Fluorescence Energy Transfer Measurements Research Articles

Dynamic Shape Transformation of a DNA Scaffold Applied for an Enzyme Nanocarrier.

Structural programmability and accurate addressability of DNA nanostructures are ideal characteristics for the platform of arranging enzymes with the nanoscale precision. In this study, a three-dimensional DNA scaffold was designed to enable a dynamic shape transition from an open plate-like structure to its closed state of a hexagonal prism structure. The two domains in the open state were folded together to transform into the closed state by hybridization of complementary short DNA closing keys at both of the facing edges in over 90% yield. The shape transformation of the DNA scaffold was extensively studied by means of the fluorescence energy transfer measurement, atomic force microscope images, and agarose gel electrophoretic analyses. A dimeric enzyme xylitol dehydrogenase was assembled on the DNA scaffold in its open state in a high-loading yield. The enzyme loaded on the scaffold was subsequently transformed to its closed state by the addition of short DNA closing keys. The enzyme encapsulated in the closed state displayed comparable activity to that in the open state, ensuring that the catalytic activity of the enzyme was well maintained in the DNA nanocarrier. The nanocarrier with efficient encapsulation ability is potentially applicable for drug delivery, biosensing, biocatalytic, and diagnostic tools.

New in Vitro Assay Measuring Direct Interaction of Nonsense Suppressors with the Eukaryotic Protein Synthesis Machinery.

Nonsense suppressors (NonSups) induce "readthrough", i.e., the selection of near cognate tRNAs at premature termination codons and insertion of the corresponding amino acid into nascent polypeptide. Prior readthrough measurements utilized contexts in which NonSups can promote readthrough directly, by binding to one or more of the components of the protein synthesis machinery, or indirectly, by several other mechanisms. Here we utilize a new, highly purified in vitro assay to measure exclusively direct nonsense suppressor-induced readthrough. Of 16 NonSups tested, 12 display direct readthrough, with results suggesting that such NonSups act by at least two different mechanisms. In preliminary work we demonstrate the potential of single molecule fluorescence energy transfer measurements to elucidate mechanisms of NonSup-induced direct readthrough, which will aid efforts to identify NonSups having improved clinical efficacy.

Deprotonation of a Single Amino Acid Residue Induces Significant Stability in an α-Helical Heteropeptide.

Stability of secondary structural elements is an integral component of a structurally stable protein. Presence of protons in the residue sequence and their immediate environment play a significant role in conformational stability. In this study, we show that removing a proton from a single amino acid residue significantly increases the stability of an α-helical heteropeptide in comparison with the unprotonated form. Far-UV circular dichroism spectroscopy, fluorescence spectroscopy, fluorescence energy transfer measurements, and over 10 μs of all-atom molecular dynamics simulations are used to provide an atomically detailed characterization of this event. There is a single histidine residue in the studied α-helical peptide sequence toward the N-terminal that interacts with a tryptophan located four residues away and quenches the fluorescence when protonated. Removing a proton from this histidine residue dequenches the tryptophan fluorescence and contributes to a significant increase in the helix stability. Atomically detailed analysis of individual residue conformations shows that the protonated histidine tends to be in closer proximity to the tryptophan, which correlates with higher helix content in the N and C termini and lower helix content in the central region of the peptide. In the presence of a neutral histidine, when tryptophan fluorescence is no longer quenched and histidine moves further away from tryptophan, the helix content remains mostly unchanged in the N-and-C termini and significantly increases in the central region. Our results strongly suggest that interactions of the tryptophan with a protonated histidine downregulate the helix population in the central segment of the helical structure compared to a neutral histidine residue. Upregulation of helix population of the central segment of this α-helical heteropeptide in the presence of a neutral histidine residue significantly increases the peptide structural stability.

Uncoupling PIP2-calmodulin regulation of Kv7.2 channels by an assembly destabilizing epileptogenic mutation.

