The myristoylation of the neuronal Ca2+ -sensors guanylate cyclase-activating protein 1 and 2

Guanylate cyclase-activating proteins (GCAPs) are Ca(2+)-binding proteins with a fatty acid (mainly myristic acid) that is covalently attached at the N terminus. Myristoylated forms of GCAP were produced in E. coli by coexpression of yeast N-myristoyl-transferase. Proteins with nearly 100% degree of myristoylation were obtained after chromatography on a reversed phase column. Although proteins were denatured by this step, they could be successfully refolded. Nonmyristoylated GCAPs activated bovine photoreceptor guanylate cyclase 1 less efficiently than the myristoylated forms. Maximal activity of guanylate cyclase at low Ca(2+)-concentration decreased about twofold, when GCAPs lacked myristoylation. In addition, the x-fold activation of cyclase was lower with nonmyristoylated GCAPs. Myristoylation of GCAP-2 had no influence on the apparent affinity for photoreceptor guanylate cyclase 1, but GCAP-1 has an about sevenfold higher affinity for cyclase, when it is myristoylated. We conclude that myristoylation of GCAP-1 and GCAP-2 is important for fine tuning of guanylate cyclase activity.     

Internal genomic sequence of human CYP1A1 gene is involved in superinduction of dioxin-induced CYP1A1 transcription by cycloheximide

Guanylate cyclase-activating protein 2 (GCAP2) is expressed in vertebrate photoreceptors cells where it regulates the activity of membrane bound guanylate cyclases in a Ca(2+)-dependent manner. The essential trigger step involves a Ca(2+)-induced conformational change in GCAP2. We investigated these Ca(2+)-dependent changes by probing the cysteine accessibility in wild type and mutant GCAP2 forms with the thiol-modifying reagent 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB). Cysteine residues in position 35 and 111 displayed a restricted accessibility in the presence of Ca(2+), whereas cysteine in position 131 reacted with DTNB in the presence and absence of Ca(2+). Our data indicate that the Ca(2+)-sensitivity of GCAP2 is significantly controlled by its third Ca(2+)-binding site, EF-hand 3.     

Differential Ca(2+) sensor guanylate cyclase activating protein modes of photoreceptor rod outer segment membrane guanylate cyclase signaling

Photoreceptor ROS-GC1 (rod outer segment membrane guanylate cyclase) is a vital component of phototransduction. It is a bimodal Ca(2+) signal transduction switch, operating between 20 and ～1000 nM. Modulated by Ca(2+) sensors guanylate cyclase activating proteins 1 and 2 (GCAP1 and GCAP2, respectively), decreasing [Ca(2+)](i) from 200 to 20 nM progressively turns it "on", as does the modulation by the Ca(2+) sensor S100B, increasing [Ca(2+)](i) from 100 to 1000 nM. The GCAP mode plays a vital role in phototransduction in both rods and cones and the S100B mode in the transmission of neural signals to cone ON-bipolar cells. Through a programmed domain deletion, expression, in vivo fluorescence spectroscopy, and in vitro reconstitution experiments, this study demonstrates that the biochemical mechanisms modulated by two GCAPs in Ca(2+) signaling of ROS-GC1 activity are totally different. (1) They involve different structural domains of ROS-GC1. (2) Their signal migratory pathways are opposite: GCAP1 downstream and GCAP2 upstream. (3) Importantly, the isolated catalytic domain, translating the GCAP-modulated Ca(2+) signal into the generation of cyclic GMP, in vivo, exists as a homodimer, the two subunits existing in an antiparallel conformation. Furthermore, the findings demonstrate that the N-terminally placed signaling helix domain is not required for the catalytic domain's dimeric state. The upstream GCAP2-modulated pathway is the first of its kind to be observed for any member of the membrane guanylate cyclase family. It defines a new model of Ca(2+) signal transduction.     

