are 38 and 42 A, respectively. (In calculating these distances, random orientation of the donor-acceptor dipoles was assumed.) The results are consistent with a previously described structural model of the intact enzyme and can be used to gain insight into the overall structural organization or alpha-, beta-, and gamma-polypeptides within the coupling factor.     

Structural mapping of chloroplast coupling factor

Fluorescence resonance energy transfer measurements have been used to investigate the spatial relationships between the nucleotide binding sites and the gamma-subunit of the H+-ATPase from chloroplasts and the orientation of these sites with respect to the membrane surface. Fluorescent maleimides reacted covalently at specific sulfhydryl sites on the gamma-subunit served as energy donors. One sulfhydryl site can be labeled only under energized conditions on the thylakoid membrane surface (light site). The two gamma-sulfhydryls exposed after catalytic activation served as a second donor site (disulfide site). In one set of experiments, the nucleotide analogue 2'(3')-(trinitrophenyl)adenosine triphosphate, selectively bound at each of the three nucleotide binding sites of the solubilized coupling factor, was used as an energy acceptor; in another, octadecylrhodamine with its acyl chain inserted in the vesicle bilayer and the rhodamine fluorophore exposed along the membrane surface was the energy acceptor. The distance between the sulfhydryl and disulfide sites was also obtained by sequentially labeling the sites with coumarin (donor) and fluorescein (acceptor) maleimide derivatives, respectively. The results indicate that all three nucleotide sites are approximately equal to 50 A from the light-labeled gamma-sulfhydryl. Two of the nucleotide sites are very far from the gamma-disulfide (greater than 74 A), while the third site, which binds nucleotides reversibly under all conditions, is 62 A from this sulfhydryl. The light-labeled sulfhydryl and disulfide sites are about 42-47 A apart. Finally, the distance of closest approach between the membrane surface of the reconstituted system and the gamma-disulfide is 31 A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural mapping of nucleotide binding sites on chloroplast coupling factor

Fluorescence resonance energy transfer was used to measure the distances between three nucleotide binding sites on solubilized chloroplast coupling factor from spinach and between each nucleotide site and two tyrosine residues which are important for catalytic activity. The nucleotide energy donor was 1,N6-ethenoadenosine di- or triphosphate, and the nucleotide energy acceptor was 2'(3')-(trinitrophenyl)adenosine diphosphate. The tyrosine residues were specifically labeled with 7-chloro-4-nitro-2,1,3-benzoxadiazole, which served as an energy acceptor. The results obtained indicate the three nucleotide binding sites form a triangle with sides of 44, 48, and 36 A. (The assumption has been made in calculating these distances that the energy donor and acceptor rotate rapidly relative to the fluorescence lifetime.) Two of the nucleotide sites are approximately equidistant from each of the two tyrosines: one of the nucleotide sites is about 37 A and the other about 41 A from each tyrosine. The third nucleotide site is about 41 A from one of the tyrosines and greater than or equal to 41 A from the other tyrosine.     

Fluorescence energy transfer experiments with Escherichia coli carbamoyl-phosphate synthetase

Fluorescence energy transfer experiments were used to measure distances between three fluorescently labeled sulfhydryl sites on Escherichia coli carbamoyl-phosphate synthetase, an unsymmetrical dimer. When five different combinations of fluorescent donor-acceptor pairs are used, the distance between site 1, located on the large subunit, and site 2, located on the small subunit, is in the range of 27-33 A. Similarly, the distance between site 1 and site 3 (large subunit) was approximately 27 A and between site 2 and site 3 was approximately 21 A. A similar approach was employed to determine distances between each sulfhydryl group and the ATP site(s), and in all cases no fluorescence quenching was observed using Cr3+ATP or Co(NH3)4ATP as substrate analogues. A lower limit could be calculated from these data, resulting in a distance of greater than or equal to 21 A from each sulfhydryl site to the ATP site. Additional experiments were performed to evaluate if the substrates ATP, HCO3(-), or glutamine or the allosteric modifiers ornithine, IMP, and UMP altered the distance relationships among the sulfhydryl sites. IMP and UMP produced a slight decrease in fluorescence between sites while glutamine and ATP produced a slight increase in fluorescence.     

