ATP-dependent conformational dynamics in a photoactivated adenylate cyclase revealed by fluorescence spectroscopy and small-angle X-ray scattering.

Structural insights into the photoactivated adenylate cyclases can be used to develop new ways of controlling cellular cyclic adenosine monophosphate (cAMP) levels for optogenetic and other applications. In this work, we use an integrative approach that combines biophysical and structural biology methods to provide insight on the interaction of adenosine triphosphate (ATP) with the dark-adapted state of the photoactivated adenylate cyclase from the cyanobacterium Oscillatoria acuminata (OaPAC). A moderate affinity of the nucleotide for the enzyme was calculated and the thermodynamic parameters of the interaction have been obtained. Stopped-flow fluorescence spectroscopy and small-angle solution scattering have revealed significant conformational changes in the enzyme, presumably in the adenylate cyclase (AC) domain during the allosteric mechanism of ATP binding to OaPAC with small and large-scale movements observed to the best of our knowledge for the first time in the enzyme in solution upon ATP binding. These results are in line with previously reported drastic conformational changes taking place in several class III AC domains upon nucleotide binding.

In vivo direct interaction of the antibiotic primycin on a Candida albicans clinical isolate and its ergosterol-less mutant.

Interaction of primycin antibiotic with plasma membrane, and its indirect biological effects were investigated in this study. The antifungal activity of primycin against 13 human pathogenic Candida ATCC and CBS reference species and 74 other Candida albicans clinical isolates was investigated with a microdilution technique. No primycin-resistant strain was detected. Direct interaction of primycin with the plasma membrane was demonstrated for the first time by using an ergosterol-producing strain 33erg+ and its ergosterol-less mutant erg-2. In growth inhibition tests, the 33erg+ strain proved to be more sensitive to primycin than its erg-2 mutant, indicating the importance of the plasma membrane composition in primycin-induced processes. The 64 µg ml-1 (56.8 nM) primycin treatment induced an enhanced membrane fluidity and altered plasma membrane dynamics, as measured by steady-state fluorescence anisotropy applying a trimethylammonium-diphenylhexatriene (TMA-DPH) fluorescence polarization probe. The following consequences were detected. The plasma membrane of the cells lost its barrier function, and the efflux of 260-nm-absorbing materials from treated cells of both strains was 1.5-1.8 times more than that for the control. Depending on the primycin concentration, the cells exhibited unipolar budding, pseudohyphae formation, and a rough cell surface visualized by scanning electron microscopy.

Screening of common Plantago species in Hungary for bioactive molecules and antioxidant activity.

Five species of Plantago genus, namely P. lanceolata, P. major, P. media, P. altissima and P. maritima were screened for iridoid content (CE-MEKC), total caffeoyl phenylethanoid glycoside (CPG) content and antioxidant activity (CUPRAC assay). The five species could be distinguished by TLC pattern analysis in a single run in a system commonly used for quality management of P. lanceolata leaves, as shown by cluster analysis of major bands; with the exception, that P. altissima and P. lanceolata did not show enough pattern difference to be fully separated. P. maritima was shown to have the highest antioxidant capacity (0.42 µmol ascorbic acid equivalent (AAE)/g DW), and the highest level of CPGs (4.29%). P. altissima was shown to be chemically indistinguishable from P. lanceolata with repsect to iridoid content (aucubin 0.55 ± 0.04%, 0.68 ± 0.23%, catalpol 0.66 ± 0.13% and 0.89 ± 0.22%, respectively), CPG content (2.40 ± 0.38% and 2.54 ± 0.56%, respectively) and antioxidant capacity (0.2206 ± 0.0290 and 0.2428 ± 0.0191 µmol AAEAC/g DW). The presented data show the potency of medicinal use of Hungarian wild populations of the studied five species, especially in the case of P. maritima, and that P. altissima can be a potential replacement of P. lanceolata in herbal mixtures.

Protein flexibility as revealed by fluorescence resonance energy transfer: an extension of the method for systems with multiple labels.

The temperature profile of the normalized fluorescence resonance energy transfer efficiency is capable of monitoring the relative change of flexibility and/or conformational state of macromolecules [Biochemistry 23 (1984) 3403]. The method described earlier for one donor-one acceptor systems is extended to multiple fluorophore systems when the energy transfer occurs between either one donor-m acceptors, or n donors-one acceptor or n donors-m acceptors (where n and m are integer values). It is shown that the normalized energy transfer efficiency obtained for systems containing multiple labels is a linear combination of the normalized transfer efficiency assigned to individual donor-acceptor pairs of the system, thus its temperature profile is capable of monitoring the change of intramolecular flexibility and/or conformational state.

