Study of the phosphorescent bases of yeast phenylalanine transfer RNA with the aid of optical detection of magnetic resonance

The phosphorescence of brewers' yeast phenylalanine transfer RNA has been investigated at 77 °K and at 1.2 °K in pumped liquid helium. Although the phosphorescence at 77 °K originates almost completely from the Y base in the anticodon loop, independent of excitation wavelength, the phosphorescence originates from normal bases with 270 nm excitation at temperatures in the helium range. The low-temperature phosphorescence is assigned to the triplet state of adenosine by optical detection of magnetic resonance measurements. The adenosine phosphorescence at 1.2 °K is quenched by the binding of the codon poly(U), as well as by the removal of Mg²⁺. The former result indicates that the adenosine phosphorescence originates from the anticodon, -Gm-A-A-, while the second shows that a conformational change introduced by removing Mg²⁺ (possibly involving unstacking of the anticodon) prevents energy trapping in the anticodon triplet state. The lack of triplet energy transfer from anticodon to Y indicates that Y cannot be stacked with the anticodon in the conformation that is stable at helium temperature. The adenosine phosphorescence of transfer RNA^Phe is nearly completely quenched at 77 °K, at least partially due to energy transfer to Y. We think that the thermally activated energy transfer is associated with some mobility of the Y base at 77 °K. Our observations are in contrast with previous results on bakers' yeast tRNA^Phe where there is apparently little, if any, energy transfer to Y from the normal nucleotides at 80 °K with 265 nm excitation. Optically detected magnetic resonance measurements on the triplet state of Y base in various environments indicate that removal of Mg²⁺ causes a shift of the Y base in tRNA^Phe to a more solvent-exposed position, whereas the binding of poly(U) has little effect on the environment of Y.     

Antisense activity detection by inhibition of fluorescence resonance energy transfer.

Use of antisense nucleic acids to modulate expression of particular genes is a promising approach to the therapy of human papillomavirus type 16 (HPV-16)-associated cervical cancer. Understandably, evaluation of the in vivo performance of synthetic antisense oligodeoxynucleotides (AS-ODNs) or ribozymes is of ultimate importance to development of effective antisense tools. Here we report the use of a bacterial reporter system based on the inhibition of fluorescence resonance energy transfer (FRET) to measure the interaction of AS-ODNs with HPV-16 target nt 410-445, using variants of the green fluorescent protein (GFP). An optimal FRET-producing pair was selected with GFP as the donor and yellow fluorescent protein (YFP) as the acceptor molecule. Hybridization of AS-ODNs with a chimaeric mRNA containing the antisense target site flanked by GFP variants resulted in the inhibition of the FRET effect. Use of different linkers suggested that the amino acid content of the linker has no significant effect on FRET effect. Antisense accessibility, tested by RNaseH assays with phosphorothioated target-specific and mutant AS-ODNs, suggested a specific effect on the chimaeric mRNA. FRET inhibition measurements correlated with the presence of truncated proteins confirming true antisense activity over the target. Therefore, FRET inhibition may be used for the direct measurement of AS-ODNs activity in vivo.     

Cofilin, actin and their complex observed in vivo using fluorescence resonance energy transfer

Actin is the principal component of microfilaments. Its assembly/disassembly is essential for cell motility, cytokinesis, and a range of other functions. Recent evidence suggests that actin is present in the nucleus where it may be involved in the regulation of gene expression and that cofilin binds actin and can translocate into the nucleus during times of stress. In this report, we combine fluorescence resonance energy transfer and confocal microscopy to analyze the interactions of cofilin and G-actin within the nucleus and cytoplasm. By measuring the rate of photobleaching of fluorescein-labeled actin in the presence and absence of Cy5-labeled cofilin, we determined that almost all G-actin in the nucleus is bound to cofilin, whereas approximately (1/2) is bound in the cytoplasm. Using fluorescence resonance energy transfer imaging techniques we observed that a significant proportion of fluorescein-labeled cofilin in both the nucleus and cytoplasm binds added tetramethylrhodamine-labeled G-actin. Our data suggest there is significantly more cofilin-G-actin complex and less free cofilin in the nucleus than in the cytoplasm.     

Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy.

Fluorescence resonance energy transfer (FRET) is a technique used for quantifying the distance between two molecules conjugated to different fluorophores. By combining optical microscopy with FRET it is possible to obtain quantitative temporal and spatial information about the binding and interaction of proteins, lipids, enzymes, DNA, and RNA in vivo. In conjunction with the recent development of a variety of mutant green fluorescent proteins (mtGFPs), FRET microscopy provides the potential to measure the interaction of intracellular molecular species in intact living cells where the donor and acceptor fluorophores are actually part of the molecules themselves. However, steady-state FRET microscopy measurements can suffer from several sources of distortion, which need to be corrected. These include direct excitation of the acceptor at the donor excitation wavelengths and the dependence of FRET on the concentration of acceptor. We present a simple method for the analysis of FRET data obtained with standard filter sets in a fluorescence microscope. This method is corrected for cross talk (any detection of donor fluorescence with the acceptor emission filter and any detection of acceptor fluorescence with the donor emission filter), and for the dependence of FRET on the concentrations of the donor and acceptor. Measurements of the interaction of the proteins Bcl-2 and Beclin (a recently identified Bcl-2 interacting protein located on chromosome 17q21), are shown to document the accuracy of this approach for correction of donor and acceptor concentrations, and cross talk between the different filter units.     

Direct detection of phospholamban and sarcoplasmic reticulum Ca-ATPase interaction in membranes using fluorescence resonance energy transfer

We used fluorescence resonance energy transfer (FRET) to detect and quantitate the interaction of the sarcoplasmic reticulum Ca-ATPase (SERCA) with phospholamban (PLB) in membranes. PLB inhibits SERCA only at submicromolar Ca. It has been proposed that relief of inhibition at micromolar Ca is due to dissociation of the inhibitory complex. To test this hypothesis, we co-reconstituted donor-labeled SERCA and acceptor-labeled I40A-PLB (superinhibitory, monomeric PLB mutant) in membranes of defined lipid and protein composition, with full retention of Ca-dependent ATPase activity and inhibitory regulation by PLB. FRET from SERCA to PLB was measured as a function of membrane concentrations of PLB and SERCA, and functional activity was measured on the same samples. The data revealed clearly that the stoichiometry of binding is one PLB per SERCA, and that binding is a strict function of the ratio of total PLB to SERCA in the membrane. We conclude that the dissociation constant of PLB binding to SERCA is far less than physiological PLB membrane concentrations. Binding at low Ca (pCa 6.5), where I40A-PLB inhibits SERCA, was virtually identical to that at high Ca (pCa 5.0), where no inhibition was observed. However, the limiting energy transfer at saturating PLB was less at high Ca, indicating a greater donor-acceptor distance. We conclude that (a) the affinity of PLB for SERCA is so great that PLB is essentially a SERCA subunit under physiological conditions and (b) relief of inhibition at micromolar Ca is due to a structural rearrangement within the SERCA-PLB complex, rather than dissociation.     

Improving lanthanide-based resonance energy transfer detection by increasing donor-acceptor distances

Lanthanide-based resonance energy transfer (LRET) is an established method for measuring or detecting proximity between a luminescent lanthanide (energy donor) and an organic fluorophore (energy acceptor). Because resonance energy transfer is a distance-dependent phenomenon that increases in efficiency to the 6th power of the distance between the donor and the acceptor, assay systems are often designed to minimize donor-acceptor distances. However, the authors show that because of the R(6) relationship between transfer efficiency and sensitized emission lifetime, energy transfer can be difficult to measure in a time-gated manner when the donor-acceptor distance is small relative to the F?rster radius. In such systems, the advantages inherent in time-resolved, ratiometric measurements are lost but can be regained by designing the system such that the average donor-acceptor distance is increased.     

