Conformational distributions of melittin in water/methanol mixtures from frequency-domain measurements of nonradiative energy transfer

We used fluorescence energy transfer to examine the effects of solvent composition on the distribution of distances between the single tryptophan residue of melittin (residue 19) to the N-terminal alpha-amino group, which was labeled with a dansyl residue. The tryptophan intensity decays, with and without the dansyl acceptor, were measured by the frequency-domain method. The data were analyzed by a least-squares algorithm which accounts for correlation between the parameters. A wide distribution of tryptophan to dansyl distances was found for the random-coil state, with a Gaussian half-width of 25 A. Increasing concentrations of methanol, which were shown to induce and alpha-helical conformation, resulted in a progressive decrease in the width of the distribution, reaching a limiting half-width of 3 A at 80% (v/v) methanol. The distance from the indole moiety of Trp-19 to the dansyl group in 80% (v/v) methanol/water was found to be 25 A, as assessed from the center of the distance distribution. A distance of 24-25 A was recovered from the X-ray crystal structure of the tetramer, which is largely alpha-helical. At low ionic strength (less than 0.01) the CD spectra revealed a small fraction or amount of alpha-helix for melittin in water, which implies a small fraction of residual structure. This residual structure is apparently lost in guanidine hydrochloride as demonstrated by a further broadening in the distribution of distances. These results demonstrate the usefulness of frequency-domain measurements of resonance transfer for resolution of conformational distributions of proteins.     

Distance distributions in proteins recovered by using frequency-domain fluorometry. Applications to troponin I and its complex with troponin C

We used resonance energy transfer to examine the distribution of distances between two sites on troponin I (TnI). The donor (D) was the single tryptophan residue at site 158 (Trp 158), and the acceptor (A) was cysteine 133 (Cys 133) which was labeled with N-(iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine (IE). A distribution of D-A distances results in a distribution of donor decay times, which were resolved by using frequency-domain fluorometry. In the native state we recovered a relatively narrow distribution of D-A distances. The widths of the distance distributions were found to increase progressively and dramatically with increasing concentrations of guanidine hydrochloride. Binding of calcium-free troponin C (TnC) to troponin I did not alter the distance distribution. Addition of Ca2+ to the TnI.TnC complex resulted in a sharper distance distribution and protected against the guanidine hydrochloride induced increase in the width of the distance distribution. Additionally, the same distance distributions were recovered for native and denatured TnI when the Forster distance for energy transfer was decreased by acrylamide quenching. These results demonstrate that distance distributions can be recovered with good accuracy, to the extent of revealing modest changes due to binding of other components. This technique should have widespread applications in studies of protein folding.     

Distance distributions from the tyrosyl to disulfide residues in the oxytocin and [Arg8]-vasopressin measured using frequency-domain fluorescence resonance energy transfer

We have examined the fluorescence intensity decays of oxytocin and [Arg⁸]-vasopressin resulting from the single tyrosyl residue in each peptide, and the intensity decay of the Asu ^1,6-analogues in which the disulfide bridge is substituted by a CH₂-CH₂ bridge. Viscosity-dependent steady state and intensity decay measurements indicated that fluorescence resonance energy transfer (FRET) from tyrosyl phenol to the disulfide bridge is responsible for the decrease in fluorescence relative to the Asu-analogues. The frequency-domain phase and modulation data for the tyrosyl donor were interpreted in terms of fluorescence resonance energy transfer (FRET) to the weakly absorbing disulfide bridge and a distribution of donor-to-acceptor distances. Energy transfer efficiencies were determined from both time-resolved and steady-state measurements. Fitting the frequency-domain phase and modulation data to a Gaussian distance distribution indicated that the average inter-chromophoric distance (R_av) is similar in both compounds, R_av=7.94 Å for oxytocin and R_av = 8.00 Å for vasopressin. However, the width of the distance distribution is narrower for vasopression (hw =2.80 Å) than for oxytocin (hw =3.58 Å), which is consistent with restriction of the tyrosine phenol motion due to its stacking with the Phe³ side chain of vasopressin. Finally, the recovered distance distribution functions are compared with histograms describing the distance between the chromophores during the course of long, in vacuo, molecular dynamics runs using the computer program CHARMm and the QUANTA 3.0 parameters.Abbreviations AVP [Arg⁸]-vasopressin - FRET fluorescence resonance energy transfer - FD frequency-domain - D donor - A acceptor - DTT dithiothreitol Correspondence to: J. R. Lakowicz     