Rotation of nucleotide sites is not required for the enzymatic activity of chloroplast coupling factor 1

New heterobifunctional photoaffinity cross-linking reagents, 6-maleimido-N-(4-benzoylphenyl)hexanamide, 12-maleimido-N-(4-benzoylphenyl)dodecanamide, and 12-[14C]maleimido-N-(4-benzoylphenyl)dodecanamide, were synthesized to investigate the mechanism of ATP hydrolysis by chloroplast coupling factor 1. These reagents react with sulfhydryl groups on the gamma-polypeptide. Subsequent photolysis cross-links the gamma-polypeptide covalently to alpha- and beta-polypeptides. The cross-linkers prevent major movements of the gamma-polypeptide with respect to the alpha- and beta-polypeptides but are sufficiently long to permit some flexibility in the enzyme structure. When approximately 50% of the gamma-polypeptide was cross-linked to alpha- and beta-polypeptides, a 7% loss in ATPase activity was observed for the longer cross-linker and a 12% loss for the shorter. These results indicate that large movements of alpha- and beta-polypeptides with respect to the gamma-polypeptide are not essential for catalysis. In particular, rotation of the polypeptide chains to create structurally equivalent sites during catalysis is not a required feature of the enzyme mechanism.     

Binding stoichiometry and structural mapping of the epsilon polypeptide of chloroplast coupling factor 1

Fluorescent probes were attached to the single sulfhydryl residue on the isolated epsilon polypeptide of chloroplast coupling factor 1 (CF1), and the modified polypeptide was reconstituted with the epsilon-deficient enzyme. A binding stoichiometry of one epsilon polypeptide per CF1 was obtained. This stoichiometry corresponded to a maximum inhibition of the Ca2+-dependent ATPase activity of the enzyme induced by epsilon removal. Resonance energy transfer between the modified epsilon polypeptide and fluorescent probes attached to various other sites on the enzyme allowed distance measurements between these sites and the epsilon polypeptide. The epsilon-sulfhydryl is nearly equidistant from both the disulfide (23 A) and the dark-accessible sulfhydryl (26 A) of the gamma subunit. Measurement of the distance between epsilon and the light-accessible gamma-sulfhydryl was not possible due to an apparent exclusion of modified epsilon from epsilon-deficient enzyme after modification of the light-accessible site. The distances measured between epsilon and the nucleotide binding sites on the enzyme were 62, 66, and 49 A for sites 1, 2, and 3, respectively. These measurements place the epsilon subunit in close physical proximity to the sulfhydryl-containing domains of the gamma subunit and approximately 40 A from the membrane surface. Enzyme activity measurements also indicated a close association between the epsilon and gamma subunits: epsilon removal caused a marked increase in accessibility of the gamma-disulfide bond to thiol reagents and exposed a trypsin-sensitive site on the gamma subunit. Either disulfide bond reduction or trypsin cleavage of gamma significantly enhanced the Ca2+-ATPase activity of the epsilon-deficient enzyme. Thus, the epsilon and gamma polypeptides of coupling factor 1 are closely linked, both physically and functionally.     

Investigation of quercetin binding sites on chloroplast coupling factor 1.

