Transconformations of the SERCA1 Ca-ATPase: a normal mode study

The transport of Ca(2+) by Ca-ATPase across the sarcoplasmic reticulum membrane is accompanied by several transconformations of the protein. Relying on the already established functional importance of low-frequency modes in dynamics of proteins, we report here a normal mode analysis of the Ca(2+)-ATPase based on the crystallographic structures of the E1Ca(2) and E2TG forms. The lowest-frequency modes reveal that the N and A(+Nter) domains undergo the largest amplitude movements. The dynamical domain analysis performed with the DomainFinder program suggests that they behave as rigid bodies, unlike the highly flexible P domain. We highlight two types of movements of the transmembrane helices: i), a concerted movement around an axis perpendicular to the membrane which "twists open" the lumenal side of the protein and ii), an individual translational and rotational mobility which is of lower amplitude for the helices hosting the calcium binding sites. Among all modes calculated for E1Ca, only three are enough to describe the transition to E2TG; the associated movements involve almost exclusively the A and N domains, reflecting the closure of the cytoplasmic headpiece and high displacement of the L7-8 lumenal loop. Subsequently, we discuss the potential contribution of the remaining low-frequency normal modes to the transconformations occurring within the overall calcium transport cycle.     

A stopped-flow fluorescence study of the native and modified lysozyme

The protein folding kinetics of hen egg white lysozyme (HEWL) was studied using experimental and bioinformatics tools. The structure of the transition state in the unfolding pathway of lysozyme was determined with stopped-flow kinetics using intact HEWL and its chemically modified derivative, in which six lysine residues have been modified. The overall consistency of φ-value (φ ≈ 1) indicates that lysine side chains interactions are subject to breaking in the structure of the transition state. Following experimental evidences, multiple sequence alignment of lysozyme family in vertebrates and exact structural examination of lysozyme, showed that the α-helix in the structure of lysozyme has critical role in the unfolding kinetics.     

Steady-state tyrosine fluorescence to study the lipid-binding properties of a wheat non-specific lipid-transfer protein (nsLTP1)

The binding properties of a wheat non-specific lipid-transfer protein (nsLTP1) for different mono- and diacylated lipids was investigated. Lipids varied by their chain length, unsaturation and/or polar head group. In the case of fatty acid or lysophospholipid with a C10 chain length, no interaction can be measured, while poor affinity is reported for a C12 chain length. The dissociation constant (Kd) is about 0.5 microM independent of chain length from C14 to C18. The same affinity is obtained for C18 fatty acids with one or two unsaturations, whatever the cis-trans double bond isomery. In all cases, the number of binding sites, n, by protein ranges between 1.6 and 1.9, suggesting that two lipids can fit within the protein. omega-Hydroxy-palmitic acid, a natural monomer of cutin polymer, is found to interact with nsLTP1 with a Kd of 1 microM and n = 2. In contrast with previous data that reported the binding of the anionic diacylated phospholipid, DMPG (Sodano et al., FEBS Lett. 416 (1997) 130-134), nsLTP1 is not able to bind dimyristoylphosphatidylcholine, dimyristoylphosphatidic acid, palmitoyl-oleoylphosphatidylcholine or palmitoyl-oleoylphosphatidylglycerol added as liposomes or solubilized in ethanol. However, when both nsLTP1 and lipids are first solubilized in methanol, and then in the buffer, it was evidenced that the protein can bind these lipids. These results suggest that lipid-lipid interactions play an essential role in the binding process of plant nsLTP1 as previously mentioned for other lipid-transfer proteins.     

