Structural mapping of membrane-bound immunoglobulin E-receptor complexes: use of monoclonal anti-IgE antibodies to probe the conformation of receptor-bound IgE

Previous resonance energy-transfer measurements have suggested that immunoglobulin E (IgE) may bend near the junction of its Fc and Fab segments in order to bind to its high-affinity receptor on rat basophilic leukemia cells. In order to test this possibility, two monoclonal antibodies were employed that bind specifically to rat IgE (IgER) when IgER is in solution and when it is bound to receptors on the plasma membrane. The F(ab')2 fragment of one monoclonal (B5) that is specific for the Fab region of IgER was labeled with donor probes and bound to IgER, and the quenching of the fluorescence of these donors due to simultaneous binding of the Fab' fragment of an anti-Fc monoclonal (A2) that was labeled with an acceptor probe at its interchain disulfide bond was measured. Significantly less energy transfer between these probes was observed when IgER was bound to its receptor on membrane vesicles than when it was free in solution, and this result is interpreted in light of other energy-transfer measurements using A2 and B5 that were preferentially labeled near their combining sites with donors and acceptors, respectively, as well as measurements of the distance of closest approach between these sites and the membrane surface. These results along with previous energy-transfer measurements and other biochemical information form the basis for a working model of the conformation and orientation of receptor-bound IgE. This study demonstrates the use of fluorescently labeled monoclonal antibodies as highly selective energy-transfer probes in assessing structures of macromolecular complexes on the plasma membrane.     

Location of the epidermal growth factor binding site on the EGF receptor. A resonance energy transfer study

As a first step toward developing a structural map of key sites on the epidermal growth factor (EGF) receptor, we have used resonance energy transfer to measure the distance of closest approach between the receptor-bound growth factor molecule and lipid molecules at the surface of the plasma membrane. EGF, specifically labeled at its amino terminus with fluorescein 5-isothiocyanate, was used as an energy donor in these experiments, while either octadecylrhodamine B or octadecylrhodamine 101, inserted into plasma membranes isolated from human epidermoid carcinoma (A431) cells, served as the energy acceptors. The energy transfer measurements indicate that the amino terminus of the bound growth factor is about 67 A away from the plasma membrane. On the basis of the dimensions of the EGF molecule, this suggests that EGF binds to a site on its receptor that is a considerable distance (52-82 A) from the surface of these cells. Identical results were obtained under conditions where the receptor functions as an active tyrosine kinase, suggesting that the relative juxtaposition of the EGF binding domain to the membrane surface does not change with receptor autophosphorylation or with the activation of the receptor tyrosine kinase activity.     

Segmental flexibility of receptor-bound immunoglobulin E

The segmental flexibility of mouse immunoglobulin E (IgE) bound to its high-affinity receptor on membrane vesicles from rat basophilic leukemia cells was compared to that of IgE in solution by measuring the steady-state anisotropy as a function of temperature and viscosity. A monoclonal IgE was used to bind the fluorescent probe N-[5-(dimethylamino)naphthalene-1-sulfonyl]-L-lysine (DNS-Lys) rigidly and specifically in the antigen combining site at the tip of the Fab region. The average rotational correlation time, phi, of 74-89 ns for the receptor-bound IgE is only slightly longer than that for IgE in solution where phi = 54 ns. Another mouse monoclonal IgE was covalently labeled in the Fab region with N-(1-pyrenyl)maleimide. Anisotropy measurements with this derivative yielded results that are very similar to those found with anti-DNS IgE and DNS-Lys. These findings are strikingly different from that expected for a rigid IgE bound to its receptor since in this case phi is likely to be very much larger. Evidently, the segmental flexibility of IgE is not greatly altered upon binding to its receptor.     

