The interactions of horse heart apocytochromec with phospholipid vesicles and surfactant micelles: time-resolved fluorescence study of the single tryptophan residue (Trp-59)

The interactions of horse heart apocytochromec with membrane interfaces were studied on membrane models including micelles of the anionic surfactant sodium dodecyl sulfate (SDS), the micelle forming lipid analogs dodecylphosphoglycol (Cl2PG), tetradecylphosphoglycol (Cl4PG), and dodecylphosphocholine (Cl2PN), and the negatively charged phospholipid 1-palmitoyl-2-oleoylsn-glycero phosphocholine (POPS) forming small unilamellar vesicles (SUV). The time-resolved fluorescence of the single tryptophan residue (Trp-59) emission was monitored to characterize the modifications of the conformational equilibrium and of the internal dynamics of the protein, which can be brought about by its binding to these model membranes. In most of the cases, as for the protein in solution, the excited state lifetime distribution of the Trp emission was described by four discrete classes, whose relative proportions and barycenters vary significantly in the different complexes formed. In the complex with POPS, however, the decay analysis showed only 3 lifetime classes: the long lifetime class displayed a bary-center value smaller than that observed for the protein in aqueous solution but with a much higher proportion, indicating a stabilization of this conformer in the membrane-bound form of the protein. A similar sensitivity of theTrp-59 excited state to deactivation by thermal collisions in water and in the protein/POPS complex was observed, indicating a probable location ofTrp-59 at the membrane/water interface. The effects of protein binding to C12PN, C12PG and C14PG micelles on the long life-time class proportion were similar to that of POPS but, in addition, there was a large contribution of a short lifetime component which was absent in POPS vesicles. The bary-center values of the excited state lifetime classes were comparable in these membrane systems, suggesting thatTrp-59 is not transferred to a non-polar environment. Binding of apocytochrome c to SDS micelles strongly reduced the lifetime class barycenters and, in contrast to the other membrane systems, strongly favored the contribution of the shortest lifetime class at the expense of the c3 class. This suggests an interaction of theTrp-59 with the sulfur containing head-group of this surfactant. The indole ring mobility is reduced at the interface contacts. A fastTrp-59 mobility with a large amplitude is suggested in the complex with POPS by an initial anisotropy value lower than the expected one of 0.295 measured in vitrified medium. These observations can be correlated with the induction of -helical structure after interactions of apocytochromec with membrane model systems (de Jongh and de Kruijff 1990).Abbreviations AOT sodium bis-(2-ethylhexyl)sulfosuccinate - C12PG dodecylphosphoglycol - C14PG tetradecylphosphoglycol - C12PN dodecylphosphocholine - MEM Maximum Entropy Method - NMR nuclear magnetic resonance - POPS 1-palmitoyl, 2-oleoyl-sn-glycerophosphoserine - SUV small unilamellar vesicles - Trp tryptophan     

Mechanism of the efficient tryptophan fluorescence quenching in human gammaD-crystallin studied by time-resolved fluorescence

