Structural and mechanistic insights into the transport of aristolochic acids and their active metabolites by human serum albumin.

Aristolochic acids I and II (AA-I/II) are carcinogenic principles of Aristolochia plants, which have been employed in traditional medicinal practices and discovered as food contaminants. While the deleterious effects of AAs are broadly acknowledged, there is a dearth of information to define the mechanisms underlying their carcinogenicity. Following bioactivation in the liver, N-hydroxyaristolactam and N-sulfonyloxyaristolactam metabolites are transported via circulation and elicit carcinogenic effects by reacting with cellular DNA. In this study, we apply DNA adduct analysis, X-ray crystallography, isothermal titration calorimetry, and fluorescence quenching to investigate the role of human serum albumin (HSA) in modulating AA carcinogenicity. We find that HSA extends the half-life and reactivity of N-sulfonyloxyaristolactam-I with DNA, thereby protecting activated AAs from heterolysis. Applying novel pooled plasma HSA crystallization methods, we report high-resolution structures of myristic acid-enriched HSA (HSAMYR) and its AA complexes (HSAMYR/AA-I and HSAMYR/AA-II) at 1.9 Å resolution. While AA-I is located within HSA subdomain IB, AA-II occupies subdomains IIA and IB. ITC binding profiles reveal two distinct AA sites in both complexes with association constants of 1.5 and 0.5 · 106 M-1 for HSA/AA-I versus 8.4 and 9.0 · 105 M-1 for HSA/AA-II. Fluorescence quenching of the HSA Trp214 suggests variable impacts of fatty acids on ligand binding affinities. Collectively, our structural and thermodynamic characterizations yield significant insights into AA binding, transport, toxicity, and potential allostery, critical determinants for elucidating the mechanistic roles of HSA in modulating AA carcinogenicity.

Instructional Models for Course-Based Research Experience (CRE) Teaching.

The course-based research experience (CRE) with its documented educational benefits is increasingly being implemented in science, technology, engineering, and mathematics education. This article reports on a study that was done over a period of 3 years to explicate the instructional processes involved in teaching an undergraduate CRE. One hundred and two instructors from the established and large multi-institutional SEA-PHAGES program were surveyed for their understanding of the aims and practices of CRE teaching. This was followed by large-scale feedback sessions with the cohort of instructors at the annual SEA Faculty Meeting and subsequently with a small focus group of expert CRE instructors. Using a qualitative content analysis approach, the survey data were analyzed for the aims of inquiry instruction and pedagogical practices used to achieve these goals. The results characterize CRE inquiry teaching as involving three instructional models: 1) being a scientist and generating data; 2) teaching procedural knowledge; and 3) fostering project ownership. Each of these models is explicated and visualized in terms of the specific pedagogical practices and their relationships. The models present a complex picture of the ways in which CRE instruction is conducted on a daily basis and can inform instructors and institutions new to CRE teaching.

Prophage-mediated defence against viral attack and viral counter-defence.

Temperate phages are common, and prophages are abundant residents of sequenced bacterial genomes. Mycobacteriophages are viruses that infect mycobacterial hosts including Mycobacterium tuberculosis and Mycobacterium smegmatis, encompass substantial genetic diversity and are commonly temperate. Characterization of ten Cluster N temperate mycobacteriophages revealed at least five distinct prophage-expressed viral defence systems that interfere with the infection of lytic and temperate phages that are either closely related (homotypic defence) or unrelated (heterotypic defence) to the prophage. Target specificity is unpredictable, ranging from a single target phage to one-third of those tested. The defence systems include a single-subunit restriction system, a heterotypic exclusion system and a predicted (p)ppGpp synthetase, which blocks lytic phage growth, promotes bacterial survival and enables efficient lysogeny. The predicted (p)ppGpp synthetase coded by the Phrann prophage defends against phage Tweety infection, but Tweety codes for a tetrapeptide repeat protein, gp54, which acts as a highly effective counter-defence system. Prophage-mediated viral defence offers an efficient mechanism for bacterial success in host-virus dynamics, and counter-defence promotes phage co-evolution.

RNA-induced silencing attenuates G protein-mediated calcium signals.