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 map of the dicyclohexylcarbodiimide site of chloroplast coupling factor determined by resonance energy transfer

Fluorescence resonance energy-transfer measurements were made on the membrane-bound chloroplast coupling factor. The distances from the N,N'-dicyclohexylcarbodiimide-binding site on the membrane-bound portion of the enzyme (CF0) to the vesicle surface and to two sulfhydryl sites on the gamma-polypeptide were determined. The dicyclohexylcarbodiimide-binding site was labeled with the fluorescent species N-cyclohexyl-N'-pyrenylcarbodiimide. The vesicle surface was labeled with N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine. Steady-state energy transfer between the fluorescent-labeled enzyme (energy donor) and varying concentrations of the ethanolamine derivative (energy acceptor) indicated that the distance of closest approach between the energy donor and the outer vesicle surface is 16-24 A. Two specific sites on the gamma-polypeptide were reacted with a coumarinylmaleimide derivative; one is a sulfhydryl that can be labeled only on the thylakoids under energized conditions (the "light" site), while the other is the disulfide site that regulates enzymatic activity. Energy-transfer measurements utilizing steady-state fluorescence and fluorescence lifetime methods indicated that the dicyclohexylcarbodiimide site is approximately 41 A from the light site and approximately 50 A from the gamma-disulfide site. These distances are used to extend the current structural model of the chloroplast coupling factor.     

Resonance energy transfer between the active sites of myocardial-type creatine kinase (isozyme MB)

The single reactive sulfhydryl group, located in the active site of each subunit of dimeric creatine kinase from rabbit muscle (isozyme MM), was selectively labeled with 3-(4-maleimidylphenyl)-7-(diethylamino)-4-methylcoumarin (CPM). Isozyme BB, purified to homogeneity from rabbit brain, was conjugated with the sulfhydryl-specific reagent 5'-(iodoacetamido)fluorescein (5'-IAF). Spectral analyses demonstrated that 1.8 mol of CPM and 1.9 mol of 5'-IAF had reacted per mol of protein. Labeled isozymes were combined, denatured in 8 M urea, and renatured by dialysis, producing the parent labeled homodimers and forming the heterolabeled hybrid dimer, creatine kinase MB. Similar hybridizations were performed to prepare singly labeled hybrids, starting with labeled and unlabeled homodimers. The hybrid isozymes were isolated by ion-exchange chromatography, and spectral analyses of singly labeled heterodimers revealed overlap between the absorption spectrum of MB labeled with acetamidofluorescein on the B subunit and the corrected fluorescence emission spectrum of MB labeled with CPM on the M subunit. Analyses included evaluation of the quantum yield of the CPM-labeled hybrid, estimation of the range of the orientation factor K2 from fluorescence polarization and anisotropy studies, and determination of J, the spectral overlap integral for the fluorescence donor (CPM-labeled MB) and acceptor (acetamidofluorescein-labeled MB). Results of these experiments permitted an estimation of R0, the distance between the donor and the acceptor at which energy transfer is 50% efficient. Comparison of the relative fluorescence of the donor in the presence (heterolabeled hybrid) and absence (hybrid conjugated with CPM on the M subunit) of the acceptor or determination of the normalized sensitization of the acceptor fluorescence led to an evaluation of the transfer efficiency and the actual transfer distance of between 27 and 52 A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescence energy transfer studies on lima bean lectin. Distance between the subunit hydrophobic binding site and the thiol group essential for carbohydrate binding

Measurements of the efficiency of singlet-singlet energy transfer were used to determine the distance between the hydrophobic binding site and the thiol group required for carbohydrate-binding activity of lima bean lectin. 1-Anilino-8-naphthalenesulfonate, bound to the hydrophobic binding site by noncovalent interactions, was used as the donor. Two different nonfluorescent probes were used as the acceptors: a mercurial, 2-chloromercuri-4-nitrophenol, and a maleimide, 4-dimethylaminophenylazophenyl-4'-maleimide. Acceptor was covalently attached to the thiol group at the putative carbohydrate binding site. The efficiency of energy transfer in both the 1-anilino-8-naphthalenesulfonate/2-chloromercuri-4-nitrophenol and and 1-anilino-8-naphthalenesulfonate/4-dimethylaminophenylazophenyl-4' -maleimide donor-acceptor systems indicated an apparent distance of 28 A between the two sites, assuming that the transition dipole of the donor is not correlated with respect to that of the acceptor and that each donor is quenched by a single acceptor. Using an alternate model wherein each donor is equally quenched by two acceptors on adjacent subunits, an apparent distance of 33.4 A was calculated. The fact that two donor-acceptor pairs with different F?rster's critical distance parameters yielded the same distance between the sites is consistent with our assumption of uncorrelated donor-acceptor transition dipoles.     