The quercetin binding sites on spinach chloroplast coupling factor 1 (CF1) have been investigated using direct and competitive binding, stopped-flow, temperature-jump, and fluorescence resonance energy transfer measurements. It was found that 8-anilino-1-naphthalensulfonic acid (ANS) competes with quercetin binding at two sites on the solubilized enzyme which are distinct from the two tight nucleotide binding sites and the 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) reactive site. The bimolecular association of quercetin with CF1 is too fast to measure directly and is followed by two slower conformational changes. The distances from the tight nucleotide sites to the quercetin-ANS sites were estimated as 40-48 A by fluorescence resonance energy transfer using 1,N6-ethenoadenosine diphosphate and 1,N6-ethenoadenylyl imidodiphosphate as donors and quercetin as the acceptor. The distance from the quercetin-ANS site to the NBD-C1 reactive site was found to be about 30 A using ANS as a donor and NBD-C1 reacted with a tyrosine group on CF1 as the energy acceptor. A model is proposed for the relative location of these sites on CF1.     

Fluorescent derivatives of the pyruvate dehydrogenase component of the Escherichia coli pyruvate dehydrogenase complex.

One sulfhydryl group per polypeptide chain of the pyruvate dehydrogenase component of the pyruvate dehydrogenase multienzyme complex from Escherichia coli was selectively labeled with N-[P-(2-benzoxazoyl)phenyl]-maleimide (NBM), 4-dimethylamino-4-magnitude of-maleimidostilbene (NSM), and N-(4-dimethylamino-3,5-dinitrophenyl)maleimide (DDPM) in 0.05 M potassium phosphate (pH 7). Modification of the sulfhydryl group did not alter the enzymatic activity or the binding of 8-anilino-1-naphthalenesulfonate (ANS) or thiochrome diphosphate to the enzyme. The fluorescence of the NBM or NSM coupled to the sulfhydryl group on the enzyme was quenched by binding to the enzyme of the substrate pyruvate the coenzyme thiamine diphosphate, the coenzyme analogue thiochrome diphosphate, the regulatory ligands acetyl-CoA, GTP, and phosphoenolpyruvate, and the acetyl-CoA analogue, ANS. Fluorescence energy transfer measurements were carried out for the enzyme-bound donor-acceptor pairs NBM-ANS, NBM-thiochrome diphosphate ANS-DDPM, and thiochrome diphosphate-DDM. The results indicate that the modified sulfhydryl group is more than 40 A from the active site and approximately 49 A from the acetyl-CoA regulatory site. Thus, a conformational change must accompany the binding of ligands to the regulatory and catalytic sites. Anisotropy depolarization measurements with ANS bound on the isolated pyruvate dehydrogenase in 0.05 M potassium phosphate (pH 7.0) suggest that under these conditions the enzyme is dimeric.     

Selective modification of an alpha subunit of chloroplast coupling factor 1

Lucifer yellow (4-amino-N-[3-(vinylsulfonyl)phenyl]naphthalimide-3,6-disulf onate), a fluorescent probe that can react covalently with sulfhydryl or amino groups, has been used to modify chloroplast coupling factor 1 (CF1). Conditions are described under which Lucifer yellow selectively labels the alpha subunit of CF1 to the extent of about 1 mol of probe per mole of CF1. An especially reactive amino group is apparently labeled, and modification has little effect on the ATPase activity of the enzyme. Lucifer yellow is a useful probe for fluorescence energy transfer measurements. The distances between this probe and fluorescent and absorbing molecules attached to seven specific sites on the beta, gamma, and epsilon subunits were determined. These distances converge to a single location. In addition to providing further information about the structure of CF1, these results suggest that the alpha subunits of CF1 are not structurally equivalent.     