Phospholipase Cß (PLCß) is activated by G protein subunits in response to environmental stimuli to increase intracellular calcium. In cells, a significant portion of PLCß is cytosolic, where it binds a protein complex required for efficient RNA-induced silencing called C3PO (component 3 promoter of RISC). Binding between C3PO and PLCß raises the possibility that RNA silencing activity can affect the ability of PLCß to mediate calcium signals. By use of human and rat neuronal cell lines (SK-N-SH and PC12), we show that overexpression of one of the main components of C3PO diminishes Ca(2+) release in response to Gαq/PLCß stimulation by 30 to 40%. In untransfected SK-N-SH or PC12 cells, the introduction of siRNA(GAPDH) [small interfering RNA(glyceraldehyde 3-phosphate dehydrogenase)] reduces PLCß-mediated calcium signals by ∼30%, but addition of siRNA(Hsp90) (heat shock protein 90) had little effect. Fluorescence imaging studies suggest an increase in PLCß-C3PO association in cells treated with siRNA(GAPDH) but not siRNA(Hsp90). Taken together, our studies raise the possibility that Ca(2+) responses to extracellular stimuli can be modulated by components of the RNA silencing machinery.-Philip, F., Sahu, S., Golebiewska, U., Scarlata, S. RNA-induced silencing attenuates G protein-mediated calcium signals.

Phospholipase Cß connects G protein signaling with RNA interference.

Phosphoinositide-specific-phospholipase Cß (PLCß) is the main effector of Gαq stimulation which is coupled to receptors that bind acetylcholine, bradykinin, dopamine, angiotensin II as well as other hormones and neurotransmitters. Using a yeast two-hybrid and other approaches, we have recently found that the same region of PLCß that binds Gαq also interacts with Component 3 Promoter of RNA induced silencing complex (C3PO), which is required for efficient activity of the RNA-induced silencing complex. In purified form, C3PO competes with Gαq for PLCß binding and at high concentrations can quench PLCß activation. Additionally, we have found that the binding of PLCß to C3PO inhibits its nuclease activity leading to reversal of RNA-induced silencing of specific genes. In cells, we found that PLCß distributes between the plasma membrane where it localizes with Gαq, and in the cytosol where it localizes with C3PO. When cells are actively processing small interfering RNAs the interaction between PLCß and C3PO gets stronger and leads to changes in the cellular distribution of PLCß. The magnitude of attenuation is specific for different silencing RNAs. Our studies imply a direct link between calcium responses mediated through Gαq and post-transcriptional gene regulation through PLCß.

High pressure promotes alpha-synuclein aggregation in cultured neuronal cells.

α-Synuclein is found in plaques associated with Parkinson's and other neurodegenerative diseases. Changes in α-synuclein oligomerization are thought to give rise to nucleation of neurodegenerative plaques. Here, we investigated the effect of hydrostatic pressure on the aggregation of α-synuclein in cultured neuronal cells. We found that hydrostatic pressure is associated with a transition from monomeric to higher order α-synuclein aggregates. We then tested whether this aggregation is associated with the loss of binding partners, such as phospholipase Cß. We found that increased pressure reduces the level of PLCß1 and the amount of α-synuclein/PLCß1 complexes. These studies suggest that pressure promotes release of α-synuclein from protein partners promoting its oligomerization.

Comparative genomics of Cluster O mycobacteriophages.

Mycobacteriophages--viruses of mycobacterial hosts--are genetically diverse but morphologically are all classified in the Caudovirales with double-stranded DNA and tails. We describe here a group of five closely related mycobacteriophages--Corndog, Catdawg, Dylan, Firecracker, and YungJamal--designated as Cluster O with long flexible tails but with unusual prolate capsids. Proteomic analysis of phage Corndog particles, Catdawg particles, and Corndog-infected cells confirms expression of half of the predicted gene products and indicates a non-canonical mechanism for translation of the Corndog tape measure protein. Bioinformatic analysis identifies 8-9 strongly predicted SigA promoters and all five Cluster O genomes contain more than 30 copies of a 17 bp repeat sequence with dyad symmetry located throughout the genomes. Comparison of the Cluster O phages provides insights into phage genome evolution including the processes of gene flux by horizontal genetic exchange.

A broadly implementable research course in phage discovery and genomics for first-year undergraduate students.