Opposite effects of Ca(2+) and GTP binding on tissue transglutaminase tertiary structure

Tissue transglutaminase (tTG) belongs to a class of enzymes that catalyze a cross-linking reaction between proteins or peptides. The protein activity is known to be finely tuned by Ca(2+) and GTP binding. In this study we report the effects of these ligands on the enzyme structure, as revealed by circular dichroism, and steady-state and dynamic fluorescence measurements. We have found that calcium and GTP induced opposite conformational changes at the level of the protein tertiary structure. In particular the metal ions were responsible for a small widening of the protein molecule, as indicated by anisotropy decay measurements and by the binding of a hydrophobic probe such as 1-anilino-8-naphthalenesulfonic acid (ANS). Unlike Ca(2+), the nucleotide binding increased the protein dynamics, reducing its rotational correlation lifetime from 32 to 25 ns, preventing also the binding of ANS into the protein matrix. Unfolding of tTG by guanidinium hydrochloride yielded a three-state denaturation mechanism, involving an intermediate species with the characteristics of the so-called "molten globule" state. The effect of GTP binding (but not that of Ca(2+)) had an important consequence on the stability of tissue transglutaminase, increasing the free energy change from the native to the intermediate species by at least approximately 0.7 kcal/mol. Also a greater stability of tTG to high hydrostatic pressure was obtained in presence of GTP. These findings suggest that the molecular mechanism by which tTG activity is inhibited by GTP is essentially due to a protein conformational change which, decreasing the accessibility of the protein matrix to the solvent, renders more difficult the exposure of the active site.     

Regulatory modes of rod outer segment membrane guanylate cyclase differ in catalytic efficiency and Ca(2+)-sensitivity.

In rod phototransduction, cyclic GMP synthesis by membrane bound guanylate cyclase ROS-GC1 is under Ca(2+)-dependent negative feedback control mediated by guanylate cyclase-activating proteins, GCAP-1 and GCAP-2. The cellular concentration of GCAP-1 and GCAP-2 approximately sums to the cellular concentration of a functional ROS-GC1 dimer. Both GCAPs increase the catalytic efficiency (kcat/Km) of ROS-GC1. However, the presence of a myristoyl group in GCAP-1 has a strong impact on the regulation of ROS-GC1, this is in contrast to GCAP-2. Catalytic efficiency of ROS-GC1 increases 25-fold when it is reconstituted with myristoylated GCAP-1, but only by a factor of 3.4 with nonmyristoylated GCAP-1. In contrast to GCAP1, myristoylation of GCAP-2 has only a minor effect on kcat/Km. The increase with both myristoylated and nonmyristoylated GCAP-2 is 10 to 13-fold. GCAPs also confer different Ca(2+)-sensitivities to ROS-GC1. Activation of the cyclase by GCAP-1 is half-maximal at 707 nM free [Ca(2+)], while that by GCAP-2 is at 100 nM. The findings show that differences in catalytic efficiency and Ca(2+)-sensitivity of ROS-GC1 are conferred by GCAP-1 and GCAP-2. The results further indicate the concerted operation of two 'GCAP modes' that would extend the dynamic range of cyclase regulation within the physiological range of free cytoplasmic Ca(2+) in photoreceptor cells.     

Conformational changes in the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum detected using phosphorescence polarization

The technique of time-averaged phosphorescence has been used to study the interaction of calcium ions and ATP with the (Ca²⁺ + Mg²⁺)-ATPase in sarcoplasmic reticulum vesicles. The presence of excess calcium ions was found to cause a 20% decrease in the phosphorescence emission anisotropy. This is interpreted as being due to a conformational change in the protein and is supported by data from time-resolved phosphorescence measurements which also show a lowering of the anisotropy. This change in the decay of the emission anisotropy is associated with only minor changes in the rotational relaxation time of the protein and is again suggestive of a conformational change in the protein. In some cases ATP was also observed to lower the time-averaged phosphorescence anisotropy possibly via an interaction with the low-affinity regulatory site of the protein.     

