Distribution of distances between the tryptophan and the N-terminal residue of melittin in its complex with calmodulin, troponin C, and phospholipids.

We used frequency-domain measurements of fluorescence resonance energy transfer to measure the distribution of distances between Trp-19 of melittin and a 1-dimethylamino-5-sulfonylnaphthalene (dansyl) residue on the N-terminal-alpha-amino group. Distance distributions were obtained for melittin free in solution and when complexed with calmodulin (CaM), troponin C (TnC), or palmitoyloleoyl-L-alpha-phosphatidylcholine (POPC) vesicles. A wide range of donor (Trp-19)-to-acceptor (dansyl) distances was found for free melittin, which is consistent with that expected for the random coil state, characterized by a Gaussian width (full width at half maxima) of 28.2 A. In contrast, narrow distance distributions were found for melittin complexed with CaM, 8.2 A, or with POPC vesicles, 4.9 A. A somewhat wider distribution was found for the melittin complex with TnC, 12.8 A, suggesting the presence of heterogeneity in the mode of binding between melittin and TnC. For all the complexes the mean Trp-19 to dansyl distance was near 20 A. This value is somewhat smaller than expected for the free alpha-helical state of melittin, suggesting that binding with CaM or TnC results in a modest decrease in the length of the melittin molecule.     

Distance distributions in native and random-coil troponin I from frequency-domain measurements of fluorescence energy transfer

We used time-dependent fluorescence energy transfer to determine the distribution of donor-to-acceptor distances in native and denatured troponin I(TnI). The single tryptophan residue (Trp 158) of TnI served as the donor (D), and the acceptor (A) was a labeled cysteine residue (Cys 133). The time-dependent intensity decays of the donor were measured by the frequency-domain method from 10 to 320 MHz. The frequency response of the donor emission, in the absence and presence of acceptor, was used to recover the distribution of D to A distances, using an algorithm that accounts for the intrinsic multiexponential decay of the donor. In the native state the D–A distribution is characterized by an average distance of 23 Å and a half-width of 12 Å. Denaturation results in a modest increase in the average distance to 27 Å, and a dramatic increase in half-width to 47 Å. We believe the ability to recover distance distributions will have numerous applications in the characterization of biological macromolecules.     

Resolution of end-to-end distance distributions of flexible molecules using quenching-induced variations of the Forster distance for fluorescence energy transfer.

We describe a new method to recover the distribution of donor-to-acceptor (D-A) distances in flexible molecules using steady-state measurements of the efficiency of fluorescence energy transfer. The method depends upon changes in the Forster distance (Ro) induced by collisional quenching of the donor emission. The Ro-dependent transfer efficiencies are analyzed using nonlinear least squares to recover the mean D-A distance and the width of the distribution. The method was developed and tested using three synthetic D-A pairs, in which the chromophores were separated by alkyl chains of varying lengths. As an example application we also recovered the distribution of distances from the single tryptophan residue in troponin I (trp 158) to acceptor-labeled cysteine 133. The half-width of the distribution increases from 12 A in the native state to 53 A when unfolded by guanidine hydrochloride. For both TnI and the three model compounds the distance distributions recovered from the steady-state transfer efficiencies were in excellent agreement with the distributions recovered using the more sophisticated frequency-domain method (Lakowicz, J.R., M.L. Johnson, W. Wiczk, A. Bhat, and R.F. Steiner. 1987. Chem. Phys. Lett. 138:587-593). The method was found to be reliable and should be generally useful for studies of conformational distributions of macromolecules.     

Contribution of proline-14 to the structure and actions of melittin

The structure and dynamic properties of bee venom melittin and a synthetic analogue, [Ala14]-melittin (melittin P14A), are compared, using high resolution 1H nuclear magnetic resonance (NMR) spectroscopy and amide exchange measurements in methanol. P14A is shown to adopt a regular, stable alpha-helical conformation in solution without the flexibility around the Pro-14 residue found in melittin. P14A has twice the hemolytic activity of melittin but is less able to induce voltage-dependent ion conductance in planar bilayers. The results indicate that helix flexibility afforded by the Pro-14 residue promotes the ability of melittin to adopt the transbilayer associates thought to underlie ion translocation.     

