Bibliometric analysis of the brazilian periodical journal of biochemistry education.

The bibliometric analysis of the Brazilian periodical Journal of Biochemistry Education (JBE) covered the 117 articles published in 15 volumes in the period 2001-2017. Our results showed a positive trend in JBE publications with a significant increase in the number of articles since 2014, which can be related to the increase in research groups working in this area. The Southeast region of Brazil was the most productive one mainly due to the contribution of papers from institutions located in the State of São Paulo. Only four articles aimed the undergraduate courses (87.0%) showing methodological approaches to teach biochemistry (51.3%) and laboratory exercises (18.8%) among others. Most of the 332 authors contributed to a single article (87.7%) and just 3% of them published more than twice in JBE. The majority of the JBE articles had at least one citation in Google Scholar. There is also a great variety in the references used by the authors. Our analysis showed that JBE is an important peer reviewed publication aimed to improve teaching and learning of Biochemistry in Brazil. © 2019 International Union of Biochemistry and Molecular Biology, 47(3):249-256, 2019.

Charge neutralization as the major factor for the assembly of nucleocapsid-like particles from C-terminal truncated hepatitis C virus core protein.

BACKGROUND: Hepatitis C virus (HCV) core protein, in addition to its structural role to form the nucleocapsid assembly, plays a critical role in HCV pathogenesis by interfering in several cellular processes, including microRNA and mRNA homeostasis. The C-terminal truncated HCV core protein (C124) is intrinsically unstructured in solution and is able to interact with unspecific nucleic acids, in the micromolar range, and to assemble into nucleocapsid-like particles (NLPs) in vitro. The specificity and propensity of C124 to the assembly and its implications on HCV pathogenesis are not well understood. METHODS: Spectroscopic techniques, transmission electron microscopy and calorimetry were used to better understand the propensity of C124 to fold or to multimerize into NLPs when subjected to different conditions or in the presence of unspecific nucleic acids of equivalent size to cellular microRNAs. RESULTS: The structural analysis indicated that C124 has low propensity to self-folding. On the other hand, for the first time, we show that C124, in the absence of nucleic acids, multimerizes into empty NLPs when subjected to a pH close to its isoelectric point (pH ≈ 12), indicating that assembly is mainly driven by charge neutralization. Isothermal calorimetry data showed that the assembly of NLPs promoted by nucleic acids is enthalpy driven. Additionally, data obtained from fluorescence correlation spectroscopy show that C124, in nanomolar range, was able to interact and to sequester a large number of short unspecific nucleic acids into NLPs. DISCUSSION: Together, our data showed that the charge neutralization is the major factor for the nucleocapsid-like particles assembly from C-terminal truncated HCV core protein. This finding suggests that HCV core protein may physically interact with unspecific cellular polyanions, which may correspond to microRNAs and mRNAs in a host cell infected by HCV, triggering their confinement into infectious particles.

Avoiding Buffer Interference in ITC Experiments: A Case Study from the Analysis of Entropy-Driven Reactions of Glucose-6-Phosphate Dehydrogenase.

Isothermal titration calorimetry (ITC) is a label-free technique that allows the direct determination of the heat absorbed or released in a reaction. Frequently used to determining binding parameters in biomolecular interactions, it is very useful to address enzyme-catalyzed reactions as both kinetic and thermodynamic parameters can be obtained. Since calorimetry measures the total heat effects of a reaction, it is important to consider the contribution of the heat of protonation/deprotonation that is possibly taking place. Here, we show a case study of the reaction catalyzed by the glucose-6-phosphate dehydrogenase (G6PD) from Leuconostoc mesenteroides. This enzyme is able to use either NAD(+) or NADP(+) as a cofactor. The reactions were done in five buffers of different enthalpy of protonation. Depending on the buffer used, the observed calorimetric enthalpy (ΔH(cal)) of the reaction varied from -22.93 kJ/mol (Tris) to 19.37 kJ/mol (phosphate) for the NADP(+)-linked reaction, and -11.67 kJ/mol (Tris) to 7.32 kcal/mol or 30.63 kJ/mol (phosphate) for the NAD(+) reaction. We will use this system as an example of how to extract proton-independent reaction enthalpies from kinetic data to ensure that the reported accurately represent the intrinsic heat of reaction.