Distance distributions and anisotropy decays of troponin C and its complex with troponin I

We used frequency domain measurements of fluorescence resonance energy transfer to recover the distribution of distances between Met 25 and Cys 98 in rabbit skeletal troponin C. These residues were labeled with dansylaziridine as energy donor and 5-(iodoacetamido)eosin as acceptor and are located on the N- and C-terminal lobes of the two-domain protein, respectively. We developed a procedure to correct for the fraction of the sample that was incompletely labeled with the acceptor independent of chemical data. At pH 7.5 and in the presence of Mg2+, the mean distance was near 15 A with a half-width of the distribution of 15 A; when Mg2+ was replaced by Ca2+, the mean distance increased to 22 A with a decrease in the half-width by 4 A. Similar but less pronounced differences in the mean distance and half-width between samples containing Mg2+ and Ca2+ were also observed with troponin C complexed to troponin I. The results suggest that the conformation of troponin C is altered by Ca2+ binding to the Ca(2+)-specific sites and displacing bound Mg2+ at the Ca2+/Mg2+ sites. This alteration may play an important role in Ca2+ signaling in muscle. At pH 7.5, the anisotropy decays of the donor-labeled troponin C showed two components, with the long rotational correlation time (12 ns) reflecting the overall motion of the protein. When the pH was lowered from 7.5 to 5.2, the mean distribution distance of apotroponin C increased from 22 to 32 A and the half-width decreased by a factor of 2 from 13 to 7 A. The long correlation time of apotroponin C increased to 19 ns at the acidic pH. These results are discussed in terms of a model in which skeletal troponin C is a dimer at low pH and enable comparison of the solution conformation of the protein at neutral pH with a crystal structure obtained at pH 5.2. While the conformation of the monomeric unit of troponin C dimer at pH 5.2 is extended and consistent with the crystal structure, the conformation at neutral pH is likely more compact than the crystal structure predicts.     

Distribution of distances in thiopeptides by fluorescence energy transfer and frequency-domain fluorometry

Frequency-domain fluorescence spectroscopy was employed to examine the decays of tryptophan in Boc-Trp-Met-Asp-Phe-NH2 (donor) and (Formula: see text) (donor-acceptor pair). The efficiency of energy transfer in the thiopeptide amounted to 60%. The measured dispersion of fluorescence decay times was used to recover the donor-acceptor distance distribution. The parameters of the Gaussian distance distribution obtained for this peptide (r, the mean distance (9 A); hw, the halfwidth (25 A)) indicate the lack of a distinct favorable conformation.     

Distance measurements in cardiac troponin C

Intramolecular distance measurements were made in cardiac troponin C (cTnC) by fluorescence energy transfer using Eu3+ or Tb3+ as energy donors and Nd3+ or an organic chromophore as acceptors. The laser-induced luminescence of bound Eu3+ is quenched in Eu1Nd1cTnC with a lifetime of 0.328 ms, compared with 0.43 ms for Eu2cTnC. The enhanced decay corresponds to an energy transfer efficiency of 0.25, or a distance of 1.1 nm between the two high affinity sites. We have also labeled cTnC with 4-dimethylaminophenylazophenyl-4'-maleimide (DAB-Mal) at the two cysteine residues (Cys-35 and Cys-84). Energy transfer measurements were carried out between Tb3+ bound to the high affinity sites and the labels attached to the domain containing the low affinity site. Upon uv irradiation at pH 6.7, Tb1cTnCDAB emits tyrosine-sensitized Tb3+ luminescence that decays bioexponentially with lifetimes of 1.29 and 0.76 ms. The shorter lifetime is ascribed to energy transfer from Tb3+ to the DAB labels, yielding an average distance of 3.4 nm between the donor and the acceptors. At pH 5.0, however, the luminescence decays exclusively with a single lifetime of 1.31 ms, suggesting that under these conditions all Tb3+ ions are more than 5.2 nm away from the label. Thus cTnC, like skeletal TnC, undergoes a pH-dependent conformational transition which converts an elongated structure at lower pH's to a rather compact conformation in a more physiological medium.     

Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy.

