Examination of lipid-bound conformation of apolipoprotein E4 by pyrene excimer fluorescence

Apolipoprotein E (apoE) is a 34-kDa resident of lipoproteins that plays a key role in cholesterol homeostasis in plasma and in brain. It is composed of an N-terminal (NT) domain (residues 1-191) and a C-terminal (CT) domain (residues 201-299). Of the three major isoforms (apoE2, -E3, and -E4), apoE4 is considered a risk factor for both cardiovascular and Alzheimer disease. Compared with apoE3, domain interaction between NT and CT domains is believed to direct the lipoprotein distribution preference of apoE4 for very low density lipoprotein-sized particles. We examined the relative disposition of apoE4 NT and CT domains in lipid-free and lipid-bound forms by monitoring pyrene excimer fluorescence emission as a direct indicator of spatial proximity. Site-specific labeling of apoE4 by N-(1-pyrene)maleimide was accomplished after substitution of Cys residues for Arg-61 in NT domain and Glu-255 in CT domain. Pyrene labeling did not alter the lipoprotein distribution pattern of apoE4 in plasma. Pyrene excimer fluorescence was noted in lipid-free pyrene-R61C/E255C/apoE4 in mixtures containing excess wild-type apoE4, which was attributed to intramolecular spatial proximity between these specified sites. Upon disruption of tertiary interaction, a large decrease in excimer fluorescence emission was noted in pyrene-R61C/E255C/apoE4. In dimyristoylphosphatidylcholine/pyrene-R61C/E255C/apoE4 discoidal complexes, pyrene excimer fluorescence emission was retained. Taken together with fluorescence quenching and cross-linking analysis, a looped-back model of apoE4 is proposed in lipid-bound state, including spherical lipoprotein particles, wherein residues Arg-61 and Glu-255 are proximal to one another.     

Pressure dependence of pyrene excimer fluorescence in human erythrocyte membranes

The intensity of pyrene excimer fluorescence in human erythrocyte membranes and in sonicated dispersions of the membrane lipid (liposomes) was examined as a function of pressure (1–2080 bar) and temperature (5–40°C). Higher pressure or lower temperature decreased the excimer/monomer intensity ratios. A thermotropic transition was detected in both membranes and liposomes by plots of the logarithm of the excimer/monomer intensity ratio versus 1/K. The transition temperature of the membranes was 19–21°C at 1 bar and 28–31°C at 450 bar, a shift with pressure of approx. 20–22 K per kbar. Corresponding transition temperatures of the liposomes were 21°C at 1 bar and 33°C at 450 bar, a shift of approx. 27 K per kbar. The observed pressure dependence of the thermotropic transition temperature is similar to that reported for phospholipid bilayers and greatly exceeds that of protein conformation changes. In concert with the liposome studies the results provide direct evidence for a lipid transition in the erythrocyte membrane.     

Conformational changes of troponin C within the thin filaments detected by neutron scattering

Regulation of skeletal and cardiac muscle contraction is associated with structural changes of the thin filament-based proteins, troponin consisting of three subunits (TnC, TnI, and TnT), tropomyosin, and actin, triggered by Ca2+-binding to TnC. Knowledge of in situ structures of these proteins is indispensable for elucidating the molecular mechanism of this Ca2+-sensitive regulation. Here, the in situ structure of TnC within the thin filaments was investigated with neutron scattering, combined with selective deuteration and the contrast matching technique. Deuterated TnC (dTnC) was first prepared, this dTnC was then reconstituted into the native thin filaments, and finally neutron scattering patterns of these reconstituted thin filaments containing dTnC were measured under the condition where non-deuterated components were rendered "invisible" to neutrons. The obtained scattering curves arising only from dTnC showed distinct difference in the absence and presence of Ca2+. These curves were analyzed by model calculations using the Monte Carlo method, in which inter-dTnC interference was explicitly taken into consideration. The model calculation showed that in situ radius of gyration of TnC was 23 A (99% confidence limits between 22 A and 23 A) and 24 A (99% confidence limits between 23 A and 25 A) in the absence and presence of Ca2+, respectively, indicating that TnC within the thin filaments assumes a conformation consistent with the extended dumbbell structure, which is different from the structures found in the crystals of various Tn complexes. Elongation of TnC by binding of Ca2+ was also suggested. Furthermore, the radial position of TnC within the thin filament was estimated to be 53 A (99% confidence limits between 49 A and 57 A) and 49 A (99% confidence limits between 44 A and 53 A) in the absence and presence of Ca2+, respectively, suggesting that this radial movement of TnC by 4A is associated with large conformational changes of the entire Tn molecule by binding of Ca2+.     