UNLABELLED: Engaging large numbers of undergraduates in authentic scientific discovery is desirable but difficult to achieve. We have developed a general model in which faculty and teaching assistants from diverse academic institutions are trained to teach a research course for first-year undergraduate students focused on bacteriophage discovery and genomics. The course is situated within a broader scientific context aimed at understanding viral diversity, such that faculty and students are collaborators with established researchers in the field. The Howard Hughes Medical Institute (HHMI) Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) course has been widely implemented and has been taken by over 4,800 students at 73 institutions. We show here that this alliance-sourced model not only substantially advances the field of phage genomics but also stimulates students' interest in science, positively influences academic achievement, and enhances persistence in science, technology, engineering, and mathematics (STEM) disciplines. Broad application of this model by integrating other research areas with large numbers of early-career undergraduate students has the potential to be transformative in science education and research training. IMPORTANCE: Engagement of undergraduate students in scientific research at early stages in their careers presents an opportunity to excite students about science, technology, engineering, and mathematics (STEM) disciplines and promote continued interests in these areas. Many excellent course-based undergraduate research experiences have been developed, but scaling these to a broader impact with larger numbers of students is challenging. The Howard Hughes Medical Institute (HHMI) Science Education Alliance Phage Hunting Advancing Genomics and Evolutionary Science (SEA-PHAGES) program takes advantage of the huge size and diversity of the bacteriophage population to engage students in discovery of new viruses, genome annotation, and comparative genomics, with strong impacts on bacteriophage research, increased persistence in STEM fields, and student self-identification with learning gains, motivation, attitude, and career aspirations.

Linking alpha-synuclein properties with oxidation: a hypothesis on a mechanism underling cellular aggregation.

α-Synuclein is a small, natively unstructured protein with propensity to aggregate. α-Synuclein fibrils are major components of Lewy bodies that are hallmarks of many neurodegenerative diseases. The solution properties and aggregation behavior of α-synuclein has been well characterized, but despite numerous studies that address the role of α-synuclein in cells, a clear physiological function of this protein remains a mystery. Over a hundred review articles of α-synuclein have been written in the last decade, making it difficult to list all of the important studies that have added to our insight of α-synuclein physiology. Instead, we briefly review the status of α-synuclein research and propose a model based on the idea that α-synuclein may not have an intrinsic activity in cells but rather, it modifies the function of a group of protein partners that in turn affect cell processes. We propose that it is the loss of its cellular partners under oxidative conditions that promotes α-synuclein aggregation accelerating neuronal death.

Defining the oligomerization state of γ-synuclein in solution and in cells.

γ-Synuclein is expressed at high levels in neuronal cells and in multiple invasive cancers. Like its family member α-synuclein, γ-synuclein is thought to be natively unfolded but does not readily form fibrils. The function of γ-synuclein is unknown, but we have found that it interacts strongly with the enzyme phospholipase Cß (PLCß), altering its interaction with G proteins. As a first step in determining its role, we have characterized its oligomerization using fluorescence homotransfer, photon-counting histogram analysis, and native gel electrophoresis. We found that when its expressed in Escherichia coli and purified, γ-synuclein appears monomeric on chromatographs under denaturing conditions, but under native conditions, it appears as oligomers of varying sizes. We followed the monomer-to-tetramer association by labeling the protein with fluorescein and following the concentration-dependent loss in fluorescence anisotropy resulting from fluorescence homotransfer. We also performed photon-counting histogram analysis at increasing concentrations of fluorescein-labeled γ-synuclein and found concentration-dependent oligomerization. Addition of PLCß2, a strong γ-synuclein binding partner whose cellular expression is correlated with γ-synuclein, results in disruption of γ-synuclein oligomers. Similarly, its binding to lipid membranes promotes the monomer form. When we exogenously express γ-synuclein or microinject purified protein into cells, the protein appears monomeric. Our studies show that even though purified γ-synuclein form oligomers, when binding partners are present, as in cells, it dissociates to a monomer to bind these partners, which in turn may modify protein function and integrity.

γ-Synuclein interacts with phospholipase Cß2 to modulate G protein activation.

