Anisotropy decays of indole, melittin monomer and melittin tetramer by frequency-domain fluorometry and multi-wavelength global analysis

We used frequency-domain fluorescence spectroscopy to measure the fluorescence lifetime and anisotropy decays of indole in propylene glycol, and of the tryptophan emission of melittin monomer and tetramer in water solutions at 5 degrees C. We obtained an increase in resolution of the anisotropy decays by using multiple excitation wavelengths, chosen to provide a range of fundamental anisotropy values. The multi-excitation wavelength anisotropy decays were analyzed globally to recover a single set of correlation times with wavelength-dependent anisotropy amplitudes. Simulated data and kappaR2 surfaces are shown to reveal the effect of multi-wavelength data on the resolution of complex anisotropy decays. For both indole and melittin, the anisotropy decays are heterogeneous and require two correlation times to fit the frequency-domain data. For indole in propylene glycol at 5 degrees C we recovered correlation times of 0.59 and 4.10 ns, which appear to be characteristic of the rigid and asymmetric indole molecule. For melittin monomer the correlation times were 0.13 and 1.75 ns, and for melittin tetramer 0.12 and 3.96 ns. The shorter and longer correlation times of melittin are due to segmental motions and overall rotational diffusion of the polypeptide.     

Organization and dynamics of tryptophan residues in tetrameric and monomeric soybean agglutinin: Studies by steady-state and time-resolved fluorescence,phosphorescence and chemical modification

We have investigated the organization and dynamics of tryptophan residues in tetrameric, monomeric and unfolded states of soybean agglutinin (SBA) by selective chemical modification, steady-state and time-resolved fluorescence, and phosphorescence. Oxidation with N-bromosuccinimide (NBS) modifies two tryptophans (Trp 60 and Trp 132) in tetramer, four (Trp 8, Trp 203 and previous two) in monomer, and all six (Trp 8, Trp 60, Trp 132, Trp 154, Trp 203 and Trp 226) in unfolded state. Utilizing wavelength-selective fluorescence approach, we have observed a red-edge excitation shift (REES) of 10 and 5 nm for tetramer and monomer, respectively. A more pronounced REES (21 nm) is observed after NBS oxidation. These results are supported by fluorescence anisotropy experiments. Acrylamide quenching shows the Stern–Volmer constant (K_SV) for tetramer, monomer and unfolded SBA being 2.2, 5.0 and 14.6 M⁻¹, respectively. Time-resolved fluorescence studies exhibit biexponential decay with the mean lifetime increasing along tetramer (1.0 ns) to monomer (1.9 ns) to unfolded (3.6 ns). Phosphorescence studies at 77 K give more structured spectra, with two (0,0) bands at 408.6 (weak) and 413.2 nm for tetramer. However, a single (0,0) band appears at 411.8 and 407.2 nm for monomer and unfolded SBA, respectively. The exposure of hydrophobic surface in SBA monomer has been examined by 8-anilino-1-naphthalenesulfonate (ANS) binding, which shows ∼20-fold increase in ANS fluorescence compared to that for tetramer. The mean lifetime of ANS also shows a large increase (12.0 ns) upon binding to monomer. These results may provide important insight into the role of tryptophans in the folding and association of SBA, and oligomeric proteins in general.     

Fluorescence studies of the restricted motion of tryptophan in α-cobratoxin

The fluorescence lifetime and rotational correlation time of the single tryptophan residue in -cobratoxin have been measured between pH 2 and 10. The fluorescence decays are non-exponential and give lifetimes that are shorter than normally observed in small proteins (0.3 ns and 1 ns). This emission is consistent with a model in which the tryptophan residue is in slightly different environments in the protein. Fluorescence anisotropy decays show that the tryptophan residue is almost completely immobilised by neighbouring groups in the protein. The range of the wobbling motion is slightly pH dependent and limited to between 5° and 10°.     