Fluorescence quenching of the tryptophan emission from the F- and G-forms of actin.

The parameters characterizing the quenching of fluorescence emitted by the four tryptophans of actin were measured by time resolved techniques in the monomeric (G) and the polymerized (F) forms of the protein. Acrylamide as a neutral and cesium ions as positively charged quenchers were used to characterize the subdomain 1, which contains all the four tryptophan residues. Use of acrylamide did not reveal any difference between the G- and F-forms, cesium, however, did so. The value of the quenching rate constant, k+, is significantly higher for the F-form than that for the G-form. This difference is present independently of the model (discrete or continuous lifetime distribution) used to process the data. These results are compatible with the conclusion that the charge distribution of the microenvironment around at least one of the four tryptophans is changed. This means that this region becomes more attracted to the cesium ion as a result of transition from the G- to the F-form.

EFFECT OF PHALLOIDIN ON FILAMENTS POLYMERIZED FROM HEART MUSCLE ADP-ACTIN MONOMERS.

The effect of phalloidin on filaments polymerized from ADP-actin monomers of the heart muscle was investigated with differential scanning calorimetry. Heart muscle contains alpha-skeletal and alpha-cardiac actin isoforms. In the absence of phalloidin the melting temperature was 55 degrees C for the alpha-cardiac actin isoform and 58 degrees C for the alpha-skeletal one when the filaments were generated from ADP-actin monomers. After the binding of phalloidin the melting temperature was isoform independent (85.5 degrees C). We concluded that phalloidin stabilized the actin filaments of alpha-skeletal and alpha-cardiac actin isoforms to the same extent when they were polymerized from ADP-actin monomers.

Effect of Ca2+-Mg2+ exchange on the flexibility and/or conformation of the small domain in monomeric actin.

A fluorescence resonance energy transfer (FRET) parameter, f' (defined as the average transfer efficiency, (E), normalized by the actual fluorescence intensity of the donor in the presence of acceptor, F(DA)), was previously shown to be capable of monitoring both changes in local flexibility of the protein matrix and major conformational transitions. The temperature profile of this parameter was used to detect the change of the protein flexibility in the small domain of the actin monomer (G-actin) upon the replacement of Ca2+ by Mg2+. The Cys-374 residue of the actin monomer was labeled with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS) to introduce a fluorescence donor and the Lys-61 residue with fluorescein-5-isothiocyanate (FITC) to serve as an acceptor. The f' increases with increasing temperature over the whole temperature range for Mg-G-actin. This parameter increases similarly in the case of Ca-G-actin up to 26 degrees C, whereas an opposite tendency appears above this temperature. These data indicate that there is a conformational change in Ca-G-actin above 26 degrees C that was not detected in the case of Mg-G-actin. In the temperature range between 6 degrees C and 26 degrees C the slope of the temperature profile of f' is the same for Ca-G-actin and Mg-G-actin, suggesting that the flexibility of the protein matrix between the two labels is identical in the two forms of actin.

The influence of divalent cations on the dynamic properties of actin filaments: a spectroscopic study.

The principal aim of this investigation was to study the change of the protein flexibility and/or conformational properties of actin filaments upon the replacement of Ca2+ by Mg2+. The temperature dependence of the fluorescence lifetime and the anisotropy decay of N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS) attached covalently to the Cys374 residue of actin were measured. Saturation transfer electron paramagnetic resonance (ST-EPR) experiments were also carried out using N-(1-oxyl-2,2,6, 6-tetramethyl-4-piperidinyl)-maleimide (MSL) attached to the same residue (Cys374). The Arrhenius analysis of the temperature dependence of the fluorescence lifetimes shows that for Mg-F-actin, both the activation energy (E*) and the frequency factor (A) are smaller than they are for Ca-F-actin. The longer rotational correlation times resolved in the fluorescence experiments are larger in the Mg2+-loaded form of the actin filament between 6 degreesC and 28 degreesC, but this difference becomes negligible above 28 degreesC. The results of saturation transfer electron paramagnetic resonance measurements on maleimide spin-labeled actin filaments indicate that the replacement of Ca2+ by Mg2+ induced a decrease of the mobility of the label on the sub-millisecond time scale. Based upon these results, we concluded that the filaments polymerized from Ca-actin are more flexible than the filaments of Mg-actin.

Spectroscopic study of conformational changes in subdomain 1 of G-actin: influence of divalent cations.