Use of a fluorescence spectroscopy technique to study the adsorption of sodium dodecylsulfonate on liposomes

The fluorescent probe 2-(p-toluidinyl)-naphthalene-6-sodium sulfonate (TNS) was used to study the surface adsorption of sublytic concentrations of the anionic surfactant sodium dodecylsulfonate (C(12)-SO(3)) on phosphatidylcholine (PC) bilayers. The number of adsorbed molecules was quantified by determination of the electrostatic potential (psi(o)) of the bilayers. The abrupt decrease in the fluorescence intensity detected even 10 s after the surfactant addition and the slight fluorescence variations with time indicated that the surfactant adsorption was very fast and almost complete. For a given number of monomers adsorbed a linear dependence between the lipid and C12-SO3 concentrations was obtained, indicating similar adsorption mechanism regardless of the surfactant concentration. Hence, a monomeric adsorption is assumed even in systems with a C12-SO3 concentration above its CMC. In addition, this linear correlation allowed us to determine the surfactant/lipid molar ratios (Re) (inversely related to the C12-SO3 ability to be adsorbed on liposomes) and the bilayer/aqueous phase coefficients (K). The fact that the lowest values for Re were always reached after 10 s of incubation corroborates the rapid kinetics of the process. The decrease in the C12-SO3 partitioning (K) when the number of surfactant molecules exceeded 15000 was possibly due to the electrostatic repulsion between the free and the adsorbed monomers, which could hinder the incorporation of new monomers on the charged surface of liposomes.     

Use of fluorescence resonance energy transfer to analyze oligomerization of G-protein-coupled receptors expressed in yeast

Oligomerization or dimerization of G-protein-coupled receptors (GPCRs) has emerged as an important theme in signal transduction. This concept has recently gained widespread interest due to the application of direct and noninvasive biophysical techniques such as fluorescence resonance energy transfer (FRET), which have shown unequivocally that several types of GPCR can form dimers or oligomers in living cells. Current challenges are to determine which GPCRs can self-associate and/or interact with other GPCRs, to define the molecular principles that govern these specific interactions, and to establish which aspects of GPCR function require oligomerization. Although these questions ultimately must be addressed by using GPCRs expressed endogenously in their native cell types, analysis of GPCR oligomerization in heterologous expression systems will be useful to survey which GPCRs can interact, to conduct structure-function studies, and to identify peptides or small molecules that disrupt GPCR oligomerization and function. Here, we describe methods employing scanning fluorometry to detect FRET between GPCRs tagged with enhanced cyan and yellow fluorescent proteins (CFP and YFP) in living yeast cells. This approach provides a powerful means to analyze oligomerization of a variety of GPCRs that can be expressed in yeast, such as adrenergic, adenosine, C5a, muscarinic acetylcholine, vasopressin, opioid, and somatostatin receptors.     

Estimation of tyrosine-40-DNA distance in the filamentous phage Pf1 by analysis of its intrinsic fluorescence properties

Iodination of the exposed Tyr-25 in the coat protein decreases the fluorescence intensity of the filamentous phage Pf1 to less than 3% of its original fluorescence. If one assumes that the total residual fluorescence originates from the non-iodinated, buried Tyr-40, one can estimate the distance between Tyr-40 and the DNA bases in Pf1 to be less than 7 A, making use of the Foerster law for fluorescence energy transfer. The result is consistent with the idea that Tyr-40-DNA interaction is responsible for the unusually large axial base separation in Pf1-DNA.     