Distance relationships between the catalytic site labeled with 4-(iodoacetamido)salicylic acid and regulatory sites of glutamate dehydrogenase

The distance between the catalytic site on bovine liver glutamate dehydrogenase labeled with 4-(iodoacetamido)salicylic acid (ISA) and the adenosine 5'-diphosphate (ADP) activatory site occupied by the analogue 2',3'-O-(2,4,6-trinitrocyclohexadienylidene)adenosine 5'-diphosphate (TNP-ADP) was evaluated by energy transfer. Native enzyme and enzyme containing about 1 mol of acetamidosalicylate/mol of subunit bind about 0.5 mol of TNP-ADP/mol of subunit, and TNP-ADP competes for binding with ADP to native and modified enzyme, indicating that the analogue is a satisfactory probe of the ADP site. From the quenching of acetamidosalicylate donor fluorescence upon addition of TNP-ADP, an average distance of 33 A was determined between the catalytic and ADP sites. The fluorescent nucleotide analogue 5'-[p-(fluorosulfonyl)benzoyl]-2-aza-1,N6-ethenoadenosine (5'-FSBa epsilon A) reacts covalently with glutamate dehydrogenase to about 1 mol/peptide chain. As compared to native enzyme, the SBa epsilon A-enzyme exhibits decreased sensitivity to GTP inhibition but retains its catalytic activity as well as its ability to be activated by ADP and inhibited by high concentrations of NADH. Complete protection against decreased sensitivity to GTP inhibition is provided by GTP in the presence of NADH. It is concluded that 5'-FSBa epsilon A modifies a GTP site on glutamate dehydrogenase. The distance of 23 A between the catalytic site labeled with ISA and a GTP site labeled with 5'-FSBa epsilon A was measured from the quenching of salicylate donor fluorescence in the presence of the SBa epsilon A acceptor on a doubly labeled enzyme. The average distance between the ADP and GTP sites was previously measured as 18 A [Jacobson, M. A., & Colman, R. F. (1983) Biochemistry 22, 4247-4257], indicating that the regulatory sites of glutamate dehydrogenase are closer to each other than to the catalytic site.     

Localization of site-specific probes on the Ca-ATPase of sarcoplasmic reticulum using fluorescence energy transfer

Highly reactive sulfhydryls, previously labeled with an iodoacetamide spin label on the Ca-ATPase of sarcoplasmic reticulum, were labeled with the fluorescent probe, 5-(2-[iodoacetyl)amino)ethyl)aminonaphthalene-1-sulfonic acid (IAEDANS), without loss of enzymatic activity. We have selectively measured the apparent distance of the more reactive site, relative to other site-specific probes at both the nucleotide and the high affinity calcium binding sites. Fluorescence energy transfer efficiencies from the donor IAEDANS to two acceptors: fluorescein 5'-isothiocyanate or 2',3'-O-(2,4,3-trinitrophenyl)adenosine monophosphate, situated at or near the nucleotide site, were measured using fluorescence lifetimes and yields. Fluorescence on polyacrylamide gels shows that the IAEDANS and fluorescein 5'-isothiocyanate labels are both associated with the B tryptic fragment. The energy transfer measurements are consistent with distances of 56 and 68 A between IAEDANS and these respective binding sites. On the other hand, energy transfer measurements using the lanthanide, praseodymium (Pr3+), as an acceptor indicate that IAEDANS is located 16-18 A from the binding site(s) of this calcium analog. Pr3+ is shown to be a good analog for calcium binding to the high affinity sites on the enzyme since it competitively displaces calcium, as evidenced by the reversal of the specific calcium-dependent intrinsic fluorescent signal and inactivation of ATPase activity, over the same narrow range in Pr3+ concentration where energy transfer is observed. Our observations suggest that the portion of the B fragment spanning the cytoplasmic portion of the ATPase is folded onto the A fragment, bringing the IAEDANS label in close proximity to the high affinity calcium binding domain.     