Ca(2+)-binding proteins in the retina: from discovery to etiology of human disease(1)

Examination of the role of Ca(2+)-binding proteins (CaBPs) in mammalian retinal neurons has yielded new insights into the function of these proteins in normal and pathological states. In the last 8 years, studies on guanylate cyclase (GC) regulation by three GC-activating proteins (GCAP1-3) led to several breakthroughs, among them the recent biochemical analysis of GCAP1(Y99) mutants associated with autosomal dominant cone dystrophy. Perturbation of Ca(2+) homeostasis controlled by mutant GCAP1 in photoreceptor cells may result ultimately in degeneration of these cells. Here, detailed analysis of biochemical properties of GCAP1(P50L), which causes a milder form of autosomal dominant cone dystrophy than constitutive active Y99C mutation, showed that the P50L mutation resulted in a decrease of Ca(2+)-binding, without changes in the GC activity profile of the mutant GCAP1. In contrast to this biochemically well-defined regulatory mechanism that involves GCAPs, understanding of other processes in the retina that are regulated by Ca(2+) is at a rudimentary stage. Recently, we have identified five homologous genes encoding CaBPs that are expressed in the mammalian retina. Several members of this subfamily are also present in other tissues. In contrast to GCAPs, the function of this subfamily of calmodulin (CaM)-like CaBPs is poorly understood. CaBPs are closely related to CaM and in biochemical assays CaBPs substitute for CaM in stimulation of CaM-dependent kinase II, and calcineurin, a protein phosphatase. These results suggest that CaM-like CaBPs have evolved into diverse subfamilies that control fundamental processes in cells where they are expressed.     

The homogeneous fluorescence anisotropic sensing of salivary lysozyme using the 6-carboxyfluorescein-labeled DNA aptamer

A simple and sensitive fluorescence anisotropy method was developed for lysozyme, employing the coupling of fluorophore, 6-carboxyfluorescein (FAM), with lysozyme upon recognition between the target molecule and its DNA aptamer. It was found in this study that the rotational dynamic of the detecting system is crucial to obtain a high anisotropy signal that cannot always be achieved by simply increasing the molecular volume, because molecular volume increase may not be able to efficiently retard the rotational movement of the fluorophore. FAM was selected as the label of the ssDNA aptamer to effectively facilitate the change of the fluorophore from a primarily independent segmental movement to slow global rotation. The time-resolved measurements, including lifetime and dynamic fluorescence anisotropy, were conducted to study the recognition interaction and to better understand the methodology. The proposed method had a wide linear dynamic range of 12.5-300 nM and a high sensitivity with the limit of detection of 4.9 nM (3S/N). This proposed method was successfully applied to assay of human salivary lysozyme. The results based on the standard addition recovery and comparison with enzyme-linked immunosorbent assay (ELISA) demonstrated the feasibility of this method for biological samples. Using coupling between the fluorophore and the analyte can be one of the approaches working toward expanding the application of fluorescence anisotropy based on aptamer-target and antibody-antigen recognitions.     

Modulation by Ca(2+) and by membrane binding of the dynamics of domain III of annexin 2 (p36) and the annexin 2-p11 complex (p90): implications for their biochemical properties

The modulation of the local structure and dynamics of domain III of annexin 2 (Anx2), in both the monomeric (p36) and heterotetrameric forms (p90), by calcium and by membrane binding was studied by time-resolved fluorescence intensity and anisotropy measurements of the single tryptophan residue (W212). The results yield the same dominant excited-state lifetime (1.4 ns) in both p36 and p90, suggesting that the conformation and environment of W212 are very similar. The fluorescence anisotropy decay data were analyzed by associative (two-dimensional) as well as nonassociative (one-dimensional) models. Although no statistical criterion is decisive for one model versus the other, only the associative model allows recovery of a physically relevant value of the Brownian rotational correlation of the protein. Using the associative model, a nanosecond flexibility is detectable in p90 but not in p36. When Ca(2+) binds in the millimolar concentration range to both forms of Anx2, a conformational change takes place leading to an increase of the major excited-state lifetime (2.6 ns) and to a suppression of the W212 local flexibility of p90. Binding to membranes of either p36 or p90 in the presence of Ca(2+) does not induce any conformational change other than that provoked by Ca(2+) binding alone. The W212 local flexibility in both proteins increases significantly, however, in their membrane-bound forms. In the presence of membranes, the conformation change of domain III in p90 displays a sensitivity to Ca(2+) 2 orders of magnitude higher than that of p36, reaching intracellular sub-micromolar concentration ranges. This higher Ca(2+) sensitivity correlates with the Ca(2+)-dependent membrane aggregation but not with their Ca(2+)-dependent binding to membranes. The significance of these structural and dynamical changes for the function of the protein is discussed.     