Human gammaD-crystallin (HgammaD-Crys) is a two-domain, beta-sheet eye lens protein found in the lens nucleus. Its long-term solubility and stability are important to maintain lens transparency throughout life. HgammaD-Crys has four highly conserved buried tryptophans (Trps), with two in each of the homologous beta-sheet domains. In situ, these Trps will be absorbing ambient UV radiation that reaches the lens. The dispersal of the excited-state energy to avoid covalent damage is likely to be physiologically relevant for the lens crystallins. Trp fluorescence is efficiently quenched in native HgammaD-Crys. Previous steady-state fluorescence measurements provide strong evidence for energy transfer from Trp42 to Trp68 in the N-terminal domain and from Trp130 to Trp156 in the C-terminal domain [Chen, J., et al. (2006) Biochemistry 45, 11552-11563]. Hybrid quantum mechanical-molecular mechanical (QM-MM) simulations indicated that the fluorescence of Trp68 and Trp156 is quenched by fast electron transfer to the amide backbone. Here we report additional information obtained using time-resolved fluorescence spectroscopy. In the single-Trp-containing proteins (Trp42-only, Trp68-only, Trp130-only, and Trp156-only), the highly quenched Trp68 and Trp156 have very short lifetimes, tau approximately 0.1 ns, whereas the moderately fluorescent Trp42 and Trp130 have longer lifetimes, tau approximately 3 ns. In the presence of the energy acceptor (Trp68 or Trp156), the lifetime of the energy donor (Trp42 or Trp130) decreased from approximately 3 to approximately 1 ns. The intradomain energy transfer efficiency is 56% in the N-terminal domain and is 71% in the C-terminal domain. The experimental values of energy transfer efficiency are in good agreement with those calculated theoretically. The absence of a time-dependent red shift in the time-resolved emission spectra of Trp130 proves that its local environment is very rigid. Time-resolved fluorescence anisotropy measurements with the single-Trp-containing proteins, Trp42-only and Trp130-only, indicate that the protein rotates as a rigid body and no segmental motion is detected. A combination of energy transfer with electron transfer results in short excited-state lifetimes of all Trps, which, together with the high rigidity of the protein matrix around Trps, could protect HgammaD-Crys from excited-state reactions causing permanent covalent damage.     

Unfolding of ubiquitin studied by picosecond time-resolved fluorescence of the tyrosine residue

The photophysics of the single tyrosine in bovine ubiquitin (UBQ) was studied by picosecond time-resolved fluorescence spectroscopy, as a function of pH and along thermal and chemical unfolding, with the following results: First, at room temperature (25 degrees C) and below pH 1.5, native UBQ shows single-exponential decays. From pH 2 to 7, triple-exponential decays were observed and the three decay times were attributed to the presence of tyrosine, a tyrosine-carboxylate hydrogen-bonded complex, and excited-state tyrosinate. Second, at pH 1.5, the water-exposed tyrosine of either thermally or chemically unfolded UBQ decays as a sum of two exponentials. The double-exponential decays were interpreted and analyzed in terms of excited-state intramolecular electron transfer from the phenol to the amide moiety, occurring in one of the three rotamers of tyrosine in UBQ. The values of the rate constants indicate the presence of different unfolded states and an increase in the mobility of the tyrosine residue during unfolding. Finally, from the pre-exponential coefficients of the fluorescence decays, the unfolding equilibrium constants (KU) were calculated, as a function of temperature or denaturant concentration. Despite the presence of different unfolded states, both thermal and chemical unfolding data of UBQ could be fitted to a two-state model. The thermodynamic parameters Tm = 54.6 degrees C, DeltaHTm = 56.5 kcal/mol, and DeltaCp = 890 cal/mol//K, were determined from the unfolding equilibrium constants calculated accordingly, and compared to values obtained by differential scanning calorimetry also under the assumption of a two-state transition, Tm = 57.0 degrees C, DeltaHm= 51.4 kcal/mol, and DeltaCp = 730 cal/mol//K.     

Melittin-phospholipid interaction studied by employing the single tryptophan residue as an intrinsic fluorescent probe

