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)     

Liquid-crystalline phases of cholesterol/lipid bilayers as revealed by the fluorescence of trans-parinaric acid.

The presence of two liquid-crystalline phases, alpha and beta, in mixed bilayers of dimyristoylphosphatidylcholine/cholesterol was detected by the changes in the distribution of the fluorescence lifetimes of t-PnA, as analyzed by the Maximum Entropy Method. The formation of the liquid-ordered beta-phase, in the 30-40 degrees C temperature range as a function of cholesterol concentration (0-40 mol%), could be related quantitatively to the relative amplitude of a long lifetime component of the probe (10-14 ns). Based on this evidence, the phase behavior of mixtures of the unsaturated lipid palmitoyloleoylphosphatidylcholine and cholesterol was determined using the same technique, for cholesterol concentrations in the 0-50 mol% range, between 10 and 40 degrees C. It was found that two liquid-crystalline phases are also formed in this system, with physical properties reminiscent of the alpha- and beta-phases formed with saturated lipids. However, in this case it was determined that, for temperatures in the physiological range, the alpha- and beta-phases coexist up to 40 mol% cholesterol. This finding may be of significant biological relevance, because it supports the long held notion that cholesterol is responsible for the lipid packing heterogeneity of several natural membranes rich in unsaturated lipid components.     

Ligand binding and protein dynamics: a fluorescence depolarization study of aspartate transcarbamylase from Escherichia coli

The polarization of the fluorescence and the real-time fluorescence intensity decay of the two tryptophan residues of aspartate transcarbamylase from Escherichia coli were studied as a function of temperature. The protein was dissolved in an 80% glycerol/buffer mixture, and temperatures were varied between -40 and 20 degrees C in order to limit the depolarization to local rotations of the tryptophans. Two fluorescent species contribute to over 95% of the emission. They differ in their fluorescence lifetimes by approximately 4 ns depending upon the temperature observed and their fractional contributions to the total intensity. The Y-plot analysis of the polarization and lifetime data allows for the distinction of two rotational species by their critical amplitude of rotation, the first being component 1 and the second being component 2. We suggest that these two species correspond to the two tryptophan residues of the protein. The polarization and lifetime experiments were carried out for ATCase in presence of the bisubstrate analogue N-(phosphonoacetyl)-L-aspartate (PALA) and in presence of the nucleotide effector molecules ATP and CTP. The binding of PALA results in an increase in the thermal coefficient of frictional resistance to rotation of tryptophan 1 and a decrease in that of tryptophan 2. ATP binding does not affect the degree to which the protein hinders tryptophan rotation but does result in a change in the critical amplitude of rotation of tryptophan 2. The results obtained in the presence of CTP are similar to those obtained with PALA.     

Pulse fluorimetry study of beef liver glutamate dehydrogenase reduced nicotinamide adenine dinucleotide phosphate complexes.

Single photon counting pulse fluorimetry has been used in order to study the two ternary complexes GDH-GTP-NADPH and GDH-L-glutamate-NADPH and the quaternary complex GDH-GTP-L-glutamate-NADPH. The fluorescence decay of the enzyme-bound NADPH is not monoexponential in any of these complexes. Moreover, it does not seem to be dependent on the coenzyme concentration. The experimental curves can be satisfactorily fitted with the sum of two exponentials, the relative amplitudes of which significantly depend on the complex studied. Thus, for dihydronicotinamide two possible environments might exist in the enzyme active sites. It is also shown that the fluorescence decay times of the enzyme are shortened by the bound NADPH.     

Relationship between the oligomeric status of HIV-1 integrase on DNA and enzymatic activity

The 3'-processing of the extremities of viral DNA is the first of two reactions catalyzed by HIV-1 integrase (IN). High order IN multimers (tetramers) are required for complete integration, but it remains unclear which oligomer is responsible for the 3'-processing reaction. Moreover, IN tends to aggregate, and it is unknown whether the polymerization or aggregation of this enzyme on DNA is detrimental or beneficial for activity. We have developed a fluorescence assay based on anisotropy for monitoring release of the terminal dinucleotide product in real-time. Because the initial anisotropy value obtained after DNA binding and before catalysis depends on the fractional saturation of DNA sites and the size of IN.DNA complexes, this approach can be used to study the relationship between activity and binding/multimerization parameters in the same assay. By increasing the IN:DNA ratio, we found that the anisotropy increased but the 3'-processing activity displayed a characteristic bell-shaped behavior. The anisotropy values obtained in the first phase were predictive of subsequent activity and accounted for the number of complexes. Interestingly, activity peaked and then decreased in the second phase, whereas anisotropy continued to increase. Time-resolved fluorescence anisotropy studies showed that the most competent form for catalysis corresponds to a dimer bound to one viral DNA end, whereas higher order complexes such as aggregates predominate during the second phase when activity drops off. We conclude that a single IN dimer at each extremity of viral DNA molecules is required for 3'-processing, with a dimer of dimers responsible for the subsequent full integration.     