CALCIUM-INDUCED LIPID PEROXIDATION IS MEDIATED BY RHODNIUS HEME-BINDING PROTEIN (RHBP) AND PREVENTED BY VITELLIN.

Lipid peroxidation is promoted by the quasi-lipoxygenase (QL) activity of heme proteins and enhanced by the presence of free calcium. Unlike mammalian plasma, the hemolymph of Rhodnius prolixus, a vector of Chagas disease, contains both a free heme-binding protein (RHBP) and circulating lipoproteins. RHBP binds and prevents the heme groups of the proteins from participating in lipid peroxidation reactions. Herein, we show that despite being bound to RHBP, heme groups promote lipid peroxidation through a calcium-dependent QL reaction. This reaction is readily inhibited by the presence of ethylene glycol tetraacetic acid (EGTA), the antioxidant butylated hydroxytoluene or micromolar levels of the main yolk phosphoprotein vitellin (Vt). The inhibition of lipid peroxidation is eliminated by the in vitro dephosphorylation of Vt, indicating that this reaction depends on the interaction of free calcium ions with negatively charged phosphoamino acids. Our results demonstrate that calcium chelation mediated by phosphoproteins occurs via an antioxidant mechanism that protects living organisms from lipid peroxidation.

rBPI21 interacts with negative membranes endothermically promoting the formation of rigid multilamellar structures.

rBPI21 belongs to the antimicrobial peptide and protein (AMP) family. It has high affinity for lipopolysaccharide (LPS), acting mainly against Gram-negative bacteria. This work intends to elucidate the mechanism of action of rBPI21 at the membrane level. Using isothermal titration calorimetry, we observed that rBPI21 interaction occurs only with negatively charged membranes (mimicking bacterial membranes) and is entropically driven. Differential scanning calorimetry shows that membrane interaction with rBPI21 is followed by an increase of rigidity on negatively charged membrane, which is corroborated by small angle X-ray scattering (SAXS). Additionally, SAXS data reveal that rBPI21 promotes the multilamellarization of negatively charged membranes. The results support the proposed model for rBPI21 action: first it may interact with LPS at the bacterial surface. This entropic interaction could cause the release of ions that maintain the packed structure of LPS, ensuring peptide penetration. Then, rBPI21 may interact with the negatively charged leaflets of the outer and inner membranes, promoting the interaction between the two bacterial membranes, ultimately leading to cell death.

The structural dynamics of the flavivirus fusion peptide-membrane interaction.

Membrane fusion is a crucial step in flavivirus infections and a potential target for antiviral strategies. Lipids and proteins play cooperative roles in the fusion process, which is triggered by the acidic pH inside the endosome. This acidic environment induces many changes in glycoprotein conformation and allows the action of a highly conserved hydrophobic sequence, the fusion peptide (FP). Despite the large volume of information available on the virus-triggered fusion process, little is known regarding the mechanisms behind flavivirus-cell membrane fusion. Here, we evaluated the contribution of a natural single amino acid difference on two flavivirus FPs, FLA(G) ((98)DRGWGNGCGLFGK(110)) and FLA(H) ((98)DRGWGNHCGLFGK(110)), and investigated the role of the charge of the target membrane on the fusion process. We used an in silico approach to simulate the interaction of the FPs with a lipid bilayer in a complementary way and used spectroscopic approaches to collect conformation information. We found that both peptides interact with neutral and anionic micelles, and molecular dynamics (MD) simulations showed the interaction of the FPs with the lipid bilayer. The participation of the indole ring of Trp appeared to be important for the anchoring of both peptides in the membrane model, as indicated by MD simulations and spectroscopic analyses. Mild differences between FLA(G) and FLA(H) were observed according to the pH and the charge of the target membrane model. The MD simulations of the membrane showed that both peptides adopted a bend structure, and an interaction between the aromatic residues was strongly suggested, which was also observed by circular dichroism in the presence of micelles. As the FPs of viral fusion proteins play a key role in the mechanism of viral fusion, understanding the interactions between peptides and membranes is crucial for medical science and biology and may contribute to the design of new antiviral drugs.

Thermodynamic and structural characterization of zwitterionic micelles of the membrane protein solubilizing amidosulfobetaine surfactants ASB-14 and ASB-16.