The rotational correlation time of melittin, obtained from the nanosecond anisotropy of the emission from its single tryptophan residue, has been found to increase considerably in phosphate solution relative to that in aqueous solution, consistent with protein aggregation. The steady-state fluorescence spectra as well as the absorption spectra in phosphate solution exhibit a very good degree of similarity with those of the protein bound to egg phosphatidylcholine (PC) and distearoylphosphatidylcholine (DSPC) bilayer liposomes. The value of the second-order rate constant for dynamic quenching, $\text{k}{}_{\mathrm{<mtext>q</mtext>}}\text{= 1.4·10}{}^9\text{M}{}^{\mathrm{?1}}\text{·}\text{s}{}^{\mathrm{?1}}$, by acrylamide in 0.5 M phosphate solution is comparable to those for the protein-phospholipids complexes (1·10⁹ and 0.7·10⁹ M^?1·s^?1 for egg PC and DSPC, respectively). Similarities are also found in the nanosecond properties. There is a much stronger and quite similar dependence of the fluorescence spectra on time in the nanosecond range and of the fluorescence decay times on the emission wavelength in both cases as compared to the case in aqueous solution. These observations support the notion that melittin binds to the phospholipids in an aggregated form. The results suggest that the reduction in the $\text{k}{}_{\mathrm{<mtext>q</mtext>}}$ values of bound melittin relative to that in aqueous solution and the blue shift of the fluorescence spectrum (from 352 to 337 nm) are brought about by shielding of the tryptophan residue from the solvent through a combination of protein aggregation and enhancement of its α-helical content (suggested by published CD data). The magnitude of the $\text{k}{}_{\mathrm{<mtext>q</mtext>}}$ values for bound melittin, however, is still relatively high implying the occurrence of rather frequent encounters between the tryptophan residue and the hydrophilic acrylamide molecules. Thus, the residue is found not to penetrate deep into the phospholipid bilayer.     

Study of apocytochrome c from the horse heart by high resolution 1H NMR

The horse heart apocytochrome c was investigated by high resolution 1H-NMR. The secondary chemical shifts of 1H-NMR spectra are not observed, which is typical for disordered molecules. However NOE is characterized by the appearance of spin diffusion which decreases with a temperature increase. The presence of spin diffusion assumes an existence of rigid sites and thus some ordered conformation of the molecule.     

Cytochrome c maturation. The in vitro reactions of horse heart apocytochrome c and Paracoccus dentrificans apocytochrome c550 with heme

C-type cytochromes are characterized by having the heme moiety covalently attached via thioether bonds between the heme vinyl groups and the thiols of conserved cysteine residues of the polypeptide chain. Previously, we have shown the in vitro formation of Hydrogenobacter thermophilus cytochrome c(552) (Daltrop, O., Allen, J. W. A., Willis, A. C., and Ferguson, S. J. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7872-7876). In this work we report that thioether bonds can form spontaneously in vitro between heme and the apocytochromes c from horse heart and Paracoccus denitrificans via b-type cytochrome intermediates. Both apocytochromes, but not the holo forms, bind 8-anilino-1-naphthalenesulfonate, indicating that the apoproteins each have an affinity for a hydrophobic ligand. Furthermore, for both apocytochromes c an intramolecular disulfide can form between the cysteines of the CXXCH motif that is characteristic of c-type cytochromes. In vitro reaction of these apocytochromes c with heme to yield holocytochromes c, and the tendency to form a disulfide, have implications for the different systems responsible for cytochrome c maturation in vivo in various organisms.     

Conformational dynamics of bovine Cu, Zn superoxide dismutase revealed by time-resolved fluorescence spectroscopy of the single tyrosine residue.