Time resolved fluorescence of bacteriophage Pfl DNA binding protein and its complex with DNA

The DNA binding protein of the filamentous bacteriophage Pfl exhibits fluorescence from a single tryptophan residue. The location of the emission maximum at 340 nm ist quite common for proteins, but the single lifetime of 7.8 ns is one of the longest yet reported. Protein fluorescence is quenched more efficiently by Cs⁺ than by I^-; the Trp is located in a partially exposed pocket, in the vicinity of a negative charge.In the native complex of the binding protein with Pfl DNA the fluorescence emission maximum is at 330 nm, indicating a more apolar environment for Trp 14. The native nucleoprotein complex exhibits a similar fluorescence lifetime (6.5 ns) and an approximately equal fluorescence yield, indicating the absence of Trp-DNA stacking. The tryptophan in the complex is virtually inaccessible to ionic quenchers, and thus appears to be buried.Fluorescence depolarisation measurements have been used to examine the rotational mobility of the tryptophan in the protein and in the nucleoprotein complex. In the protein alone a single rotational correlation time () of 19 ns is observed, corresponding to rotation of the entire dimeric molecule; in the native nucleoprotein complex with Pfl DNA, a of 500 ns is observed, corresponding to a rigid unit of at least 50 subunits. In neither case does the tryptophan exhibit any detectable flexibility on the subnanosecond time scale.     

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.     

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.     

A study of protein dynamics from anisotropy decays obtained by variable frequency phase-modulation fluorometry: internal motions of N-methylanthraniloyl melittin

Internal motions of melittin and its lipid complexes were studied by anisotropy decays determined by frequency-domain fluorometry. A covalent anthraniloyl probe was attached, probably to lysine-21. The emission spectra indicate that the anthraniloyl moiety is exposed to solvent in both monomeric and tetrameric forms and is present at the lipid-water interfacial region in the lipid complexes. The fluorescence intensity decay of melittin in solution and its lipid complexes was characterized by three lifetimes. The lifetimes were near 1-2 ns, 6-7 ns and 10 ns. At increased temperatures there was an increase in the amplitude of the intermediate lifetime and a decrease in that of the longer lifetime. For all the melittin systems, at least three correlation times were required to fit the anisotropy data. Of the three correlation times, the shortest correlation time represents the local motions of the probe, while the longest represents global motions of the whole molecule. The intermediate correlation time probably represents the dynamics of domains/helices within the molecule. The melittin monomer is highly flexible, with greater than 90% of its anisotropy being lost by the local motions. Even though it is well organized (greater than 75% helical), the tetramer is still a highly flexible molecule, with 70% of its anisotropy being lost by the local motions. The internal motions of melittin decrease upon binding to lipids and are sensitive to the phase state of the lipid complexes.     

Fluorescence and 13C NMR determination of side-chain and backbone dynamics of synthetic melittin and melittin analogues in isotropic solvents

The dynamics in isotopic solvents of selectively 13C labeled synthetic melittin and three analogues have been investigated by using NMR and fluorescence techniques both separately and in combination. In conjunction with the "model-free" approach to interpretation of NMR relaxation data [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4570], the availability of steady-state fluorescence anisotropy and lifetime data augment T1, T2, and NOE data to provide quantitative information about fluorophore dynamics in these peptides. A method is presented for using combined fluorescence and NMR data to obtain technique- and model-independent values for parameters describing local motion of 13C-labeled fluorophores in peptides and proteins. The dynamics of melittin and melittin analogues are found to be consistent with structural characteristics inferred from CD, fluorescence, and NMR spectral information presented in the preceding paper (Weaver et al., 1989). In particular, the mobility of the random coil peptide monomers is shown to be quite similar, while side-chain as well as peptide backbone motion in the aggregated or oligomeric species differs markedly among the analogues. For melittin itself, experimentally determined overall rotational correlation times for the monomer and tetramer agree very well with values predicted on the basis of solvent-accessible protein surface area. The local dynamics of selectively 13C-labeled Trp-19 and Gly-12 residues of melittin are also found to be consistent with peptide structure. In random coil melittin monomer, a specific model for the motion indicates that the Trp side chain moves through an approximate angle of +/- 71 degrees about the beta-gamma bond with a correlation time of 159 +/- 24 ps. In melittin tetramer, the indole moiety is spatially more confined with a flip angle of +/- 37 degrees, yet demonstrates an increased rate of motion with a correlation time of 56 +/- 8 ps. The constrained mobility of the Trp-19 side chain is consistent with motional constraints inferred from the X-ray structure of melittin tetramer. These results show that protein side-chain motion, even of moieties as large as indole, can occur on the picosecond time scale and that these motions are reasonably similar to those inferred from molecular dynamics simulations.     