We show that the combination of an intracellular bi-partite calmodulin (CaM)-binding site and a distant assembly region affect how an ion channel is regulated by a membrane lipid. Our data reveal that regulation by phosphatidylinositol(4,5)bisphosphate (PIP2) and stabilization of assembled Kv7.2 subunits by intracellular coiled-coil regions far from the membrane are coupled molecular processes. Live-cell fluorescence energy transfer measurements and direct binding studies indicate that remote coiled-coil formation creates conditions for different CaM interaction modes, each conferring different PIP2 dependency to Kv7.2 channels. Disruption of coiled-coil formation by epilepsy-causing mutation decreases apparent CaM-binding affinity and interrupts CaM influence on PIP2 sensitivity.

The assembly and distribution in vivo of the Escherichia coli RNA degradosome

We report an analysis in vivo of the RNA degradosome assembly of Escherichia coli. Employing fluorescence microscopy imaging and fluorescence energy transfer (FRET) measurements, we present evidence for in vivo pairwise interactions between RNase E-PNPase (polynucleotide phosphorylase), and RNase E-Enolase. These interactions are absent in a mutant strain with genomically encoded RNase E that lacks the C-terminal half, supporting the role of the carboxy-end domain as the scaffold for the degradosome. We also present evidence for in vivo proximity of Enolase-PNPase and Enolase-RhlB. The data support a model for the RNA degradosome (RNAD), in which the RNase E carboxy-end is proximal to PNPase, more distant to Enolase, and more than 10 nm from RhlB helicase. Our measurements were made in strains with mono-copy chromosomal fusions of the RNAD enzymes with fluorescent proteins, allowing measurement of the expression of the different proteins under different growth and stress conditions.

State-Dependent FRET Reports Large Gating-Ring Motions in BK Channels

Large conductance voltage- and calcium-dependent potassium channels (BK channels) are key regulators of many important physiological processes and a key feature to their physiological role is that the channel's open probability is regulated both by changes in transmembrane voltage and by intracellular calcium concentration. The voltage sensor resides within the transmembrane region of the channel, while Ca2+ binding is sensed by a large C-terminal intracellular region, where eight Regulator of Conductance for K+ (RCK) domains form a “gating ring”. Calcium binding to this region reduces the energy required to open the channel, but the exact mechanism underlying this process is still uncertain. Structural studies using isolated gating rings from prokaryotic channels and a biochemical study of the isolated gating ring from the human channel suggest that Ca2+ binding expands the gating ring. The large movement of the gating ring would physically pull and open the gate located at the pore domain. In the present study we investigate the calcium and voltage-dependence of conformational changes in the intact human BK channel by patch-clamp recordings and simultaneous measurements of fluorescence energy transfer between CFP and YFP variants of the green fluorescent protein, inserted into three sites in the BK gating ring. Depending of the site studied, different movements are detected that differ in their Ca- and V-dependence. Here we show that Ca2+ binding produces surprisingly large structural changes that, contrary to current theories, are not obligatorily coupled to the opening of the pore and are not strictly cooperative. Instead, a mechanism such as the “flip” transitions that have been identified in pentameric neurotransmitter receptor-channels is operative.

Methodological considerations for global analysis of cellular FLIM/FRET measurements

Global algorithms can improve the analysis of fluorescence energy transfer (FRET) measurement based on fluorescence lifetime microscopy. However, global analysis of FRET data is also susceptible to experimental artifacts. This work examines several common artifacts and suggests remedial experimental protocols. Specifically, we examined the accuracy of different methods for instrument response extraction and propose an adaptive method based on the mean lifetime of fluorescent proteins. We further examined the effects of image segmentation and a priori constraints on the accuracy of lifetime extraction. Methods to test the applicability of global analysis on cellular data are proposed and demonstrated. The accuracy of global fitting degrades with lower photon count. By systematically tracking the effect of the minimum photon count on lifetime and FRET prefactors when carrying out global analysis, we demonstrate a correction procedure to recover the correct FRET parameters, allowing us to obtain protein interaction information even in dim cellular regions with photon counts as low as 100 per decay curve.