Phospholipase Cß2 (PLC ß2) is activated by G proteins and generates calcium signals in cells. PLCß2 is absent in normal breast tissue, but is highly expressed in breast tumors where its expression is correlated with the progression and migration of the tumor. This pattern of expression parallels the expression of the breast cancer specific gene protein 1 which is also known as γ-synuclein. The cellular function of γ-synuclein and the role it plays in proliferation are unknown. Here, we determined whether γ-synuclein can interact with PLCß2 and affect its activity. Using co-immunprecitation and co-immunofluorescence, we find that in both benign and aggressive breast cancer cell lines γ-synuclein and PLCß2 are associated. In solution, purified γ-synuclein binds to PLCß2 with high affinity as measured by fluorescence methods. Protease digestion and mass spectrometry studies show that γ-synuclein binds to a site on the C-terminus of PLCß2 that overlaps with the Gαq binding site. Additionally, γ-synuclein competes for Gαq association, but not for activators that bind to the N-terminus (i.e. Rac1 and Gßγ). Binding of γ-synuclein reduces the catalytic activity of PLCß2 by mechanism that involves inhibition of product release without affecting membrane interactions. Since activated Gαq binds more strongly to PLCß2 than γ-synuclein, addition of Gαq(GTPγS) to the γ-synuclein -PLCß2 complex allows for relief of enzyme inhibition along with concomitant activation. We also find that Gßγ can reverse γ-synuclein inhibition without dissociating the γ-synuclein- PLCß2- complex. These studies point to a role of γ-synuclein in promoting a more robust G protein activation of PLCß2.

Measuring fast calcium fluxes in cardiomyocytes.

Cardiomyocytes have multiple Ca(2+) fluxes of varying duration that work together to optimize function (1,2). Changes in Ca(2+) activity in response to extracellular agents is predominantly regulated by the phospholipase Cß- Gα(q;) pathway localized on the plasma membrane which is stimulated by agents such as acetylcholine (3,4). We have recently found that plasma membrane protein domains called caveolae(5,6) can entrap activated Gα(q;)(7). This entrapment has the effect of stabilizing the activated state of Gα(q;) and resulting in prolonged Ca(2+) signals in cardiomyocytes and other cell types(8). We uncovered this surprising result by measuring dynamic calcium responses on a fast scale in living cardiomyocytes. Briefly, cells are loaded with a fluorescent Ca(2+) indicator. In our studies, we used Ca(2+) Green (Invitrogen, Inc.) which exhibits an increase in fluorescence emission intensity upon binding of calcium ions. The fluorescence intensity is then recorded for using a line-scan mode of a laser scanning confocal microscope. This method allows rapid acquisition of the time course of fluorescence intensity in pixels along a selected line, producing several hundreds of time traces on the microsecond time scale. These very fast traces are transferred into excel and then into Sigmaplot for analysis, and are compared to traces obtained for electronic noise, free dye, and other controls. To dissect Ca(2+) responses of different flux rates, we performed a histogram analysis that binned pixel intensities with time. Binning allows us to group over 500 traces of scans and visualize the compiled results spatially and temporally on a single plot. Thus, the slow Ca(2+) waves that are difficult to discern when the scans are overlaid due to different peak placement and noise, can be readily seen in the binned histograms. Very fast fluxes in the time scale of the measurement show a narrow distribution of intensities in the very short time bins whereas longer Ca(2+) waves show binned data with a broad distribution over longer time bins. These different time distributions allow us to dissect the timing of Ca(2+)fluxes in the cells, and to determine their impact on various cellular events.

Evidence for a fence that impedes the diffusion of phosphatidylinositol 4,5-bisphosphate out of the forming phagosomes of macrophages.

To account for the many functions of phosphatidylinositol 4,5-bisphosphate (PIP(2)), several investigators have proposed that there are separate pools of PIP(2) in the plasma membrane. Recent experiments show the surface concentration of PIP(2) is indeed enhanced in regions where phagocytosis, exocytosis, and cell division occurs. Kinases that produce PIP(2) are also concentrated in these regions. However, how is the PIP(2) produced by these kinases prevented from diffusing rapidly away? First, proteins could act as "fences" around the perimeter of these regions. Second, some factor could markedly decrease the diffusion coefficient, D, of PIP(2) within these regions. We used fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) to investigate these two possibilities in the forming phagosomes of macrophages injected with fluorescent PIP(2). FCS measurements show that PIP(2) diffuses rapidly (D ~ 1 µm(2)/s) in both the forming phagosomes and unengaged plasma membrane. FRAP measurements show that the fluorescence from PIP(2) does not recover (>100 s) after photobleaching the entire forming phagosome but recovers rapidly (~10 s) in a comparable area of membrane outside the cup. These results (and similar data for a plasma membrane-anchored green fluorescent protein) support the hypothesis that a fence impedes the diffusion of PIP(2) into and out of forming phagosomes.

A dynamic model of membrane-bound phospholipase Cß2 activation by Gßγ subunits.