Fluorescence resonance energy transfer (FRET) is a technique used for quantifying the distance between two molecules conjugated to different fluorophores. By combining optical microscopy with FRET it is possible to obtain quantitative temporal and spatial information about the binding and interaction of proteins, lipids, enzymes, DNA, and RNA in vivo. In conjunction with the recent development of a variety of mutant green fluorescent proteins (mtGFPs), FRET microscopy provides the potential to measure the interaction of intracellular molecular species in intact living cells where the donor and acceptor fluorophores are actually part of the molecules themselves. However, steady-state FRET microscopy measurements can suffer from several sources of distortion, which need to be corrected. These include direct excitation of the acceptor at the donor excitation wavelengths and the dependence of FRET on the concentration of acceptor. We present a simple method for the analysis of FRET data obtained with standard filter sets in a fluorescence microscope. This method is corrected for cross talk (any detection of donor fluorescence with the acceptor emission filter and any detection of acceptor fluorescence with the donor emission filter), and for the dependence of FRET on the concentrations of the donor and acceptor. Measurements of the interaction of the proteins Bcl-2 and Beclin (a recently identified Bcl-2 interacting protein located on chromosome 17q21), are shown to document the accuracy of this approach for correction of donor and acceptor concentrations, and cross talk between the different filter units.     

Myosin structure. Proximity measurements by fluorescence energy transfer

The results of energy transfer experiments on the proximity of six sites on the globular head region of myosin are discussed. A large hydrophobic crevice has been detected on each myosin head which is sufficiently large to accommodate six aromatic rings simultaneously. In the crevice is located a thiol residue not involved in activation of myosin Ca²⁺ ATPase and a lysine residue which is specifically trinitrophenylated with 2, 4, 6-trinitrobenzenesulfonic acid. A second sulfhydryl whose modification activates the Ca²⁺ ATPase is located near the hydrophobic thiol site. The tryptophan whose fluorescence is enhanced by ATP binding is sufficiently close to the thiols and lysine residue to quantitatively transfer its energy to probes at these sites. The site of myosin ATPase has been tentatively located as being near the other five sites by energy transfer to or from synthetic chromophoric substrates. Implications of these results on the possibility of determining the location of the myosin light chain and actin binding sites are discussed.     

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.     

Statistical interpretation of fluorescence energy transfer measurements in macromolecular systems.

A statistical method is presented for the interpretation of intramolecular distance measurements by the fluorescence energy transfer technique in systems for which the detailed geometries of the donor-acceptor pairs are unknown. This method enables calculation of the probability that a specified distance range corresponds to the actual distance to be measured. It makes use of the numerically calculated probability density function for the distance of interest. The two general systems considered are the single donor-acceptor pair and the multi-donor-single-acceptor transfer. In both systems, the statistical method incorporates the uncertainty in the orientation of the donor and acceptor dipoles. In addition, it can take into account the rotational mobility of the donor dipoles determined by time-dependent emission anisotropy measurements. When more than one donor is involved in the transfer process, the uncertainties associated with the number and location of individual donors and the size and shape of the donor distribution are also incorporated in calculating the distance ranges. Application of the method was demonstrated for a wide range of transfer efficiency and Ro values for the single donor-acceptor system. Specific examples are also presented for interpretation of both single donor-acceptor and multi-donor-single-acceptor energy transfer measurements performed in order to reveal the spatial relationship of the sigma subunit and the rifampicin binding site in the Escherichia coli RNA polymerase (see Wu, C.-W., Yarbrough, L. R., Wu, F. Y.-H., and Hillel, Z. (1976), Biochemistry, preceding paper in this issue). Analysis of these energy transfer data by methods which use average values of the unknown geometrical parameters of the system yielded results similar to those obtained by the statistical method. However, the statistical method represents a more realistic approach to the interpretation of energy transfer measurements since it provides information concerning the entire range of possible distances and their relative likelihood.     

Flow cytometric measurements of fluorescence energy transfer using single laser excitation

Flow cytometric energy transfer (FCET) measurements between labeled specific sites of cell surface elements (Sz?llosi et al., Cytometry, 5:210-216, 1984) have been extended in a simplified form using a flow cytometer equipped with single excitation beam. This versatile and easily applicable method has several advantages over any nonflow cytometric (i.e., spectrofluorimetric) energy transfer measurements on cell surfaces: The labeled ligands can be applied in excess, without washing, thereby enabling the investigation of relatively labile receptor-ligand complexes. Contributions of signals from cell debris, from cell aggregates, or from nonviable cells can be avoided by gating the data collection on the light scatter signal. The heterogeneity of the cell population with respect to the proximity of the labeled binding sites can be studied. In the cases of homologous ligands or of ligands binding to the same molecule but at different epitopes, the determination of fluorescence resonance energy transfer efficiency values can be carried out on a cell-by-cell basis, offering data on intramolecular conformational changes. This modified FCET method enabled us to demonstrate the uniform density of glycoproteins, specific for Con A binding, in the plasma membrane of normal and Gross virus leukemic mouse cells of different sizes. The utility of this procedure has also been demonstrated by using the mean fluorescence intensities of the distribution curves in the calculation of the fluorescence energy transfer efficiency.     