Comparison of the Dielectric Response Obtained from Fluorescence Upconversion Measurements and Molecular Dynamics Simulations for Coumarin 153−Apomyoglobin Complexes and Structural Analysis of the Complexes by NMR and Fluorescence Methods

We present a comparison of the dielectric response obtained from fluorescence upconversion experiments and from molecular dynamics simulations of the complexes of coumarin 153 with five apomyoglobins (apoMbs): wild-type horse heart (HH-WT) and those of wild-type sperm whale (SW-WT); its two triple mutants, L29F/H64Q/V68F and H64L/V68F/P88A; and its double mutant, L29F/V68L. Comparisons between experimental and simulated solvation relaxation functions, C(t)s, for the wild-type proteins range from very good to excellent. For the three mutants we investigated, however, agreement between experiment and simulation was considerably inferior. Thus, an NMR study of the complex of the HH-WT complex apoMb, and fluorescence energy transfer and anisotropy studies of the five complexes, were performed to investigate the structures upon which the simulations were based. The NMR measurements confirm our earlier conclusions that the C153 lies in the heme pocket of the HH-WT apoMb. For the wild-type complexes, fluorescence energy transfer measurements provide two rise times, suggesting a definite spatial relationship between the two Trp donors and the C153 acceptor. These results confirm the structural integrity of the wild-type complexes and validate the initial structures used for the molecular dynamics simulations. On the other hand, the three mutants provided single exponential rise times for energy transfer, suggesting that the position of the C153 used in the simulations may have been in error or that the C153 is mobile on the time scale of the energy transfer experiment. Fluorescence anisotropy studies also suggest that the double mutant was not structurally intact. Furthermore, examination of these systems demonstrates the sensitivity of C153 to its environment and permits the observation of differences in the heme pockets. These results point to the importance of structural characterization of modified proteins used in studies of the dielectric response and suggest strategies for performing molecular dynamics simulations of modified proteins.

Interaction of hnRNP A1 with telomere DNA G-quadruplex structures studied at the single molecule level

G-rich telomeric DNA sequences can form G-quadruplex structures. The heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) and a shortened derivative (UP1) are active in telomere length regulation, and it has been reported that UP1 can unwind G-quadruplex structures. Here, we investigate the interaction of hnRNP A1 with G-quadruplex DNA structures containing the human telomere repeat (TTAGGG) by gel retardation assays, ensemble fluorescence energy transfer (FRET) spectroscopy, and single molecule FRET microscopy. Our biochemical experiments show that hnRNP A1 binds well to the G-quadruplex telomeric DNA. Ensemble and single molecule FRET measurements provide further insight into molecular conformation: the telomeric DNA overhang is found to be in a folded state in the absence of hnRNP A1 and to remain predominantly in a compact state when complexed with hnRNP A1. This finding is in contrast to the previously reported crystal structures of UP1-telomere DNA complexes where the DNA oligo within the protein-DNA complex is in a fully open conformation.

Folding energy landscape of cytochrome cb 562

Cytochrome cb(562) is a variant of an Escherichia coli four-helix bundle b-type heme protein in which the porphyrin prosthetic group is covalently ligated to the polypeptide near the terminus of helix 4. Studies from other laboratories have shown that the apoprotein folds rapidly without the formation of intermediates, whereas the holoprotein loses heme before native structure can be attained. Time-resolved fluorescence energy transfer (TRFET) measurements of cytochrome cb(562) refolding triggered using an ultrafast continuous-flow mixer (150 micros dead time) reveal that heme attachment to the polypeptide does not interfere with rapid formation of the native structure. Analyses of the TRFET data produce distributions of Trp-59-heme distances in the protein before, during, and after refolding. Characterization of the moments and time evolution of these distributions provides compelling evidence for a refolding mechanism that does not involve significant populations of intermediates. These observations suggest that the cytochrome b(562) folding energy landscape is minimally frustrated and able to tolerate the introduction of substantial perturbations (i.e., the heme prosthetic group) without the formation of deep misfolded traps.