Characterization of protein triplet states by optical detection of magnetic resonance

The technique of optical detection of magnetic resonance (ODMR) is applied for the first time to the study of molecules of biological interest in frozen glassy solutions. We present results describing the triplet state properties of tryptophan, tyrosine, and the tryptophan and tyrosine residues of bovine serum albumin.     

Simulation of coherent energy transfer in an alpha-helical peptide by Fermi resonance.

A mechanism by which NH stretching quanta are coherently transported along a chain of hydrogen bonded peptide groups is demonstrated by classical simulation of a section of the alpha-helical peptide poly(L-alanine). Vibrational motion takes place on a complex energy surface constructed from earlier ab initio and empirical surfaces. A speculative hypothesis of the biological role of this mechanism is presented, and the critical parameters governing the dynamics are identified and discussed.     

Luminescence resonance energy transfer measurements in myosin.

Myosin is thought to generate force by a rotation between the relative orientations of two domains. Direct measurements of distances between the domains could potentially confirm and quantify these conformational changes, but efforts have been hampered by the large distances involved. Here we show that luminescence resonance energy transfer (LRET), which uses a luminescent lanthanide as the energy-transfer donor, is capable of measuring these long distances. Specifically, we measure distances between the catalytic domain (Cys707) and regulatory light chain domain (Cys108) of the myosin head. An energy transfer efficiency of 21.2 +/- 1.9% is measured in the myosin complex without nucleotide or actin, corresponding to a distance of 73 A, consistent with the crystal structure of Rayment et al. Upon binding to actin, the energy transfer efficiency decreases by 4.5 +/- 1.0%, indicating a conformational change in myosin that involves a relative rotation and/or translation of Cys707 relative to the light chain domain. Addition of ADP also alters the energy transfer efficiency, likely through a rotation of the probe attached to Cys707. These results demonstrate that LRET is capable of making accurate measurements on the relatively large actomyosin complex, and is capable of detecting conformational changes between the catalytic and light chain domains of myosin.     

Homogeneous detection of concanavalin A using pyrene-conjugated maltose assembled graphene based on fluorescence resonance energy transfer

In this work, we proposed a novel biosensor to homogeneously detect concanavalin A (ConA) using pyrene-conjugated maltose assembled graphene based on fluorescence resonance energy transfer (FRET). Maltose-grafted-aminopyrene (Mal-Apy) was synthesized and characterized by mass spectra, UV-vis and fluorescence spectra. The Mal-Apy was further employed for fluorescence switch and ConA recognition. When Mal-Apy was self-assembled on the surface of graphene by means of π-stacking interaction, its fluorescence was adequately quenched because the graphene acted as a "nanoquencher" of the pyrene rings due to FRET. As a result, in the presence of ConA, competitive binding of ConA with glucose destroyed the π-stacking interaction between the pyrene and graphene, thereby causing the fluorescence recovery. This method was demonstrated the selective sensing of ConA, and the linear range is 2.0 × 10?2 to 1.0 μM with the linear equation y=1.029x + 0.284 (R = 0.996). The limit of detection for ConA was low to 0.8 nM, and the detection of ConA could be performed in 5 min, indicating that this method could be used for fast, sensitive, and selective sensing of ConA. Such data suggests that the graphene FRET platform is a great potential application for protein-carbohydrate studies, and would be widely applied in drug screening, bimolecular recognition and disease diagnosis.     

Imaging protein-protein interactions using fluorescence resonance energy transfer microscopy

Fluorescence resonance energy transfer (FRET) detects the proximity of fluorescently labeled molecules over distances >100 A. When performed in a fluorescence microscope, FRET can be used to map protein-protein interactions in vivo. We here describe a FRET microscopy method that can be used to determine whether proteins that are colocalized at the level of light microscopy interact with one another. This method can be implemented using digital microscopy systems such as a confocal microscope or a wide-field fluorescence microscope coupled to a charge-coupled device (CCD) camera. It is readily applied to samples prepared with standard immunofluorescence techniques using antibodies labeled with fluorescent dyes that act as a donor and acceptor pair for FRET. Energy transfer efficiencies are quantified based on the release of quenching of donor fluorescence due to FRET, measured by comparing the intensity of donor fluorescence before and after complete photobleaching of the acceptor. As described, this method uses Cy3 and Cy5 as the donor and acceptor fluorophores, but can be adapted for other FRET pairs including cyan fluorescent protein and yellow fluorescent protein.     