Selective labelling of melittin with a fluorescent dansylcadaverine probe using guinea-pig liver transglutaminase.

Melittin, a C-terminal glutamine peptide, incorporated the fluorescent probe monodansylcadaverine (DNC) when catalysed by guinea-pig liver transglutaminase and Ca2+, as determined by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). A 1:1 adduct DNC-melittin was identified in which a single glutamine residue out of two, i.e. Gln25, acts as acyl donor. Incubation of melittin with transglutaminase in the absence of DNC originated high molecular mass complexes indicative that the peptide lysine residue can act as an acyl acceptor. The DNC-melittin was about 3 times more active in the lysis of red cell membranes than native melittin. Fluorescence study of the labelled melittin in the submicromolar range where it is active on cells showed that while totally exposed to solvent in methanol solution, both Trp and dansyl groups are buried in buffer solution. This strongly suggests that DNC-melittin is self-associated and indeed more active than the native melittin in the same conditions.     

Influence of end-to-end diffusion on intramolecular energy transfer as observed by frequency-domain fluorometry

We investigated the influence of end-to-end diffusion on intramolecular energy transfer between a naphthalene donor and dansyl acceptor linked by polymethylene chain. A range of viscosities from 0.6 to 200 cP were obtained using propylene glycol at different temperatures (0-80 degrees C) and methanol at 20 degrees C. The intensity decays of naphthalene were measured in the frequency domain. Several theoretical models, including distance distributions, were used to fit the data. The results indicate that end-to-end diffusion of flexible donor-acceptor pairs can be detected and quantified using frequency-domain fluorometry, even in the presence of a distribution of donor-to-acceptor distances.     

Effect of ionic strength on folding and aggregation of the hemolytic peptide melittin in solution

Melittin is a cationic, amphipathic, hemolytic peptide composed of 26 amino acid residues. It is intrinsically fluorescent due to the presence of a single tryptophan residue, which has been shown to be crucial for its hemolytic activity. It undergoes a structural transition from a random coil monomer to an alpha-helical tetramer at high ionic strength. Although the aggregation behavior of melittin in solution is well characterized, dynamic information associated with the aggregation of melittin is lacking. In this paper, we have monitored the effect of ionic strength on the dynamics and aggregation behavior of melittin in aqueous solution by utilizing sensitive fluorescence approaches, which include the red edge excitation shift (REES) approach. Importantly, we demonstrate that REES is sensitive to the self-association of melittin induced by ionic strength. The change in environment experienced by melittin tryptophan(s) is supported by changes in fluorescence emission maximum, polarization, and lifetime. In addition, the accessibility of the tryptophan residue was probed by fluorescence quenching experiments using acrylamide and trichloroethanol as soluble and hydrophobic quenchers, respectively. Circular dichroism studies confirm the ionic strength-induced change in the secondary structure of melittin. Taken together, these results constitute the first report showing that REES could be used as a sensitive tool to monitor the aggregation behavior of melittin in particular and other proteins and peptides in general.     

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.     

Calcium-regulated interactions of human alpha-lactalbumin with bee venom melittin

Affinity chromatography, fluorescence and circular dichroism spectroscopy methods have been used to study the interaction of melittin, a 26-residue peptide from bee venom, with Ca2(+)-binding alpha-lactalbumin from human milk. It has been revealed that melittin binds to the apo- and acidic states of alpha-lactalbumin while the presence of Ca2+ makes the interaction essentially weaker. The association constant for the complex of melittin with apo-alpha-lactalbumin determined from spectropolarimetric melittin-titration data is 2 X 10(7) M-1. The complexation of alpha-lactalbumin with melittin decreases its affinity to Ca2+ by three orders of magnitude. The interaction of apo-alpha-lactalbumin with melittin causes some changes in the environment of its aromatic amino acid residues and drastically alters the conformation of melittin, increasing its alpha-helical content but leaving its single tryptophan residue accessible to water. In the case of the acidic state of alpha-lactalbumin the interaction does not induce an increase in alpha-helical content of melittin.     