Environment-induced changes in DNA conformation as probed by ethidium bromide fluorescence

The interaction of the ethidium cation with calf thymus DNA is investigated in solutions of different ionic strength and temperature by observation of the enhancement of fluorescence of ethidium upon intercalation in the duplex structure. The quantum yield of the fluorescence of the intercalated dye is found to increase either upon lowering the Na+ concentration or upon increasing the temperature. The existence of a correlation between the geometry of the intercalation complex and the features of the secondary structure of DNA is suggested. Binding isotherms under corresponding environmental conditions are also quantitated by fluorescence enhancement and interpreted in terms of the neighbor exclusion model. Large contributions from change in hydration to the thermodynamics of binding are demonstrated by the temperature dependences of the equilibrium constants. The neighbor exclusion range is found to be practically independent of the salt concentration but its value increases from an average of 2.4 around room temperature to 4-5 at 80 degrees C, as inferred from the binding curves in 0.15 and 0.5 M [Na+] or from the DNA hypochromism vs temperature profiles of complexes at 10(-3) M [Na+]. All the data point to a possible sequence-conformation specificity in the intercalation of ethidium which in heterogeneous DNA is mediated by environmental changes.     

Tumor promoters induce membrane changes detected by fluorescence polarization.

The probe, 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to determine if tumor promoting agents alter cell membranes. The active tumor promoters TPA (12-0-tetra-decanoyl-phorbol-13-acetate), PDD (phorbol-12,13-didecanoate) and PDB (phorbol-12,13-dibenzoate) were found to decrease fluorescence polarization of DPH in rat embryo cells, whereas the inactive tumor promoting compounds phorbol and 4α-PDD failed to induce this change.     

Development of peptide substrates for trypsin based on monomer/excimer fluorescence of pyrene

An assay using fluorogenic peptides based on the monomer/excimer fluorescence features of pyrene was developed to measure the proteolytic activity of trypsin, a serine protease. Two pyrene moieties were incorporated into the respective N- and C-terminus of the peptides as (pyrene)-C-Xaa-C-(pyrene), where Xaa represents amino acid residues of 5-, 6-, 7-, or 8-mer containing the cleavage site of trypsin. The proteolytic cleavage of the substrates led to an increase in monomer fluorescence and a decrease in excimer fluorescence of pyrene. Kinetic parameters (k(cat) and K(m)) for the enzymatic hydrolysis of the substrates were successfully determined. The parameters are dependent on the chain length of the substrate and optimal catalytic activity was obtained with substrates that consisted of 9 or 10 amino acid residues. The present assay system is sensitive and the preparation of the substrate is very simple. We suggest that this method may be suitable for high-throughput screening and also applicable to the characterization of other proteases.     