Confocal and force probe imaging system for simultaneous three-dimensional optical and mechanical spectroscopic evaluation of biological samples

We present the design and operation of a novel instrument for the simultaneous three-dimensional measurements of localized properties using optical and mechanical probes. In this instrument the mechanical and optical probes are stationary relative to the instrument frame while the specimen can be navigated in three-dimensional space in the probing field, translating over a range of 64.5 microm by 49.7 microm by 31.5 microm in each axis, respectively, at closed loop speeds of 10 Hz. A large aperture is provided in the center of the moving platform so that an optical lens can image the specimen from below. An additional z-direction translator has been integrated with this instrument to independently move a force probe that contacts the specimen from above with a translation range of 16 microm. Furthermore, there is an additional seven degrees of freedom providing adjustments to independently position and/or align the scanner and force probe relative to the optical imaging lens. Initial results of both optical and mechanical scans demonstrate 6 nm localization from single molecule fluorescence measurements, as well as single pair fluorescence energy transfer measurements indicating molecular separations of about 2 nm.

Conformation of Poly(methacrylic acid) Chains in Dilute Aqueous Solution

Poly(methacrylic acid) (PMAA) undergoes a conformational transition between pH 4 and 6 from a hypercoiled structure to a water-swollen state. There has been much speculation as to the exact nature and driving force of the transition. In this paper, we present a comprehensive investigation of the conformational switch of PMAA using techniques which report on various length scales: fluorescence energy transfer experiments provide unique information on the nanometer length scale while dynamic light scattering (DLS) offers an insight into longer range interactions involved in the transition. Fluorescence energy transfer measurements demonstrate that PMAA undergoes subtle molecular rearrangements between pH 2 and 5 as short-range hydrophobic interactions between methyl groups are broken down by increasing concentrations of mutually repulsive carboxylate anions. Although such rearrangements have been proposed to account for the pH behavior of PMAA, we reveal them experimentally using techniques sensitive to na...

Novel FRET-Based Assay to Detect Reverse Transcriptase Activity Using Modified dUTP Analogues

We have developed a novel continuous assay to measure reverse transcriptase (RT) polymerase activity. The assay uses fluorescence energy transfer measurements to detect the incorporation of complementary pairs of fluorescently labeled deoxyuridine into cDNA product. The fluorescently labeled dUTP substrates were prepared using commercially available reagents with a simple coupling reaction. The fluorescent dye pairs have significant spectral overlap which allows FRET interaction between dyes incorporated into the cDNA. Using a polyA/oligo dT primer/template, the assay can readily detect DNA polymerase activity from any viral reverse transcriptase enzyme. The reaction proceeds linearly over time, and the rate is proportional to the enzyme concentration. We used the assay to compare the thermostability of a number of wild-type and mutant viral RT enzymes. Our results indicate that the wild-type AMV (avian myeloblastosis virus) enzyme is slightly more stable at 43 degrees C than the HIV-1 (human immunodeficiency virus) or MMLV (Moloney murine leukemia virus) enzymes. The thermostability of the RT enzyme was dramatically increased by the presence of primer/template with the enzyme. We also used the assay to study the effects of inhibitors on HIV-1 RT polymerase activity. This assay may be highly useful for the identification and characterization of potent RT inhibitors which could be candidates for development as therapeutic antiviral agents.

Fluorescent resonant energy transfer: Correlated fluctuations of donor and acceptor

Mounting evidence suggests that in single-molecule fluorescent resonant energy transfer (FRET) measurements, correlation between fluctuations in donor and acceptor may be important. We present a general theory to describe this correlation and its effect on the FRET rate. The correlation arises from low-energy excitations (e.g., acoustic phonons) of the molecule to which a donor-acceptor pair is attached, and results in an effective interaction between local environments or baths associated with the donor and the acceptor. The correlation is found to reduce the transfer rate, in particular, at short donor-acceptor distances. The theory can quantitatively explain recent measurements of polyproline peptides.