Distances between active site probes in glutamine synthetase from Escherichia coli: fluorescence energy transfer in free and in stacked dodecamers

Probes for fluorescence energy transfer measurements were introduced into active sites of dodecameric glutamine synthetase from Escherichia coli by substituting appropriate ATP analogues for ATP in the autoinactivation reaction of this enzyme with L-Met-(S)-sulfoximine and Mn2+ [Maurizi, M. R., & Ginsburg, A. (1986) Biochemistry (preceding paper in this issue)]. Two fluorescent donors, 8-mercapto-ATP alkylated with either 5-[[[(iodoacetyl)amino]ethyl]amino]naphthalene-1-sulfonic acid (AEDANS-ATP) or 1,N6-etheno-2-aza-ATP (aza-epsilon-ATP), and two acceptors, 6-mercaptopurine ribonucleotide triphosphate or 8-mercapto-ATP alkylated with the chromophore 4'-[[4-(dimethylamino)-phenyl]azo]-2-iodoacetanilide (6-Y- or 8-Y-ATP), were used. Fluorescence emissions of enzyme derivatives with 1 or 2 equiv of fluorescent donor per dodecamer and either an acceptor (Y) or ADP at the remaining active sites were compared at pH 7.0. The results, together with the known geometry of the enzyme, indicate that active site probes in the dodecamer are widely separated and that energy transfer occurs from a single donor to two or three acceptors on adjacent subunits. The calculated distance between equidistant active site probes on heterologously bonded subunits within the same hexagonal ring is 56-60 A. Probes on isologously bonded subunits are no closer than 60 A and may be as far apart as 78 A. Thus, active sites are away from the 6-fold axis of symmetry toward the outer edges of the dodecamer and are located greater than or equal to 30 A from the plane separating the hexagonal rings. During Zn2+-induced stacking of the same enzyme derivatives along the 6-fold axes of symmetry, additional quenches of fluorescent probes were dependent on the presence of acceptors on separate dodecamers. The Zn2+-induced face to face aggregation of dodecamers in the presence of 46 microM ZnCl2 and 9 mM MgCl2 at pH 7.0 had an Arrhenius activation energy of 22.3 +/- 0.2 kcal/mol and a second-order rate constant at 25 degrees C of approximately 10(5) M-1 s-1 at early stages. Time-dependent fluorescence quenches correlated well with the degree of linear polymer formation and reached maximum values of 47-70% quench when the average n-mer was six dodecamers. After correction for unquenched polymer ends, a fluorescent donor and an acceptor probe in layered dodecamers were estimated to be approximately 36 A apart--an average value if there is some twisting of single strands.(ABSTRACT TRUNCATED AT 400 WORDS)     

Proximity of bound Hoechst 33342 to the ATPase catalytic sites places the drug binding site of P-glycoprotein within the cytoplasmic membrane leaflet

The P-glycoprotein multidrug transporter carries out ATP-driven cellular efflux of a wide variety of hydrophobic drugs, natural products, and peptides. Multiple binding sites for substrates appear to exist, most likely within the hydrophobic membrane spanning regions of the protein. Since ATP hydrolysis is coupled to drug transport, the spatial relationship of the drug binding sites relative to the ATPase catalytic sites is of considerable interest. We have used a fluorescence resonance energy transfer (FRET) approach to estimate the distance between a bound substrate and the catalytic sites in purified P-glycoprotein. The fluorescent dye Hoechst 33342 (H33342), a high-affinity P-glycoprotein substrate, bound to the transporter and acted as a FRET donor. H33342 showed greatly enhanced fluorescence emission when bound to P-glycoprotein, together with a substantial blue shift, indicating that the drug binding site is located in a nonpolar environment. Cys428 and Cys1071 within the catalytic sites of P-glycoprotein were covalently labeled with the acceptor fluorophore NBD-Cl (7-chloro-4-nitrobenz-2-oxa-1,3-diazole). H33342 fluorescence was highly quenched when bound to NBD-labeled P-glycoprotein relative to unlabeled protein, indicating that FRET takes place from the bound dye to NBD. The distance separating the bound dye from the NBD acceptor was estimated to be approximately 38 A. Transition-state P-glycoprotein with the complex ADP*orthovanadate*Co2+ stably trapped at one catalytic site bound H33342 with similar affinity, and FRET measurements led to a similar separation distance estimate of 34 A. Since previous FRET studies indicated that a fluorophore bound within the catalytic site was positioned 31-35 A from the interfacial region of the bilayer, the H33342 binding site is likely located 10-14 A below the membrane surface, within the cytoplasmic leaflet of the membrane, in both resting-state and transition-state P-glycoprotein.     