The structural dynamics of bovine erythrocyte Cu, Zn superoxide dismutase (BSOD) was studied by time-resolved fluorescence spectroscopy. BSOD is a homodimer containing a single tyrosine residue (and no tryptophan) per subunit. Frequency-domain fluorometry revealed a heterogeneous fluorescence decay that could be described with a Lorentzian distribution of lifetimes. The lifetime distribution parameters (center and width) were markedly dependent on temperature. The distribution center (average lifetime) displayed Arrhenius behavior with an Ea of 4.2 kcal/mol, in contrast with an Ea of 7.4 kcal/mol for the single-exponential decay of L-tyrosine. This indicated that thermal quenching of tyrosine emission was not solely responsible for the effect of temperature on the lifetimes of BSOD. The distribution width was broad (1 ns at 8 degrees C) and decreased significantly at higher temperatures. Furthermore, the width of the lifetime distribution increased in parallel to increasing viscosity of the medium. The combined effects of temperature and viscosity on the fluorescence decay suggest the existence of multiple conformational substrates in BSOD that interconvert during the excited-state lifetime. Denaturation of BSOD by guanidine hydrochloride produced an increase in the lifetime distribution width, indicating a larger number of conformations probed by the tyrosine residue in the denatured state. The rotational mobility of the tyrosine in BSOD was also investigated. Analysis of fluorescence anisotropy decay data enabled resolution of two rotational correlation times. One correlation time corresponded to a fast (picosecond) rotation that contributed 62% of the anisotropy decay and likely reported local mobility of the tyrosine ring. The longer correlation time was 50% of the expected value for rotation of the whole (dimeric) BSOD molecule and appeared to reflect segmental motions in the protein in addition to overall tumbling. Comparison between rotational correlation times and fluorescence lifetimes of BSOD indicates that the heterogeneity in lifetimes does not arise from mobility of the tyrosine per se, but rather from dynamics of the protein matrix surrounding this residue which affect its fluorescence decay.     

Insight into the conformational dynamics of specific regions of porcine pancreatic phospholipase A2 from a time-resolved fluorescence study of a genetically inserted single tryptophan residue

The effects of Ca2+ and substrate analogue binding on the conformational dynamics of porcine pancreas phospholipase A2 (PLA2) in different regions was explored by combining site-directed mutagenesis and time-resolved fluorescence measurements. The single tryptophan residue (Trp-3) of the wild-type protein (W3), in the alpha-helix A, was replaced by a phenylalanine residue (W3F), whereafter Trp was substituted either for leucine-31 (W31), located in the calcium binding loop, or for phenylalanine-94 (W94), located at the "back side" of the enzyme. Furthermore, mutants lacking the 62-66 sequence were constructed with the Trp at position 3 (delta W3) or 31 (delta W31). The total fluorescence intensity decays of Trp in each protein, in the protein-calcium and the protein-calcium-substrate analogue complexes, analyzed by the maximum entropy method (MEM) can be interpreted as distributions of separated lifetime classes. In the case of the W94 mutant, a major short-lived excited-state population (tau approximately 50 ps) is observed, probably deactivated by the interaction with two proximate disulfide bridges via a radiationless process. For the four other mutants, the respective barycenters of the four lifetime classes display comparable values, but the amplitude distributions are different for Trp-3 and Trp-31. The rotational mobility of the Trp residue varies along the peptide chain. Trp-3 experiences only a fast hindered motion. Trp-31 is sensitive to an additional local flexibility that is absent in the N-terminal part of the protein. The largest wobbling angle is observed at position 94. No effect of calcium binding occurs on the lifetime distribution of the Trp-3 and Trp-94 residues. Their mobilities are not affected. In contrast, calcium binding displays a strong influence on the excited-state population distribution of Trp-31. A major population decaying with the longest lifetime is selected in the W31 protein and contributes to approximately 50% of the decay. The local flexibility and the amplitude of motion of Trp-31 is wider in the protein-calcium complex than in the unliganded protein. Binding of the monomeric substrate analogue n-dodecylphosphocholine (C12PN) in the presence of calcium slightly affects the Trp-3 excited-state population distribution and its mobility. Trp-31 is more sensitive to this binding. In particular, a more restricted rotation of the Trp-31 residue and a decrease of the peptide local flexibility as protein-calcium complexes are observed in both the W31 and delta W31 mutants.(ABSTRACT TRUNCATED AT 400 WORDS)     

Total synthesis of horse heart apocytochrome c by conformation-assisted condensation of two chemically synthesized fragments.