Measurement of subnanosecond anisotropy decays of protein fluorescence using frequency-domain fluorometry

We report the first anisotropy decays of protein fluorescence obtained using a frequency-domain fluorometer. The ultraviolet light source (300 nm) was a ring dye laser equipped with an intracavity frequency doubler, pumped by an argon ion laser. The data, measured at modulation frequencies from 2 to 200 MHz, reveal the presence of subnanosecond motions (0.1-0.2 ns) of the single tryptophan residues in melittin and monellin. For melittin the data also indicate the presence of slower motions near 1 ns, which may be the result of concerted motions of several peptide units. Smaller amplitude motions, on a similar timescale, were observed for the single tryptophan residue in staphylococcal nuclease. We demonstrate using N-acetyl-L-tryptophanamide in water that the method of frequency-domain fluorometry is capable of measuring correlation times as short as 50 ps. This method can provide data for the direct comparison of measured anisotropy decays with those predicted from molecular dynamics calculations.     

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.     

Probing alpha-helical secondary structure at a specific site in model peptides via restriction of tryptophan side-chain rotamer conformation.

The relationship between alpha-helical secondary structure and the fluorescence properties of an intrinsic tryptophan residue were investigated. A monomeric alpha-helix forming peptide and a dimeric coiled-coil forming peptide containing a central tryptophan residue were synthesized. The fluorescence parameters of the tryptophan residue were determined for these model systems at a range of fractional alpha-helical contents. The steady-state emission maximum was independent of the fractional alpha-helical content. A minimum of three exponential decay times was required to fully describe the time-resolved fluorescence data. Changes were observed in the decay times and more significantly, in their relative contributions that could be correlated with alpha-helix content. The results were also shown to be consistent with a model in which the decay times were independent of both alpha-helix content and emission wavelength. In this model the relative contributions of the decay time components were directly proportional to the alpha-helix content. Data were also analyzed according to a continuous distribution of exponential decay time model, employing global analysis techniques. The recovered distributions had "widths" that were both poorly defined and independent of peptide conformation. We propose that the three decay times are associated with the three ground-state chi 1 rotamers of the tryptophan residue and that the changes in the relative contributions of the decay times are the result of conformational constraints, imposed by the alpha-helical main-chain, on the chi 1 rotamer populations.     

Structural transitions in the protein L denatured state ensemble

We use a broad array of biophysical methods to probe the extent of structure and time scale of structural transitions in the protein L denatured state ensemble. Measurement of amide proton exchange protection during the first several milliseconds following initiation of refolding in 0.4 M sodium sulfate revealed weak protection in the first beta-hairpin and helix. A tryptophan residue was introduced into the first beta-hairpin to probe the extent of structure formation in this part of the protein; the intrinsic fluorescence of this tryptophan was found to deviate from that expected given its local sequence context in 2-3 M guanidine, suggesting some partial ordering of this region in the unfolded state ensemble. To further probe this partial ordering, dansyl groups were introduced via cysteine residues at three sites in the protein. It was found that fluorescence energy transfer from the introduced tryptophan to the dansyl groups decreased dramatically upon unfolding. Stopped-flow fluorescence studies showed that the recovery of dansyl fluorescence upon refolding occurred on a submillisecond time scale. To probe the interactions responsible for the residual structure observed in the denatured state ensemble, the conformation of a peptide corresponding to the first beta-hairpin and helix of protein L was studied using circular dichroism spectroscopy and compared to that of full-length protein L and previously characterized peptides corresponding to the isolated helix and second beta-hairpin.     

Hydrogen bonding in helical polypeptides from molecular dynamics simulations and amide hydrogen exchange analysis: alamethicin and melittin in methanol.