Time resolved spectroscgpy of tryptopkyl fluorescence of yeast 3-phosphoglycerate kinase

The tryptophyl fluorescence emission of yeast 3-phosphoglycerate kinase decreases from pH 3.9 to pH 7.2 following a normal titration curve with an apparent pK of 4.7. The fluorescence decays have been determined at both extreme pH by photocounting pulse fluorimetry and have been found to vary with the emission wavelength. A quantitative analysis of these results according to a previously described method allows to determine the emission characteristics of the two tryptophan residues present in the protein molecule. At pH 3.9, one of the tryptophan residues is responsible for only 13% of the total fluorescence emission. This first residue has a lifetime τ₁= 0.6 ns and a maximum fluorescence wavelength λ_2max = 332 nm. The second tryptophan residue exhibits two lifetimes τ₂₁= 3.1 ns and τ₂₂= 7.0 ns (λ_2max= 338 nm). In agreement with the attribution of τ₂₁and τ₃₂ to the same tryptophan residue, the ratio β = C₂₁/C₂₂ of the normalized amplitudes is constant along the fluorescence emission spectrum. At pH 7.2, the two tryptophan residues contribute almost equally tc the protein fluorescence. The decay time of tryptophan 1 is 0.4 ns. The other emission parameters are the same as those determined at pH 3.9. We conclude that the fluorescence quenching in the range pH 3.9 to pH 8.0 comes essentially from the formation of a non emitting internal ground state complex between the tryptophan having the longest decay times and a neighbouring protein chemical group. The intrinsic pK of this group and the equilibrium constant of the irternal complex can be estimated. The quenching group is thought to be a carboxylate anion. Excitation transfers between the two tryptophyl residues of the protein molecule appear to have a small efficiency.     

Conformation and dynamics of bovine brain S-100a protein determined by fluorescence spectroscopy.

We have used time-resolved laser fluorescence spectroscopy to investigate the intensity and anisotropy decays of the single tryptophan residue in bovine brain S-100a (alpha beta) protein. The steady-state and acrylamide quenching results indicated that the Trp 90 of the alpha-subunit was partially buried in a relatively nonpolar environment at pH 7.5. Both Ca2+ and pH 8.5 slightly enhanced the exposure of the residue to the solvent, but the residue remained partially buried in the calcium complex at both pH values. The best representation of the intensity decays was a linear combination of three exponential terms, regardless of solvent condition and temperature. The three lifetimes (tau i) were in the range of 0.4-5 ns and insensitive to emission wavelength, but their fractional amplitudes (alpha i) shifted in favor of the shortest component (alpha 1) when the decays were measured at the blue end of the emission spectrum. These results suggest that an excited-state interaction between the indole ring and the side chain of an adjacent residue may be responsible for the observed shortest lifetime. In the presence of Ca2+, the three lifetimes remained relatively unaltered, but the values of alpha 1 decreased by a factor of 2.3 at pH 7.2 and a factor of 1.8 at pH 8.2. This Ca(2+)-induced decrease may be attributed to disruption of the putative excited-state interaction resulting from reorientations of the alpha-helical segments flanking a Ca(2+)-binding loop (residues 62-73). At both pH 7.2 and 8.4, the anisotropy decays of the apoprotein followed a biexponential decay law.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhanced resolution of fluorescence anisotropy decays by simultaneous analysis of progressively quenched samples. Applications to anisotropic rotations and to protein dynamics.

Enhanced resolution of rapid and complex anisotropy decays was obtained by measurement and analysis of data from progressively quenched samples. Collisional quenching by acrylamide was used to vary the mean decay time of indole or of the tryptophan fluorescence from melittin. Anisotropy decays were obtained from the frequency-response of the polarized emission at frequencies from 4 to 2,000 MHz. Quenching increases the fraction of the total emission, which occurs on the subnanosecond timescale, and thereby provides increased information on picosecond rotational motions or local motions in proteins. For monoexponential subnanosecond anisotropy decays, enhanced resolution is obtained by measurement of the most highly quenched samples. For complex anisotropy decays, such as those due to both local motions and overall protein rotational diffusion, superior resolution is obtained by simultaneous analysis of data from quenched and unquenched samples. We demonstrate that measurement of quenched samples greatly reduces the uncertainty of the 50-ps correlation time of indole in water at 20 degrees C, and allows resolution of the anisotropic rotation of indole with correlation times of 140 and 720 ps. The method was applied to melittin in the monomeric and tetrameric forms. With increased quenching, the anisotropy data showed decreasing contributions from overall protein rotation and increased contribution from picosecond tryptophan motions. The tryptophan residues in both the monomeric and the tetrameric forms of melittin displayed substantial local motions with correlation times near 0.16 and 0.06 ns, respectively. The amplitude of the local motion is twofold less in the tetramer. These highly resolved anisotropy decays should be valuable for comparison with molecular dynamics simulations of melittin.     