A fully synthetic peptide, corresponding to the entire 104-residue sequence of horse heart apocytochrome c with Met65 replaced by homoserine, has been obtained by an original conformation-assisted three-fragment condensation procedure. The method involves the selective joining of two synthetic fragments, namely residues 1-65 of the apopeptide with Met65 replaced by homoserine lactone and residues 66-104 of the protein in the presence of fragment 1-25 of the native heme-containing peptide. The joining conditions have been optimized with regard to solvent, pH and possible influence of additives. The presence of radical scavengers and the complete exclusion of oxygen were found essential in order to prevent oxidative side reactions. A sensitive method based on reverse-phase HPLC has been used to monitor the course of the reaction. Condensation yields up to 80% were obtained. The data obtained by this new three-fragment rejoining approach are discussed and compared to those of a similar two-fragment condensation procedure. Our data demonstrate how the folding properties of large synthetic peptide fragments, organized in a complex, can be utilized to extend the presently improved solid-phase peptide methods to the synthesis of a functioning protein with more than 100 residues.     

Structure and dynamics of the alpha-lactalbumin molten globule: fluorescence studies using proteins containing a single tryptophan residue

The fluorescence properties of three variants of alpha-lactalbumin (alpha-LA) containing a single tryptophan residue were investigated under native, molten globule, and unfolded conditions. These proteins have levels of secondary structure and stability similar to those of the wild type. The fluorescence signal in the native state is dominated by that of W104, with the signal of W60 and W118 significantly quenched by the disulfide bonds in their vicinity. In the molten globule state, the magnitude of the fluorescence signal of W60 and W118 increases, due to the loss of rigid, specific side chain packing. In contrast, the magnitude of the signal of W104 decreases in the molten globule state, perhaps due to the protonation of H107 or quenching by D102 or K108. The solvent accessibilities of individual tryptophan residues were investigated by their fluorescence emission maximum and by acrylamide quenching studies. In the native state, the order of solvent accessibility is as follows: W118 > W60 > W104. This order changes to W60 > W104 > W118 in the molten globule state. Remarkably, the solvent accessibility of W118 in the alpha-LA molten globule is lower than that in the native state. The dynamic properties of the three tryptophan residues were examined by time-resolved fluorescence anisotropy decay studies. The overall rotation of the molecule can be observed in both the native and molten globule states. In the molten globule state, there is an increase in the extent of local backbone fluctuations with respect to the native state. However, the fluctuation is not sufficient to result in complete motional averaging. The three tryptophan residues in the native and molten globule states have different degrees of motional freedom, reflecting the folding pattern and dynamic heterogeneity of these states. Taken together, these studies provide new insight into the structure and dynamics of the alpha-LA molten globule, which serves as a prototype for partially folded proteins.     

Nanosecond dynamics of the single tryptophan reveals multi-state equilibrium unfolding of protein GB1

Unfolding of the immunoglobulin binding domain B1 of streptococcal protein G (GB1) was induced by guanidine hydrochloride (GdnHCl) and studied by circular dichroism, steady-state, and time-resolved fluorescence spectroscopy. The fluorescence methods employed the single tryptophan residue of GB1 as an intrinsic reporter. While the transitions monitored by circular dichroism and steady-state fluorescence coincided with each other, the transitions followed by dynamic fluorescence were markedly different. Specifically, fluorescence anisotropy data showed that a relaxation spectrum of tryptophan contained a slow motion with relaxation times of 9 ns in the native state and 4 ns in the unfolded state in 6 M GdnHCl. At intermediate GdnHCl concentrations of 3.8-4.2 M, however, the slow relaxation time increased to 18 ns. The fast nanosecond motion had an average time of 0.8 ns and showed no dependence on the formation of native structure. Overall, dynamic fluorescence revealed two preliminary stages in GB1 folding, which are equated with the formation of local structure in the beta(3)-strand hairpin and the initial collapse. Both stages exist without alpha-helix formation, i. e., before the appearance of any ordered secondary structure detectable by circular dichroism. Another stage in GB1 folding might exist at very low ( approximately 1 M) GdnHCl concentrations.     