Temperature dependence of the fluorescence intensity and anisotropy decay of N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine attached to Cys374 of actin monomer was investigated to characterize conformational differences between Ca- and Mg-G-actin. The fluorescence lifetime is longer in Mg-G-actin than that in Ca-G-actin in the temperature range of 5-34 degrees C. The width of the lifetime distribution is smaller by 30% in Mg-saturated actin monomer at 5 degrees C, and the difference becomes negligible above 30 degrees C. The semiangle of the cone within which the fluorophore can rotate is larger in Ca-G-actin at all temperatures. Electron paramagnetic resonance measurements on maleimide spin-labeled (on Cys374) monomer actin gave evidence that exchange of Ca2+ for Mg2+ induced a rapid decrease in the mobility of the label immediately after the addition of Mg2+. These results suggest that the C-terminal region of the monomer becomes more rigid as a result of the replacement of Ca2+ by Mg2+. The change can be related to the difference between the polymerization abilities of the two forms of G-actin.

The flexibility of actin filaments as revealed by fluorescence resonance energy transfer. The influence of divalent cations.

The temperature profile of the fluorescence resonance energy transfer efficiency normalized by the fluorescence quantum yield of the donor in the presence of acceptor, f', was measured in a way allowing the independent investigation of (i) the strength of interaction between the adjacent protomers (intermonomer flexibility) and (ii) the flexibility of the protein matrix within actin protomers (intramonomer flexibility). In both cases the relative increase as a function of temperature in f' is larger in calcium-F-actin than in magnesium-F-actin in the range of 5-40 degrees C, which indicates that both the intramonomer and the intermonomer flexibility of the actin filaments are larger in calcium-F-actin than those in magnesium-F-actin. The intermonomer flexibility was proved to be larger than the intramonomer one in both the calcium-F-actin and the magnesium-F-actin. The distance between Gln41 and Cys374 residues was found to be cation-independent and did not change during polymerization at 21 degrees C. The steady-state fluorescence anisotropy data of fluorophores attached to the Gln41 or Cys374 residues suggest that the microenvironments around these regions are more rigid in the magnesium-loaded actin filament than in the calcium-loaded form.

Conformational distributions and proximity relationships in the rigor complex of actin and myosin subfragment-1.

Cyclic conformational changes in the myosin head are considered essential for muscle contraction. We hereby show that the extension of the fluorescence resonance energy transfer method described originally by Taylor et al. (Taylor, D. L., Reidler, J., Spudich, J. A., and Stryer, L. (1981) J. Cell Biol. 89, 362-367) allows determination of the position of a labeled point outside the actin filament in supramolecular complexes and also characterization of the conformational heterogeneity of an actin-binding protein while considering donor-acceptor distance distributions. Using this method we analyzed proximity relationships between two labeled points of S1 and the actin filament in the acto-S1 rigor complex. The donor (N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonate) was attached to either the catalytic domain (Cys-707) or the essential light chain (Cys-177) of S1, whereas the acceptor (5-(iodoacetamido)fluorescein) was attached to the actin filament (Cys-374). In contrast to the narrow positional distribution (assumed as being Gaussian) of Cys-707 (5 +/- 3 A), the positional distribution of Cys-177 was found to be broad (102 +/- 4 A). Such a broad positional distribution of the label on the essential light chain of S1 may be important in accommodating the helically arranged acto-myosin binding relative to the filament axis.

Conformational and dynamic differences between actin filaments polymerized from ATP- or ADP-actin monomers.

Conformational and dynamic properties of actin filaments polymerized from ATP- or ADP-actin monomers were compared by using fluorescence spectroscopic methods. The fluorescence intensity of IAEDANS attached to the Cys(374) residue of actin was smaller in filaments from ADP-actin than in filaments from ATP-actin monomers, which reflected a nucleotide-induced conformational difference in subdomain 1 of the monomer. Radial coordinate calculations revealed that this conformational difference did not modify the distance of Cys(374) from the longitudinal filament axis. Temperature-dependent fluorescence resonance energy transfer measurements between donor and acceptor molecules on Cys(374) of neighboring actin protomers revealed that the inter-monomer flexibility of filaments assembled from ADP-actin monomers were substantially greater than the one of filaments from ATP-actin monomers. Flexibility was reduced by phalloidin in both types of filaments.

Ionic interactions play a role in the regulatory mechanism of scallop heavy meromyosin.