Calcium-induced flexibility changes in the troponin C-troponin I complex

The contraction of vertebrate striated muscle is modulated by Ca(2+) binding to the regulatory protein troponin C (TnC). Ca(2+) binding causes conformational changes in TnC which alter its interaction with the inhibitory protein troponin I (TnI), initiating the regulatory process. We have used the frequency domain method of fluorescence resonance energy transfer (FRET) to measure distances and distance distributions between specific sites in the TnC-TnI complex in the presence and absence of Ca(2+) or Mg(2+). Using sequences based on rabbit skeletal muscle proteins, we prepared functional, binary complexes of wild-type TnC and a TnI mutant which contains no Cys residues and a single Trp residue at position 106 within the TnI inhibitory region. We used TnI Trp-106 as the FRET donor, and we introduced energy acceptor groups into TnC by labeling at Met-25 with dansyl aziridine or at Cys-98 with N-(iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine. Our distance distribution measurements indicate that the TnC-TnI complex is relatively rigid in the absence of Ca(2+), but becomes much more flexible when Ca(2+) binds to regulatory sites in TnC. This increased flexibility may be propagated to the whole thin filament, helping to release the inhibition of actomyosin ATPase activity and allowing the muscle to contract. This is the first report of distance distributions between TnC and TnI in their binary complex.     

Proximity relationships between residue 6 of troponin I and residues in troponin C: further evidence for extended conformation of troponin C in the troponin complex

Skeletal muscle troponin C (TnC) adopts an extended conformation when crystallized alone and a compact one when crystallized with an N-terminal troponin I (TnI) peptide, TnI(1-47) [Vassylyev et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 4847-4852]. The N-terminal region of TnI (residues 1-40) was suggested to play a functional role of facilitating the movement of TnI's inhibitory region between TnC and actin [Tripet et al. (1997) J. Mol. Biol. 271, 728-750]. To test this hypothesis and to investigate the conformation of TnC in the intact troponin complex and in solution, we attached fluorescence and photo-cross-linking probes to a mutant TnI with a single cysteine at residue 6. Distances from this residue to residues of TnC were measured by the fluorescence resonance energy transfer technique, and the sites of photo-cross-linking in TnC were determined by microsequencing and mass spectrometry following enzymatic digestions. Our results show that in the troponin complex neither the distance between TnI residue 6 and TnC residue 89 nor the photo-cross-linking site in TnC, Ser133, changes with Ca(2+), in support of the notion that this region plays mainly a structural rather than a regulatory role. The distances to residues 12 and 41 in TnC's N-domain are both considerably longer than those predicted by the crystal structure of TnC.TnI(1-47), supporting an extended rather than a compact conformation of TnC. In the binary TnC.TnI complex and the presence of Ca(2+), Met43 in TnC's N-domain was identified as the photo-cross-linking site, and multiple distances between TnI residue 6 and TnC residue 41 were detected. This was taken to indicate increased flexibility in TnC's central helix and that TnC dynamically changes between a compact and an extended conformation when troponin T (TnT) is absent. Our results further emphasize the difference between the binary TnC.TnI and the ternary troponin complexes and the importance of using intact proteins in the study of structure-function relationships of troponin.     

Conformational changes induced in troponin I by interaction with troponin T and actin/tropomyosin.

Troponin I (TnI) is the inhibitory component of the striated muscle Ca2+ regulatory protein troponin (Tn). The other two components of Tn are troponin C (TnC), the Ca2+-binding component, and troponin T (TnT), the tropomyosin-binding component. We have used limited chymotryptic digestion to probe the local conformation of TnI in the free state, the binary TnC*TnI complex, the ternary TnC*. TnI*TnT (Tn) complex, and in the reconstituted Tn*tropomyosin*F-actin filament. The digestion of TnI alone or in the TnC*TnI complex produced initially two major fragments via a cleavage of the peptide bond between Phe100 and Asp101 in the so-called inhibitory region. In the ternary Tn complex cleavage occurred at a new site between Leu140 and Lys141. In the absence of Ca2+ this was followed by digestion of the 1-140 fragment at Leu122 and Met116. In the reconstituted thin filament the same fragments as in the case of the ternary complex were produced, but the rate of digestion was slower in the absence than in the presence of Ca2+. These results indicate firstly that in both free TnI and TnI complexed with TnC there is an exposed and flexible site in the inhibitory region. Secondly, TnT affects the conformation of TnI in the inhibitory region and also in the region that contains the 140-141 bond. Thirdly, the 140-141 region of TnI is likely to interact with actin in the reconstituted thin filament when Ca2+ is absent. These findings are discussed in terms of the role of TnI in the mechanism of thin filament regulation, and in light of our previous results [Y. Luo, J.-L. Wu, J. Gergely, T. Tao, Biochemistry 36 (1997) 13449-13454] on the global conformation of TnI.     

