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

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

Fluorescence energy transfer between Cys-10 residues in F-actin filaments

Fluorescence energy transfer was measured between Cys-10 residues in an F-actin filament using 5-[2-((iodoacetyl)amino)-ethyl]aminonaphthalene-1-sulphonic acid (1,5-IAEDANS) as a fluorescence energy donor and 4-dimethylaminophenylazophenyl-4'-maleimide (DABMI) as the acceptor. Both labels were covalently attached to Cys-10 residues in an F-actin filament. Taking the helical structure of the F-actin filament into consideration, the radial coordinate of Cys-10 was calculated to be 23 A. This corresponds to a distance between adjacent sites along the long pitch helix of 56.1 A and along the genetic helix of 53.3 A.     

Fluorescence energy transfer between subfragment-1 and actin points in the rigor complex of actosubfragment-1.

The fast-reacting thiol (SH1) of myosin subfragment-1 (S-1) was covalently and specifically labeled with (iodoacetamido)fluorescein (IAF), while Cys-373 of actin was also covalently and preferentially labeled with N-(iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine (1,5-IAEDANS). The method of fluorescence energy transfer was used to examine the spatial proximity between the two sites, i.e., SH1 and Cys-373, in the rigor complex of acto-S-1. Approximately 30% fluorescence energy transfer was observed from the 1,5-IAEDANS on actin as a donor to the IAF on S-1 as an acceptor in their rigor complex; under certain assumptions this corresponds to a distance of ca. 6.0 nm.     

Spatial relationship between the nucleotide-binding site, Lys-61 and Cys-374 in actin and a conformational change induced by myosin subfragment-1 binding

The spatial relationship between Lys-61, the nucleotide binding site and Cys-374 was studied. Lys-61 was labelled with fluorescein-5-isothiocyanate as a resonance energy acceptor, the nucleotide-binding site was labelled with the fluorescent ATP analogues epsilon ATP or formycin-A 5'-triphosphate (FTP) and Cys-374 was labelled with 5-(2-[(iodoacetyl)amino]ethyl)aminonaphthalene-1-sulfonic acid (1,5-IAEDANS) as a resonance energy donor. The distances between the nucleotide binding site and Lys-61 or between Lys-61 and Cys-374 were calculated to be 3.5 +/- 0.3 nm and 4.60 +/- 0.03 nm, respectively. (The assumption has been made in calculating these distances that the energy donor and acceptor rotate rapidly relative to the fluorescence lifetime.) On the other hand, when doubly-labelled actin with 1,5-IAEDANS at Cys-374 and FITC at Lys-61 was polymerized in the presence of a twofold molar excess of phalloidin [Miki, M. (1987) Eur. J. Biochem. 164, 229-235], the fluorescence of 1,5-IAEDANS bound to actin was quenched significantly. This could be attributed to inter-monomer energy transfer. The inter-monomer distance between FITC attached to Lys-61 in a monomer and 1,5-IAEDANS attached to Cys-374 in its nearest-neighbour monomer in an F-actin filament was calculated to be 3.34 +/- 0.06 nm, assuming that the likely change in the intra-monomer distance does not change during polymerization by more than 0.4 nm. One possible spatial relationship between Lys-61, Cys-374 and the nucleotide binding site in an F-actin filament is proposed. The effect of myosin subfragment-1 (S1) binding on the energy transfer efficiency was studied. The fluorescence intensity of AEDANS-FITC-actin decreased by 30% upon interaction with S1. The fluorescence intensity of AEDANS-FITC-actin polymer in the presence of phalloidin increased by 21% upon interaction with S1. The addition of ATP led to the fluorescence intensity returning to the initial level. Assuming that the change of fluorescence intensity can be attributed to conformational change in the actin molecule induced by S1 binding, the intra-monomer distance was reduced by 0.4 nm and the inter-monomer distance was increased by 0.2 nm.     

Fluorescence energy transfers between points in acto-subfragment-1 rigor complex

Fluorescence energy transfer was measured by time-resolved and steady-state fluorimetry in order to investigate the spatial relationships between the nucleotide binding site of actin, the Cys-373 residue of actin, and the SH1 of myosin subfragment-1 in the rigor complex of acto-subfragment-1. N-Iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS) bound to the Cys-373 of actin or the fluorescent ADP analogue 1-N6-ethenoadenosine-5'-diphosphate (epsilon-ADP) bound to F-actin was used as a donor and 4-(N-(iodoacetoxy)ethyl-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazo le (IANBD) or 5-iodoacetamidofluorescein (IAF) bound to SH1 of myosin subfragment-1 was used as an acceptor. Assuming the random orientation factor, K2, to be 2/3, the distance between Cys-373 residue of actin and SH1 of myosin subfragment-1 was calculated to be about 50 A, in agreement with the values previously reported, 60 A (Takashi, R. (1969) Biochemistry 18, 5164-69) and 50 A (Trayer, H.R. and Trayer, I.P. (1983) Eur. J. Biochem. 135, 47-59). The distance between the nucleotide binding site of actin and SH1 of myosin subfragment-1 was calculated to be about 70 A or greater.     

