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PubMed2002

A biosensor for theophylline based on fluorescence detection of ligand-induced hammerhead ribozyme cleavage.

Recently, Breaker and coworkers engineered hammerhead ribozymes that rearrange from a catalytically inactive to an active conformation upon allosteric binding of a specific ligand. To monitor cleavage activity in real time, we have coupled a donor-acceptor fluorophore pair to the termini of the substrate RNA of such a hammerhead ribozyme, modified to cleave in trans in the presence of the bronchodilator theophylline. In the intact substrate, the fluorophores interact by fluorescence resonance energy transfer (FRET). The specific FRET signal breaks down as the effector ligand binds, the substrate is cleaved, and the products dissociate, with a rate constant dependent on the concentration of the ligand. Our biosensor cleaves substrate at 0.46 min(-1) in 1 mM theophylline and 0.04 min(-1) without effector, and discriminates against caffeine, a structural relative of theophylline. We have measured the theophylline-dependence profile of this biosensor, showing that concentrations as low as 1 microM can be distinguished from background. To probe the mechanism of allosteric regulation, a single nucleotide in the communication domain between the catalytic and ligand-binding domains was mutated to destabilize the inactive conformation of the ribozyme. As predicted, this mutant shows the same activity (0.3 min(-1)) in the presence and absence of theophylline. Additionally, time-resolved FRET measurements on the biosensor ribozyme in complex with a noncleavable substrate analog reveal no significant changes in fluorophore distance distribution upon binding of effector.

Base Pairing↗

PubMed2005

Method for detection of specific nucleic acids by recombinant protein with fluorescent resonance energy transfer.

Detection of specific nucleic acids is important to understand cellular mechanisms and functions of gene regulation. Here, we demonstrated a novel method to detect specific nucleic acids using recombinant protein and oligonucleotides. A recombinant protein YRGnC-11ad, which has a Rev-peptide between enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP) was constructed and expressed in HeLa cells. Rev-peptide, which corresponds to amino acids 34-50 of the HIV-1 Rev protein, indicates disordered structure in solution but forms alpha-helical and elongated conformation upon binding to Rev response element RNA (RRE-RNA) and Rev-aptamer, respectively. We confirmed that YRGnC-11ad could specifically bind to RRE-RNA and Rev-aptamer in cell lysate, and fluorescent resonance energy transfer (FRET) signal was changed upon binding following the conformational change of Rev-peptide. To utilize this FRET signal change toward the detection of specific nucleic acids, we split the RRE-RNA sequence and connected to the complementary oligonucleotide for target nucleic acids. When each two oligonucleotides hybridized to an adjacent region of target nucleic acids correctly, a Rev-peptide binding site was reformed on the hybridized complex. And we could confirm that YRGnC-11ad recombinant protein indicated FRET increase upon binding to the hybridized complex in cell lysate. These results suggest that the recombinant protein probe is available for specific nucleic acid detection.

Fluorescence Resonance Energy Transfer↗

PubMed2005

Solid-phase immobilized tripod for fluorescent renewable immunoassay. A concept for continuous monitoring of an immunoassay including a regeneration of the solid phase.

A new concept of immunoassay based on the use of a trifunctional reagent (tripod) and fluorescence resonance energy transfer (FRET) phenomenon is described. This procedure involves differential steps: (1) the tripod bearing (i) a fluorophore, (ii) a molecule structurally close to the target, and (iii) a linker reacts with the solid phase; (2) the solid phase is further activated with an anti-target antibody labeled with a quencher molecule, generating the decrease of the fluorophore emission via FRET; (3) FRET being distance dependent, the presence of the target by competing with the tripod for binding the quencher-labeled antibody leads to a rise of the fluorescence signal; (4) the solid phase is reactivated simply, by adding the quencher-labeled antibody. This method was evaluated in microtiter plates using the susbtance P as model while fluorescein and TAMRA were used as donor and acceptor, respectively. Results clearly illustrated the interest of the method, by allowing (i) a simple regeneration procedure, without requiring any drastic treatment, (ii) a direct fluorescence measurement onto the solid support, leading to a localized and cumulative signal, (iii) an increase of the signal when detecting the target, unlike classical competitive immunoassays, and (iv) a real-time monitoring of the competition and regeneration steps.