Global structure of four-way RNA junctions studied using fluorescence resonance energy transfer.

Four-way helical junctions are found widely in natural RNA species. In this study, we have studied the conformation of two junctions by fluorescence resonance energy transfer. We show that the junctions are folded by pairwise coaxial helical stacking, forming one predominant stacking conformer in both examples studied. At low magnesium ion concentrations, the helical axes of both junctions are approximately perpendicular. One junction undergoes a rotation into a distorted antiparallel structure induced by the binding of a single magnesium ion. By contrast, the axes of the four-way junction of the U1 snRNA remain approximately perpendicular under all conditions examined, and we have determined the stacking conformer adopted.     

Structural determination of lipid-bound ApoA-I using fluorescence resonance energy transfer

Based on the x-ray crystal structure of lipid-free Delta43 apoA-I, two monomers of apoA-I were suggested to bind to a phospholipid bilayer in an antiparallel paired dimer, or "belt orientation." This hypothesis challenges the currently held model in which each of the two apoA-I monomers fold as antiparallel alpha-helices or "picket fence orientation." When apoA-I is bound to a phospholipid disc, the first model predicts that the glutamine at position 132 on one apoA-I molecule lies within 16 A of glutamine 132 in the second monomer, whereas, the second model predicts glutamines at position 132 to be 104 A apart. To distinguish between these models, glutamine at position 132 was mutated to cysteine in wild-type apoA-I to produce Q132C apoA-I, which were labeled with thiol-reactive fluorescent probes. Q132C apoA-I was labeled with either fluorescein (donor probe) or tetramethylrhodamine (acceptor probe) and then used to make recombinant phospholipid discs (recombinant high density lipoprotein (rHDL)). The rHDL containing donor- and acceptor-labeled Q132C apoA-I were of similar size, composition, and lecithin:cholesterol acyltransferase reactivity when compared to rHDL-containing human plasma apoA-I. Analysis of donor probe fluorescence showed highly efficient quenching in rHDL containing one donor- and one acceptor-labeled Q132C apoA-I. rHDL containing only acceptor probe-labeled Q132C apoA-I showed rhodamine self-quenching. Both of these observations demonstrate that position 132 in two lipid-bound apoA-I monomers were in close proximity, supporting the "belt conformation" hypothesis for apoA-I on rHDL.     

Quantitative analysis of membrane protein-amphiphile interactions using resonance energy transfer

This work describes a simple method for determining the association constant of amphiphiles to membrane proteins. The method uses a fluorescent phospholipid probe, which senses the competition among unlabeled amphiphiles for positions on the transmembrane surface of the protein. The contact between the probe and the protein surface is detected through resonance energy transfer. We have analyzed theoretically this process deriving a general equation for the dependence of the energy transfer efficiency on the composition of the micelles/bilayers in which the protein is inserted. This equation includes an exchange constant for each amphiphile, which gives a measure of its affinity for the protein with respect to that of an amphiphile set as the reference. We applied this method to determine the exchange constant of different phospholipids for the plasma membrane calcium pump.     

Live cell imaging of protein interactions in poliovirus RNA replication complex using fluorescence resonance energy transfer (FRET)