Fluorescent Properties of Firefly Luciferases and Their Complexes with Luciferin

Fluorescence of luciferases from Luciola mingrelica (single tryptophanresidue, Trp-419) and Photinus pyralis (two tryptophan residues, Trp-417,Trp-426) was studied. Analysis of quenching of tryptophan fluorescenceshowed that the tryptophan residue conserved in all luciferases is notaccessible for charged quenchers, which is explained by the presence ofpositively and negatively charged amino acid residues in the close vicinityto it. An effective energy transfer from tryptophan to luciferin wasobserved during quenching of tryptophan fluorescence of both luciferaseswith luciferin. From the data on the energy transfer, the distance betweenthe luciferin molecule and Trp-417 (419) in the luciferin–luciferasecomplex was calculated: 11–15 for P. pyralis and 12–17 for L. mingrelica luciferases. The role of the conserved Trp residuein the catalysis is discussed.     

Structural-dynamic properties of the tryptophan residue environment in melittin

The structural dynamics of the environment of the single tryptophan residue in melittin was studied by site-selective red-edge-excitation fluorescence spectroscopy. The dependence of the spectral shift on transition from excitation in a maximum (at 280 nm) to long-wavelength edge (305 nm) was studied as a function of temperature. It was shown, that for melittin at high ionic strength (tetramer), the three regions of temperature dependence of the red-edge effect are observed: retarded relaxation (up to +30 degrees C), relaxational changes of spectra (from +30 to +50 degrees C) and thermal changes of structure (above +50 degrees C). The dipolar-re-orientational relaxation time and activation energy of orientation motions in the environment of indolic ring in the tetrameric melittin structure were estimated. Extrapolation from relaxational region to room temperature results in relaxation time 40 ns. In monomeric melittin (at low ionic strength) the red-edge shift of spectra is absent. The distinct differences in character of thermal quenching of fluorescence between monomeric and tetrameric forms of melittin are observed. It follows, that the short-wave-length fluorescence shift on monomer-tetramer transition is due to both the reduction of polarity, and the increase in rigidity of tryptophan environment, the absence of relaxation motions at nanosecond times.     

Resolution of the fluorescence decay of the two tryptophan residues of lac repressor using single tryptophan mutants.

We have studied the time-resolved intrinsic tryptophan fluorescence of the lac repressor (a symmetric tetramer containing two tryptophan residues per monomer) and two single-tryptophan mutant repressors obtained by site-directed mutagenesis, lac W201Y and lac W220Y. These mutant repressor proteins have tyrosine substituted for tryptophan at positions 201 and 220, respectively, leaving a single tryptophan residue per monomeric subunit at position 220 for the W201Y mutant and at position 201 in the W220Y mutant. It was found that the two decay rates recovered from the analysis of the wild type data do not correspond to the rates recovered from the analysis of the decays of the mutant proteins. Each of these residues in the mutant repressors displays at least two decay rates. Global analysis of the multiwavelength data from all three proteins, however, yielded results consistent with the fluorescence decay of the wild type lac repressor corresponding simply to the weighted linear combination of the decays from the mutant proteins. The effect of ligation by the antagonistic ligands, inducer and operator DNA, was similar for all three proteins. The binding of the inducer sugar resulted in a quenching of the long-lived species, while binding by the operator decreased the lifetime of the short components. Investigation of the time-resolved anisotropy of the intrinsic tryptophan fluorescence in these three proteins revealed that the depolarization of fluorescence resulted from a fast motion and the global tumbling of the macromolecule. Results from the simultaneous global analysis of the frequency domain data sets from the three proteins revealed anisotropic rotations for the macromolecule, consistent with the known elongated shape of the repressor tetramer. In addition, it appears that the excited-state dipole of tryptophan 220 is alighed with the long axis of the repressor.     