Fluorescence energy transfer detects changes in fibronectin structure upon surface binding

We report here the changes in intramolecular distances in human plasma fibronectin (Fn) detected, upon adsorption of the protein to the surface of the Cytodex dextran microcarrier, using a fluorescence energy transfer technique. The glutamine-3 residue, near the amino terminus of each chain, was labeled enzymatically with either monodansylcadaverine (dansyl) or monofluoresceinyl-cadaverine (fluorescein) by use of coagulation factor XIIIa. Using this donor (dansyl)-acceptor (fluorescein) pair, and steady-state measurements, we demonstrated previously that the two amino termini of plasma fibronectin in solution were juxtaposed and separated by 23 A (C. Wolff and C.-S. Lai (1988) Biochemistry 27, 3483-3487). Upon adsorption to the microcarrier, the energy transfer was found to be completely abolished, suggesting that the surface binding induces a conformational change by which the distance between the two amino termini is increased to more than 70 A. Moreover, we have labeled the amino terminus of each chain with fluorescein and the two free sulfhydryl groups of each chain with coumarinyl-phenylmaleimide which serves as an energy donor. The emission spectra of the double-labeled protein in solution showed the occurrence of energy transfer, indicating that the relative distances between the amino termini and the free sulfhydryl group(s) are within 70 A. Upon surface binding, a decrease in the energy transfer between this donor-acceptor pair was also noted. The results presented here are consistent with the notion that plasma Fn undergoes a drastic conformational change upon surface binding, perhaps changing from a compact form to an extended form. This process may be important for the surface activation of the fibronectin molecule.     

Determination of the transbilayer distribution of fluorescent lipid analogues by nonradiative fluorescence resonance energy transfer.

We measured the nonradiative fluorescence resonance energy transfer between 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD) labeled lipids (amine labeled phosphatidylethanolamine or acyl chain labeled phosphatidylcholine) and rhodamine labeled lipids in large unilamellar dioleoylphosphatidylcholine vesicles. Two new rhodamine labeled lipid analogues, one a derivative of monolauroylphosphatidylethanolamine and the other of sphingosylphosphorylcholine, were found to exchange through the aqueous phase between vesicle populations but not to be capable of rapid transbilayer movement between leaflets. Energy transfer from NBD to rhodamine was measured using liposomes with symmetric or asymmetric distributions of these new rhodamine labeled lipid analogues to determine the relative contributions of energy transfer between donor and acceptor fluorophores in the same (cis) and opposite (trans) leaflets. Since the characteristic R0 values for energy transfer ranged from 47 to 73 A in all cases, significant contributions from both cis and trans energy transfer were observed. Therefore, neither of these probes acts strictly as a half-bilayer quencher of NBD lipid fluorescence. The dependence of transfer efficiency on acceptor density was fitted to a theoretical treatment of energy transfer to determine the distances of closest approach for cis and trans transfer. These parameters set limits on the positions of the fluorescent groups relative to the bilayer center, 20-31 A for NBD and 31-55 A for rhodamine, and provide a basis for future use of these analogues in measurements of transbilayer distribution and transport.     

Structural studies and mechanism of Saccharomyces cerevisiae dolichyl-phosphate-mannose synthase: insights into the initial step of synthesis of dolichyl-phosphate-linked oligosaccharide chains in membranes of endoplasmic reticulum

Dolichyl-phosphate-mannose (Dol-P-Man) synthase catalyzes the reversible formation of a key intermediate that is involved as a mannosyl donor in at least three different pathways for the synthesis of glycoconjugates important for eukaryotic development and viability. The enzyme is found associated with membranes of the endoplasmic reticulum (ER), where it transfers mannose from the water soluble cytoplasmic donor, guanosine 5'-diphosphate (GDP)-Man, to the membrane-bound, extremely hydrophobic, and long-chain polyisoprenoid acceptor, dolichyl-phosphate (Dol-P). The enzyme from Saccharomyces cerevisiae has been utilized to investigate the structure and activity of the protein and interactions of the enzyme with Dol-P and synthetic Dol-P analogs containing fluorescent probes. These interactions have been explored utilizing fluorescence resonance energy transfer (FRET) to establish intramolecular distances within the protein molecule as well as intermolecular distances to determine the localization of the active site and the hydrophobic substrate on the enzyme's surface. A three-dimensional (3D) model of the enzyme was produced with bound substrates, Dol-P, GDP-Man, and divalent cations to delineate the binding sites for these substrates as well as the catalytic site. The FRET analysis was used to characterize the functional properties of the enzyme and to evaluate its modeled structure. The data allowed for proposing a molecular mechanism of catalysis as an inverting mechanism of mannosyl residue transfer.     