Surface tension and isothermal titration calorimetry (ITC) were used to determine the critical micelle concentration (cmc) of the zwitterionic amidosulfobetaine surfactants ASB-14 and ASB-16 (linear-alkylamidopropyldimethylammoniopropanosulfonates) at 25 °C. The cmc and the heat of micellization were determined from 15 to 75 °C by ITC for both surfactants. The increase in temperature caused significant changes in the enthalpy and in the entropy of micellization, with small changes in the standard Gibbs energy (ΔG(mic)), which is consistent to an enthalpy−entropy compensation with a compensatory temperature of 311 K (ASB-14) and 314 K (ASB-16). In the studied temperature range, the heat capacity of micellization (ΔC(p)(mic)) was essentially constant. The experimental ΔC(p)(mic) was lower than that expected if only hydrophobic interactions were considered, suggesting that polar interactions at the head groups are of significant importance in the thermodynamics of micelle formation by these surfactants. Indeed, a NMR NOESY spectrum showed NOEs that are improbable to occur within the same monomer, resulting from interactions at the polar head groups involving more than one monomer. The ITC and NMR results indicate a tilt in the polar headgroup favoring the polar interactions. We have also observed COSY correlations typical of dipolar interactions that could be recovered with the partial alignment of the molecule in solution, which results in an anisotropic tumbling. The anisotropy suggested an ellipsoidal shape of the micelles, which results in a positive magnetic susceptibility, and ultimately in orientation induced by the magnetic field. Such an ellipsoidal shape was confirmed from results obtained by SAXS experiments that revealed aggregation numbers of 108 and 168 for ASB-14 and ASB-16 micelles, respectively. This study characterizes an interesting micelle system that can be used in the study of membrane proteins by solution NMR spectroscopy.

Measuring the strength of interaction between the Ebola fusion peptide and lipid rafts: implications for membrane fusion and virus infection.

The Ebola fusion peptide (EBO16) is a hydrophobic domain that belongs to the GP2 membrane fusion protein of the Ebola virus. It adopts a helical structure in the presence of mimetic membranes that is stabilized by the presence of an aromatic-aromatic interaction established by Trp8 and Phe12. In spite of its infectious cycle becoming better understood recently, several steps still remain unclear, a lacuna that makes it difficult to develop strategies to block infection. In order to gain insight into the mechanism of membrane fusion, we probed the structure, function and energetics of EBO16 and its mutant W8A, in the absence or presence of different lipid membranes, including isolated domain-resistant membranes (DRM), a good experimental model for lipid rafts. The depletion of cholesterol from living mammalian cells reduced the ability of EBO16 to induce lipid mixing. On the other hand, EBO16 was structurally sensitive to interaction with lipid rafts (DRMs), but the same was not observed for W8A mutant. In agreement with these data, W8A showed a poor ability to promote membrane aggregation in comparison to EBO16. Single molecule AFM experiments showed a high affinity force pattern for the interaction of EBO16 and DRM, which seems to be a complex energetic event as observed by the calorimetric profile. Our study is the first to show a strong correlation between the initial step of Ebola virus infection and cholesterol, thus providing a rationale for Ebola virus proteins being co-localized with lipid-raft domains. In all, the results show how small fusion peptide sequences have evolved to adopt highly specific and strong interactions with membrane domains. Such features suggest these processes are excellent targets for therapeutic and vaccine approaches to viral diseases.

Conformational selection, dynamic restriction and the hydrophobic effect coupled to stabilization of the BIR3 domain of the human X-linked inhibitor of apoptosis protein by the tetrapeptide AVPI.

The XIAP-BIR3 domain blocks a substantial portion of the apoptosis pathway and is an attractive target for novel anticancer agents. The tetrapeptide AVPI, from the protein Smac/DIABLO, binds to the XIAP-BIR3 domain, allowing the cancer cells to die. Here we characterize the binding parameters of AVPI to XIAP-BIR3 and analyze its effects on the thermodynamic stability of this domain. XIAP-BIR3 was exceptionally stable against physical and chemical treatments and became even more stable by interaction with AVPI. Nuclear magnetic resonance experiments demonstrated that conformational selection is taking place upon AVPI interaction with XIAP-BIR3. Molecular dynamics simulations corroborate that the flexibility of XIAP-BIR3 is significantly reduced. The positive binding entropy associated with a loss of conformational entropy involved in the binding indicates that hydrophobic interactions play an important role in the interaction and domain stabilization. The mechanism of XIAP-BIR3 stabilization and its implications for drug affinity optimization are discussed.