Fluorescence characterization of Trp 21 in rat glutathione S-transferase 1–1: Microconformational changes induced by S-hexyl glutathione

The glutathione S-transferase (GST) isoenzyme A1–1 from rat contains a single tryptophan, Trp 21, which is expected to lie within α-helix 1 based on comparison with the X-ray crystal structures of the pi- and mu-class enzymes. Steady-state and multifrequency phase/modulation fluorescence studies have been performed in order to characterize the fluorescence parameters of this tryptophan and to document ligand-induced conformational changes in this region of the protein. Addition of S-hexyl glutathione to GST isoenzyme A1–1 causes an increase in the steady-state fluorescence intensity, whereas addition of the substrate glutathione has no effect. Frequency-domain excited-state lifetime measurements indicate that Trp 21 exhibits three exponential decays in substrate-free GST. In the presence of S-hexyl glutathione, the data are also best described by the sum of three exponential decays, but the recovered lifetime values change. For the substrate-free protein, the short lifetime component contributes 9–16% of the total intensity at four wavelengths spanning the emission. The fractional intensity of this lifetime component is decreased to less than 3% in the presence of S-hexyl glutathione. Steady-state quenching experiments indicate that Trp 21 is insensitive to quenching by iodide, but it is readily quenched by acrylamide. Acrylamide-quenching experiments at several emission wavelengths indicate that the long-wavelength components become quenched more easily in the presence of S-hexyl glutathione. Differential fluorescence polarization measurements also have been performed, and the data describe the sum of two anisotropy decay rates. The recovered rotational correlation times for this model are 26 ns and 0.81 ns, which can be attributed to global motion of the protein dimer, and fast local motion of the tryptophan side chain. These results demonstrate that regions of GST that are not in direct contact with bound substrates are mobile and undergo microconformational rearrangement when the “H-site” is occupied.     

Comparison of 15N- and 13C-determined parameters of mobility in melittin

Backbone and tryptophan side-chain mobilities in the 26-residue, cytolytic peptide melittin (MLT) were investigated by ¹⁵N and ¹³C NMR. Specifically, inverse-detected ¹⁵N T₁ and steady-state NOE measurements were made at 30 and 51 MHz on MLT at 22 °C enriched with ¹⁵N at six amide positions and in the Trp¹⁹ side chain. Both the disordered MLT monomer (1.2 mM peptide at pH 3.6 in neat water) and -helical MLT tetramer (4.0 mM peptide at pH 5.2 in 150 mM phosphate buffer) were examined. The relaxation data were analyzed in terms of the Lipari and Szabo model-free formalism with three parameters: _m, the correlation time for the overall rotation; S², a site-specific order parameter which is a measure of the amplitude of the internal motion; and _e, a local, effective correlation time of the internal motion. A comparison was made of motional parameters from the ¹⁵N measurements and from ¹³C measurements on MLT, the latter having been made here and previously [Kemple et al. (1997) Biochemistry, 36, 1678–1688]. _m and _e values were consistent from data on the two nuclei. In the MLT monomer, S² values for the backbone N-H and C-H vectors in the same residue were similar in value but in the tetramer the N-H order parameters were about 0.2 units larger than the C-H order parameters. The Trp side-chain N-H and C-H order parameters, and _e values were generally similar in both the monomer and tetramer. Implications of these results regarding the dynamics of MLT are examined.     

Similarity of decay-associated spectra for tryptophan fluorescence of proteins with different structures

Tryptophan fluorescence lifetimes were analyzed for three proteins: human serum albumin, bovine serum albumin, and bacterial luciferase, which contain one, two, and seven tryptophan residues, respectively. For all of the proteins, the fluorescence decays were fitted by three lifetimes: τ₁ = 6–7 ns, τ₂ = 2.0–2.3 ns, and τ₃ ≤ 0.1 ns (the native state), and τ₁ = 4.4–4.6 ns, τ₂ = 1.7–1.8 ns, and τ₃ ≤ 0.1 ns (the denatured state). Corresponding decay-associated spectra had similar peak wavelengths and spectrum half-widths both in the native state (\(\lambda _{\max }^{{\tau _1}} = 324nm\), \(\lambda _{\max }^{{\tau _2}} = 328nm\), and \(\lambda _{\max }^{{\tau _3}} = 315nm\)), and in the denatured state (\(\lambda _{\max }^{{\tau _1}} = 350nm\), \(\lambda _{\max }^{{\tau _2}} = 343nm\), and \(\lambda _{\max }^{{\tau _3}} = 317nm\)). The differences in the steady-state spectra of the studied proteins were accounted for the individual ratio of the lifetime component contributions. The lifetime components were compared with a classification of tryptophan residues in the structure of these proteins within the discrete states model.     