Pressure effects on the physical properties of lipid bilayers detected by trans-parinaric acid fluorescence decay.

The effects of hydrostatic pressure on the physical properties of large unilamellar vesicles of single lipids dipalmitoyl phosphatidylcholine (DPPC) and dimyristoyl phosphatidylcholine (DMPC) and lipid mixtures of DMPC/DPPC have been studied from time-resolved fluorescence of trans-parinaric acid. Additional experiments were carried out using diphenylhexatriene to compare the results extracted from both probes. Fluorescence decays were analyzed by the maximum entropy method. Pressure does not influence the fluorescence lifetime distribution of trans-parinaric acid in isotropic solvents. However, in pressurized lipid bilayers an abrupt change was observed in the lifetime distribution which was associated with the isothermal pressure-induced phase transition. The pressure to temperature equivalence values, dT/dP, determined from the midpoint of the phase transitions, were 24 and 14.5 degrees C kbar-1 for DMPC and POPC, respectively. Relatively moderate pressures of about 500 bar shifted the DMPC/DPPC phase diagram 11.5 degrees C to higher temperatures. The effects of pressure on the structural properties of these lipid vesicles were investigated from the anisotropy decays of both probes. Order parameters for all systems increased with pressure. In the gel phase of POPC the order parameter was smaller than that obtained in the same phase of saturated phospholipids, suggesting that an efficient packing of the POPC hydrocarbon chains is hindered.     

Oligomeric states of the HIV-1 integrase as measured by time-resolved fluorescence anisotropy

Self-assembly properties of HIV-1 integrase were investigated by time-resolved fluorescence anisotropy using tryptophanyl residues as a probe. From simulation analyses, we show that suitable photon counting leads to an accurate determination of long rotational correlation times in the range of 20-80 ns, permitting the distinction of the monomer, dimer, and tetramer from higher oligomeric forms of integrase. The accuracy of correlation times higher than 100 ns is too low to distinguish the octamer from other larger species. The oligomeric states of the widely used detergent-solubilized integrase were then studied in solution under varying parameters known to influence the activity. In the micromolar range, integrase exists as high-order multimers such as an octamer and/or aggregates and a well-defined tetramer, at 25 and 35 degrees C, respectively. However, integrase is monomeric at catalytically active concentrations (in the sub-micromolar range). Detergents (NP-40 and CHAPS) and divalent cation cofactors (Mg(2+) and Mn(2+)) have a clear dissociative effect on the high multimeric forms of integrase. In addition, we observed that Mg(2+) and Mn(2+) have different effects on both the oligomeric state and the conformation of the monomer. This could explain in part why these two metal cations are not equivalent in terms of catalytic activity in vitro. In contrast, addition of Zn(2+) stimulates dimerization. Interestingly, this role of Zn(2+) in the multimerization process was evident only in the presence of Mg(2+) which by itself does not induce oligomerization. Finally, it is highly suggested that the presence of detergent during the purification procedure plays a negative role in the proper self-assembly of integrase. Accordingly, the accompanying paper [Leh, H., et al. (2000) Biochemistry 39, 9285-9294] shows that a detergent-free integrase preparation has self-assembly and catalytic properties different from those of the detergent-solubilized enzyme.     