Animals↗

PubMed2004

General strategy for biosensor design and construction employing multifunctional surface-tethered components.

Biosensors function by reversibly linking bioreceptor-target analyte binding with closely integrated signal generation and can either continuously monitor analyte concentrations or be returned to baseline readout values by removal of analyte. We present an approach for producing fully reversible, reagentless, self-assembling biosensors on surfaces. In the prototype biosensor, quencher-dye-labeled biotin-linked E. coli maltose binding protein (MBP) bound in a specific orientation to a NeutrAvidin-coated surface is employed as a bioreceptor. To complete sensor formation, a modular tether arm consisting of a flexible biotinylated DNA oligonucleotide, a fluorescence resonance energy-transfer (FRET) donor dye, and a distal beta-cyclodextrin (beta-CD) analyte analogue is bound in an equimolar amount to the same surface by means of DNA-directed immobilization. After self-assembly, a baseline level of FRET quenching is observed due to specific interaction between the beta-CD of the flexible tether arm and the sugar binding site of MBP, which brings the two dyes into proximity. Addition of the target analyte, the nutrient maltose, displaces the linked beta-CD-dye of the DNA-based tether arm, and a concentration-dependent change in FRET results. Biosensor sensitivity and dynamic range can be controlled by either using MBP variants having different binding constants or by binding of modulator DNA oligonucleotides that are complementary to the flexible DNA tether. The sensor can be regenerated and returned to baseline quenching levels by washing away analyte. A complex set of interactions apparently exists on the sensing surface that may contribute to sensor behavior and range. This approach may represent a general way to assemble a wide range of useful biosensors.

Base Sequence↗

PubMed2006

Upconversion fluorescence resonance energy transfer in a homogeneous immunoassay for estradiol.

We recently described a novel homogeneous assay principle based on upconversion fluorescence resonance energy transfer (UC-FRET), where an upconverting phosphor (UCP) is utilized as a donor. The UC-FRET has now been applied to a competitive homogeneous immunoassay for 17beta-estradiol (E2) in serum, using a small-molecular dye as an acceptor. The assay was constructed by employing an UCP coated with an E2-specific recombinant antibody Fab fragment as a donor and an E2-conjugated small-molecular dye, Oyster-556, as an acceptor. Standard curves for the assay were produced both in buffer and in male serum. Sensitized acceptor emission was measured at 600 nm under continuous laser diode excitation at 980 nm. In buffer, the IC50 value of the assay was 1 nM and in serum 3 nM. The lower limits of detection (mean of zero calibrators, 3 SD) were 0.4 and 0.9 nM, respectively. The measurable concentration range extended up to 3 nM in buffer and 9 nM in serum. Equilibrium in the assay was reached in 30 min. The novel principle of UC-FRET has unique advantages compared to present homogeneous luminescence-based methods and can enable an attractive assay system platform for clinical diagnostics and for high-throughput screening approaches.

Estradiol↗

PubMedDate not supplied

Multistep fluorescence resonance energy transfer in sequential chromophore array constructed on oligo-DNA assemblies.

Sequential arrays of chromophores at regulated distances were constructed on a noncovalent DNA molecular assembly system in aqueous media. Photoinduced fluorescence resonance energy transfer (FRET) behaviors were then observed. We designed a number of chromophore/oligo-DNA conjugates with varying sequences. The chromophores eosin (Eo), TexasRed (TR), and tetramethylrhodamine (Rho) were employed as the energy donor, acceptor, and mediator, respectively, based on overlapping excitation and emission spectra. The chromophores were attached via aminolinkers to the 5'-terminals of 10mer oligo-DNAs consisting of AT rich sequences. The arrangement of Eo-Rho or Rho-TR with 10-residue (1 pitch of duplex) distances was ensured by duplex formation of the conjugates with a 20mer matrix oligo-DNA composed of complementary sequences to the conjugates. Single-step FRET from Eo to Rho and from Rho to TR was confirmed on the duplex. The three chromophore conjugates were then mixed with longer matrix oligo-DNAs (30 or 40mer) consisting of complementary sequences to the conjugates, producing Eo-(Rho)(n)-TR (n = 1 or 2) arrays with 10-residue distances. Multistep FRET from Eo to TR through the Rho mediator(s) was observed on the molecular assemblies. This photoenergy transmission system offers a good model for a photoenergy transmission system mimicking photosynthetic systems.