Fluorescence resonance energy transfer between points on actin and the C-terminal region of tropomyosin in skeletal muscle thin filaments

Fluorescence resonance energy transfer between points on tropomyosin (positions 87 and 190) and actin (Gln-41, Lys-61, Cys-374, and the ATP-binding site) showed no positional change of tropomyosin relative to actin on the thin filament in response to changes in Ca2+ concentration (Miki et al. (1998) J. Biochem. 123, 1104-1111). This is consistent with recent electron cryo-microscopy analysis, which showed that the C-terminal one-third of tropomyosin shifted significantly towards the outer domain of actin, while the N-terminal half of tropomyosin shifted only a little (Narita et al. (2001) J. Mol. Biol. 308, 241-261). In order to detect any significant positional change of the C-terminal region of tropomyosin relative to actin, we generated mutant tropomyosin molecules with a unique cysteine residue at position 237, 245, 247, or 252 in the C-terminal region. The energy donor probe was attached to these positions on tropomyosin and the acceptor probe was attached to Cys-374 or Gln-41 of actin. These probe-labeled mutant tropomyosin molecules retain the ability to regulate the acto-S1 ATPase activity in conjunction with troponin and Ca2+. Fluorescence resonance energy transfer between these points of tropomyosin and actin showed a high transfer efficiency, which should be very sensitive to changes in distance between probes attached to actin and tropomyosin. However, the transfer efficiency did not change appreciably upon removal of Ca2+ ions, suggesting that the C-terminal region of tropomyosin did not shift significantly relative to actin on the reconstituted thin filament in response to the change of Ca2+ concentration.     

Fluorescence energy transfer between Tyr 69 and Cys 374 in actin

Modification of Tyr 69 with the chromophore dansyl chloride was found to completely block exchange of the bound ATP on actin. No significant conformational change was detected in the actin after labelling thus indicating that the dansyl chloride is close to and probably sterically blocks the ATP site. The distance separating dansyl chloride attached to Tyr 69 and IAEDANS attached to Cys 374 on actin was found using fluorescence energy transfer to be 3.9 nm. This result is consistent with the known distance between the ATP site and Cys 374.     

Fluorescence energy transfer between active sites in aspartate transaminase.

The method of fluorescence energy transfer has been used to measure the distance between the active sites in a dimeric enzyme, aspartate aminotransferase. The procedure involves the prior preparation of a hybrid enzyme with the natural chromophore, pyridoxal phosphate, in one subunit as the aldimine and of the reduced aldimine in the other subunit. The two active site chromophores are used as donor and acceptor of the energy transfer and a distance of 21 Å is obtained for the separation of the active sites.     

Fluorescence resonance energy transfer mapping of the fourth of six nucleotide-binding sites of chloroplast coupling factor 1.

Equilibrium dialysis measurements of adenine nucleotide binding to chloroplast coupling factor 1 suggest that the enzyme has six binding sites for ADP, adenylyl-beta,gamma-imidodiphosphate (AMP-PNP), and 2'(3')-O-2,4,6-trinitrophenyl-ATP (TNP-ATP). High affinity binding at all six sites requires the divalent cation, Mg2+. Three of the nucleotide-binding sites, sites 1, 2, and 3, have been mapped by fluorescence resonance energy transfer distance measurements (see McCarty, R. E., and Hammes, G. G. (1987) Trends Biochem. Sci. 12, 234-237). Using the same technique, we mapped the location of nucleotide-binding site 4, a tight, exchangeable site (Shapiro, A. B., Huber, A. H., and McCarty, R. E. (1991) J. Biol. Chem. 266, 4194-4200). Two arrangements of the energy transfer map of coupling factor 1 were found which are compatible with the available data. The two arrangements make different predictions about which sites interact in cooperative catalysis.     

Fluorescence energy transfer between nucleotide binding sites in an F-actin filament

Fluorescence energy transfer between nucleotide binding sites in an F-actin filament was measured using 1-N6-ethenoadenosine diphosphate (epsilon-ADP) as a fluorescent donor and 2'(or 3')-O-(2,4,6-trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP) as an acceptor, both of which were bound to F-actin. Taking into consideration the helical structure of the F-actin filament, the radial coordinate of the nucleotide binding site was calculated to be 25 A, which corresponds to a distance between these sites along the long-pitch helix of 56.3 A and along the genetic helix of 56.7 A.     

