Detection of conformationally changed MBP using intramolecular FRET

The principal objective of this study was to explore protein conformational changes using fluorescence resonance energy transfer (FRET) technology. Maltose binding protein (MBP) was adopted as a target model, due to its well-characterized structure and ligand specificity. To the best of our knowledge, this is the first report to provide information regarding the biological distance between the two lobes of MBP upon maltose binding. For the FRET pair, ECFP and EYFP were used as the donor and the acceptor, and were linked genetically to the C-terminal and N-terminal regions of MBP (ECFP:MBP:EYFP), respectively. After the FRET reaction, maltose-treated MBP was shown to exhibit a considerable energy transfer (FRET efficiency (E) = ∼0.11, Distance (D) = ∼6.93 nm) at the ensemble level, which was regarded as reflective of the increase in donor quenching and the upshift in acceptor emission intensity, thereby suggesting that the donor and the acceptor had been brought close together as the result of structural alterations in MBP. However, upon glucose treatment, no FRET phenomenon was detected, thereby implying the specificity of interaction between MBP and maltose. The in vitro FRET results were also confirmed via the acceptor photobleaching method. Therefore, our data showed that maltose-stimulated conformational changes of MBP could be measured by FRET, thereby providing biological information, including the FRET efficiency and the intramolecular distance.     

Dynamic Regulation of N-Methyl-d-aspartate (NMDA) and α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors by Posttranslational Modifications

Many molecular mechanisms underlie the changes in synaptic glutamate receptor content that are required by neuronal networks to generate cellular correlates of learning and memory. During the last decade, posttranslational modifications have emerged as critical regulators of synaptic transmission and plasticity. Notably, phosphorylation, ubiquitination, and palmitoylation control the stability, trafficking, and synaptic expression of glutamate receptors in the central nervous system. In the current review, we will summarize some of the progress made by the neuroscience community regarding our understanding of phosphorylation, ubiquitination, and palmitoylation of the NMDA and AMPA subtypes of glutamate receptors.     

Vesicular GABA transporter (VGAT) transports β‐alanine

Vesicular GABA transporter (VGAT) is expressed in GABAergic and glycinergic neurons, and is responsible for vesicular storage and subsequent exocytosis of these inhibitory amino acids. In this study, we show that VGAT recognizes β‐alanine as a substrate. Proteoliposomes containing purified VGAT transport β‐alanine using Δψ but not ΔpH as a driving force. The Δψ‐driven β‐alanine uptake requires Cl^?. VGAT also facilitates Cl^? uptake in the presence of β‐alanine. A previously described VGAT mutant (Glu213Ala) that disrupts GABA and glycine transport similarly abrogates β‐alanine uptake. These findings indicated that VGAT transports β‐alanine through a mechanism similar to those for GABA and glycine, and functions as a vesicular β‐alanine transporter.

     

Ratiometric detection of Zn(II) using chelating fluorescent protein chimeras

Fluorescent indicators for the real-time imaging of small molecules or metal ions in living cells are invaluable tools for understanding their physiological function. Genetically encoded sensors based on fluorescence resonance energy transfer (FRET) between fluorescent protein domains have important advantages over synthetic probes, but often suffer from a small ratiometric change. Here, we present a new design approach to obtain sensors with a large difference in emission ratio between the bound and unbound states. De novo Zn(II)-binding sites were introduced directly at the surface of both fluorescent domains of a chimera of enhanced cyan and yellow fluorescent protein, connected by a flexible peptide linker. The resulting sensor ZinCh displayed an almost fourfold change in fluorescence emission ratio upon binding of Zn(II). Besides a high affinity for Zn(II), the sensor was shown to be selective over other physiologically relevant metal ions. Its unique biphasic Zn(II)-binding behavior could be attributed to the presence of two distinct Zn(II)-binding sites and allowed ratiometric fluorescent detection of Zn(II) over a concentration range from 10 nM to 1 mM. Size-exclusion chromatography and fluorescence anisotropy were used to provide a detailed picture of the conformational changes associated with each Zn(II)-binding step. The high affinity for Zn(II) was mainly due to a high effective concentration of the fluorescent proteins and could be understood quantitatively by modeling the peptide linker between the fluorescent proteins as a random coil. The strategy of using chelating fluorescent protein chimeras to develop FRET sensor proteins with a high ratiometric change is expected to be more generally applicable, in particular for other metal ions and small molecules.     