Base Sequence↗

PubMedDate not supplied

Competitive binding assay using fluorescence resonance energy transfer for the identification of calmodulin antagonists.

The ubiquitous calcium regulating protein calmodulin (CaM) has been utilized as a model drug target in the design of a competitive binding fluorescence resonance energy transfer assay for pharmacological screening. The protein was labeled by covalently attaching the thiol-reactive fluorophore, N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC) to an engineered C-terminal cysteine residue. Binding of the environmentally sensitive hydrophobic probe 2,6-anilinonaphthalene sulfonate (2,6-ANS) to CaM could be monitored by an increase in the fluorescence emission intensity of the 2,6-ANS. Evidence of fluorescence resonance energy transfer (FRET) from 2,6-ANS (acting as a donor) to MDCC (the acceptor in this system) was also observed; fluorescence emission representative of MDCC could be seen after samples were excited at a wavelength specific for 2,6-ANS. The FRET signal was monitored as a function of the concentration of calmodulin antagonists in solution. Calibration curves for both a selection of small molecules and a series of peptides based upon known CaM-binding domains were obtained using this system. The assay demonstrated dose-dependent antagonism by analytes known to hinder the biological activity of CaM. These data indicate that the presence of molecules known to bind CaM interfere with the ability of FRET to occur, thus leading to a concentration-dependent decrease of the ratio of acceptor:donor fluorescence emission. This assay can serve as a general model for the development of other protein binding assays intended to screen for molecules with preferred binding activity.

Amino Acid Sequence↗

PubMed2001

The rate of lipid transfer during fusion depends on the structure of fluorescent lipid probes: a new chain-labeled lipid transfer probe pair.

A number of fluorescent probes have been used to follow membrane fusion events, particularly intermixing of lipids. None of them is ideal. The most popular pair of probes is NBD-PE and Rh-PE, in which the fluorescent groups are attached to the lipid headgroups, making them sensitive to changes in the surrounding medium. Here we present a new assay for monitoring lipid transfer during membrane fusion using the acyl chain tagged fluorescent probes BODIPY500-PC and BODIPY530-PE. Like the NBD-PE/Rh-PE assay, this assay is based on fluorescence resonance energy transfer (FRET) between the donor, BODIPY500, and the acceptor, BODIPY530. The magnitude of FRET is sensitive to the probe surface concentration, allowing one to detect movement of probes from labeled to unlabeled vesicles during fusion. The high quantum yield of fluorescence, high efficiency of FRET (R(o) is estimated to be approximately 60 A), photostability, and localization in the central hydrophobic region of a bilayer all make this pair of probes quite promising for detecting fusion. We have compared this and two other lipid mixing assays for their abilities to detect the initial events of poly(ethylene glycol) (PEG)-mediated fusion of small unilamellar vesicles (SUVs). We found that the BODIPY500/530 assay showed lipid transfer rates consistent with those obtained using the DPHpPC self-quenching assay, while lipid mixing rates measured with the NBD-PE/Rh-PE RET assay were significantly slower. We speculate that the bulky labeled headgroups of NBD-PE and especially Rh-PE molecules hamper movement of probes through the stalk between fusing vesicles, and thus reduce the apparent rate of lipid mixing.

Boron Compounds↗

PubMed2002

DNA bending by bZIP charge variants: a unified study using electrophoretic phasing and fluorescence resonance energy transfer.

The role of asymmetric charge neutralization as a primary determinant of protein-induced DNA helical bending remains controversial. Electrophoretic phasing experiments have been conducted previously for peptides derived from the yeast basic leucine zipper (bZIP) transcription factor GCN4 bound to AP-1 sites in duplex DNA. Mutations altering the electrostatic character of amino acids close to the DNA backbone result in phase-dependent gel mobility changes, interpreted as evidence of DNA bending. However, alternate interpretations are possible. The effect of electrostatic interactions on DNA conformation has now been investigated further, using purified peptides having indistinguishable AP-1 DNA affinity. Two independent techniques have been employed: electrophoretic phasing and fluorescence resonance energy transfer (FRET). The phasing results imply DNA bending by bZIP charge variants, consistent with earlier findings. FRET studies yield the mean 5' end to 3' end distance of AP-1 DNA when free or bound to neutral or charged bZIP peptides. These distances were reduced in the charged variant complexes relative to those in the free duplex and the wild-type complex. Bending of the DNA helical axis is shown by molecular modeling to be the simplest interpretation of these results. The electrophoretic phasing and FRET results thus offer two mutually supportive lines of evidence for induced bending of the DNA helical axis due to asymmetric changes in charge density caused by the electrostatic character of the amino acids residing near the DNA backbone.