RNA and DNA binding properties of HIV-1 Vif protein: a fluorescence study

The HIV-1 viral infectivity factor (Vif) is a small basic protein essential for viral fitness and pathogenicity. Some "non-permissive" cell lines cannot sustain replication of Vif(-) HIV-1 virions. In these cells, Vif counteracts the natural antiretroviral activity of the DNA-editing enzymes APOBEC3G/3F. Moreover, Vif is packaged into viral particles through a strong interaction with genomic RNA in viral nucleoprotein complexes. To gain insights into determinants of this binding process, we performed the first characterization of Vif/nucleic acid interactions using Vif intrinsic fluorescence. We determined the affinity of Vif for RNA fragments corresponding to various regions of the HIV-1 genome. Our results demonstrated preferential and moderately cooperative binding for RNAs corresponding to the 5'-untranslated region of HIV-1 (5'-untranslated region) and gag (cooperativity parameter omega approximately 65-80, and K(d) = 45-55 nM). In addition, fluorescence spectroscopy allowed us to point out the TAR apical loop and a short region in gag as primary strong affinity binding sites (K(d) = 9.5-14 nM). Interestingly, beside its RNA binding properties, the Vif protein can also bind the corresponding DNA oligonucleotides and their complementary counterparts with an affinity similar to the one observed for the RNA sequences, while other DNA sequences displayed reduced affinity. Taken together, our results suggest that Vif binding to RNA and DNA offers several non-exclusive ways to counteract APOBEC3G/3F factors, in addition to the well documented Vif-induced degradation by the proteasome and to the Vif-mediated repression of translation of these antiviral factors.     

Use of fluorescence resonance energy transfer to study serpin-proteinase interactions

One of the important questions in the serpin mechanism of inhibition of serine and cysteine proteinases of different specificities and structural classes is whether a common "crushing" mechanism of proteinase inactivation is used in all cases. This mechanism was seen in an X-ray structure of the complex between alpha(1)-proteinase inhibitor and trypsin and required the full insertion of the reactive center loop into beta-sheet A and translocation of the proteinase from one pole of the serpin to the other. However, it has yet to be shown to be general for serine proteinases of structural classes other than the trypsin-fold or for cysteine proteinases with the papain-fold or for the caspases. Fluorescence resonance energy transfer offers a potential means of obtaining an answer to this question for each of these classes, without the concern for the effect that increasing size has on the observed signal that applies to NMR spectroscopy. However, care must be taken to ensure that measurements made represent sufficient overdetermination that the answer obtained is unambiguous.     

Use of fluorescence flow cytometry to study the binding of various ligands to platelets

Fluorescence flow cytometry can be used to analyze the binding of different ligands to platelets. However careful choice of volume gates is essential in selecting the population of platelets for analysis. The use of fluorescein isothiocyanate conjugated to staphylococcal protein A or F(ab')2 fragments of immunoglobulin G anti-immunoglobulin offers no advantage in sensitivity or specificity in fluorescence studies of platelets and prefixation of washed platelets with paraformaldehyde has no effect on nonspecific fluorescence. The application of this technology to platelets facilitates quantitation of fluorescence intensity and may yield additional useful information.     

The dissociation properties of native C1

The first component of human complement (C1) readily dissociates under physiologic conditions into two subunits - C1q and C1r₂C1s₂. The equilibrium constant for this reaction has been determined for native C1 in fresh normal human serum by hemolytic titration. Standard technology was modified to simulate physiologic conditions. Furthermore, assays were carried out at numerous poncentrations of sensitized erythrocytes, thereby allowing the calculation of the percent of associated C1 at different total C1 concentration. Increased C1 dissociation was observed with dilution. From these data, an association constant of 4.5 × 10⁸ M^?1 was calculated for native C1. Thus in normal human serum approximately ten percent of the C1 is present as free C1q and C1r₂C1s₂.     

Use of virus-like particles as a native membrane model to study the interaction of insulin with the insulin receptor