Fluorescence energy transfer between cobra alpha-toxin molecules bound to the acetylcholine receptor

An approach was developed with steady state fluorescence energy transfer measurements to examine the spatial relationship between the two alpha-toxins bound to the acetylcholine receptor. By taking advantage of the slow dissociation rates of alpha-toxins (Naja naja siamensis 3) from the receptor and of the equal probability with which alpha-toxins bind to the two alpha-toxin-binding sites, we derived an equation which allows prediction of a true efficiency of transfer based on the relationship between fractional site occupancy and the observed transfer efficiency ascertained from donor quenching. Using this approach, we examined the efficiency of energy transfer between two fluorescently labeled alpha-toxins, N epsilon-fluorescein isothiocyanate lysine 23 alpha-toxin and monolabeled tetramethylrhodamine isothiocyanate alpha-toxin bound to the receptor from the Torpedo californica electric organ. Significantly greater (32 versus 14%) energy transfer was observed with the membrane-associated than with the solubilized receptor, suggesting that transfer between fluorophores on separate receptor molecules is greater than that occurring intramolecularly between the two sites on the receptor. The magnitude of the distances calculated from the intrareceptor energy transfer efficiency combined with the considerable inter-receptor energy transfer indicate that the fluorophores would reside on the outer perimeter of the receptor molecule rather than near the central axis perpendicular to the plane of the membrane.     

Ca2+ dependence of the distance between Cys-98 of troponin C and Cys-133 of troponin I in the ternary troponin complex. Resonance energy transfer measurements

We have used resonance energy transfer to study the spatial relationship between Cys-98 of rabbit skeletal troponin C and Cys-133 of rabbit skeletal troponin I in the reconstituted ternary troponin complex. The donor was introduced by labeling either troponin C or troponin I with N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine, while the acceptor was introduced by labeling either protein with N-[4-(dimethylamino)phenyl-4'-azophenyl]maleimide. The extent of energy transfer was determined by measuring the quenching of the donor fluorescence decay. The results indicate first that the distance between these two sites is not fixed, suggesting that the protein regions involved possess considerable segmental flexibility. Second, the mean distance between the two sites is dependent on the metal-binding state of troponin C, being 39.1 A when none of the metal-binding sites are occupied, 41.0 A when Mg2+ ions bind at the high-affinity sites, and 35.5 A when Ca2+ ions bind to the low-affinity sites. Neither the magnitude of the distances nor the trend of change with metal ions differs greatly when the locations of the probes are switched or when steady-state fluorometry was used to determine the transfer efficiency. Since the low-affinity sites have been implicated as the physiological triggering sites, our findings suggest that one of the key events in Ca2+ activation of skeletal muscle contraction is a approximately 5-A decrease in the distance between the Cys-98 region of troponin C and the Cys-133 region of troponin I.     

Cys-113 and Cys-422 form a high affinity metalloid binding site in the ArsA ATPase

The arsRDABC operon of Escherichia coli plasmid R773 encodes the ArsAB extrusion pump for the trivalent metalloids As(III) and Sb(III). ArsA, the catalytic subunit has two homologous halves, A1 and A2. Each half has a consensus signal transduction domain that physically connects the nucleotide-binding domain to the metalloid-binding domain. The relation between metalloid binding by ArsA and transport through ArsB is unclear. In this study, direct metalloid binding to ArsA was examined. The results show that ArsA binds a single Sb(III) with high affinity only in the presence of Mg(2+)-nucleotide. Mutation of the codons for Cys-113 and Cys-422 eliminated Sb(III) binding to purified ArsA. C113A/C422A ArsA has basal ATPase activity similar to that of the wild type but lacks metalloid-stimulated activity. Accumulation of metalloid was assayed in intact cells, where reduced uptake results from active extrusion by the ArsAB pump. Cells expressing the arsA(C113A/C422A)B genes had an intermediate level of metalloid resistance and accumulation between those expressing only arsB alone and those expressing wild type arsAB genes. The results indicate that, whereas metalloid stimulation of ArsA activity enhances the ability of the pump to reduce the intracellular concentration of metalloid, high affinity binding of metalloid by ArsA is not obligatory for transport or resistance. Yet, in mixed populations of cells bearing either arsAB or arsA(C113A/C422A)B growing in subtoxic concentrations of arsenite, cells bearing wild type arsAB replaced cells with mutant arsA(C113A/C422A)B in less than 1 week, showing that the metalloid binding site confers an evolutionary advantage.     