Comparative Study of the Fatty Acid Binding Process of a New FABP from Cherax quadricarinatus by Fluorescence Intensity,Lifetime and Anisotropy

Fatty acid-binding proteins (FABPs) are small cytosolic proteins, largely distributed in invertebrates and vertebrates, which accomplish uptake and intracellular transport of hydrophobic ligands such as fatty acids. Although long chain fatty acids play multiple crucial roles in cellular functions (structural, energy metabolism, regulation of gene expression), the precise functions of FABPs, especially those of invertebrate species, remain elusive. Here, we have identified and characterized a novel FABP family member, Cq-FABP, from the hepatopancreas of red claw crayfish Cherax quadricarinatus. We report the characterization of fatty acid-binding affinity of Cq-FABP by four different competitive fluorescence-based assays. In the two first approaches, the fluorescent probe 8-Anilino-1-naphthalenesulfonate (ANS), a binder of internal cavities of protein, was used either by directly monitoring its fluorescence emission or by monitoring the fluorescence resonance energy transfer occurring between the single tryptophan residue of Cq-FABP and ANS. The third and the fourth approaches were based on the measurement of the fluorescence emission intensity of the naturally fluorescent cis-parinaric acid probe or the steady-state fluorescence anisotropy measurements of a fluorescently labeled fatty acid (BODIPY-C16), respectively. The four methodologies displayed consistent equilibrium constants for a given fatty acid but were not equivalent in terms of analysis. Indeed, the two first methods were complicated by the existence of non specific binding modes of ANS while BODIPY-C16 and cis-parinaric acid specifically targeted the fatty acid binding site. We found a relationship between the affinity and the length of the carbon chain, with the highest affinity obtained for the shortest fatty acid, suggesting that steric effects primarily influence the interaction of fatty acids in the binding cavity of Cq-FABP. Moreover, our results show that the binding affinities of several fatty acids closely parallel their prevalences in the hepatopancreas of C. quadricarinatus as measured under specific diet conditions.     

The effects of ligands on the conformation of phosphoglycerate kinase: fluorescence anisotropy decay and theoretical interpretation

Horse muscle phosphoglycerate kinase (PGK) is a monomer folded into two widely distant domains. In the glycolytic pathway, this enzyme catalyzes the first reaction that produces ATP. It was suggested, by analogy with yeast hexokinase, that a hinge-bending motion may be induced by the binding of specific substrates to the protein. To analyze such a motion, or any structural changes induced by ligand binding, fluorescence anisotropy decay of tryptophan residues in free and liganded PGK was studied. At 293 K, for the free protein and the binary complex with 3-phosphoglycerate, a single correlation time of 26 ns was observed, corresponding to the rotation of the overall protein, whereas upon addition of MgADP, this correlation time decreased to 10 ns. Such a decrease cannot be merely due to a change of the protein's shape and volume. To explain this, it was suggested that the fluorescence anisotropy decay of the PGK-MgADP complex corresponded to the rotation of the only buried tryptophan (Trp 335). The rotational paths of this tryptophan, in the presence and absence of the nucleotide, were established by potential energy minimization calculations. The results indicated that MgADP induces a displacement of helix alpha-13 that decreases the rotational energy barrier of Trp 335 from 16 kcal/mol in the free protein to 8 kcal/mol in the complex.     

Comparison of the dynamical structures of lipoamide dehydrogenase and glutathione reductase by time-resolved polarized flavin fluorescence.

Time-resolved polarized fluorescence spectroscopy has been applied to the bound FAD in the structurally related flavoproteins lipoamide dehydrogenase from Azotobacter vinelandii (LipDH-AV) and glutathione reductase (GR) from human erythrocytes. The fluorescence parameters as obtained from the maximum entropy analysis differ considerably in both enzymes, reflecting the unique properties of the flavin microenvironment. Three conformational substates are revealed in LipDH-AV and five in GR. Almost 90% of the population of GR molecules has a fluorescence lifetime in the order of 30 ps which originates from efficient exciplex formation with Tyr197. Equilibrium fluctuations between conformational substates are observed for LipDH-AV on a nanosecond time scale in the temperature range 277-313 K. Interconversion between conformational substates in GR is slow, indicating that large activation barriers exist between the states. In agreement with these results, a model is postulated which ascribes a role in catalysis to equilibrium fluctuations between conformational substates in GR and LipDH-AV. From time-resolved fluorescence anisotropy as a function of temperature, distinction can be made between flavin reorientational motion and interflavin energy transfer. In both proteins intersubunit energy transfer between the prosthetic groups is observed. Furthermore, it is revealed that only the flavin in glutathione reductase exhibits rapid restricted reorientational motion. Geometric information concerning the relative orientation and distance of the flavins can be extracted from the parameters describing the energy-transfer process. The obtained spatial arrangement of the flavins is in excellent agreement with crystallographic data.