Heavy meromyosin from scallop (scHMM) striated muscle is regulated by calcium binding to the essential light chain. The regulation can be modeled with a calcium-dependent equilibrium between on and off scHMM conformations. The observed rate constant for mant-ADP dissociation from scHMM is calcium dependent, and we show here that it can be used to define the equilibrium constant (K(eq)) between on and off conformations. The data show that K(eq) is markedly ionic strength dependent, with high salt (>/=200 mM) abolishing the off state even in the absence of calcium and low salt (<50 mM) favoring the off state even in the presence of calcium. Debye-Huckel plots of the equilibrium constant (K(eq)) for the on and off forms gave parallel slopes (5.94 +/- 0.33 and 6.36 +/- 0.17 M(-0.5)) in the presence and absence of calcium. The presence of an equilibrium mixture of two conformations was confirmed by sedimentation data and the effects of ADP, calcium and ionic strength were in qualitative agreement. Thus scHMM exists in two conformations that can be distinguished in sedimentation profiles and by the rate of release of mant-ADP. Increasing salt concentrations biases the system toward the on state, suggesting a role for ionic interactions in stabilizing the off state.

Flexibility of myosin-subfragment-1 in its complex with actin as revealed by fluorescence resonance energy transfer.

The flexibility of the acto-myosin complex in rigor conditions was characterized by measuring the temperature profile of normalized fluorescence resonance energy transfer efficiency, f' [Somogyi, B., Matkó, J., Papp, S., Hevessy, J., Welch, G.R. & Damjanovich, S. (1984) Biochemistry 23, 3403-3411]. Fluorescence acceptors were introduced to the Cys374 residues of actin and the donors were covalently attached either to Cys707 in the catalytic domain or to Cys177 in the essential light-chain of myosin S1. Fluorescence resonance energy transfer measurements revealed that the protein matrix between Cys374 of actin and Cys707 of S1 is rigid. In contrast, the link between the catalytic and light-chain-binding domains in myosin S1 is flexible. We have recently shown that the positional distribution of Cys707 was narrow relative to the actin filament, while that of the Cys177 was broad. Accordingly, the broad positional distribution of Cys177 is likely to be due to the large flexibility of the link between the catalytic and light-chain-binding domains. This flexibility is probably essential for the interdomain reorganization of the myosin head during the force generation process and for accommodating the symmetry difference between actin and myosin filaments to allow the formation of cross-bridges.

9-Anthroylnitrile binding to serine-181 in myosin subfragment 1 as revealed by FRET spectroscopy and molecular modeling.

It has been shown that one of the 12 serine residues within the 23 kDa segment of myosin subfragment 1 can be covalently modified with a fluorescent probe 9-anthroylnitrile (ANN) [Hiratsuka, T. (1989) J. Biol. Chem. 264 (30), 18188-18194]. To identify the exact binding site of the probe, the distances between the bound ANN as donor and acceptors in known positions (Lys-553 or Cys-707) of the myosin head were determined by using fluorescence resonance energy transfer. Comparison of the spectroscopic results with distances obtained from the atomic model of subfragment 1 revealed that ANN binds to Ser-181. The result was in good agreement with the assumptions of Andreev and co-workers [Andreev, O. A., et al. (1995) J. Muscle Res. Cell Motil. 16 (4), 353-367]. This conclusion was further supported by protein modeling calculations. The results presented herein might bring ANN into the focus when the molecular mechanism and effects of the binding of ATP and its subsequent hydrolysis are studied.

Replacement of ATP with ADP affects the dynamic and conformational properties of actin monomer.

The effect of the replacement of ATP with ADP on the conformational and dynamic properties of the actin monomer was investigated, by means of electron paramagnetic resonance (EPR) and fluorescence spectroscopic methods. The measurement of the ATP concentration during these experiments provided the opportunity to estimate the time dependence of ADP-Mg-G-actin concentration in the samples. According to the results of the fluorescence resonance energy transfer experiments, the Gln-41 and Cys-374 residues are closer to each other in the ADP-Mg-G-actin than in the ATP-Mg-G-actin. The fluorescence resonance energy transfer efficiency increased simultaneously with the ADP-G-actin concentration and reached its maximum value within 30 min at 20 degrees C. The EPR data indicate the presence of an ADP-Mg-G-actin population that can be characterized by an increased rotational correlation time, which is similar to the one observed in actin filaments, and exists only transiently. We suggest that the conformational transitions, which were reflected by our EPR data, were coupled with the transient appearance of short actin oligomers during the nucleotide exchange. Besides these relatively fast conformational changes, there is a slower conformational transition that could be detected several hours after the initiation of the nucleotide exchange.