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.     

Distance distribution in a dye-linked oligonucleotide determined by time-resolved fluorescence energy transfer.

Fluorescence energy transfer is potentially a useful technique for obtaining structural and dynamic information on duplex and branched DNA molecules suitably labeled with donor and acceptor dyes. We have assessed the accuracy and limitations of FET measurements in nucleic acids with respect to the localization of the dyes and the flexibility of the dye-DNA linkages. A nine base-pair duplex oligonucleotide was synthesized with donor and acceptor dyes linked at the opposing 5' termini by alkyl chains. A careful analysis of the fluorescence decay of the donor revealed that the donor-acceptor distance in this molecule was not well defined, but was described by a rather broad distribution. The mean donor-acceptor distance and the distribution of distances have been recovered from the donor decay. Orientational effects on energy transfer have been included in the analysis. The implications of these findings for FET measurements in nucleic acids are considered.     

Real-time measurements of DNA hybridization on microparticles with fluorescence resonance energy transfer

When capture oligonucleotides are tethered on planar surfaces, mass transport limitations influence the kinetics of solid-phase nucleic acid hybridizations. By diffusion theory, however, hybridization of oligonucleotides on microparticles should be reaction-rate limited. In an initial effort to understand the kinetics of microparticle hybridization reactions, we developed a fluorescence resonance energy transfer method for monitoring oligonucleotide hybridization on microparticles. Microparticles were coated with a fluoresceinated oligomer at surface densities of 20, 40, and 80% saturation, hybridized to a complementary oligonucleotide labeled with tetramethylrhodamine, and monitored over time for quenching of the fluorescein signal as hybridization occurred on the particle surface. Association rate constants were compared for microparticle-based hybridization and solution-phase hybridization. Rate constants for hybridizations on the particle surface were about an order of magnitude less than those for hybridization in solution, but decreasing the surface density of the capture oligonucleotide to 20% saturation improved particle hybridization rates. Although a bimolecular reaction model adequately described solution-phase hybridization kinetics, oligonucleotide hybridization on microparticles did not fit this model but exhibited biphasic reaction kinetics. Based on two different lines of reasoning, we argue that microparticle-based oligonucleotide hybridization was indeed reaction-rate limited in our system and not diffusion-rate limited.     

ATP-induced transconformation of myosin revealed by determining three-dimensional positions of fluorophores from fluorescence energy transfer measurements

The method of fluorescence resonance energy transfer (FRET) is one of the most important techniques for measuring the distance between two fluorophores and for detecting the changes in protein structure under physiological conditions. The use of green fluorescent protein is also a powerful technology that has been used to elucidate dynamic molecular events. From these we have developed a novel method to determine the three-dimensional positions of fluorophores by combining the FRET data and other structural information available. Using this method, we could determine the ATP-induced changes of three-dimensional structure of truncated Dictyostelium myosin in solution. The myosin structure with ADP in solution was found to be similar to that of the crystal structure of MgADPBeFx-bound truncated Dictyostelium myosin (type I structure), whereas myosin with ATP in solution was similar to the crystal structure of MgAdPVi-bound one (type II structure). However, the crystal structure of MgADP-bound scallop myosin (type III structure) could not be explained by any of our FRET data under various conditions. This indicates that the type III crystal structure might represent a transient intermediate conformation that could not be detected using fluorescence energy transfer.     

Non-linear least-squares methods applied to the analysis of fluorescence energy transfer measurements.

We describe a new method for calculating the efficiency of fluorescence energy transfer on labeled macromolecules using steady-state measurements. A single estimation of the efficiency value is obtained by a global analysis of all the measurement data sets (absorption, emission and excitation spectra) using non-linear least-squares. The method was tested on simulated and experimental data obtained from three simple model compounds: an equimolar mixture of tryptophan-tyrosine and two peptides, Trp-Tyr and Trp-Gly-Gly-Tyr, in which transfer efficiencies are respectively nearly 100% and 50%. The method was found to be reliable and provides methodological and quantitative advantages in regard to the sequential methods currently used.     