Complexation study of europium(III) and curium(III) with urea in aqueous solution investigated by time-resolved laser-induced fluorescence spectroscopy

The complex formation of europium(III) and curium(III) with urea in aqueous solution has been studied at I = 0.1 M (NaClO₄), room temperature and trace metal concentrations in the pH-range of 1-8 at various ligand concentrations using time-resolved laser-fluorescence spectroscopy. While for curium(III) the luminescence maximum is red shifted upon complexation, in case of europium(III) emission wavelengths remain unaltered but a significant change in peak splitting occurs. Both heavy metals form weak complexes of the formulae ML³⁺ and MLOH²⁺ with urea. Stability constants were determined to be log β₁₁₀ = −0.12 ± 0.05 and log β_11-1 = −6.86 ± 0.15 for europium(III) and log β₁₁₀ = −0.28 ± 0.12 and log β_11-1 = −7.01 ± 0.15 for curium(III).     

Fluorescence studies of the conformational dynamics of parvalbumin in solution: lifetime and rotational motions of the single tryptophan residue

The fluorescence properties of the single tryptophan residue in whiting parvalbumin were used to probe the dynamics of the protein matrix. Ca2+ binding caused a blue-shift in the emission (from lambda max = 339 to 315 nm) and a 2.5-fold increase in quantum yield. The fluorescence decay was nonexponential in both Ca2(+)-free and Ca2(+)-bound parvalbumin and was best described by Lorentzian lifetime distributions centered around two components: a major long-lived component at 2-5 ns and a small subnanosecond component. Raising the temperature from 8 to 45 degrees C resulted in a decrease in both the center (average) and width (dispersion) of the major lifetime distribution component, whereas the center, width, and fractional intensity of the fast component increased with temperature. Arrhenius activation energies of 1.3 and 0.3 kcal/mol were obtained in the absence and in the presence of Ca2+, respectively, from the temperature dependence of the center of the major lifetime distribution component. Direct anisotropy decay measurements of local tryptophan rotations yielded an activation energy of 2.3 kcal/mol in Ca2(+)-depleted parvalbumin and indicated a correlation between rotational rates and lifetime distribution parameters (center and width). Ca2+ binding produced a decrease in the width of the major lifetime distribution component and a decrease in tryptophan rotational mobility within the protein. There was a rough correlation between these two parameters with changes in Ca2+ and temperature, so that both measurements may be taken to indicate that the structure of Ca2(+)-bound parvalbumin was more rigid than in Ca2(+)-depleted parvalbumin.     

Nanosecond motions of the single tryptophan residues in apolipoproteins C-I and C-II: a study by oxygen quenching and fluorescence depolarization

Rotational freedom of the single tryptophan residue in human plasma apolipoproteins C-I (apo C-I) and C-II (apo C-II) was investigated by oxygen quenching and lifetime-resolved anisotropies. The tryptophan in both apo C-I and C-II was highly accessible to oxygen quenching. The tryptophan residue in both apo C-I and C-II and their sodium dodecyl sulfate (SDS) or dimyristoylphosphatidylcholine (DMPC) complexes displayed significant motional freedom on the nanosecond time scale. Lifetime-resolved anisotropies of tryptophan residues under conditions of oxygen quenching revealed an increase in the amplitude of the segmental motions at 40 degrees C as compared to that at 5 degrees C. It was concluded from these studies that both the apoprotein C-I and C-II are highly flexible molecules, and that the nanosecond motions of the tryptophan residue are sensitive to the fluidity of its environment in both SDS and DMPC complexes.     

Nanosecond fluorometry of the single tryptophan in cytochrome P-450e (P450IIB2)

Properties of the single tryptophan residue in rat liver microsomal phenobarbital-inducible cytochrome P-450e (P450IIB2) were studied by the nanosecond time-resolved fluorometry. The tryptophan fluorescence decay time was found to be 3.6 ns and it was not affected by the addition of substrate (perhydrophenanthrene). This result strongly indicates that the tryptophan residue is not a part of the substrate-binding site.