Basic-Leucine Zipper Transcription Factors↗

PubMed2002

Spatial relationship between the prodan site, Trp-214, and Cys-34 residues in human serum albumin and loss of structure through incremental unfolding.

Prodan (6-propionyl-2-(dimethylamino)-naphthalene), a competitive inhibitor of warfarin binding to human serum albumin (HSA) at drug site I, was used to determine the inter- and intradomain distances of HSA. The fluorescence resonance energy transfer (FRET) distances between prodan and Trp-214, prodan and 7-(diethyl amino)-4-methylcoumarin 3-maleimide (CM)-modified Cys-34, and Trp-214 and CM-Cys-34 were determined to be 25.5 +/- 0.5 A, 33.1 +/- 0.8 A, and 32.4 +/- 1 A, respectively. FRET analysis showed that low concentration of palmitic acid (5 microM) increased the interdomain distance between the Trp-214 in domain II and CM-Cys-34 in domain I by approximately 5 A without perturbing the secondary structure of HSA and the immediate environment of Trp-214. Palmitic acid (5 microM) increased the prodan fluorescence by increasing the quantum yield of bound prodan without altering the tryptophan environment. However, palmitic acid (>10 microM) decreased the prodan fluorescence and increased the tryptophan fluorescence. Our results indicate that the high affinity palmitic acid binding site is located at the interface of domains I and II. On the basis of our measurements, a schematic model representing the drug site-1, Trp-214, and Cys-34 along with the palmitic acid sites has been constructed. In addition, prodan fluorescence, FRET, and ligand binding were used to monitor guanidine hydrochloride-induced denaturation of HSA. An analysis of the equilibrium unfolding data suggests that HSA undergoes a two-state unfolding transition with no detectable intermediate. However, kinetic analysis using multiple probes and thermal denaturation studies showed that the unfolding of the prodan site in HSA preceded the unfolding of tryptophan environment. In addition, the separation of domain I and II occurred before the global unfolding of the protein. The data support the idea that HSA loses its structure incrementally during its unfolding.

2-Naphthylamine↗

PubMed2003

Rational design of genetically encoded fluorescence resonance energy transfer-based sensors of cellular Cdc42 signaling.

The temporal and spatial control of Rho GTPase signaling pathways is a central issue in understanding the molecular mechanisms that generate complex cellular movements. The Rho protein Cdc42 induces a significant conformational change in its downstream effector, the Wiskott-Aldrich syndrome protein (WASP). On the basis of this conformational change, we have created a series of single-molecule sensors for both active Cdc42 and Cdc42 guanine nucleotide exchange factors (GEFs) that utilize fluorescence resonance energy transfer (FRET) between cyan and yellow fluorescent proteins. In vitro, the Cdc42 sensors produce up to 3.2-fold FRET emission ratio changes upon binding active Cdc42. The GEF sensors yield up to 1.7-fold changes in FRET upon exchange of GDP for GTP. The GEF-catalyzed rate of nucleotide exchange for the GEF sensor is indistinguishable from that of wild-type Cdc42, but GAP-catalyzed nucleotide hydrolysis is slowed approximately 16-fold. In vivo, both sensors faithfully report on Cdc42 and/or Cdc42-GEF activity. These results establish the successful creation of rationally designed and genetically encoded tools that can be used to image the activity of biologically and medically important molecules in living systems.

Energy Transfer↗

PubMed2003

Spectroscopic studies of structural changes in two beta-sheet-forming peptides show an ensemble of structures that unfold noncooperatively.