Extremely low polymerizability of a highly-divergent Chlamydomonas actin (NAP)

Novel actin-like protein (NAP) is a highly divergent actin expressed in Chlamydomonas. With its low sequence similarity, it is uncertain whether NAP can polymerize into filaments. Here I assessed it by ectopically expressing enhanced green fluorescent protein-tagged NAP (EGFP-NAP) in cultured cells. EGFP-NAP was excluded from stress fibres but partially co-localized with endogenous actin in the cell periphery. In fluorescence recovery after photobleaching experiment, turnover rate of EGFP-NAP was similar to the estimated diffusion rate of monomeric actin. Therefore, EGFP-NAP likely accumulates by diffusion. These findings suggest that NAP has extremely poor ability to polymerize.     

Cell signaling: What is the signal and what information does it carry?

This paper reviews key findings from quantitative study of the yeast pheromone response system. Most come from single cell experiments that quantify molecular events the system uses to operate. After induction, signal propagation is relatively slow; peak activity takes minutes to reach the nucleus. At each measurement point along the transmission chain, signal rises, overshoots, peaks, and declines toward steady state. At at least one measurement point, this decline depends on negative feedback. The system senses and relays percent receptor occupancy, and one effect of the feedback is to maximize precision of this transmitted information. Over time, the system constantly adjusts quantitative behaviors to convey extracellular ligand concentration faithfully. These behaviors and mechanisms that control them are likely to be general for metazoan signaling systems.     

Nitric oxide inhibits mitochondrial movement in forebrain neurons associated with disruption of mitochondrial membrane potential

Nitric oxide (NO) has a number of physiological and pathophysiological effects in the nervous system. One target of NO is the mitochondrion, where it inhibits respiration and ATP synthesis, which may contribute to NO-mediated neuronal injury. Our recent studies suggested that impaired mitochondrial function impairs mitochondrial trafficking, which could also contribute to neuronal injury. Here, we studied the effects of NO on mitochondrial movement and morphology in primary cultures of forebrain neurons using a mitochondrially targeted enhanced yellow fluorescent protein. NO produced by two NO donors, papa non-oate and diethylamine/NO complex, caused a rapid cessation of mitochondrial movement but did not alter morphology. Movement recovered after removal of NO. The effects of NO on movement were associated with dissipation of the mitochondrial membrane potential. Increasing cGMP levels using 8-bromoguanosine 3',5'-cyclic monophosphate, did not mimic the effects on mitochondrial movement. Furthermore, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of NO-induced activation of soluble guanylate cyclase, did not block the effects of NO. Thus, neither increasing nor decreasing cGMP levels had an effect on mitochondrial movement. Based on these data, we conclude that NO is a novel modulator of mitochondrial trafficking in neurons, which may act through the inhibition of mitochondrial function.     

Bimolecular Fluorescence Complementation Analysis of Inducible Protein Interactions: Effects of Factors Affecting Protein Folding on Fluorescent Protein Fragment Association