Spectrally and time-resolved fluorescence spectroscopic study on melittin-calmodulin interaction

The origin of multi-exponential fluorescence decay property of tryptophan (Trp) in protein has been in controversy, and dielectric relaxation is thought to be one of the most plausible candidates of that origin. In this study, we studied melittin-calmodulin interaction on the concept of dielectric relaxation by spectrally and time-resolved fluorescence spectroscopy. Trp residue in melittin demonstrated drastic change in its dielectric relaxation rate and scale by binding with calmodulin. Expected change of relaxation rate suggested that dielectric relaxation accounts for multi-exponential property of fluorescence decay. We also examined the time variation of radiative and non-radiative decay rates. That result demonstrated the distinct difference profiles of non-radiative decay rate of Trp in melittin and the complex.     

Spectroscopic studies on human serum albumin and methemalbumin: optical, steady-state, and picosecond time-resolved fluorescence studies, and kinetics of substrate oxidation by methemalbumin

The nature of the heme environment in methemalbumin, the Fe(III) protoporphyrin IX (heme)-human serum albumin (HSA) complex, was investigated by optical spectroscopy. Comparison of the optical spectra of methemalbumin, ferro-hemalbumin in the absence and presence of 2-methylimidazole, and their carbon monoxide derivatives with horseradish peroxidase (HRP) and its corresponding derivatives indicates that histidine is not present in the first coordination sphere of heme in methemalbumin and that the protein is devoid of a well-defined heme cavity. The complex exhibits peroxidase activity by catalyzing oxidation of 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) by hydrogen peroxide. Its activity ( K(M)=433 microM, molar catalytic activity=0.33 s(-1)), however, is considerably lower compared to HRP, indicating differences in the heme environments. Fluorescence intensity decays of Trp214 in HSA and methemalbumin, best fitted to a three-exponential model, gave the lifetimes 7.03 ns (30%), 3.17 ns (38%), and 0.68 ns (32%) for HSA and 8.04 ns (1.7%), 2.42 ns (19.7%), and 0.64 ns (78.6%) for methemalbumin. These lifetime values were further confirmed by a model-independent maximum entropy method. Similarity in the lifetimes and variations in the amplitudes suggest that while conformational heterogeneity of HSA is unperturbed on heme binding, redistribution of the populations of the three conformations occurs and the sub-state associated with the shortest lifetime dominates the total population by approximately 80%. Decay associated spectra (DAS) indicate that the observed lifetime variation with wavelength is predominantly due to ground state heterogeneity, though solvent dipolar relaxation also contributes. Time-resolved fluorescence anisotropy measurements of the Trp214 residue yielded information on motion within the protein together with the whole protein molecule. The binding of heme did not affect the rotational correlation time of the albumin molecule (approximately 20 ns). However, the motion of tryptophan within the protein matrix increased by a factor of approximately 3 (0.46 ns to 0.15 ns). This indicates that while the overall hydrodynamic volume of the albumin molecule remained the same, tryptophan underwent a more rapid internal rotation because of the efficient energy transfer to the bound heme. Optical studies, analysis of lifetime measurements, DAS, and anisotropy measurements together suggest that heme binds to a surface residue. The rapid internal motion of Trp214 during its excited state lifetime for the approximately 80% populated conformer of methemalbumin allows the orientation factor, kappa(2), to approach the average value of 2/3. From the time-resolved fluorescence measurements and the energy transfer calculations on methemalbumin, a Trp214-heme distance of 22 A was deduced.     

Investigation of the membrane-active peptides melittin and glucagon by photochemically induced dynamic-nuclear-polarization (photo-CIDNP) NMR

The photochemically induced dynamic-nuclear-polarization (photo-CIDNP) NMR technique was used to investigate the membrane-active peptides melittin and glucagon. The experiments were performed both in the absence and presence of phospholipid vesicles in order to study the topography of the membrane-bound state. From the results it can be concluded that the melittin peptide chain is oriented in such a way that the single tryptophan residue (Trp19) reaches into the membrane. In the case of glucagon, a binding interaction with vesicle membranes is indicated within the pH range 2-10, whereby the single tryptophan residue (Trp25) is buried in the lipid bilayer and the tyrosine and histidine residues are exposed to the aqueous solvent.