Distance measurements between metal-binding sites of calmodulin and from these sites to Cys-133 of troponin I in the binary complex

Distances between the four Ca2+-binding sites of calmodulin (CaM) have been measured by fluorescence energy transfer techniques using Eu3+ and Tb3+ as energy donors and a number of other lanthanide ions (Ln3+) as acceptors. It was shown previously that lanthanide ions preferentially bind to sites I and II of CaM with an affinity higher than that for sites III and IV (Kilhoffer, M.-C., Demaille, J. G., and Gerald, D. (1980) FEBS Lett. 116, 269-272; Wang, C.-L. A., Aquaron, R. R., Leavis, P. C., and Gergely, J. (1982) Eur. J. Biochem. 124, 7-12). Thus upon direct excitation with a laser the luminescence lifetimes of Eu1Ln1CaM and Tb1Ln1CaM provide information on the distance between sites I and II. On the other hand, since Tb3+ ions bound to sites III and IV are sensitizable through tyrosine residues, lifetime measurements of Tb2Ln2CaM excited by UV light yield the distance between sites III and IV. Both pairs of sites were found to be separated by a distance of 1.05 +/- 0.07 nm. Binding of Ca2+ to sites III and IV does not alter the distance between sites I and II. We have also attached a chromophoric label, dimethylaminophenylazobenzene, to Cys-133 of skeletal troponin I and carried out distance measurements on its complex with CaM by both direct and indirect excitation. The averaged distances from sites I and II in the N-terminal half and from sites III and IV in the C-terminal half of the CaM molecule to the label on troponin I are 2.7 and 2.5 nm, respectively.     

Peptide folding, metal-binding mechanisms, and binding site structures in metallothioneins

This minireview specifically focuses on recent studies carried out on structural aspects of metal-free metallothionein (MT), the mechanism of metal binding for copper and arsenic, structural studies using x-ray absorption spectroscopy and molecular mechanics modeling, and speciation studies of a novel cadmium and arsenic binding algal MT. Molecular mechanics-molecular dynamics calculations of apo-MT show that significant secondary structural features are retained by the polypeptide backbone upon sequential removal of the metal ions, which is stabilized by a possible H-bonding network. In addition, the cysteinyl sulfurs were shown to rotate from within the domain core, where they are found in the metallated state, to the exterior surface of the domain, suggesting an explanation for the rapid metallation reactions that were measured. Mixing Cu6beta-MT with Cd4alpha-MT and Cu6alpha-MT with Cd3beta-MT resulted in redistribution of the metal ions to mixed metal species in each domain; however, the Cu+ ions preferentially coordinated to the beta domain in each case. Reaction of As3+ with the individual metal-free beta and alpha domains of MT resulted in three As3+ ions coordinating to each of the domains, respectively, in a proposed distorted trigonal pyramid structure. Kinetic analysis provides parameters that allow simulation of the binding of each of the As3+ ions. X-ray absorption spectroscopy provides detailed information about the coordination environment of the absorbing element. We have combined measurement of x-ray absorption near edge structure (XANES) and extended x-ray absorption fine structure (EXAFS) data with extensive molecular dynamics calculations to determine accurate metal-thiolate structures. Simulation of the XANES data provides a powerful technique for probing the coordination structures of metals in metalloproteins. The metal binding properties of an algal MT, Fucus vesiculosus, has been investigated by UV absorption and circular dichroism spectroscopy and electrospray ionization-mass spectrometry. The 16 cysteine residues of this algal MT were found to coordinate six Cd2+ ions in two domains with stoichiometries of a novel Cd3S7 cluster and a beta-like Cd3S9 cluster.     

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.     

Fluorescence resonance energy transfer on the voltage-dependent sodium channel. Spatial relationship and site coupling between the batrachotoxin and Leiurus quinquestriatus quinquestriatus alpha-scorpion toxin receptors

A fluorescent N- methylanthraniloyl derivative of the potent depolarizing agent batrachotoxin has been used to probe the structural and conformational properties of the neurotoxin receptor site on the voltage-dependent sodium channel. Batrachotoxin A 20-alpha-N- methylanthranilate (BTX-NMA) retains high affinity for its receptor site on the synaptosomal sodium channel with a Kd between 78 and 91 nM and an average site capacity of 2 pmol/mg of synaptosomal protein in the presence of Leiurus quinquestriatus quinquestriatus alpha-scorpion toxin. The fluorescence emission of BTX-NMA upon binding to synaptosomes indicates a hydrophobic environment. Toxin V from L. quinquestriatus, an allosteric activator, effects a 20-nm red shift in the spectrum of bound BTX-NMA and a 4-fold enhancement in the fluorescence quantum yield disclosing a conformational change into a hydrophilic environment. Fluorescence resonance energy transfer measurements show that the distance separating the receptor sites is 37 +/- 10 A. Thus, the binding of alpha-scorpion toxin must involve conformational changes that extend over large distances from the batrachotoxin-binding locus. This information together with the distance measurements between the tetrodotoxin and alpha-scorpion toxin receptors and the conformational transition associated with this distance upon batrachotoxin addition indicate a conformationally flexible channel with coupling of sites through the polyatomic framework of individual subunits or through extensive alterations in subunit/subunit interactions.