The myristoylation of guanylate cyclase-activating protein-2 causes an increase in thermodynamic stability in the presence but not in the absence of Ca²⁺

Guanylate cyclase activating protein‐2 (GCAP‐2) is a Ca²⁺‐binding protein of the neuronal calcium sensor (NCS) family. Ca²⁺‐free GCAP‐2 activates the retinal rod outer segment guanylate cyclases ROS‐GC1 and 2. Native GCAP‐2 is N‐terminally myristoylated. Detailed structural information on the Ca²⁺‐dependent conformational switch of GCAP‐2 is missing so far, as no atomic resolution structures of the Ca²⁺‐free state have been determined. The role of the myristoyl moiety remains poorly understood. Available functional data is incompatible with a Ca²⁺‐myristoyl switch as observed in the prototype NCS protein, recoverin. For the homologous GCAP‐1, a Ca²⁺‐independent sequestration of the myristoyl moiety inside the proteins structure has been proposed. In this article, we compare the thermodynamic stabilities of myristoylated and non‐myristoylated GCAP‐2 in their Ca²⁺‐bound and Ca²⁺‐free forms, respectively, to gain information on the nature of the Ca²⁺‐dependent conformational switch of the protein and shed some light on the role of its myristoyl group. In the absence of Ca²⁺, the stability of the myristoylated and non‐myristoylated forms was indistinguishable. Ca²⁺ exerted a stabilizing effect on both forms of the protein, which was significantly stronger for myr GCAP‐2. The stability data were corroborated by dye binding experiments performed to probe the solvent‐accessible hydrophobic surface of the protein. Our results strongly suggest that the myristoyl moiety is permanently solvent‐exposed in Ca²⁺‐free GCAP‐2, whereas it interacts with a hydrophobic part of the protein's structure in the Ca²⁺‐bound state.     

Time-resolved fluorescence microscopy.

In fluorescence microscopy, the fluorescence emission can be characterised not only by intensity and position, but also by lifetime, polarization and wavelength. Fluorescence lifetime imaging (FLIM) can report on photophysical events that are difficult or impossible to observe by fluorescence intensity imaging, and time-resolved fluorescence anisotropy imaging (TR-FAIM) can measure the rotational mobility of a fluorophore in its environment. We compare different FLIM methods: a chief advantage of wide-field time-gating and phase modulation methods is the speed of acquisition whereas for time-correlated single photon counting (TCSPC) based confocal scanning it is accuracy in the fluorescence decay. FLIM has been used to image interactions between proteins such as receptor oligomerisation and to reveal protein phosphorylation by detecting fluorescence resonance energy transfer (FRET). In addition, FLIM can also probe the local environment of fluorophores, reporting, for example, on the local pH, refractive index, ion or oxygen concentration without the need for ratiometric measurements.     