Glucose induces membrane changes detected by fluorescence polarization in endocrine pancreatic cells

Pancreatic endocrine cells prepared from rat islets were labelled with 1,6-diphenyl-1,3,5-hexatriene and examined in a microviscosimeter. Glucose caused a dose-related decrease in fluorescence polarization. This decrease was observed within 1 min after increasing the concentration of glucose. These findings suggest that glucose affects membrane viscosity in pancreatic endocrine cells. At a glucose concentration of 16.7 mM, the estimated viscosity was 15 ± 3 per cent lower than basal value (2.01 ± 0.12 P).     

Modulation of cardiac troponin C-cardiac troponin I regulatory interactions by the amino-terminus of cardiac troponin I

Multidimensional heteronuclear magnetic resonance studies of the cardiac troponin C/troponin I(1-80)/troponin I(129-166) complex demonstrated that cardiac troponin I(129-166), corresponding to the adjacent inhibitory and regulatory regions, interacts with and induces an opening of the cardiac troponin C regulatory domain. Chemical shift perturbation mapping and (15)N transverse relaxation rates for intact cardiac troponin C bound to either cardiac troponin I(1-80)/troponin I(129-166) or troponin I(1-167) suggested that troponin I residues 81-128 do not interact strongly with troponin C but likely serve to modulate the interaction of troponin I(129-166) with the cardiac troponin C regulatory domain. Chemical shift perturbations due to troponin I(129-166) binding the cardiac troponin C/troponin I(1-80) complex correlate with partial opening of the cardiac troponin C regulatory domain previously demonstrated by distance measurements using fluorescence methodologies. Fluorescence emission from cardiac troponin C(F20W/N51C)(AEDANS) complexed to cardiac troponin I(1-80) was used to monitor binding of cardiac troponin I(129-166) to the regulatory domain of cardiac troponin C. The apparent K(d) for cardiac troponin I(129-166) binding to cardiac troponin C/troponin I(1-80) was 43.3 +/- 3.2 microM. After bisphosphorylation of cardiac troponin I(1-80) the apparent K(d) increased to 59.1 +/- 1.3 microM. Thus, phosphorylation of the cardiac-specific N-terminus of troponin I reduces the apparent binding affinity of the regulatory domain of cardiac troponin C for cardiac troponin I(129-166) and provides further evidence for beta-adrenergic modulation of troponin Ca(2+) sensitivity through a direct interaction between the cardiac-specific amino-terminus of troponin I and the cardiac troponin C regulatory domain.     

Calcium-induced changes in the location and conformation of troponin in skeletal muscle thin filaments.

Troponin is the regulatory protein of striated muscle. Without Ca2+, the contraction of striated muscle is inhibited. Binding of Ca2+ to troponin activates contraction. The location of troponin on the thin filaments and its relation to the regulatory mechanism has been unknown, though the Ca2+-induced dislocation of tropomyosin has been studied. By binding troponin(C+I) to actin in an almost stoichiometric ratio and reconstituting actin-tropomyosin-troponin(C+I) filaments, we reconstructed the three-dimensional structure of actin-tropomyosin-troponin(C+I) with or without Ca2+ from electron cryomicrographs to about 2.5 or 3 nm resolution, respectively. Without Ca2+, the three-dimensional map reveals the extra-density region due to troponin(C+I), which extends perpendicularly to the helix axis and covers the N-terminal and C-terminal regions of actin. In the presence of Ca2+, the C-terminal region of actin became more exposed, and troponin(C+I) became V-shaped with one arm extending towards the pointed end of the actin filament. This structure can be considered to show the location of troponin(C+I) in at least one of the states of skeletal muscle thin filaments. These Ca2+-induced changes of troponin(C+I) provide a clue to the regulatory mechanism of contraction.     