Molecular dynamics simulations of ion channel peptides alamethicin and melittin, solvated in methanol at 27 degrees C, were run with either regular alpha-helical starting structures (alamethicin, 1 ns; melittin 500 ps either with or without chloride counterions), or with the x-ray crystal coordinates of alamethicin as a starting structure (1 ns). The hydrogen bond patterns and stabilities were characterized by analysis of the dynamics trajectories with specified hydrogen bond angle and distance criteria, and were compared with hydrogen bond patterns and stabilities previously determined from high-resolution NMR structural analysis and amide hydrogen exchange measurements in methanol. The two alamethicin simulations rapidly converged to a persistent hydrogen bond pattern with a high level of 3(10) hydrogen bonding involving the amide NH's of residues 3, 4, 9, 15, and 18. The 3(10) hydrogen bonds stabilizing amide NH's of residues C-terminal to P2 and P14 were previously proposed to explain their high amide exchange stabilities. The absence, or low levels of 3(10) hydrogen bonds at the N-terminus or for A15 NH, respectively, in the melittin simulations, is also consistent with interpretations from amide exchange analysis. Perturbation of helical hydrogen bonding in the residues before P14 (Aib10-P14, alamethicin; T11-P14, melittin) was characterized in both peptides by variable hydrogen bond patterns that included pi and gamma hydrogen bonds. The general agreement in hydrogen bond patterns determined in the simulations and from spectroscopic analysis indicates that with suitable conditions (including solvent composition and counterions where required), local hydrogen-bonded secondary structure in helical peptides may be predicted from dynamics simulations from alpha-helical starting structures. Each peptide, particularly alamethicin, underwent some large amplitude structural fluctuations in which several hydrogen bonds were cooperatively broken. The recovery of the persistent hydrogen bonding patterns after these fluctuations demonstrates the stability of intramolecular hydrogen-bonded secondary structure in methanol (consistent with spectroscopic observations), and is promising for simulations on extended timescales to characterize the nature of the backbone fluctuations that underlie amide exchange from isolated helical polypeptides.     

The aggregation state of mellitin in lipid bilayers. An energy transfer study

We used fluorescence non-radiative energy transfer to measure the self-association of melittin in solution and when bound to lipid bilayers. Energy transfer occurred from the tryptophan residue of unlabeled melittin to an N-methyl anthraniloyl residue covalently bound to a basic lysine residue on melittin. The extent of energy transfer from tryptophan to the label was found to increase severalfold upon the salt-induced tetramerization of melittin. When bound to vesicles of dimyristoyl-L-alpha-phosphatidylcholine, the extent of energy transfer was found to be equivalent to that of monomeric melittin, irrespective of the presence of monomeric or tetrameric melittin in the aqueous phase. We conclude that membrane-bound melittin is monomeric.     

Melittin and a chemically modified trichotoxin form alamethicin-type multi-state pores

The bee venom constituent, melittin, is structurally and functionally related to alamethicin. By forming solvent-free planar bilayers of small area (approx. 100 microns 2) on the tip of fire-polished glass pipettes we could observe single melittin pores in these membranes. An increase in the applied voltage induced further non-integral conductance levels. This indicates that melittin forms multi-level pores similar to those formed by alamethicin. Trichotoxin A40, an antibiotic analogue of alamethicin, also induces a voltage-dependent bilayer conductivity, but no stable pore states are resolved. However, chemical modification of the C-terminal molecule part by introduction of a dansyl group leads to a steeper voltage-dependence and pore state stabilization. Comparing structure and activity of several natural and synthetic amphiphilic polypeptides, we conclude that a lipophilic, N-terminal alpha-helical part of adequate length (dipole moment) and a large enough hydrophilic, C-terminal region are sufficient prerequisites for voltage-dependent formation of multi-state pores.     