Structural mapping of rabbit muscle phosphofructokinase. Distance between the adenosine cyclic 3',5'-phosphate binding site and a reactive sulfhydryl group

The cAMP binding site of rabbit muscle phosphofructokinase has been labeled with the fluorescent molecule 5'-(p-fluorosulfonylbenzoyl)-2-aza-1,N6-ethenoadenosine. The most reactive sulfhydry- group of this modified enzyme, which is catalytically active, has been labeled with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and with N-]4-(dimethylamino)-3,5-dinitrophenyl]maleimide. The calculated distances between the cAMP binding site and the most reactive sulfhydryl group, as determined by resonance energy transfer measurements, are 31 and 26 A, respectively, for the two sulfhydryl group labels. Both steady-state and fluorescent -ifetime techniques were used to measure the energy transfer efficiencies in 50 mM potassium phosphate (pH 8.0) and 1 mM ethylenediaminetetraacetic acid, and a value of 2/3 was assumed for the donor-acceptor orientation factor. If the difference in calculated distances is attributed to a difference in the orientation factor for the two donor-acceptor ,airs, the actual distance between the cAMP ligand binding site and the most reactive sulfhydryl group on phosphofructokinase is shown to be 28 +/- 6 A.     

FRET analysis indicates that the two ATPase active sites of the P-glycoprotein multidrug transporter are closely associated

Members of the ABC superfamily carry out the transport of various molecules and ions across cellular membranes, powered by ATP hydrolysis. Substantial evidence indicates that the two catalytic sites of the nucleotide binding domains function in a highly cooperative, alternating sites mode, which suggests the possibility that they interact with each other physically. In this study, fluorescence energy transfer experiments were used to estimate the distance between two fluors, each covalently linked to a highly conserved Cys residue (Cys428 and Cys1071) within the Walker A motif of the catalytic site. The vanadate.ADP.Mg(2+) complex was trapped in one catalytic site of membrane-bound or highly purified P-glycoprotein, and the other site was labeled with MIANS [2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid]. Following loss of the trapped vanadate complex, the newly vacant site was then labeled with NBD-Cl (7-chloro-4-nitrobenzo-2-oxa-1,3-diazole). The fluorescence properties of the singly labeled P-glycoproteins showed that no energy transfer occurred between MIANS (the donor) and NBD (the acceptor) when they were simply mixed together. On the other hand, the fluorescence emission of the MIANS group in doubly labeled P-glycoprotein was highly quenched as a result of energy transfer to NBD, leading to an estimate of a donor-acceptor separation distance of approximately 16 A for P-glycoprotein labeled in the native plasma membrane and approximately 22 A for P-glycoprotein labeled in detergent solution. The separation of the two fluorophores is compatible with the recently reported crystal structure of the Rad50cd dimer, but not with that of the HisP dimer. These results suggest that the two catalytic sites of the P-glycoprotein nucleotide binding domains are relatively close together, which would facilitate cooperation between them during the catalytic cycle.     

Resonance energy transfer measurements between substrate binding sites within the large (Klenow) fragment of Escherichia coli DNA polymerase I