Ca(2+) induces an extended conformation of the inhibitory region of troponin I in cardiac muscle troponin.

The inhibitory region of troponin I (TnI) plays a central regulatory role in the contraction and relaxation cycle of skeletal and cardiac muscle through its Ca(2+)-dependent interaction with actin. Detailed structural information on the interface between TnC and this region of TnI has been long in dispute. We have used fluorescence resonance energy transfer (FRET) to investigate the global conformation of the inhibitory region of a full-length TnI mutant from cardiac muscle (cTnI) in the unbound state and in reconstituted complexes with the other cardiac troponin subunits. The mutant contained a single tryptophan residue at the position 129 which was used as an energy transfer donor, and a single cysteine residue at the position 152 labeled with IAEDANS as energy acceptor. The sequence between Trp129 and Cys152 in cTnI brackets the inhibitory region (residues 130-149), and the distance between the two sites was found to be 19.4 A in free cTnI. This distance was insensitive to reconstitution of cTnI with cardiac troponin T (cTnT), cTnC, or cTnC and cTnT in the absence of bound regulatory Ca(2+) in cTnC. An increase of 9 A in the Trp129-Cys152 separation was observed upon saturation of the Ca(2+) regulatory site of cTnC in the complexes. This large increase suggests an extended conformation of the inhibitory region in the interface between cTnC and cTnI in holo cardiac troponin. This extended conformation is different from a recent model of the Ca(2+)-saturated skeletal TnI-TnC complex in which the inhibitory region is modeled as a beta-turn. The observed Ca(2+)-induced conformational change may be a switch mechanism by which movement of the regulatory region of cTnI to the exposed hydrophobic patch of the open regulatory N-domain of cTnC pulls the inhibitory region away from actin upon Ca(2+) activation in cardiac muscle.     

Solution conformation of the C-terminal domain of skeletal troponin C. Cation, trifluoperazine and troponin I binding effects

Proton magnetic resonance spectroscopy has been used to study the cation (Mg2+, Ca2+)-dependent conformational states of the C-terminal domain of rabbit skeletal troponin C under a variety of solution conditions. Nuclear Overhauser data and paramagnetic probe observations provide definition of the configuration of this region of troponin C. Comparative study of homologous proteins identify common features of the tertiary structure relevant to the cation binding reaction. Complex formation with troponin I and the drug trifluoperazine is observed to adjust the solution conformation of the C-terminal domain of troponin C. The interactive conformational response to cation coordination and the binding of the drug and troponin I are discussed.     

Cooperative binding of myosin subfragment one to regulated actin as measured by fluorescence changes of troponin I modified with different fluorophores

Binding studies of myosin subfragment one (S-1) to regulated actin in the presence and absence of Ca2+ indicate that, as S-1 binds to regulated actin, tropomyosin-actin units undergo a cooperative transition from a weak to a strong S-1-binding form. Trybus and Taylor (Trybus, K.M., and Taylor, E.W. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 7209-7213) suggested that this transition could be measured by the change in fluorescence of troponin I modified with 4-(N-iodoacetoxyethyl-N-methyl)-7-nitrobenz-2-oxa-1,3-diazole (IANBD). In the present study, this was tested by determining whether the change in fluorescence was proportional to the fraction of tropomyosin-actin units in the strong S-1-binding form as predicted by our model on the cooperative binding of S-1 to regulated actin (Hill, T.L., Eisenberg, E., and Greene, L.E. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 3186-3190). Experiments were performed both in the presence and absence of Ca2+ by using troponin I modified with either IANBD or 5'-iodoacetamidofluorescein. In the presence of Ca2+, it was found, in agreement with the suggestion of Trybus and Taylor, that the change in fluorescence induced by S-1 was proportional to the fraction of tropomyosin-actin units shifting into the strong S-1 binding form, rather than to the fraction of actin sites having bound S-1. In the absence of Ca2+, the change in fluorescence induced by S-1 also did not reflect the binding of S-1 to regulated actin. However, in contrast to the results in the presence of Ca2+, the change in fluorescence induced by S-1 binding in the absence of Ca2+ was not in agreement with the fraction of tropomyosin-actin units calculated to be in the strong S-1 binding form by the model of Hill et al. Although a more complex model than that of Hill et al. may account for the observed fluorescence changes, it seems equally likely that at least in the absence of Ca2+, the change in fluorescence may be reflecting a more complex behavior than only the transition of tropomyosin-actin units between the weak and strong S-1-binding forms.     