Bimolecular fluorescence complementation (BiFC) analysis enables visualization of the subcellular locations of protein interactions in living cells. Using fragments of different fluorescent proteins, we investigated the temporal resolution and the quantitative accuracy of BiFC analysis. We determined the kinetics of BiFC complex formation in response to the rapamycin-inducible interaction between the FK506 binding protein (FKBP) and the FKBP-rapamycin binding domain (FRB). Fragments of yellow fluorescent protein fused to FKBP and FRB produced detectable BiFC complex fluorescence 10 min after the addition of rapamycin and a 10-fold increase in the mean fluorescence intensity in 8 h. The N-terminal fragment of the Venus fluorescent protein fused to FKBP produced constitutive BiFC complexes with several C-terminal fragments fused to FRB. A chimeric N-terminal fragment containing residues from Venus and yellow fluorescent protein produced either constitutive or inducible BiFC complexes depending on the temperature at which the cells were cultured. The concentrations of inducers required for half-maximal induction of BiFC complex formation by all fluorescent protein fragments tested were consistent with the affinities of the inducers for unmodified FKBP and FRB. Treatment with the FK506 inhibitor of FKBP-FRB interaction prevented the formation of BiFC complexes by FKBP and FRB fusions, but did not disrupt existing BiFC complexes. Proteins synthesized before the addition of rapamycin formed BiFC complexes with the same efficiency as did newly synthesized proteins. Inhibitors of protein synthesis attenuated BiFC complex formation independent of their effects on fusion protein synthesis. The kinetics at which they inhibited BiFC complex formation suggests that they prevented association of the fluorescent protein fragments, but not the slow maturation of BiFC complex fluorescence. Agents that induce the unfolded protein response also reduced formation of BiFC complexes. The effects of these agents were suppressed by cellular adaptation to protein folding stress. In summary, BiFC analysis enables detection of protein interactions within minutes after complex formation in living cells, but does not allow detection of complex dissociation. Conditional BiFC complex formation depends on the folding efficiencies of fluorescent protein fragments and can be affected by the cellular protein folding environment.     

Characterization of four mammalian 3-hydroxyacyl-CoA dehydratases involved in very long-chain fatty acid synthesis

Very long-chain fatty acids are produced through a four-step cycle. However, the 3-hydroxyacyl-CoA dehydratase catalyzing the third step in mammals has remained unidentified. Mammals have four candidates, HACD1-4, based on sequence similarities to the recently identified yeast Phs1, although HACD3 and HACD4 share relatively weak similarity. We demonstrate that all four of these human proteins are indeed 3-hydroxyacyl-CoA dehydratases, in growth suppression experiments using a PHS1-shut off yeast strain and/or in vitro 3-hydroxypalmitoyl-CoA dehydratase assays. HACD proteins exhibit distinct tissue-expression patterns. We also establish that HACD proteins interact with the condensation enzymes ELOVL1-7, with some preferences.     

Modulation of the dopamine transporter by interaction with Secretory Carrier Membrane Protein 2

The monoamine transporters for dopamine (DAT), norepinephrine (NET) and serotonin (SERT) facilitate the homeostatic balance of neurotransmitters in the synaptic cleft and thus, play a fundamental role in regulating neuronal activity. Despite the importance of these monoamine transporters in controlling brain function, only relatively little information is available regarding the cellular and molecular regulation of these proteins. The monoamine transporters have been found to associate with a number of different proteins that regulate the function and subcellular localization of the transporters. We recently reported a functional interaction between SERT and the Secretory Carrier Membrane Protein 2 (SCAMP2). Here, we demonstrate that SCAMP2 also plays a role in the functional regulation of DAT. DAT and SCAMP2 interaction is here verified by co-immunoprecipitation and fluorescence resonance energy transfer (FRET) microscopy. Moreover, co-expression of DAT and SCAMP2 results in a decrease in DAT-mediated dopamine uptake caused by reduced levels of DAT molecules on the cell surface. Our finding that SCAMP2 interacts with and regulates the subcellular distribution of both DAT and SERT suggests that interaction with SCAMP2 may constitute an important mechanism for coordinating cell surface expression of monoamine transporters.     

Recent insights into the building and cycling of synaptic vesicles

The synaptic vesicle is currently the most well-characterized cellular organelle. During neurotransmitter release it undergoes multiple cycles of exo- and endocytosis. Despite this the vesicle manages to retain its protein and lipid composition. How does this happen? Here we provide a brief overview of the molecular architecture of the synaptic vesicle, and discuss recent studies investigating single vesicle behavior and the mechanisms controlling the vesicle’s molecular contents.     