Spatial relationship and conformational changes between the cardiac glycoside site and beta-subunit oligosaccharides in sodium plus potassium activated adenosinetriphosphatase

(Na,K)-ATPase, the enzyme responsible for active transport of Na and K across the plasma membranes of animal cells, consists of a catalytic subunit (alpha) and a glycoprotein subunit (beta) with unknown function. We have determined the distance between fluorescent probes directed to specific sites on the alpha- and beta-subunits and ligand-induced changes in the fluorescence of a probe specifically attached to the beta-subunit. The cardiac glycoside site on the alpha-subunit was labeled with anthroylouabain [Fortes, P. A. G. (1977) Biochemistry 16, 531-540]. The oligosaccharides on the beta-subunit were labeled with lucifer yellow carbohydrazide [Lee, J. A., & Fortes, P. A. G. (1985) Biochemistry 24, 322-330]. Resonance energy transfer from anthroylouabain to lucifer yellow was measured by steady-state and time-resolved fluorescence spectroscopy. The distance between these probes was determined from the efficiency of energy transfer. The average distance between anthroylouabain and lucifer yellow was 47 A and was independent of the number of acceptor molecules attached to the beta-subunit. The measured distance corresponds to the distance between the cardiac glycoside site and the center of the labeled oligosaccharides on the beta-subunit within one alpha beta dimer. The distance was the same (47 A) when anthroylouabain was bound with ATP or Pi as phosphorylating ligands but increased to 49 A in the presence of vanadate. The change in average distance provides quantitative evidence of a conformational difference between the complexes of cardiac glycosides with (Na,K)-ATPase induced by phosphorylating ligands or by vanadate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Location of the stilbenedisulfonate binding site of the human erythrocyte anion-exchange system by resonance energy transfer.

The stilbenedisulfonate inhibitory site of the human erythrocyte anion-exchange system has been characterized by using serveral fluorescent stilbenedisulfonates. The covalent inhibitor 4-benzamido-4'-isothiocyanostilbene-2,2'-disulfonate (BIDS) reacts specifically with the band 3 protein of the plasma membrane when added to intact erythrocytes, and the reversible inhibitors 4,4'-dibenzamidostilbene-2,2'-disulfonate (DBDS) and 4-benzamido-4'-aminostilbene-2,2'-disulfonate (BADS) show a fluorescence enhancement upon binding to the inhibitory site on erythrocyte ghosts. The fluorescence properties of all three bound probes indicate a rigid, hydrophobic site with nearby tryptophan residues. The Triton X-100 solublized and purified band 3 protein has similar affinities for DBDS, BADS, and 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS) to those observed on intact erythrocytes and erythrocyte ghosts, showing that the anion binding site is not perturbed by the solubilization procedure. The distance between the stilbenedisulfonate binding site and a group of cysteine residues on the 40 000-dalton amino-terminal cytoplasmic domain of band 3 was measured by the fluorescence resonance energy transfer technique. Four different fluorescent sulfhydryl reagents were used as either energy transfer donors or energy transfer acceptors in combination with the stilbenedisulfonates (BIDS, DBDS, BADS, and DNDS). Efficiencies of transfer were measured by sensitized emisssion, donor quenching, and donor lifetime changes. Although these sites are approachable from opposite sides of the membrane by impermeant reagents, they are separated by only 34--42 A, indicating that the anion binding site is located in a protein cleft which extends some distance into the membrane.     

The active site of factor IXa is located far above the membrane surface and its conformation is altered upon association with factor VIIIa. A fluorescence study.