Crystal Structure of Human Estrogen-related Receptor α in Complex with a Synthetic Inverse Agonist Reveals Its Novel Molecular Mechanism

Inverse agonists of the constitutively active human estrogen-related receptor alpha (ERRalpha, NR3B1) are of potential interest for several disease indications (e.g. breast cancer, metabolic diseases, or osteoporosis). ERRalpha is constitutively active, because its ligand binding pocket (LBP) is practically filled with side chains (in particular with Phe(328), which is replaced by Ala in ERRbeta and ERRgamma). We present here the crystal structure of the ligand binding domain of ERRalpha (containing the mutation C325S) in complex with the inverse agonist cyclohexylmethyl-(1-p-tolyl-1H-indol-3-ylmethyl)-amine (compound 1a), to a resolution of 2.3A(.) The structure reveals the dramatic multiple conformational changes in the LBP, which create the necessary space for the ligand. As a consequence of the new side chain conformation of Phe(328) (on helix H3), Phe(510)(H12) has to move away, and thus the activation helix H12 is displaced from its agonist position. This is a novel mechanism of H12 inactivation, different from ERRgamma, estrogen receptor (ER) alpha, and ERbeta. H12 binds (with a surprising binding mode) in the coactivator groove of its ligand binding domain, at a similar place as a coactivator peptide. This is in contrast to ERRgamma but resembles the situation for ERalpha (raloxifene or 4-hydroxytamoxifen complexes). Our results explain the novel molecular mechanism of an inverse agonist for ERRalpha and provide the basis for rational drug design to obtain isotype-specific inverse agonists of this potential new drug target. Despite a practically filled LBP, the finding that a suitable ligand can induce an opening of the cavity also has broad implications for other orphan nuclear hormone receptors (e.g. the NGFI-B subfamily).

Luminescence study of G-quadruplex formation in the presence of Tb 3+ ion

The interactions of Tb 3+ with the quadruplex-forming oligonucleotide bearing human telomeric repeat sequence d(G 3T 2AG 3T 2AG 3T 2AG 3), (htel21), have been studied using luminescence spectroscopy and circular dichroism (CD). Enhanced luminescence of Tb 3+, resulting from energy transfer from guanines, indicated encapsulation of Tb 3+ ion in the central cavity of quadruplex core. The ability of lanthanide ions (Eu 3+ and Tb 3+) to mediate formation of quadruplex structure has been further evidenced by the fluorescence energy transfer measurements with the use of oligonucleotide probe labeled with fluorescein and rhodamine FRET partners, FAM-htel21-TAMRA. The CD spectra revealed that Tb 3+/htel21 quadruplex possesses antiparallel strand orientation, similarly as sodium quadruplex. Tb 3+ binding equilibria have been investigated in the absence and the presence of competing metal cations. At low Tb 3+ concentration (8 μM) Tb 3+/htel21 quadruplex stability is very high (5 × 10 6 M −1) and stoichiometry of 5–7 Tb 3+ ions per one quadruplex molecule is observed. Luminescence and CD titration experiments suggested that the cavity of quadruplex accommodates two Tb 3+ ions and the remaining Tb 3+ ions bind probably to TTA loops of quadruplex. Higher concentration of Tb 3+ (above 10 μM) results in the excessive binding of Tb 3+ ions that finally destabilizes quadruplex, which undergoes transformation into differently organized assemblies. Such assemblies (probably possessing multiple positive charge) exhibit kinetic stability, which is manifested by a very slow kinetics of displacement of Tb 3+ ion by competing cations (Li +, Na +, K +).