Resonance energy transfer was used to determine separation distances between fluorescent derivatives of substrates for Klenow fragment and a unique sulfhydryl, cysteine 907, on the enzyme. Fluorescent derivatives of duplex DNA, deoxynucleotide triphosphates (dNTP), and deoxynucleotide monophosphates (dNMP), modified with aminonaphthalenesulfonates (ANS), served as energy-transfer donors to the fluorophore used to modify cysteine 907, 4-[N-[(iodoacetoxy)ethyl]-N-methylamino]-7-nitrobenz-2-oxa-1,3-diazole (IANBD). The labeling of cysteine 907 with NBD caused no decrease in the enzyme's polymerase activity, suggesting that the probe did not significantly alter the conformation of the enzyme. The efficiency of singlet-singlet resonance energy transfer was determined from the quantum yield of the donor in the presence and absence of acceptor. By F?rster's theory, the measured distances between cysteine 907 and binding sites for duplex DNA, dNTP, and dNMP were 25-39, 19-28, and 17-26 A, respectively. As the fluorophores, attached to the substrates via a tether arm, are separated from the substrates by approximately 12 A, the distances measured between binding sites are subject to this uncertainty. To measure the separation between binding sites for duplex DNA and dNMP, and to reduce the uncertainty introduced by the tether arm, two experiments were carried out. In the first, duplex DNA was labeled with the acceptor fluorophore NBD and used with the donor ANS-modified dNMP to yield a measured distance separating these two sites of 19-28 A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Resonance energy transfer microscopy: observations of membrane-bound fluorescent probes in model membranes and in living cells

A conventional fluorescence microscope was modified to observe the sites of resonance energy transfer (RET) between fluorescent probes in model membranes and in living cells. These modifications, and the parameters necessary to observe RET between membrane-bound fluorochromes, are detailed for a system that uses N-4-nitrobenzo-2-oxa-1,3-diazole (NBD) or fluorescein as the energy donor and sulforhodamine as the energy acceptor. The necessary parameters for RET in this system were first optimized using liposomes. Both quenching of the energy donor and sensitized fluorescence of the energy acceptor could be directly observed in the microscope. RET microscopy was then used in cultured fibroblasts to identify those intracellular organelles labeled by the lipid probe, N-SRh-decylamine (N-SRh-C10). This was done by observing the sites of RET in cells doubly labeled with N-SRh-C10 and an NBD-labeled lipid previously shown to label the endoplasmic reticulum, mitochondria, and nuclear envelope. RET microscopy was also used in cells treated with fluorescein-labeled Lens culinaris agglutinin and a sulforhodamine derivative of phosphatidylcholine to examine the internalization of plasma membrane lipid and protein probes. After internalization, the fluorescent lectin resided in most, but not all of the intracellular compartments labeled by the fluorescent lipid, suggesting sorting of the membrane-bound lectin into a subset of internal compartments. We conclude that RET microscopy can co-localize different membrane-bound components at high resolution, and may be particularly useful in examining temporal and spatial changes in the distribution of fluorescent molecules in membranes of the living cell.     

Structural mapping of cysteine-63 of the chloroplast ATP synthase beta subunit.

The single sulfhydryl residue (cysteine-63) of the beta subunit of the chloroplast ATP synthase F1 (CF1) was accessible to labeling reagents only after removal of the beta subunit from the enzyme complex. This suggests that cysteine-63 may be located at an interface between the beta and the alpha subunits of CF1, although alternative explanations such as a conformational change in beta brought about by its release from CF1 cannot be ruled out. Cysteine-63 was specifically labeled with [(diethylamino)methylcoumarinyl]-maleimide, and the distance between this site and trinitrophenyl-ADP at the nucleotide binding site on beta was mapped using fluorescence resonance energy transfer. Cysteine-63 is located in a hydrophobic pocket, 42 A away from the nucleotide binding site on beta.     