Dynamic structure of vesicle-bound melittin in a variety of lipid chain lengths by solid-state NMR

Solid-state 31P- and 13C-NMR spectra were recorded in melittin-lecithin vesicles composed of 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Highly ordered magnetic alignments were achieved with the membrane surface parallel to the magnetic field above the gel-to-liquid crystalline phase transition temperature (Tc). Using these magnetically oriented vesicle systems, dynamic structures of melittin bound to the vesicles were investigated by analyzing the 13C anisotropic and isotropic chemical shifts of selectively 13C-labeled carbonyl carbons of melittin under the static and magic-angle spinning conditions. These results indicate that melittin molecules adopt an alpha-helical structure and laterally diffuse to rotate rapidly around the membrane normal with tilt angles of the N-terminal helix being -33 degrees and -36 degrees and those of the C-terminal helix being 21 degrees and 25 degrees for DLPC and DPPC vesicles, respectively. The rotational-echo double-resonance method was used to measure the interatomic distance between [1-13C]Val8 and [15N]Leu13 to further identify the bending alpha-helical structure of melittin to possess the interhelical angles of 126 degrees and 119 degrees in DLPC and DPPC membranes, respectively. These analyses further lead to the conclusion that the alpha-helices of melittin molecules penetrate the hydrophobic cores of the bilayers incompletely as a pseudo-trans-membrane structure and induce fusion and disruption of vesicles.     

An interdomain distance in cardiac troponin C determined by fluorescence spectroscopy

The distance between Ca2+-binding site III in the C-terminal domain and Cys35 in the N-terminal domain in cardiac muscle troponin C (cTnC) was determined with a single-tryptophan mutant using bound Tb3+ as the energy donor and iodoacetamidotetramethylrhodamine linked to the cysteine residue as energy acceptor. The luminescence of bound Tb3+ was generated through sensitization by the tryptophan located in the 12-residue binding loop of site III upon irradiation at 295 nm, and this sensitized luminescence was the donor signal transferred to the acceptor. In the absence of bound cation at site II, the mean interdomain distance was found to be 48-49 A regardless of whether the cTnC was unbound or bound to cardiac troponin I, or reconstituted into cardiac troponin. These results suggest that cTnC retains its overall length in the presence of bound target proteins. The distribution of the distances was wide (half-width >9 A) and suggests considerable interdomain flexibility in isolated cTnC, but the distributions became narrower for cTnC in the complexes with the other subunits. In the presence of bound cation at the regulatory site II, the interdomain distance was shortened by 6 A for cTnC, but without an effect on the half-width. The decrease in the mean distance was much smaller or negligible when cTnC was complexed with cTnI or cTnI and cTnT under the same conditions. Although free cTnC has considerable interdomain flexibility, this dynamics is slightly reduced in troponin. These results indicate that the transition from the relaxed state to an activated state in cardiac muscle is not accompanied by a gross alteration of the cTnC conformation in cardiac troponin.     

Interaction of melittin with membrane cholesterol: a fluorescence approach

We have monitored the organization and dynamics of the hemolytic peptide melittin in membranes containing cholesterol by utilizing the intrinsic fluorescence properties of its functionally important sole tryptophan residue and circular dichroism spectroscopy. The significance of this study is based on the fact that the natural target for melittin is the erythrocyte membrane, which contains high amounts of cholesterol. Our results show that the presence of cholesterol inhibits melittin-induced leakage of lipid vesicles and the extent of inhibition appears to be dependent on the concentration of membrane cholesterol. The presence of cholesterol is also shown to reduce binding of melittin to membranes. Our results show that fluorescence parameters such as intensity, emission maximum, and lifetime of membrane-bound melittin indicate a change in polarity in the immediate vicinity of the tryptophan residue probably due to increased water penetration in presence of cholesterol. This is supported by results from fluorescence quenching experiments using acrylamide as the quencher. Membrane penetration depth analysis by the parallax method shows that the melittin tryptophan is localized at a relatively shallow depth in membranes containing cholesterol. Analysis of energy transfer results using melittin tryptophan (donor) and dehydroergosterol (acceptor) indicates that dehydroergosterol is not randomly distributed and is preferentially localized around the tryptophan residue of membrane-bound melittin, even at the low concentrations used. Taken together, our results are relevant in understanding the interaction of melittin with membranes in general, and with cholesterol-containing membranes in particular, with possible relevance to its interaction with the erythrocyte membrane.