Irreversible changes in barley leaf chlorophyll fluorescence detected by the fluorescence temperature curve in a linear heating/cooling regime

The chlorophyll fluorescence (F) temperature curves in a linear time-temperature heating/cooling regime were used to study heat-induced irreversible F changes in primary green leaves of spring barley (Hordeum vulgare L. cv. Akcent). The leaf segments were heated in a stirred water bath at heating rates of 0.0083, 0.0166, 0.0333, and 0.0500 °C s⁻¹ from room temperature up to maximal temperature T _m and then linearly cooled to 35 °C at the same rate. The F intensity was measured by a pulse-modulated technique. The results support the existence of the two critical temperatures of irreversible F changes postulated earlier, at 45–48 and 53–55 °C. The critical temperatures are slightly dependent on the heating rate. Two types of parameters were used to characterize the irreversibility of the F changes: the coefficient of irreversibility μ defined as the ratio of F intensity at 35 °C at the starting/ending parts of the cycle and the slopes of tangents of linear parts of the F temperature curve. The dependence of μ on T _m revealed a maximum, which moved from 54 to 61 °C with the increasing heating/cooling rate v from 0.0083 to 0.0500 °C s⁻¹, showing two basic phases of the irreversible changes. The Arrhenius and Eyring approaches were applied to calculate the activation energies of the initial increase in μ. The values varied between 30 and 50 kJ mol⁻¹ and decreased slightly with the increasing heating rate.     

Solvent-induced changes in photochemical activity and conformation of photosystem I particles by glycerol

It has been shown that a large number of water molecules coordinate with the pigments and subunits of photosystem I (PSI); however, the function of these water molecules remains to be clarified. In this study, the photosynthetic properties of PSI from spinach were investigated using different spectroscopic and activity measurements under conditions of decreasing water content caused by increasing concentrations of glycerol. The results show that glycerol addition caused pronounced changes in the photochemical activity of PSI particles. At low concentrations (<60%, v/v), glycerol stimulated the rate of oxygen uptake in PSI particles, while higher concentrations of glycerol cause inhibition of PSI activity. The capacity of P700 photooxidation also increased with glycerol concentrations lower than 60%. In contrast, this capacity decreased at higher glycerol concentrations. On the other hand, glycerol addition considerably affected the distribution of the bulk and red antenna chlorophyll (Chl) forms or states, with the population of red-shifted Chl forms augmented with increasing glycerol. In addition, glycerol-treated PSI particles showed a blue shift of the tryptophan fluorescence emission maximum and an increase in their capacity to bind the hydrophobic probe 1-anilino-8-naphthalene sulfonate, indicating a more non-polar environment for tryptophan residues and increased exposure of hydrophobic surfaces.     

Developmental changes in the structural organization of the lectin discoidin I detected by limited proteolysis

Digestion of discoidin I with several proteolytic enzymes reveals the existence of structural domains in this lectin. Significative differences have been detected in the pattern of fragments generated by V8 protease on discoidin I of various developmental situations. The changes observed can be related to the presence of various types of tetrameric structures in discoidin I. Together with the presence of different types of isoforms in vegetative vs. differentiated cells, the results presented here suggest the involvement of different structural organizations in discoidin I which can be related to the biological functions of this lectin.