The active site of membrane-bound meizothrombin. A fluorescence determination of its distance from the phospholipid surface and its conformational sensitivity to calcium and factor Va

The distance between the phospholipid surface and the active site of membrane-bound meizothrombin, a derivative of prothrombin, was determined directly using fluorescence energy transfer. The active site of prothrombin was exposed after a single cleavage by Echis carinatus protease in the presence of [5-(dimethylamino)-1-naphthalenesulfonyl]glutamylglycylarginyl+ ++ (DEGR) chloromethyl ketone to yield DEGR-meizothrombin and thereby minimize secondary proteolysis. When DEGR-meizothrombin was titrated with 80% phosphatidylcholine, 20% phosphatidylserine vesicles containing octadecylrhodamine, singlet-singlet energy transfer was observed between the donor dyes in the active sites of the membrane-bound proteins and the acceptor dyes at the outer surface of the phospholipid bilayer. This energy transfer required both Ca2+ and phosphatidylserine. Assuming k2 = 2/3, the dependence of the efficiency of energy transfer upon the acceptor density showed that the distance of closest approach between the active site probe and the bilayer surface was 71 +/- 2 A. In the presence of factor Va, the distance was 67 +/- 3 A. These direct measurements show that the active site of meizothrombin is located far above the membrane surface. Also, association of factor Va with meizothrombin on the phospholipid surface appears to cause a slight movement of the meizothrombin protease domain toward the membrane surface. The environment of the dansyl dye covalently attached to the active site of meizothrombin was particularly sensitive to the presence of calcium: addition of Ca2+ ions to metal-free DEGR-meizothrombin reduced the dansyl fluorescence lifetime from 11.7 to 9.0 ns and the dansyl emission intensity by 24%. Hence, the conformation of the active site changed when Ca2+ ions bound to meizothrombin. Since the intensity change was half-maximal at 0.2 mM and was also elicited by the binding of Mg2+ ions, this spectral change correlates with the calcium-dependent conformational change previously observed in fragment 1. We conclude, therefore, that the binding of Ca2+ ions to meizothrombin and, by extension, perhaps to prothrombin, elicits a conformational change that extends beyond the fragment 1 domains into the distant (cf. above) active site or protease domain. The association of factor Va with membrane-bound DEGR-meizothrombin increased both the dansyl emission intensity (by 7%) and polarization. This intensity change and the factor-Va dependent change in energy transfer indicate that the cofactor of the prothrombinase complex functions to modulate the conformation and orientation of both the substrate and the enzyme of the complex.     

The active site of blood coagulation factor Xa. Its distance from the phospholipid surface and its conformational sensitivity to components of the prothrombinase complex

The location of the active site of membrane-bound factor Xa relative to the phospholipid surface was determined both in the presence and absence of factor Va using fluorescence energy transfer. Factor Xa was reacted with 5-(dimethylamino)-1-naphthalenesulfonyl- glutamylglycylarginyl(DEGR) chloromethyl ketone to yield DEGR-Xa, an analogue of factor Xa with a fluorescent dye attached covalently to the active site. When DEGR-Xa was titrated with phosphatidylcholine/phosphatidylserine vesicles containing octadecylrhodamine, fluorescence energy transfer was observed between the donor dyes in the active sites of the membrane-bound enzymes and the acceptor dyes at the outer surface of the phospholipid bilayer. Based on the dependence of the efficiency of singlet-singlet energy transfer upon the acceptor density and assuming kappa 2 = 2/3, the distance of closest approach between the active site probe and the surface of the phospholipid bilayer averaged 61 A in the absence of factor Va and 69 A in the presence of factor Va. These direct measurements show that the active site of factor Xa is located far above the membrane surface. Also, association of factor Xa with factor Va on the membrane surface to form the prothrombinase complex results in a substantial movement of the active site of the enzyme relative to the membrane surface. The 5-(dimethylamino)-1-naphthalenesulfonyl emission in the complete prothrombinase complex was distinct from that in any other combination of components. It therefore appears that the optimum conformation of the prothrombinase active site is achieved only when factor Va, Ca2+, and a membrane surface interact simultaneously with factor Xa. Thus, in addition to its previously demonstrated ability to stimulate factor Xa binding to membranes, factor Va, upon association with factor Xa on a phospholipid surface, allosterically induces a particular active site conformation in factor Xa and also positions the active site at the correct distance above the membrane for prothrombin activation.