A characterization of the conformation and stability of model peptide systems that form beta-sheets in aqueous solutions is considerably important in gaining insights into the mechanism of beta-sheet formation in proteins. We have characterized the conformation and equilibrium folding and unfolding of two 20-residue peptides whose NMR spectra suggest a three-stranded beta-sheet topology in aqueous solution: Betanova [Kortemme, T., Ramirez-Alvarado, M., and Serrano, L. (1998) Science 281, 253-256] and (D)P(D)P with d-Pro-Gly segments at the turns [Schenck, H. L., and Gellman, S. H. (1998) J. Am. Chem. Soc. 120, 4869-4870]. Both circular dichroism (CD) and infrared measurements indicate only 20-26% beta-sheet-like structure at 5 degrees C for Betanova and 42-59% beta-sheet for (D)P(D)P. For both peptides, the CD and infrared spectra change nearly linearly with increasing temperatures (or urea concentrations) and lack a sigmoidal signature characteristic of cooperative unfolding. Fluorescence resonance energy transfer (FRET) measurements between donor and acceptor molecules attached to the two ends confirm that Betanova is largely unstructured even at 10 degrees C; the average end-to-end distance estimated from FRET is closer to that of a random coil than a structured beta-sheet. In (D)P(D)P, the FRET results indicate a more compact structure that remains compact even at high temperatures (approximately 80 degrees C) or high urea concentrations (approximately 8 M). These results indicate that both these peptides access an ensemble of conformations at all temperatures or denaturant concentrations, with no significant free energy barrier separating the "folded" and "unfolded" conformations.

Circular Dichroism↗

PubMed2003

Linkage between fructose 1,6-bisphosphate binding and the dimer-tetramer equilibrium of Escherichia coli glycerol kinase: critical behavior arising from change of ligand stoichiometry.

Escherichia coli glycerol kinase (EC 2.7.1.30; ATP-glycerol 3-phosphotransferase) is inhibited allosterically by fructose 1,6-bisphosphate (FBP), and this inhibition is a primary mechanism by which glucose controls glycerol utilization in vivo. Earlier work indicates that glycerol kinase displays a dimer-tetramer equilibrium in solution, FBP shifts the equilibrium toward the tetramer, and tetramer formation is required for FBP inhibition. However, equilibrium constants for FBP binding and dimer-tetramer assembly that describe the linkage between these processes are unknown. Here, decreased fluorescence anisotropy of extrinsic fluorophores fluorescein and 2',7'-difluorofluorescein due to homo fluorescence resonance energy transfer (homo-FRET) is used to quantitate tetramer assembly and FBP binding. Glycerol kinase is labeled with extrinsic fluorophores covalently attached to an engineered surface cysteine residue under conditions that prevent labeling of native cysteine residues. Tryptic peptide mapping and MALDI-MS verify labeling at the engineered site only. Initial velocity studies show the labeling does not alter the catalytic properties or FBP inhibition. The steady-state fluorescence anisotropy of enzyme with a labeling stoichiometry of approximately 0.1 mol of fluorophore/mol of subunit is not sensitive to increased protein concentration or binding of FBP, indicating the absence of homo-FRET. However, steady-state fluorescence anisotropy of enzyme with a labeling stoichiometry of approximately 0.4 mol of fluorophore/mol of subunit decreases with increasing protein concentration, which is consistent with depolarization due to homo-FRET. The protein concentration dependence of the decreased fluorescence anisotropy is described by a dimer-tetramer equilibrium with an apparent dissociation constant of 61 +/- 7 nM (subunits) at pH 7.0 and 25 degrees C. FBP binds to both the dimer and tetramer of glycerol kinase, and the FBP concentration dependence of the apparent dissociation constant for the dimer-tetramer equilibrium shows critical behavior. The apparent dissociation constant decreases and then increases with increasing FBP concentration, reaching a minimum at about 20 mM FBP. Critical behavior is seen also in the FBP dependence of the inhibition. The critical behavior arises because tetramer dissociation increases FBP stoichiometry from two sites per tetramer to four half-sites per two dimers. The phenomenological description of the coupling between tetramer assembly and FBP binding shows antagonistic binding of FBP to the two sites on the tetramer, indicating that the strong positive cooperativity observed for FBP inhibition of catalytic activity (Hill coefficient approximately 1.5) is due to the approximately 4000-fold higher affinity of the tetramer for FBP rather than to positive coupling between the two FBP sites.

Biopolymers↗

PubMed2005

ADAM33 enzyme properties and substrate specificity.