Spectroscopic characterization of 2-amino-N-hexadecyl-benzamide (AHBA), a new fluorescence probe for membranes

We report the results of investigation on the spectroscopic properties of a new fluorescent lipophylic probe. The fluorophore o-aminobenzoic acid was covalently bound to the acyl chain hexadecylamine, producing the compound 2-amino-N-hexadecyl-benzamide. The behavior of the probe was dependent on the polarity of the medium: absorption and emission spectral position, quantum yield and lifetime decay indicate distinct behavior in water compared to ethanol and cyclohexane. The probe dissolves in the organic solvents, as indicated by the very low value of steady state fluorescence anisotropy and the short rotational correlation times obtained from fluorescence anisotropy decay measurements. On the other hand, the probe has low solubility in water, leading to the formation of aggregates in aqueous medium. The complex absorption spectrum in water was interpreted as originating from different forms of aggregation, as deduced from the wavelength dependence of anisotropy parameters. The probe interacts with surfactants in pre-micellar and micellar forms, as observed in experiments in the presence of sodium n-dodecylsulphate (SDS), n-cetyltrimethylammonium bromide (CTAB); 3-(dodecyl-dimethylammonium) propane-1-sulphonate (DPS) and 3-(hexadecyl-dimethylammonium) propane-1-sulphonate (HPS), and with vesicles of the phospholipid dimiristoyl-phosphatidylcholine (DMPC). The results demonstrate that AHBA is able to monitor properties like surface electric potential and phase transition of micelles and vesicles.     

Missense mutations affecting Ca2+-coordination in GCAP1 lead to cone-rod dystrophies by altering protein structural and functional properties

Guanylate cyclase activating protein 1 (GCAP1) is a neuronal calcium sensor (NCS) involved in the early biochemical steps underlying the phototransduction cascade. By switching from a Ca²⁺-bound form in the dark to a Mg²⁺-bound state following light activation of the cascade, GCAP1 triggers the activation of the retinal guanylate cyclase (GC), thus replenishing the levels of 3′,5′-cyclic monophosphate (cGMP) necessary to re-open CNG channels. Here, we investigated the structural and functional effects of three missense mutations in GCAP1 associated with cone-rod dystrophy, which severely perturb the homeostasis of cGMP and Ca²⁺. Substitutions affect residues directly involved in Ca²⁺ coordination in either EF3 (D100G) or EF4 (E155A and E155G) Ca²⁺ binding motifs. We found that all GCAP1 variants form relatively stable dimers showing decreased apparent affinity for Ca²⁺ and blocking the enzyme in a constitutively active state at physiological levels of Ca²⁺. Interestingly, by corroborating spectroscopic experiments with molecular dynamics simulations we show that beside local structural effects, mutation of the bidentate glutamate in an EF-hand calcium binding motif can profoundly perturb the flexibility of the adjacent EF-hand as well, ultimately destabilizing the whole domain. Therefore, while Ca²⁺-binding to GCAP1 per se occurs sequentially, allosteric effects may connect EF hand motifs, which appear to be essential for the integrity of the structural switch mechanism in GCAP1, and perhaps in other NCS proteins.     

Effect of primary and secondary structure of oligodeoxyribonucleotides on the fluorescent properties of conjugated dyes

We studied fluorescence intensity, polarization and lifetime of some commonly used fluorophores conjugated to oligodeoxyribonucleotides with different primary and secondary structures. We found that fluorescence intensity can increase or decrease upon hybridization of the labeled strand to its complement depending on the sequence and position of the fluorophore. Up to 10-fold quenching of the fluorescence upon hybridization was observed when the dye moiety was attached close to the 3′ end and the 3′-terminal base was either dG or dC. No quenching upon hybridization was observed when the dye was positioned within the same sequence context but close to the 5′ end. The presence of a dG overhang quenches the fluorescence less efficiently than a blunt end dG-dC or dC-dG base pair. When located internally in the double strand, the dG-dC base pair does not affect the fluorescence of the nearby dye. Guanosine in a single-stranded oligonucleotide quenches the fluorescence of nearby dye by <2-fold. Upon duplex formation, this quenching is eliminated and the fluorescence increases. This increase can only be detected when the fluorophore is located at least 6 nt from the terminal dG-dC base pair. The change of fluorescence polarization upon duplex formation inversely correlates with the change of intensity. Fluorescein conjugated to a single-stranded oligonucleotide or a duplex undergoes a bi-exponential decay with ~4 and ~1 ns lifetimes.     