Phospholipase C (PLC) ß2, a well studied member of the family of enzymes that catalyze the hydrolysis of the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) into secondary messengers, can be activated by the Gßγ subunits of heterotrimeric G-proteins in a manner that depends on the presence and composition of the associated phospholipid membrane surface. The N-terminal pleckstrin homology (PH) domain of PLCß2 mediates both the response to Gßγ and membrane binding, but how these interactions are coupled to yield an activated catalytic core remains unknown. Here we propose a mechanism based on molecular models of truncated PLCß2 in its activated form complexed with Gßγ and in the catalytically inactive/membrane-bound form, obtained with the application of protein-protein docking algorithms and coarse-grained molecular dynamics simulations. These models were probed experimentally, and the inferences were confirmed by results from a combination of molecular biology and fluorescence assays. Results from the dynamic simulations of the molecular models and their interactions with various lipid bilayers identify the determinants of PLCß2-PH domain specificity for Gßγ and lipid membranes and suggest a mechanism for the previously reported dependence of Gßγ activation on the associated membrane composition. Together, these findings explain the roles of the different activators in terms of their effect on the orientations of the PH and catalytic core domains relative to the lipid membranes.

Modulation of Ca²+ activity in cardiomyocytes through caveolae-Gαq interactions.

Cardiomyocytes have a complex Ca(2+) behavior and changes in this behavior may underlie certain disease states. Intracellular Ca(2+) activity can be regulated by the phospholipase Cß-Gα(q) pathway localized on the plasma membrane. The plasma membranes of cardiomycoytes are rich in caveolae domains organized by caveolin proteins. Caveolae may indirectly affect cell signals by entrapping and localizing specific proteins. Recently, we found that caveolin may specifically interact with activated Gα(q), which could affect Ca(2+) signals. Here, using fluorescence imaging and correlation techniques we show that Gα(q)-Gßγ subunits localize to caveolae in adult ventricular canine cardiomyoctyes. Carbachol stimulation releases Gßγ subunits from caveolae with a concurrent stabilization of activated Gα(q) by caveolin-3 (Cav3). These cells show oscillating Ca(2+) waves that are not seen in neonatal cells that do not contain Cav3. Microinjection of a peptide that disrupts Cav3-Gα(q) association, but not a control peptide, extinguishes the waves. Furthermore, these waves are unchanged with rynaodine treatment, but not seen with treatment of a phospholipase C inhibitor, implying that Cav3-Gα(q) is responsible for this Ca(2+) activity. Taken together, these studies show that caveolae play a direct and active role in regulating basal Ca(2+) activity in cardiomyocytes.

Differential response to morphine of the oligomeric state of µ-opioid in the presence of δ-opioid receptors.

Prolonged morphine treatment induces extensive desensitization of the µ-opioid receptor (µOR) which is the G-protein-coupled receptor that primarily mediates the cellular response to morphine. To date, the molecular mechanism underlying this process is unknown. Here, we have used live cell fluorescence imaging to investigate whether prolonged morphine treatment affects the physical environment of µOR, or its coupling with G-proteins, in two neuronal cell lines. We find that chronic morphine treatment does not change the amount of enhanced yellow fluorescence protein (eYFP)-tagged µOR on the plasma membrane, and only slightly decreases its association with G-protein subunits. Additionally, morphine treatment does not have a detectable effect on the diffusion coefficient of eYFP-µOR. However, in the presence of another family member, the δ-opioid receptor (δOR), prolonged morphine exposure results in a significant increase in the diffusion rate of µOR. Number and brightness measurements suggest that µOR exists primarily as a dimer that will oligomerize with δOR into tetramers, and morphine promotes the dissociation of these tetramers. To provide a plausible structural context to these data, we used homology modeling techniques to generate putative configurations of µOR-δOR tetramers. Overall, our studies provide a possible rationale for morphine sensitivity.

Wnt-dependent assembly of supermolecular Dishevelled-3-based complexes.