Calorimetry of enzyme-catalyzed reactions.

This mini-review shows the valuable contributions of Professor Julian Sturtevant to the current applications of calorimetry to the study of enzyme-catalyzed reactions. The more recent applications of calorimetric techniques such as isothermal titration calorimetry and flow calorimetry to the study of enzyme kinetics, as well as the advantages on using calorimetric techniques in the determination of kinetic parameters of enzymes, is also discussed here.

Identification of the Aedes aegypti peritrophic matrix protein AeIMUCI as a heme-binding protein.

The gene Aedes aegypti intestinal mucin 1 (AeIMUC1) encodes a putative peritrophic matrix (PM) protein that is expressed in the midgut of mosquito larvae and adults and is upregulated in response to exposure to heavy metals. The AeIMUC1 protein has a predicted secretory signal peptide and three putative chitin-binding domains (CBDs) with an intervening mucin-like domain. Immunofluorescence and immunoelectron microscopy experiments established that AeIMUC1 is a bona fide PM protein, and binding of the recombinant protein to chitin was demonstrated in vitro. Previous experiments suggested that the Ae. aegypti PM can bind toxic heme molecules generated during blood digestion. However, the identity of the binding molecule(s) was unknown. Using of heme-agarose beads and spectrophotometric and microcalorimetric titrations, we show that recombinant AeIMUC1 can bind large amounts of heme in vitro, suggesting for the first time a role for a PM protein in heme detoxification during blood digestion. Binding of heme to AeIMUC1 was accompanied by an altered circular dichroism spectrum indicating a change in protein conformation, consistent with an increase in secondary structure. Heme-binding activity was mapped to the AeIMUC1 CBDs, suggesting that these domains possess dual chitin- and heme-binding activity.

Differential scanning calorimetry as a tool to estimate binding parameters in multiligand binding proteins.

The stability of proteins and their interactions with other molecules is a topic of special interest in biochemistry because many cellular processes depend on that. New methods and approaches are constantly developed to elucidate the energetics of biomolecular recognition. In this sense, the application of the theory of macromolecular unfolding linked to ligand binding to differential scanning calorimetry (DSC) has proved to be a useful tool to simultaneously characterize the energetics of unfolding and binding. Although the general theory is well known, the applicability of DSC to study the interaction of biomolecules is not common. In the current work, we estimated the binding parameters of 8-anilinonaphthalene-1-sulfonic acid to human serum albumin using DSC. This model system was chosen due to both the complex stoichiometry and the moderate binding constants. From DSC curves acquired at different ligand concentrations, we obtained the number of bound ligands, the binding constants, and the binding enthalpy for each independent binding site. Compared with those parameters determined by titration calorimetry, the results highlight the potentiality of DSC to estimate binding parameters in multiligand binding proteins.

Probing the interaction between vesicular stomatitis virus and phosphatidylserine.

The entry of enveloped animal viruses into their host cells always depends on membrane fusion triggered by conformational changes in viral envelope glycoproteins. Vesicular stomatitis virus (VSV) infection is mediated by virus spike glycoprotein G, which induces membrane fusion between the viral envelope and the endosomal membrane at the acidic environment of this compartment. In this work, we evaluated VSV interactions with membranes of different phospholipid compositions, at neutral and acidic pH, using atomic force microscopy (AFM) operating in the force spectroscopy mode, isothermal calorimetry (ITC) and molecular dynamics simulation. We found that the binding forces differed dramatically depending on the membrane phospholipid composition, revealing a high specificity of G protein binding to membranes containing phosphatidylserine (PS). In a previous work, we showed that the sequence corresponding amino acid 164 of VSV G protein was as efficient as the virus in catalyzing membrane fusion at pH 6.0. Here, we used this sequence to explore VSV-PS interaction using ITC. We found that peptide binding to membranes was exothermic, suggesting the participation of electrostatic interactions. Peptide-membrane interaction at pH 7.5 was shown to be specific to PS and dependent on the presence of His residues in the fusion peptide. The application of the simplified continuum Gouy-Chapman theory to our system predicted a pH of 5.0 at membrane surface, suggesting that the His residues should be protonated when located close to the membrane. Molecular dynamics simulations suggested that the peptide interacts with the lipid bilayer through its N-terminal residues, especially Val(145) and His(148).