Molecular targeting of inhibitor of apoptosis proteins based on small molecule mimics of natural binding partners

An assay based on a solvent-sensitive fluorogenic dye molecule, badan, is used to test the binding affinity of a library of tetrapeptide molecules for the BIR3 (baculovirus IAP repeat) domain of XIAP (X-linked inhibitor of apoptosis protein). The fluorophore is attached to a tetrapeptide, Ala-Val-Pro-Cys-NH(2), through a thiol linkage and, upon binding to XIAP, undergoes a solvatochromic shift in fluorescence emission. When a molecule (e.g., a natural protein known to bind to XIAP or a tetrapeptide mimic) displaces the dye, the emission shifts back to the spectrum observed in water. As emission intensity is related to the binding of the tetrapeptide, the intensity can be used to determine the equilibrium constant, K, for the displacement of the dye by the tetrapeptide. The results permit residue-specific analysis of the interaction. Furthermore, we show that hydrophobic effects in the fourth position are general and can effectively increase overall affinity.     

Analysis of cell membrane micro-heterogeneity using the fluorescence lifetime of DPH-type fluorophores.

Heterogeneity in the lipid organization in lipid bilayers and cell membranes was probed by using the fluorescence decay of 1,6-diphenyl-1,3,5-hexatriene (DPH) and DPH attached to the sn-2 position of phosphatidylcholine (DPH-PC). In the presence of protein, it is proposed that the bulk lipids and boundary lipids can potentially provide distinct enough fluorophore environments for two different lifetime centers to be recovered from the analysis of the fluorescence decay. To test this model experiments were performed with cytochrome b5 in 1-palmitoyl-2-oleoylphosphatidylcholine bilayers. The number of boundary lipids of cytochrome b5 is known from the literature or can be calculated from known dimensions, so that for a known protein:lipid ratio the fraction of lipids in the bulk and boundary lipid regions is known. These values were found to closely correspond to the fractions associated with the lifetime centers recovered from an analysis of the fluorescence decay assuming two major fluorophore populations. This indicated that the DPH distributed in a similar manner to the lipids and that its boundary lipid residency time was greater than the excited state lifetime, showing the validity of the approach. An important requirement was that the protein should influence the fluorophore decay sufficiently enough to enable separate lifetime centers for the bulk and boundary lipid fluorophores to be recovered by the analysis. Attempts were made to analyze the fluorescence decay of DPH in liver plasma membranes and microsomes as arising from two distinct fluorophore populations, however, the basic condition was not satisfied. By contrast, using DPH-PC it was possible to extract two separate lifetime centers. The limitations and potential of this approach are critically assessed and it is concluded that in certain circumstances information pertaining to the protein-lipid interfacial region of membranes can be extracted from fluorescence decay heterogeneity properties.     

Green-fluorescent protein from the bioluminescent jellyfish Clytia gregaria is an obligate dimer and does not form a stable complex with the Ca(2+)-discharged photoprotein clytin

Green-fluorescent protein (GFP) is the origin of the green bioluminescence color exhibited by several marine hydrozoans and anthozoans. The mechanism is believed to be Fo?rster resonance energy transfer (FRET) within a luciferase-GFP or photoprotein-GFP complex. As the effect is found in vitro at micromolar concentrations, for FRET to occur this complex must have an affinity in the micromolar range. We present here a fluorescence dynamics investigation of the recombinant bioluminescence proteins from the jellyfish Clytia gregaria, the photoprotein clytin in its Ca(2+)-discharged form that is highly fluorescent (λ(max) = 506 nm) and its GFP (cgreGFP; λ(max) = 500 nm). Ca(2+)-discharged clytin shows a predominant fluorescence lifetime of 5.7 ns, which is assigned to the final emitting state of the bioluminescence reaction product, coelenteramide anion, and a fluorescence anisotropy decay or rotational correlation time of 12 ns (20 °C), consistent with tight binding and rotation with the whole protein. A 34 ns correlation time combined with a translational diffusion constant and molecular brightness from fluorescence fluctuation spectroscopy all confirm that cgreGFP is an obligate dimer down to nanomolar concentrations. Within the dimer, the two chromophores have a coupled excited-state transition yielding fluorescence depolarization via FRET with a transfer correlation time of 0.5 ns. The 34 ns time of cgreGFP showed no change upon addition of a 1000-fold excess of Ca(2+)-discharged clytin, indicating no stable complexation below 0.2 mM. It is proposed that any bioluminescence FRET complex with micromolar affinity must be one formed transiently by the cgreGFP dimer with a short-lived (millisecond) intermediate in the clytin reaction pathway.     