The topography of membrane-bound blood coagulation factor IXa (fIXa) and the nature of its interaction with its cofactor, factor VIIIa (fVIIIa), were examined using fluorescent derivatives of fIXa. A fluorescein dye was covalently attached to the active-site histidine of fIXa via a D-Phe-Pro-Arg tripeptide tether to form Fl-A-FPR-fIXa; similarly, a 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) dye was covalently attached via Glu-Gly-Arg to form DEGR-fIXa. When either Fl-A-FPR-fIXa or DEGR-fIXa was titrated with phosphatidylcholine-phosphatidylserine vesicles containing octadecylrhodamine in the presence of Ca2+, fluorescence energy transfer was observed. Assuming a random orientation of dyes, the distance of closest approach between the donor dyes in the active sites of the membrane-bound enzymes and the acceptor dyes at the membrane surface was found to be 89 +/- 3 A for Fl-A-FPR-fIXa and 73 +/- 4 A for DEGR-fIXa. Although the exact distance remains uncertain, it is clear that the active site of fIXa is positioned more than 70 A above the surface, and hence that the elongated fIXa molecule projects approximately perpendicularly from the surface when bound to the membrane. The binding of fVIIIa to membrane-bound Fl-A-FPR-fIXa or DEGR-fIXa did not alter the location of the active site relative to the membrane surface, but did alter both the emission intensity and anisotropy of the fluorescein and dansyl probes and hence their environments. Cofactor stimulation of fIXa activity therefore appears to be mediated, at least in part, by a conformational change in the active site that occurs when fVIIIa binds to the enzyme on the phospholipid surface.     

Resonance energy transfer between the active sites of rabbit muscle creatine kinase: analysis by steady-state and time-resolved fluorescence

Resonance energy transfer between the reactive thiols of rabbit muscle creatine kinase was evaluated. The reactive thiols are located at the active site, one occurring on each subunit of the dimeric protein that is known to be a constituent of the M-line structure of the myofibril. Transfer efficiency was evaluated from energy donor quenching of fluorescence by steady-state and phase-modulation lifetime measurements and determination of sensitized emission of the acceptor. Several sulfhydryl-specific donor fluorophores were used including 5-[[[(iodoacetyl)amino]ethyl]amino]naphthalene-1-sulfonic acid, 7-(dimethylamino)-3-(4-maleimidylphenyl)-4-methylcoumarin, and 2-[4-(iodoacetamido)anilino]naphthalene-6-sulfonic acid (IAANS). Energy transfer acceptors included 5-(iodoacetamido)fluorescein and the nonfluorescent dye [4-[[4-(dimethylamino)phenyl]azo]phenyl]iodoacetamide. In order to prepare the necessary homodimer labeled with both donor and acceptor, advantage was taken of the biphasic reaction between creatine kinase and IAANS. In some instances, donor/acceptor hybrids were prepared by denaturation/renaturation procedures, and possible deviations from expected hybridization stoichiometry were considered. Disproportionation of singly labeled dimers (to unlabeled and doubly labeled dimers) was not observed when the brain isozyme of creatine kinase was used to trap dissociated dye-conjugated or unlabeled muscle-type subunits of creatine kinase. From studies of five different donor/acceptor combinations, the efficiency of energy transfer was found to occur over a range of 5-14%, indicating that the reactive thiols are well separated. Overlap integrals and quantum yields were evaluated, and estimates of the range of orientation factor were obtained to determine a range for the distance between the active sites of creatine kinase. When the ranges are overlapped, a limited distance of 48.6-60.4 A is obtained.(ABSTRACT TRUNCATED AT 250 WORDS)     

The catecholamine binding site of the beta-adrenergic receptor is formed by juxtaposed membrane-spanning domains

The catecholamine binding domain of the turkey erythrocyte beta-adrenergic receptor was mapped by determining the sites of covalent labeling of the purified receptor by two beta-adrenergic photoaffinity reagents, [125I]iodocyanopindolol-diazirine (ICYP-da) and [125I] iodoazidobenzylpindolol (IABP). Both labels were incorporated at two separate sites. By sequencing a labeled peptide, one site of labeling was found to lie at Trp330 in the extracellular half of the seventh membrane span. This position is homologous to the retinal attachment site in rhodopsin. The second labeled site was isolated on an 8000-Da peptide and immunoprecipitated using sequence-directed antibodies. This site lies in membrane spans 3-5. Labeling of the two sites was equal using ICYP-da and 3-10-fold greater in the span 7 site using IABP. These data indicate that the catecholamine binding site is formed from the juxtaposition of span 7 and spans 3-5 in a tertiary structure probably similar to that of rhodopsin.     