There is emerging evidence of the utility of virus-like particles (VLPs) as a novel model for the study of receptor-ligand interactions in a native plasma membrane environment. VLPs consist of a viral core protein encapsulated by portions of the cell membrane with membrane proteins and receptors expressed in their native conformation. VLPs can be generated in mammalian cells by transfection with the retroviral core protein (gag). In this study, we used Chinese hamster ovary (CHO T10) cells stably overexpressing the insulin receptor (IR) to generate IR bearing VLPs. The diameter and size uniformity of VLPs were estimated by dynamic light scattering and morphological features examined by scanning electron microscopy. The presence of high affinity IR on VLPs was demonstrated by competitive binding assays (K_D: 2.3 ± 0.4 nM, n = 3), which was similar to that on the parental CHO T10 cells (K_D: 2.1 ± 0.4 nM, n = 3). We also report that increases or decreases in membrane cholesterol content by treatment with methyl-β-cyclodextrin (MBCD) or cholesterol pre-loaded methyl-β-cyclodextrin (cMBCD), respectively, substantially decreased insulin binding (> 30%) to both VLPs and cells, and we speculate this is due to a change in receptor disposition. We suggest that this novel finding of decreases in insulin binding in response to changes in membrane cholesterol content may largely account for the unexplained decreases in insulin signalling events previously reported elsewhere. Finally, we propose VLPs as a viable membrane model for the study of insulin-IR interactions in a native membrane environment.     

The intrinsic fluorescence of the recombinant human leukocyte interferon-alpha A and fibroblast interferon-beta 1

The environment of Trp residues of the recombinant human interferons has been studied by the analysis of the emission spectra of native and denatured proteins at pH 1.5-8.5 and temperature 10-65 degrees C in the presence and absence of the anionic, cationic and neutral to charge contact quenchers--KI, CsCl and acrylamide, respectively. The obtained data allow to suppose that in IFN-alpha A and IFN-beta 1 Trp141 interacts with Arg145 and one or several from the following residues--Phe124, Ile127, Tyr130, Leu131, whereas Trp77--with Arg33 and Phe36, Phe78, Leu81 or Leu82 (Ile81 or Val82 for IFN-beta 1).     

Interdomain fluorescence resonance energy transfer in SERCA probed by cyan-fluorescent protein fused to the actuator domain

We have used a biosynthetically incorporated fluorescent probe to monitor domain movements involved in ion transport by the sarcoendoplasmic reticulum Ca-ATPase (SERCA) from rabbit fast-twitch skeletal muscle. X-ray crystal structures suggest that the nucleotide-binding (N) and actuator (A) domains of SERCA move apart by several nanometers upon Ca binding. To test this hypothesis, cDNA constructs were created to fuse cyan-fluorescent protein (CFP) to the N terminus of SERCA (A domain). This CFP-SERCA fluorescent fusion protein retained activity when expressed in Sf21 insect cells using the baculovirus system. Fluorescence resonance energy transfer (FRET) was used to monitor the A-N interdomain distance for CFP-SERCA selectively labeled with fluorescein isothiocyanate (FITC) at Lys 515 in the N domain. At low [Ca (2+)] (E2 biochemical state), the measured FRET efficiency between CFP (donor in A domain) and FITC (acceptor in N domain) was 0.34 +/- 0.03, indicating a mean distance of 61.6 +/- 2.0 A between probes on the two domains. An increase of [Ca (2+)] to 0.1 mM (E1-Ca biochemical state) decreased the FRET efficiency by 0.06 +/- 0.03, indicating an increase in the mean distance by 3.0 +/- 1.2 A. Quantitative molecular modeling of dual-labeled SERCA, including an accurate calculation of the orientation factor, shows that the FRET data observed in the absence of Ca is consistent with the E2 crystal structure, but the increase in distance (decrease in FRET) induced by Ca is much less than predicted by the E1 crystal structure. We conclude that the E1 crystal structure does not reflect the predominant structure of SERCA under physiological conditions in a functional membrane bilayer.     