Quantitative analysis of multi-protein interactions using FRET: application to the SUMO pathway

Protein-protein binding and signaling pathways are important fields of biomedical science. Here we report simple optical methods for the determination of the equilibrium binding constant K(d) of protein-protein interactions as well as quantitative studies of biochemical cascades. The techniques are based on steady-state and time-resolved fluorescence resonance energy transfer (FRET) between ECFP and Venus-YFP fused to proteins of the SUMO family. Using FRET has several advantages over conventional free-solution techniques such as isothermal titration calorimetry (ITC): Concentrations are determined accurately by absorbance, highly sensitive binding signals enable the analysis of small quantities, and assays are compatible with multi-well plate format. Most importantly, our FRET-based techniques enable us to measure the effect of other molecules on the binding of two proteins of interest, which is not straightforward with other approaches. These assays provide powerful tools for the study of competitive biochemical cascades and the extent to which drug candidates modify protein interactions.     

A monopartite nuclear localization sequence regulates nuclear targeting of the actin binding protein myopodin

Myopodin is an actin bundling protein that shuttles between nucleus and cytoplasm in response to cell stress or during differentiation. Here, we show that the myopodin sequence 58KKRRRRARK66, when tagged to either enhanced green fluorescent protein (EGFP) or to enhanced cyan fluorescent protein-CapG (ECFPCapG), is able to target these proteins to the nucleolus in HeLa or HEK293T cells. By contrast, 58KKRR61-ECFP-CapG accumulates in the nucleus. Mutation of 58KKRRRRARK66 into alanine residues blocks myopodin nuclear import and promotes formation of cytoplasmic actin filaments. A second putative nuclear localization sequence, 612KTSKKKGKK620, displays much weaker activity in a heterologous context, and appears not to be functional in the full length protein. Thus myopodin nuclear translocation is dependent on a monopartite nuclear localization sequence.     

Direct evidence for coupling between bacterial chemoreceptors

Bacterial chemoreceptors form mixed trimers of homodimers that cluster further in the presence of other cytoplasmic components. The physical proximity between receptors is thought to promote conformational coupling that enhances sensitivity, dynamic range, and collaboration between receptors of different types. We investigated conformational coupling between neighboring dimers by co-expressing two types of receptors, only one of which was labeled with yellow fluorescent protein. The two types of receptors were stimulated independently, and changes in the relative orientation of the labeled receptors were followed by fluorescence anisotropy. Possible coupling via cytoplasmic components of the taxis system was avoided by working with strains lacking those components. We find that binding of ligand to one type of receptor affects the conformation of the other type of receptor but not in the same way as binding of ligand to that receptor directly does. Thus, different receptors are coupled but not as simply as previously thought.     

Mutations in p97/VCP induce unfolding activity

A comparison of the protein sequences of various two-domain AAA+ ATPases revealed a striking difference in the residues lining the central pore of the D1 domain. The protein unfoldases of the bacterial Clp family and the archaeal VAT protein have at least one aromatic residue in the central D1 pore. In contrast, none of the members of the eukaryotic p97/VCP protein family has an aromatic residue in the D1 pore. The protein unfolding activity of VAT and other AAA+ ATPases is critically dependent on the presence of aromatic residues in this central pore. Unfoldase activity has not been demonstrated for the p97/VCP family in vitro. Thus, we exchanged the two aliphatic residues leucine and alanine of the D1 pore for aromatic tyrosine residues in full length p97 and in p97DeltaN, a truncated form of p97 lacking the N domain. We found that the mutant p97DeltaN variants with a single tyrosine or with two tyrosine residues in the central pore of D1 unfold the Clp family and VAT model substrate YFP-ssrA, whereas full length p97 with aromatic pore residues and wild-type p97 or p97DeltaN do not. Thus, p97 can exert unfoldase activity in vitro, provided that a single tyrosine residue is introduced into the D1 pore and that the N domain is deleted.     