Excitation energy transfer in native and unstacked thylakoid membranes studied by low temperature and ultrafast fluorescence spectroscopy

In this work, the transfer of excitation energy was studied in native and cation-depletion induced, unstacked thylakoid membranes of spinach by steady-state and time-resolved fluorescence spectroscopy. Fluorescence emission spectra at 5 K show an increase in photosystem I (PSI) emission upon unstacking, which suggests an increase of its antenna size. Fluorescence excitation measurements at 77 K indicate that the increase of PSI emission upon unstacking is caused both by a direct spillover from the photosystem II (PSII) core antenna and by a functional association of light-harvesting complex II (LHCII) to PSI, which is most likely caused by the formation of LHCII-LHCI-PSI supercomplexes. Time-resolved fluorescence measurements, both at room temperature and at 77 K, reveal differences in the fluorescence decay kinetics of stacked and unstacked membranes. Energy transfer between LHCII and PSI is observed to take place within 25 ps at room temperature and within 38 ps at 77 K, consistent with the formation of LHCII-LHCI-PSI supercomplexes. At the 150–160 ps timescale, both energy transfer from LHCII to PSI as well as spillover from the core antenna of PSII to PSI is shown to occur at 77 K. At room temperature the spillover and energy transfer to PSI is less clear at the 150 ps timescale, because these processes compete with charge separation in the PSII reaction center, which also takes place at a timescale of about 150 ps.     

Lifetime distributions and anisotropy decays of indole fluorescence in cyclohexane/ethanol mixtures by frequency-domain fluorometry

We used frequency-domain fluorometry to measure intensity and anisotropy decay of indole fluorescence in cyclohexane/ethanol mixtures at 20 degrees C. In 100% cyclohexane or 100% ethanol the intensity decay of indole appears to be a single exponential with decay times of 7.66 and 4.10 ns, respectively. In cyclohexane containing a small percentage of ethanol (up to 10%), we observed increased heterogeneity in intensity decay, resulting in a 10-fold increase in chi 2R for the single-exponential fit, as compared with the double-exponential model. We obtained comparable or better fits using unimodal Lorentzian and Gaussian lifetime distributions (two floating parameters) than for the two-exponential model (three floating parameters). We believe that the distribution of decay times reflects a range of indole solvation states in the dominately nonpolar solutions. This result suggests that a variety of hydrogen-bonding configurations could be one origin of the distributions of decay times observed for tryptophan emission from proteins. We also measured rotational diffusion of indole in cyclohexane, ethanol and its mixtures at 20 degrees C. The picosecond correlation times required that the mean decay times be decreased by acrylamide quenching (in ethanol) or energy transfer (in cyclohexane). In ethanol we observed nearly isotropic rotation of indole; in cyclohexane we obtained two correlation times of 17 and 73 ps. The shorter correlation time in cyclohexane appears to be due to the slip boundary condition, which was found to be progressively eliminated by small percentages of ethanol. Hence, hydrogen-bonding interactions appear to have a substantial effect on the rotational dynamics of indole.     

Selecting the right fluorophores and flow cytometer for fluorescence resonance energy transfer measurements.

BACKGROUND: Fluorescence resonance energy transfer applied in flow cytometry (FCET) is an excellent tool for determining supramolecular organization of biomolecules at the cell surface or inside the cell. Availability of new fluorophores and cytometers requires the establishment of fluorophore dye pairs most suitable for FCET measurements. METHODS: A gastric tumor cell line (N87) was labeled for major histocompatibility complex class I heavy chain and beta2-microglobulin with antibodies conjugated with fluorescein- and indocarbocyanine-like fluorophores and analyzed in FCET measurements on a cell-by-cell basis using three flow cytometers: FACSCalibur, FACSDiVa, and FACSArray. RESULTS: Normalized fluorescence intensity values were measured and normalized energy transfer efficiencies, spectral overlap integrals, and crucial dye- and instrument-dependent parameters were calculated for all matching pairs of seven fluorophores on the three commercial cytometers. The most crucial parameter in determining the applicability of the donor-acceptor pairs was the normalized fluorescence intensity and the least important one was the spectral overlap. CONCLUSIONS: On the basis of available laser lines, the optimal dye pair for all three cytometers is the Alexa546-Alexa647 pair, which produces high energy transfer efficiency values and has the best spectral characteristics with regard to laser excitation, detection of emission, and spectral overlap.