Use of fluorescence enhancement technique to study bilirubin–albumin interaction

Bilirubin–albumin solution gave an emission spectrum in the wavelength range 500–600 nm with emission maxima at 528 nm when excited at 487 nm. The magnitude of fluorescence intensity increased on increasing bilirubin/albumin molar ratio. At three different albumin concentrations, namely, 1.0, 2.5 and 10.0 μM, there was an initial linear increase in fluorescence up to a molar ratio 1.0 in all cases beyond which it sloped off or decreased. This fluorescence enhancement was used to calculate the binding parameters of bilirubin–albumin interaction and the value of binding constant was found to be 1.72×10⁷ l/mol similar to the published values obtained with other methods. Different serum albumins, namely, human (HSA), goat (GSA), pig (PSA) and dog serum albumins (DSA) bound bilirubin with almost the same affinity when studied by the technique of fluorescence enhancement. Bilirubin–albumin interaction was also studied at different pH and ionic strengths. There was a decrease in bilirubin–albumin complex formation on either decreasing the pH from 9.0 to 7.0 or increasing the ionic strength from 0.15 to 1.0. These results suggest that the technique of fluorescence enhancement can be used successfully to study the bilirubin–albumin interaction.     

Accessibilities of the sulfhydryl groups of native and photooxidized lens crystallins: a fluorescence lifetime and quenching study

Fluorescence lifetime and acrylamide quenching studies on the N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-IAEDANS)-labeled sulfhydryl groups of bovine lens alpha-, beta H-, and gamma-crystallins were carried out to characterize the microenvironment of the sulfhydryls and changes produced by singlet oxygen mediated photooxidation. For the untreated proteins, the lifetimes of the major decay component of the fluorescence-labeled crystallins were 15.2, 14.4, and 13.0 ns, and the quenching rate constant, kq, values were 16.6 x 10(7), 26.9 x 10(7), and 32.7 x 10(7) M-1 s-1 for alpha-, beta H-, and gamma-crystallins, respectively. The results indicate that as the polarity of the sulfhydryl site increased (i.e., its lifetime decreased), its accessibility to collisional quenching by acrylamide also increased. The minor decay component of the fluorescence label was not significantly quenched by acrylamide for all three classes of crystallins. When the proteins were irradiated in the presence of methylene blue, in a system generating singlet oxygen, the kq value for acrylamide quenching of the major decay component of alpha-crystallin decreased to zero, while its lifetime decreased to 6 ns. Neither the lifetime nor the kq of alpha-crystallin recovered completely in the presence of the singlet oxygen quencher sodium azide. Light-induced binding of the photosensitizer methylene blue to the crystallins was observed by absorption spectroscopy. The bound photosensitizer partially quenches the fluorescence lifetime of the N-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (AEDANS) label in irradiated alpha-crystallin. Further decrease in the lifetime occurs as a result of the singlet oxygen mediated conformational change. The results suggest that the fluorescence lifetime of the AEDANS is fully quenched in the irradiated alpha-crystallin and there is no further quenching by acrylamide. An increase in the fraction of the minor component of beta H-crystallin which was inaccessible to acrylamide quenching was observed after irradiation. There was no effect of irradiation on the kq for acrylamide quenching of the major component of the decay of AEDANS bound to beta H- or gamma-crystallins. Static quenching was found to contribute significantly to the steady-state quenching plots of the polar sulfhydryl sites of irradiated alpha-crystallin and of untreated and irradiated beta H- and gamma-crystallins, but it had no detectable role in the case of untreated alpha-crystallin. Fluorescence anisotropy of the AEDANS label bound to the crystallins was higher in the irradiated crystallins as compared with the controls.     

Structural changes in the myelin sheath membranes exposed to anisotonic media. A fluorescence polarization study

Extrinsic fluorescence polarization (P parallel and P perpendicular) of intact myelinated nerve fibres in anisotonic media has been investigated. Fluorescent probes of Acridine orange and Perylen were used for the experiment. Changes in fluorescent polarization of nerve fibres, associated with osmotic movement of water, arise immediately after the incubation of fibres in hypo- or hypertonic solutions. During exosmotic and endosmotic movement of water P parallel and P perpendicular and in quite a different manner. This fact demonstrates an asymmetry of structural membrane alterations. The results obtained are explained by asymmetry of swelling and shrinking of carbohydrate gel between two adjacent extracellular membrane surfaces of the myelin sheath.