Computed Pore Potentials of the Nicotinic Acetylcholine Receptor

Electrostatic surface potentials in the vestibule of the nicotinic acetylcholine receptor (nAChR) were computed from structural models using the University of Houston Brownian Dynamics program to determine their effect on ion conduction and ionic selectivity. To further determine whether computed potentials accurately reflect the electrostatic environment of the channel, the potentials were used to predict the rate constants for diffusion-enhanced fluorescence energy transfer; the calculated energy transfer rates are directly comparable with those determined experimentally (see companion article by Meltzer et al. in this issue). To include any effects on the local potentials by the bound acceptor fluorophore crystal violet, its binding site was first localized within the pore by fluorescence energy transfer measurements from dansyl-C6-choline bound to the agonist sites and also by simulations of binding using Autodock. To compare the computed potentials with those determined experimentally, we used the predicted energy transfer rates from Tb 3+ chelates of varying charge to calculate an expected potential using the Boltzmann relationship. This expected potential (from −20 to −40 mV) overestimates the values determined experimentally (from −10 to −25 mV) by two- to fourfold at similar conditions of ionic strength. Although the results indicate a basic discrepancy between experimental and computed surface potentials, both methods demonstrate that the vestibular potential has a relatively small effect on conduction and selectivity.

Evidence for Assembly of Small Multidrug Resistance Proteins by a “Two-faced” Transmembrane Helix

Clinically significant bacterial resistance to drugs and cytotoxic compounds can be conferred by the energy-dependent efflux of toxicants, catalyzed by proteins embedded in the bacterial cell membrane. One such group of proteins, the small multidrug resistance family, are drug/proton antiporters that must oligomerize to function, a process that requires the assembly of at least two inactive monomers by intermolecular association of their four transmembrane helices. Here, we have used peptides that correspond to each of the four wild type transmembrane helices of the Halobacterium salinarum protein Hsmr and a corresponding library of mutant peptides to determine the interactive surfaces that likely contribute to protein oligomerization. Hsmr peptides were examined for strong (sodium dodecyl sulfate-resistant) and weaker (perfluorooctanoate-resistant) helix-helix interactions, in conjunction with circular dichroism, fluorescence energy transfer measurements, and molecular modeling. The results are compatible with a scheme in which two faces of helix four permit self-assembly via a higher affinity asymmetric pairing and a lower affinity symmetric interaction, resulting in a discrete tetramer. Our finding that two surfaces of helix four can contribute to the stability of small multidrug resistance protein assembly provides a molecular basis for the design of therapeutics that target this antibiotic resistance mechanism.

The biotin-capture lipid affinity assay: a rapid method for determining lipid binding parameters for apolipoproteins

The lipid affinity of plasma apolipoproteins is an important modulator of lipoprotein metabolism. Mutagenesis techniques have been widely used to modulate apolipoprotein lipid affinity for studying biological function, but the approach requires rapid and reliable lipid affinity assays to compare the mutants. Here, we describe a novel method that measures apolipoprotein binding to a standardized preparation of small unilamellar vesicles (SUVs) containing trace biotinylated and fluorescent phospholipids. After a 30 min incubation at various apolipoprotein concentrations, vesicle-bound protein is rapidly separated from free protein on columns of immobilized streptavidin in a 96-well microplate format. Vesicle-bound protein and lipid are eluted and measured in a fluorescence microplate reader for calculation of a dissociation constant and the maximum number of potential binding sites on the SUVs. Using human apolipoprotein A-I (apoA-I), apoA-IV, and mutants of each, we show that the assay generates binding constants that are comparable to other methods and is reproducible across time and apolipoprotein preparations. The assay is easy to perform and can measure triplicate binding parameters for up to 10 separate apolipoproteins in 3.5 h, consuming only 120 microg of apolipoprotein in total. The benefits and potential drawbacks of the assay are discussed.

Familial Mutants of α-Synuclein with Increased Neurotoxicity Have a Destabilized Conformation

A30P and A53T mutations of the presynaptic protein alpha-synuclein are associated with familial forms of Parkinson disease. NMR spectroscopy demonstrates that Parkinsonism-linked mutations greatly perturb specific tertiary interactions essential for the native state of alpha-synuclein. However, alpha-synuclein is not completely unfolded but exhibits structural fluctuations on the time scale of secondary structure formation and loses its native conformation gradually when protein stability decreases. The redistribution of the ensemble of alpha-synuclein conformers may underlie toxic gain-of-function by fostering self-association and altered binding affinity to ligands and receptors.