Recognition and Binding of a Helix-Loop-Helix Peptide to Carbonic Anhydrase Occurs via Partly Folded Intermediate Structures

We have studied the association of a helix-loop-helix peptide scaffold carrying a benzenesulfonamide ligand to carbonic anhydrase using steady-state and time-resolved fluorescence spectroscopy. The helix-loop-helix peptide, developed for biosensing applications, is labeled with the fluorescent probe dansyl, which serves as a polarity-sensitive reporter of the binding event. Using maximum entropy analysis of the fluorescence lifetime of dansyl at 1:1 stoichiometry reveals three characteristic fluorescence lifetime groups, interpreted as differently interacting peptide/protein structures. We characterize these peptide/protein complexes as mostly bound but unfolded, bound and partly folded, and strongly bound and folded. Furthermore, analysis of the fluorescence anisotropy decay resulted in three different dansyl rotational correlation times, namely 0.18, 1.2, and 23 ns. Using the amplitudes of these times, we can correlate the lifetime groups with the corresponding fluorescence anisotropy component. The 23-ns rotational correlation time, which appears with the same amplitude as a 17-ns fluorescence lifetime, shows that the dansyl fluorophore follows the rotational diffusion of carbonic anhydrase when it is a part of the folded peptide/protein complex. A partly folded and partly hydrated interfacial structure is manifested in an 8-ns dansyl fluorescence lifetime and a 1.2-ns rotational correlation time. This structure, we believe, is similar to a molten-globule-like interfacial structure, which allows segmental movement and has a higher degree of solvent exposure of dansyl. Indirect excitation of dansyl on the helix-loop-helix peptide through Förster energy transfer from one or several tryptophans in the carbonic anhydrase shows that the helix-loop-helix scaffold binds to a tryptophan-rich domain of the carbonic anhydrase. We conclude that binding of the peptide to carbonic anhydrase involves a transition from a disordered to an ordered structure of the helix-loop-helix scaffold.     

Effect of Adriamycin on the boundary lipid structure of cytochrome c oxidase: pico-second time-resolved fluorescence depolarization studies

The fluorescence dynamics of the dye 3,3'-diethyloxadicarbocyanine iodide (DODCI) was used to probe the microenvironment of cytochrome c oxidase (CcO) and cardiolipin. The dye was partitioned between an aqueous and a hydrophobic phase. The 'bound' and 'free' populations of DODCI could be separated by analysis of the time-resolved fluorescence decay of the dye. The anisotropy decay of the DODCI bound to CcO showed a unique 'dip and rise' shape that was analyzed by a combination of rotational correlation times with time-dependent weight factors for each lifetime component. Rotational dynamics studies revealed the existence of a restricted motion of the dye bound at the enzyme surface. Adriamycin, an anticancer, albeit cardiotoxic drug, was previously proposed to affect the surface structure of CcO, most likely by causing a disorder to the surface lipid arrangement. A drastic change in the rotational correlation time of the dye bound to the enzyme surface was observed, which suggested a depletion of cardiolipin layer due to complexation with the drug. The effect of Adriamycin on cardiolipin was drastic, leading to its phase separation. The present study suggests that the effect of Adriamycin on CcO is primarily a segregation of the cardiolipins.