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.     

Mapping the molecular structure of the voltage-dependent sodium channel. Distances between the tetrodotoxin and Leiurus quinquestriatus quinquestriatus scorpion toxin receptors

The Leiurus quinquestriatus quinquestriatus receptor site of the voltage-dependent sodium channel has been characterized using several fluorescent scorpion toxins. The derivatives show fluorescence enhancements upon binding to the receptor site on the channel together with blue shifts. The fluorescence properties of the bound probes indicate a conformationally flexible, hydrophobic site. Binding of tetrodotoxin has no effect on the fluorescence spectra of the bound derivatives, whereas binding of the allosteric activator batrachotoxin enhances the fluorescence about 2-fold and causes a red shift in the emission spectra, suggesting a batrachotoxin-induced conformational change in the scorpion toxin receptor. The distance between the tetrodotoxin receptor and the Leiurus scorpion toxin receptor on the channel was measured by fluorescence resonance energy transfer. Five different chromophoric scorpion toxin derivatives were used as energy transfer acceptors or donors with anthraniloyltetrodotoxin or N-methylanthraniloylglycine-tetrodotoxin as the energy donor or acceptor. Because of the presence of three tetrodotoxin receptors for each Leiurus receptor, the positions of the donors and acceptors were exchanged. Efficiencies of transfer were measured by both donor quenching and sensitized emission. The average distance of separation between these sites is 35 A. Upon batrachotoxin addition, this distance changes to 42 A indicating a conformational change in one subunit of the channel or a change in the interaction between two subunits coupled to the batrachotoxin-binding site. On the basis of these studies, we present a model suggesting that tetrodotoxin binds to a subunit/site which is extracellularly placed and is 35 A from the Leiurus subunit/site which is located in a protein cleft of the channel which extends partly into the membrane, and undergoes a neurotoxin and voltage-dependent conformational change.     

Quinacrine binds to the lipid-protein interface of the Torpedo acetylcholine receptor: a fluorescence study.

It has been argued both that there is a high affinity noncompetitive inhibitor binding site in the lumen of the acetylcholine receptor and that this lumen exists on the central axis of the receptor. Such a site would be expected to be 20-40 A from the membrane lipids. We tested whether, in fact, quinacrine, a potent fluorescent noncompetitive inhibitor, binds to such a site. We measured quenching of receptor-bound quinacrine fluorescence by fluorescence dipolar energy transfer to lipid probes, 5-(N-dodecanoylamino)eosin and N-(3-sulfopropyl)-4-(p-didecylaminostyryl)pyridinium, or by collision with paramagnetic lipid probes 2,2,6,6-tetramethylpiperidine-1-oxyl and 3-doxyl-17 beta-hydroxy-5 alpha-androstane (spin-labeled androstane). Initial control experiments established that in the presence of carbamylcholine, quinacrine binds to a phencyclidine-sensitive site on the Torpedo receptor with a Kd equal to 0.14 microM and with a quantum yield of 0.18. Fluorescence energy transfer from receptor-bound quinacrine had a magnitude consistent with quinacrine being less than 10 A from the lipid fluorescent probes. 2,2,6,6-Tetramethylpiperidine-1-oxyl and spin-labeled androstane were two to five times more effective at quenching receptor-bound quinacrine fluorescence than the fluorescence from membrane-partitioned 5-(dodecanoylamino)fluorescein. These results suggest that the quinacrine binding site is too close to the lipid domain to be in the lumen of the receptor, and therefore it is probably located on the outer surface of the membrane-spanning domain of the acetylcholine receptor.