Ligand-induced thermostability in proteins: thermodynamic analysis of ANS-albumin interaction.

A comparative thermodynamic study of the interaction of anilinonaphthalene sulfonate (ANS) derivatives with bovine serum albumin (BSA) was performed by using differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC). The chemically related ligands, 1,8-ANS and 2,6-ANS, present a similar affinity for BSA with different binding energetics. The analysis of the binding driving forces suggests that not only hydrophobic effect but also electrostatic interactions are relevant, even though they have been extensively used as probes for non-polar domains in proteins. Ligand association leads to an increase in protein thermostability, indicating that both dyes interact mainly with native BSA. ITC data show that 1,8-ANS and 2,6-ANS have a moderate affinity for BSA, with an association constant of around 1-9x10(5) M(-1) for the high-affinity site. Ligand binding is disfavoured by conformational entropy. The theoretical model used to simulate DSC data satisfactorily reproduces experimental thermograms, validating this approach as one which provides new insights into the interaction between one or more ligands with a protein. By comparison with 1,8-ANS, 2,6-ANS appears as a more "inert" probe to assess processes which involve conformational changes in proteins.

Conformation of a synthetic antigenic peptide from HIV-1 p24 protein induced by ionic micelles.

We studied the interaction of the peptide AAMQMLKETINEEAAEWDRVHPVHAGPIA from the HIV-1 p24 protein in the presence of SDS (anionic) and CTABr (cationic) micelles at pH 7.0 by circular dichroism, fluorescence, and electron spin resonance (ESR). The micelles induced secondary structure as well as a blue shift in the tryptophan fluorescence emission, indicating an interaction between the peptide and the micelles. However, different contents of secondary structure elements were found when the peptide interacts with SDS or CTABr micelles. Steady-state anisotropy indicates a constraint on the rotational mobility of the tryptophan residue of the peptide upon interaction with micelles. ESR studies pointed to different locations for the peptide in either micelle. Our results suggested that at least part of the peptide might be located at the hydrophobic core of the CTABr micelles, probably at the C-terminal region, while it is more inserted into the SDS micelles.

A highly active ATP-insensitive K+ import pathway in plant mitochondria.

We describe here a regulated and highly active K+ uptake pathway in potato (Solanum tuberosum), tomato (Lycopersicon esculentum), and maize (Zea mays) mitochondria. K+ transport was not inhibited by ATP, NADH, or thiol reagents, which regulate ATP-sensitive K+ channels previously described in plant and mammalian mitochondria. However, K+ uptake was completely prevented by quinine, a broad spectrum K+ channel inhibitor. Increased K+ uptake in plants leads to mitochondrial swelling, respiratory stimulation, heat release, and the prevention of reactive oxygen species formation. This newly described ATP-insensitive K+ import pathway is potentially involved in metabolism regulation and prevention of oxidative stress.

Suramin interaction with human alpha-thrombin: inhibitory effects and binding studies.

Suramin is a hexasulfonated naphthylurea commonly used as antitrypanosomial drug and more recently for the treatment of malignant tumors. Here we show that suramin binds to human alpha-thrombin inhibiting both the hydrolysis of the synthetic substrate S-2238 (IC50 = 40 microM), and the thrombin-induced fibrinogen clotting (IC50 = 20 microM). The latter is completely reversed by albumin (30 mg mL(-1)) suggesting that, at therapeutic concentrations, suramin is unable to affect alpha-thrombin activity in the plasma. Kinetic analysis showed that suramin acts as a non-competitive inhibitor decreasing Vmax without changing the Km for S-2238 hydrolysis. Calorimetric studies revealed two distinct binding sites for suramin in alpha-thrombin. In addition, circular dichroism studies showed that suramin causes significant changes in alpha-thrombin tertiary structure, without affecting the secondary structure content. Interaction with alpha-thrombin resulted in an increased fluorescence emission of the drug. Complex formation was strongly affected by high ionic strength suggesting the involvement of electrostatic interactions. Altogether our data suggest that part of the biological activities of suramin might be related to alpha-thrombin inhibition at extra-vascular sites.

Superactivity and conformational changes on alpha-chymotrypsin upon interfacial binding to cationic micelles.