Poliovirus RNA replication is directed by a replication complex on the rosette-like arrangement of membranous vesicles. Proteins derived from the p3 region of the polioviral genome, such as 3D, 3AB, and 3B (VPg), play key roles in the formation and function of the replication complex. In the present study, by using an acceptor photobleaching protocol for fluorescence resonance energy transfer (FRET) imaging, we visualized the interactions of 3D, 3AB, and VPg in living cells. The interaction of 3AB-VPg was determined by live cell FRET analysis. Quantitative analyses showed that the FRET efficiencies of 3AB-3D, VPg-3D, and 3AB-VPg were 3.9 ± 0.4% (n = 36), 4.5 ± 0.4% (n = 39), and 8.3 ± 0.6% (n = 44), respectively, in the cell cytoplasm where viral replication complexes are formed and function. Poliovirus infection enhanced the protein interactions of VPg-3D and 3AB-3D, with FRET efficiencies in the virus-infected cells of 10.7 ± 1.1% (n = 39) and 9.0 ± 0.9% (n = 37), respectively. This method of live cell analysis of protein interactions in the poliovirus RNA replication complex lays the foundation for further understanding of the real-time process of poliovirus RNA replication.     

Flexibility of myosin-subfragment-1 in its complex with actin as revealed by fluorescence resonance energy transfer.

The flexibility of the acto-myosin complex in rigor conditions was characterized by measuring the temperature profile of normalized fluorescence resonance energy transfer efficiency, f' [Somogyi, B., Matkó, J., Papp, S., Hevessy, J., Welch, G.R. & Damjanovich, S. (1984) Biochemistry 23, 3403-3411]. Fluorescence acceptors were introduced to the Cys374 residues of actin and the donors were covalently attached either to Cys707 in the catalytic domain or to Cys177 in the essential light-chain of myosin S1. Fluorescence resonance energy transfer measurements revealed that the protein matrix between Cys374 of actin and Cys707 of S1 is rigid. In contrast, the link between the catalytic and light-chain-binding domains in myosin S1 is flexible. We have recently shown that the positional distribution of Cys707 was narrow relative to the actin filament, while that of the Cys177 was broad. Accordingly, the broad positional distribution of Cys177 is likely to be due to the large flexibility of the link between the catalytic and light-chain-binding domains. This flexibility is probably essential for the interdomain reorganization of the myosin head during the force generation process and for accommodating the symmetry difference between actin and myosin filaments to allow the formation of cross-bridges.     

Calculation of resonance energy transfer in crowded biological membranes.

Analytical and numerical models were developed to describe fluorescence resonance energy transfer (RET) in crowded biological membranes. It was assumed that fluorescent donors were linked to membrane proteins and that acceptors were linked to membrane lipids. No restrictions were placed on the location of the donor within the protein or the partitioning of acceptors between the two leaflets of the bilayer; however, acceptors were excluded from the area occupied by proteins. Analytical equations were derived that give the average quantum yield of a donor at low protein concentrations. Monte Carlo simulations were used to generate protein and lipid distributions that were linked numerically with RET equations to determine the average quantum yield and the distribution of donor fluorescence lifetimes at high protein concentrations, up to 50% area fraction. The Monte Carlo results show such crowding always reduces the quantum yield, probably because crowding increases acceptor concentrations near donor-bearing proteins; the magnitude of the reduction increases monotonically with protein concentration. The Monte Carlo results also show that the distribution of fluorescence lifetimes can differ markedly, even for systems possessing the same average lifetime. The dependence of energy transfer on acceptor concentration, protein radius, donor position within the protein, and the fraction of acceptors in each leaflet was also examined. The model and results are directly applicable to the analysis of RET data obtained from biological membranes; their application should result in a more complete and accurate determination of the structures of membrane components.     

Investigation of phospholipase-lipid interactions by optical detection of triplet state magnetic resonance spectroscopy

We have investigated the binding of porcine pancreatic phospholipase A2 (PA2) to n-hexadecylphosphocholine (C16PN) micelles using optical detection of triplet state magnetic resonance (ODMR) spectroscopy. The zero field splittings (zfs) of the single Trp3 residue undergo significant changes upon binding of PA2 to C16PN micelles. Zfs titrations of PA2 vs C16PN indicate that the binding stoichiometry is C16PN:PA2 approximately 25. A reduction of the (E) parameter from 1.227 to 1.135 GHz is postulated to result from Stark effects caused by the binding of a polar group (possibly phosphocholine) near Trp3 in the PA2-C16PN micelle complex.