Dishevelled-3 (Dvl3) is a multivalent scaffold protein that is essential to Wnt signaling during development. Although Dvl-based punctae have been visualized by fluorescence microscopy; the physical nature and dynamic character of the such complexes are enigmatic. We use steric-exclusion chromatography, affinity pull-downs, proteomics and fluorescence correlation microscopy to characterize supermolecular Dvl3-based complexes of totipotent mouse F9 cells. The molecular mass of the complexes ranges from that of homodimeric Dvl3 to well-defined peaks harboring supermolecular complexes of 0.4 to 2.0 MDa. Addition of Wnt3a stimulates the formation of Dvl3-based complexes of greater molecular mass within 30 minutes. The presence of DKK1 and knockdown of Dishevelled proteins block formation of the 2 MDa Dvl3-based complexes and also block Wnt3a stimulation of the canonical pathway. Fluorescent correlation microscopy identified supermolecular Dvl3-based complexes with a molecular mass >30 MDa in live cells; these complexes were provoked to form structures with even greater molecular mass by Wnt3a. We establish for the first time the physical and functional nature of very large, supermolecular Dvl3-based complexes.

The small G protein Rac1 activates phospholipase Cdelta1 through phospholipase Cbeta2.

Rac1, which is associated with cytoskeletal pathways, can activate phospholipase Cbeta2 (PLCbeta2) to increase intracellular Ca(2+) levels. This increased Ca(2+) can in turn activate the very robust PLCdelta1 to synergize Ca(2+) signals. We have previously found that PLCbeta2 will bind to and inhibit PLCdelta1 in solution by an unknown mechanism and that PLCbeta2.PLCdelta1 complexes can be disrupted by Gbetagamma subunits. However, because the major populations of PLCbeta2 and PLCdelta1 are cytosolic, their regulation by Gbetagamma subunits is not clear. Here, we have found that the pleckstrin homology (PH) domains of PLCbeta2 and PLCbeta3 are the regions that result in PLCdelta1 binding and inhibition. In cells, PLCbeta2.PLCdelta1 form complexes as seen by Förster resonance energy transfer and co-immunoprecipitation, and microinjection of PHbeta2 dissociates the complex. Using PHbeta2 as a tool to assess the contribution of PLCbeta inhibition of PLCdelta1 to Ca(2+) release, we found that, although PHbeta2 only results in a 25% inhibition of PLCdelta1 in solution, in cells the presence of PHbeta2 appears to eliminates Ca(2+) release suggesting a large threshold effect. We found that the small plasma membrane population of PLCbeta2.PLCdelta1 is disrupted by activation of heterotrimeric G proteins, and that the major cytosolic population of the complexes are disrupted by Rac1 activation. Thus, the activity of PLCdelta1 is controlled by the amount of bound PLCbeta2 that changes with displacement of the enzyme by heterotrimeric or small G proteins. Through PLCbeta2, PLCdelta1 activation is linked to surface receptors as well as signals that mediate cytoskeletal pathways.

The effect of membrane domains on the G protein-phospholipase Cbeta signaling pathway.

The plasma membrane serves as a barrier to limit the exit and entry of components into and out of the cell, offering protection from the external environment. Communication between the cell and the external environment is mediated by multiple signaling pathways. While the plasma membrane was historically viewed as a lipid bilayer with freely diffusing proteins, the last decade has shown that the lipids and proteins in the plasma membrane are organized in a non-random manner, and that this organization can direct and modify various signaling pathways in the cell. In this review, we qualitatively discuss the ways that membrane domains can affect cell signaling. We then focus on how membrane domains can affect a specific signaling pathway--the G protein-phospholipase Cbeta pathway and show how membrane domains can play an active role in directing or redirecting G protein signals.

A self-scaffolding model for G protein signaling.

Activation of heterotrimeric G proteins is generally believed to induce dissociation of Galpha and Gbetagamma subunits, which are then free to bind to and change the catalytic activity of a variety of intracellular enzymes. We have previously found that in cells, Galphaq subunits remain complexed with its major effector, phospholipase Cbeta1, through the activation cycle. To determine whether this behavior may be operative in other systems, we carried out Förster resonance energy transfer studies and found that eYFP-Galphai and eCFP-Gbetagamma remain associated after stimulation in HEK293 cells. We also found that the level of Forster resonance energy transfer between Alexa546-phospholipase Cbeta2 and eGFP-Gbetagamma is significant and unchanged upon activation in HEK293 cells, thus showing that these proteins can localize into stable signaling complexes. To understand the basis for this stabilization, we carried out in vitro studies using a series of single-Cys mutants labeled with fluorescence tags and monitored their interaction with Gbetagamma subunits and changes in their fluorescence properties and accessibility upon activation and Gbetagamma binding. Our studies suggest a significant change in the orientation between G protein subunits upon activation that allows the G proteins to remain complexed while activating effectors.