ADAM33 is an asthma susceptibility gene recently identified through a genetic study of asthmatic families [van Eerdewegh, et al. (2002) Nature 418, 426-430]. To understand the function of the gene product, the recombinant metalloproteinase domain of human ADAM33 was purified and tested for its substrate cleavage specificity using peptides derived from beta-amyloid precursor protein (APP). A single Ala substitution at the P2 position of a 10-residue APP peptide, YEVHHQKLVF, yielded a 20-fold more efficient substrate. Terminal truncation studies identified a minimal nine-residue core (P5-P4') important for ADAM33 recognition and cleavage. Full positional scanning of the 10-mer peptide using the 19 naturally occurring l-amino acids (excluding Cys) revealed a substrate specificity profile. A strong preference for Val or Ile at P3, Ala at P2, and Gln at P1' was observed. The substrate binding model based on the X-ray structure of the ADAM33-inhibitor complex supported the observed substrate specificity profile. On the basis of this, an improved substrate was designed and a fluorescence resonance energy transfer (FRET) assay was developed using a fluorogenic derivative of this substrate. Kinetic studies confirmed that the best substrate, FRET-P2 [K(Dabcyl)YRVAFQKLAE(Edans)K], was approximately 100-fold more efficient than the wild-type APP peptide substrate, with a k(cat)/K(m) value of (3.6 +/- 0.1) x 10(4) s(-)(1) M(-)(1). Using this substrate and the FRET assay, ADAM33 enzyme activity and thermal stability were characterized. ADAM33 dependence on buffer conditions, detergents, and temperature was examined, and optimal conditions were defined. Accurate K(i) values for tissue inhibitors of metalloproteinase and small molecule compounds were obtained.

ADAM Proteins↗

PubMed2005

Reversible transition between the surface trimer and membrane-inserted monomer of annexin 12.

Under mildly acidic conditions, annexin 12 (ANX) inserts into lipid membranes to form a transbilayer pore [Langen, R., et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 14060]. In this study, we have addressed the question of the oligomeric state of ANX in this transbilayer conformation by means of Forster-type resonance energy transfer (FRET). Two single-cysteine mutants (K132C and N244C) were labeled with either Alexa-532 (donor) or Alexa-647 (acceptor). The labels were positioned at the sites thought to be on the cis side of the known transmembrane regions [Ladokhin, A. S., et al. (2002) Biochemistry 41, 13617]. If the pore were comprised of an annexin oligomer, efficient energy transfer should be observed. Fluorescence excitation spectra of several mixtures of donor- and acceptor-labeled ANX were recorded under various conditions. Spectroscopic hallmarks of oligomerization-related FRET were established by following a well-documented transition of ANX from the soluble monomer to surface trimer upon addition of calcium at neutral pH. These hallmarks, however, were not detected for the membrane-inserted form of ANX at pH 4.5, suggesting that the transbilayer form is a monomer. This implies that the pore is formed by several transmembrane regions of the same ANX molecule. FRET and other fluorescence experiments demonstrate that the transitions between the surface trimer and membrane-inserted monomer are reversible. This reversibility, in combination with the absence of oligomerization in the water-soluble and inserted state, makes ANX a good experimental model for thermodynamic studies of folding and stability of membrane proteins.

Animals↗

PubMed2004

GRP1 pleckstrin homology domain: activation parameters and novel search mechanism for rare target lipid.