Building and breeding molecules to spy on cells and tumors

Imaging of biochemical processes in living cells and organisms is essential for understanding how genes and gene products work together in space and time and in health and disease. Such imaging depends crucially on indicator molecules designed to maximize sensitivity and specificity. These molecules can be entirely synthetic, entirely genetically encoded macromolecules, or hybrid combinations, each approach having its own pros and cons. Recent examples from the author's laboratory include peptides whose uptake into cells is triggered by proteases typical of tumors, monomeric red fluorescent proteins and biarsenical-tetracysteine systems for determining the age and electron-microscopic location of proteins.     

Differences in the regulation of microtubule stability by the pro-rich region variants of microtubule-associated protein 4

We have recently reported a neural variant of microtubule-associated protein 4 with a short pro-rich region (MAP4-SP). Here, we show that the neural MAP4 has reduced microtubule-stabilizing activity, compared to the ubiquitous MAP4 with a long pro-rich region (MAP4-LP), both in vitro and in vivo. Fluorescence recovery after photobleaching analyses revealed that the interaction of MAP4-SP with the microtubules is very rapid, with a half-time of fluorescence recovery of 7 +/- 2.36 s, compared to 19.5 +/- 3.03 s in case of MAP4-LP. The dynamic interaction of MAP4-SP with microtubules in neural cells may contribute to the dynamic behaviors of extending neurites.     

Intragranular pH rapidly modulates exocytosis in adrenal chromaffin cells

Several drugs produce rapid changes in the kinetics of exocytosis of catecholamines, as measured at the single event level with amperometry. This study is intended to unveil whether the mechanism(s) responsible for these effects involve changes in the intravesicular pH. Cell incubation with bafilomycin A1, a blocker of the vesicular proton pump, caused both a deceleration in the kinetics of exocytosis and a reduction in the catecholamine content of vesicle. These effects were also observed upon reduction of proton gradient by nigericin or NH4Cl. pH measurements using fluorescent probes (acridine orange, quinacrine or enhanced green fluorescent protein-synaptobrevin) showed a strong correlation between vesicular pH and the kinetics of exocytosis. Hence, all maneuvers tested that decelerated exocytosis also alkalinized secretory vesicles and vice versa. On the other hand, calcium entry caused a transient acidification of granules. We therefore propose that the regulation of vesicular pH is, at least partially, a necessary step in the modulation of the kinetics of exocytosis and quantal size operated by some cell signals.     

Crystal structure and Raman studies of dsFP483, a cyan fluorescent protein from Discosoma striata

To better understand the diverse mechanisms of spectral tuning operational in fluorescent proteins (FPs), we determined the 2.1-Å X-ray structure of dsFP483 from the reef-building coral Discosoma. This protein is a member of the cyan class of Anthozoa FPs and exhibits broad, double-humped excitation and absorbance bands, with a maximum at 437-440 nm and a shoulder at 453 nm. Although these features support a heterogeneous ground state for the protein-intrinsic chromophore, peak fluorescence occurs at 483 nm for all excitation wavelengths, suggesting a common emissive state. Optical properties are insensitive to changes in pH over the entire range of protein stability. The refined crystal structure of the biological tetramer (space group C2) demonstrates that all protomers bear a cis-coplanar chromophore chemically identical with that in green fluorescent protein (GFP). To test the roles of specific residues in color modulation, we investigated the optical properties of the H163Q and K70M variants. Although absorbance bands remain broad, peak excitation maxima are red shifted to 455 and 460 nm, emitting cyan light and green light, respectively. To probe chromophore ground-state features, we collected Raman spectra using 752-nm excitation. Surprisingly, the positions of key Raman bands of wild-type dsFP483 are most similar to those of the neutral GFP chromophore, whereas the K70M spectra are more closely aligned with the anionic form. The Raman data provide further evidence of a mixed ground state with chromophore populations that are modulated by mutation. Possible internal protonation equilibria, structural heterogeneity in the binding sites, and excited-state proton transfer mechanisms are discussed. Structural alignments of dsFP483 with the homologs DsRed, amFP486, and zFP538-K66M suggest that natural selection for cyan is an exquisitely fine-tuned and highly cooperative process involving a network of electrostatic interactions that may vary substantially in composition and arrangement.