The catalytic behaviour of alpha-CT (alpha-chymotrypsin) is affected by cationic micelles of CTABr (hexadecyltrimethylammonium bromide). The enzyme-micelle interaction leads to an increase in both the V(max) and the affinity for the substrate p -nitrophenyl acetate, indicating higher catalytic efficiency for bound alpha-CT. The bell-shaped profile of alpha-CT activity with increasing CTABr concentrations suggests that the micelle-bound enzyme reacts with the free substrate. Although more active with CTABr micelles, the enzyme stability is essentially the same as observed in buffer only. Enzyme activation is accompanied by changes in alpha-CT conformation. Changes in tertiary structure were observed by the increase in intensity and the red shift in the alpha-CT tryptophan fluorescence spectrum, suggesting the annulment of internal quenching and a more polar location of tryptophan residues. Near-UV CD also indicated the transfer of aromatic residues to a more flexible environment. CTABr micelles also induces an increase in alpha-helix, as seen by far-UV CD and FTIR (Fourier-transform infrared) spectroscopies. The far-UV CD spectrum of alpha-CT shows an increase in the intensity of the positive band at 198 nm and in the negative band at 222 nm, indicating an increased alpha-helical content. This is in agreement with FTIR studies, where an increase in the band at 1655 cm(-1), corresponding to the alpha-helix, was shown by fitting analysis and difference spectroscopy. Spectral deconvolution indicated a reduction in the beta-sheet content in micelle-bound alpha-CT. Our data suggest that the higher catalytic efficiency of micelle-bound alpha-CT results from significant conformational changes.

Calorimetric determination of thermodynamic parameters of reaction reveals different enthalpic compensations of the yeast hexokinase isozymes.

The change in enthalpy and rate constants for the reactions of yeast hexokinase isozymes, PI (Hxk1) and PII (Hxk2), was determined at pH 7.6 and 25 degrees C by isothermal titration calorimetry. The reactions were done in five buffer systems with enthalpy of protonation varying from -1.22 kcal/mol (phosphate) to -11.51 kcal/mol (Tris), allowing the determination of the number of protons released during glucose phosphorylation. The reaction is exothermic for both isozymes with a small, but significant (p < 0.0001), difference in the enthalpy of reaction (Delta HR), with an Delta HR of -5.1 +/- 0.2 (mean +/- S.D.) kcal/mol for Hxk1, and an Delta HR of -3.3 +/- 0.3 (mean +/- S.D.) kcal/mol for Hxk2. The Km for ATP determined by ITC was very similar to those reported in the literature for both isozymes. The effect of NaCl and KCl, from 0 to 200 mM, showed that although the rate of reaction decreases with increasing ionic strength, no change in the Delta HR was observed suggesting an entropic nature for the ionic strength. The differences in Delta HR obtained here for both isozymes strongly suggest that, besides glucose phosphorylation, another side reaction such as ATP hydrolysis and/or enzyme phosphorylation is taking place.

Membrane recognition by vesicular stomatitis virus involves enthalpy-driven protein-lipid interactions.

Vesicular stomatitis virus (VSV) infection depends on the fusion of viral and cellular membranes, which is mediated by virus spike glycoprotein G at the acidic environment of the endosomal compartment. VSV G protein does not contain a hydrophobic amino acid sequence similar to the fusion peptides found among other viral glycoproteins, suggesting that membrane recognition occurs through an alternative mechanism. Here we studied the interaction between VSV G protein and liposomes of different phospholipid composition by force spectroscopy, isothermal titration calorimetry (ITC), and fluorescence spectroscopy. Force spectroscopy experiments revealed the requirement for negatively charged phospholipids for VSV binding to membranes, suggesting that this interaction is electrostatic in nature. In addition, ITC experiments showed that VSV binding to liposomes is an enthalpically driven process. Fluorescence data also showed the lack of VSV interaction with the vesicles as well as inhibition of VSV-induced membrane fusion at high ionic strength. Intrinsic fluorescence measurements showed that the extent of G protein conformational changes depends on the presence of phosphatidylserine (PS) on the target membrane. Although the increase in PS content did not change the binding profile, the rate of the fusion reaction was remarkably increased when the PS content was increased from 25 to 75%. On the basis of these data, we suggest that G protein binding to the target membrane essentially depends on electrostatic interactions, probably between positive charges on the protein surface and negatively charged phospholipids in the cellular membrane. In addition, the fusion is exothermic, indicating no entropic constraints to this process.