Pleckstrin homology (PH) domains play a central role in a wide array of signaling pathways by binding second messenger lipids of the phosphatidylinositol phosphate (PIP) lipid family. A given type of PIP lipid is formed in a specific cellular membrane where it is generally a minor component of the bulk lipid mixture. For example, the signaling lipid PI(3,4,5)P(3) (or PIP(3)) is generated primarily in the inner leaflet of the plasma membrane where it is believed to never exceed 0.02% of the bulk lipid. The present study focuses on the PH domain of the general receptor for phosphoinositides, isoform 1 (GRP1), which regulates the actin cytoskeleton in response to PIP(3) signals at the plasma membrane surface. The study systematically analyzes both the equilibrium and kinetic features of GRP1-PH domain binding to its PIP lipid target on a bilayer surface. Equilibrium binding measurements utilizing protein-to-membrane fluorescence resonance energy transfer (FRET) to detect GRP1-PH domain docking to membrane-bound PIP lipids confirm specific binding to PIP(3). A novel FRET competitive binding measurement developed to quantitate docking affinity yields a K(D) of 50 +/- 10 nM for GRP1-PH domain binding to membrane-bound PIP(3) in a physiological lipid mixture approximating the composition of the plasma membrane inner leaflet. This observed K(D) lies in a suitable range for regulation by physiological PIP(3) signals. Interestingly, the affinity of the interaction decreases at least 12-fold when the background anionic lipids phosphatidylserine (PS) and phosphatidylinositol (PI) are removed from the lipid mixture. Stopped-flow kinetic studies using protein-to-membrane FRET to monitor association and dissociation time courses reveal that this affinity decrease arises from a corresponding decrease in the on-rate for GRP1-PH domain docking with little or no change in the off-rate for domain dissociation from membrane-bound PIP(3). Overall, these findings indicate that the PH domain interacts not only with its target lipid, but also with other features of the membrane surface. The results are consistent with a previously undescribed type of two-step search mechanism for lipid binding domains in which weak, nonspecific electrostatic interactions between the PH domain and background anionic lipids facilitate searching of the membrane surface for PIP(3) headgroups, thereby speeding the high-affinity, specific docking of the domain to its rare target lipid.

Blood Proteins↗

PubMed2005

Probing the affinity of SecA for signal peptide in different environments.

SecA, the peripheral subunit of the Escherichia coli preprotein translocase, interacts with a number of ligands during export, including signal peptides, membrane phospholipids, and nucleotides. Using fluorescence resonance energy transfer (FRET), we studied the interactions of wild-type (WT) and mutant SecAs with IAEDANS-labeled signal peptide, and how these interactions are modified in the presence of other transport ligands. We find that residues on the third alpha-helix in the preprotein cross-linking domain (PPXD) are important for the interaction of SecA and signal peptide. For SecA in aqueous solution, saturation binding data using FRET analysis fit a single-site binding model and yielded a Kd of 2.4 microM. FRET is inhibited for SecA in lipid vesicles relative to that in aqueous solution at a low signal peptide concentration. The sigmoidal nature of the binding curve suggests that SecA in lipids has two conformational states; our results do not support different oligomeric states of SecA. Using native gel electrophoresis, we establish signal peptide-induced SecA monomerization in both aqueous solution and lipid vesicles. Whereas the affinity of SecA for signal peptide in an aqueous environment is unaffected by temperature or the presence of nucleotides, in lipids the affinity decreases in the presence of ADP or AMP-PCP but increases at higher temperature. The latter finding is consistent with SecA existing in an elongated form while inserting the signal peptide into membranes.

Adenosine Triphosphatases↗

PubMed2006

Metal ion dependence, thermodynamics, and kinetics for intramolecular docking of a GAAA tetraloop and receptor connected by a flexible linker.

The GAAA tetraloop-receptor motif is a commonly occurring tertiary interaction in RNA. This motif usually occurs in combination with other tertiary interactions in complex RNA structures. Thus, it is difficult to measure directly the contribution that a single GAAA tetraloop-receptor interaction makes to the folding properties of a RNA. To investigate the kinetics and thermodynamics for the isolated interaction, a GAAA tetraloop domain and receptor domain were connected by a single-stranded A(7) linker. Fluorescence resonance energy transfer (FRET) experiments were used to probe intramolecular docking of the GAAA tetraloop and receptor. Docking was induced using a variety of metal ions, where the charge of the ion was the most important factor in determining the concentration of the ion required to promote docking {[Co(NH(3))(6)(3+)] << [Ca(2+)], [Mg(2+)], [Mn(2+)] << [Na(+)], [K(+)]}. Analysis of metal ion cooperativity yielded Hill coefficients of approximately 2 for Na(+)- or K(+)-dependent docking versus approximately 1 for the divalent ions and Co(NH(3))(6)(3+). Ensemble stopped-flow FRET kinetic measurements yielded an apparent activation energy of 12.7 kcal/mol for GAAA tetraloop-receptor docking. RNA constructs with U(7) and A(14) single-stranded linkers were investigated by single-molecule and ensemble FRET techniques to determine how linker length and composition affect docking. These studies showed that the single-stranded region functions primarily as a flexible tether. Inhibition of docking by oligonucleotides complementary to the linker was also investigated. The influence of flexible versus rigid linkers on GAAA tetraloop-receptor docking is discussed.

Base Sequence↗