Fluorescence Resonance Energy Transfer Approach Research Articles

FRET Detection of CaM-RyR2 Binding Modulation by S100A1

Using fluorescence resonance energy transfer (FRET), we are testing the hypothesis that S100A1 competes with calmodulin (CaM) for binding to cardiac ryanodine receptors (RyR2). In isolated pig cardiac sarcoplasmic reticulum (SR) vesicles, we targeted a donor-labeled FKBP (D-FKBP) to the RyR2 cytosolic headpiece. We then detected FRET as a decrease of donor fluorescence in the presence of CaM labeled with acceptor within the N- or C-lobe (denoted AN-CaM and AC-CaM, respectively), thus directly and specifically indexing CaM binding in the proximity of D-FKBP. FRET between D-FKBP and A-CaMs (100 nM) was completely inhibited by unlabeled WT-CaM, with KI ≈ 100 nM, indicating that WT-CaM and A-CaMs compete with similar affinities for the same RyR2 binding site. However, S100A1 (ranging from 0.1 to 30 μM) had no significant effect on FRET when cardiac SR was concurrently incubated with S100A1 and AN-CaM. Upon sequentially incubating the SR with S100A1 first, then with AN-CaM, we found partial inhibition of FRET, but with KI ≈ 30 μM S100A1. This effect is more pronounced in nM Ca2+ (versus μM Ca2+). Intriguingly, FRET between D-FKBP and AC-CaM was not significantly affected by S100A1. S100A1 lowered maximum FRET for AN-CaM but did not significantly change its binding affinity. This suggests that S100A1 allosterically interacts with RyR-CaM binding, rather than directly competing for the same binding site as CaM. We are currently developing a complementary FRET approach, using acceptor-labeled S100A1, to specifically resolve the S100A1 binding to RyR. Ultimately, we aim to elucidate the interplay between S100A1 and CaM binding to RyR.

Cell:cell interactions in the immune system

Video podcast available Go to www.immunologicalreviews.com to watch an interview with Guest Editor Facundo Batista. The cells of the immune system are continually on the move! Naive T cells in a human collectively crawl over 10 000 miles a day in search of antigens (1). Each dendritic cell (DC) touches 5000 T cells an hour to scan repertoires over a million deep (2, 3). The adaptive immune system functions through a vast network of provisional cell–cell interactions that are continuously changing. When this frenetic scanning finds a fit between T-cell antigen receptor (TCR) and DC antigen, the interaction can be prolonged with a pronounced deceleration of the T cell and finally a long-lived interaction that has been described as an immunological synapse (2, 4). In vitro studies on the immunological synapse suggest a profound supramolecular organization that serves focused trans-synaptic and juxtracrine information transfer between cells (5–8). Immunological synapses are not restricted to T cells but are also formed by natural killer (NK) cells, B cells, granulocytes, and phagocytes using related immunotyrosine-based activation motif-containing receptors (9, 10). These interactions are profoundly influenced by the three-dimensional (3D) tissue environment (11). This volume of Immunological Reviews takes a broader look at the cell–cell interactions in the immune system from the molecular organization of antigen receptors during surveillance to the organization of stromal cell networks in lymph nodes, bone marrow, and the lung (Fig. 1). The organization of the plasma membrane is a fundamental issue in biology that sets the stage for cell–cell interaction and communication. The classical fluid mosaic model suggested membrane proteins diffusing in a lipid sea with potential for interactions with cytoplasmic and extra-cytoplasmic binding partners that account for regulation and processes like adhesion and receptor capping. Observations on lipid phases in model systems with disordered, liquidordered, and gel phases in order of increasing viscosity suggested that such phases could also exist in biological membranes on some scale (12). Biophysical observations of liquid-ordered domains enriched in various lipid-modified proteins have led to models in which lipids have a role in providing a higher level of organization (13). Most recently, evidence for protein-driven domains, referred to as Immunological Reviews 2013 Vol. 251: 7–12 Printed in Singapore. All rights reserved

Conformational Dynamics of Prothrombin Dictate Its Ordered Cleavage by Prothrombinase

Abstract 3359The prothrombinase complex (membranes/Xa/Va) binds prothrombin through exosite interactions which orient and present the substrate to the protease active site for cleavage. Although both cleavage sites within the substrate are accessible, prothrombin is preferentially activated by prothrombinase through sequential cleavage at R320 followed by cleavage at R271. We investigated the exosite-dependent interaction between prothrombin and prothrombinase using fluorescence resonance energy transfer (FRET). An acceptor fluorophore was placed onto the prothrombin cleavage site by introducing the mutation R271C and fluorescently modifying the introduced thiol (IIC271*). The active site of Xa was covalently modified with a chloromethyl ketone conjugated to a donor fluorescent compound. Titrations of the fluorescently labeled prothrombinase complex with IIC271* revealed a maximum energy transfer efficiency of 70–80 % observed with two different donor probes. Resonance energy transfer arising from the substrate docking interaction was confirmed by equivalent changes in the excited state lifetime of the donors. The calculated distance between the probe at C271 and the probe tethered at the active site of Xa ranged between 34 and 40 Å. Variation in the measured distance reflects the different lengths of the tethers used to connect the donor probes. The FRET measurements, validated with two donor-acceptor pairs, indicate that the 271 site within prothrombin docked to prothrombinase is positioned distant from the active site of Xa within the enzyme complex. This distant constraint prevents R271 from effectively engaging the active site of Xa within prothrombinase, thereby providing a physical explanation for the ordered action of prothrombinase on prothrombin. It follows that initial cleavage at R320 must reorient the substrate to allow R271 access to the Xa active site. We pursued an intramolecular FRET approach in which both donor and acceptor probes were placed on the prothrombin molecule to investigate the putative conformational change of prothrombin as a result of initial cleavage. Orthogonal labeling of prothrombin was accomplished using a prothrombin variant containing C271 and a LPETG extension at its C terminus. The optimized C271 label was used for the first modification. For the second, the sequence-specific transpeptidation activity of bacterial Sortase A was used to incorporate a fluorophore at the C-terminus (IICterm*). To assess the new fluorescent modification, FRET studies of prothrombin binding to prothrombinase were repeated with the new IICterm* construct. Titrations revealed a maximal energy transfer of 16 %, confirmed by corresponding changes in excited state lifetime and yielded a Kd = 39 nM indistinguishable from the Kd= 41 nM seen for IIC271* binding. Thus, two different approaches illustrate that the affinity of prothrombin for prothrombinase is independent of active site docking by the substrate. To examine the intramolecular dynamics of prothrombin during activation, the C271 site was labeled with BADAN (donor) and the Sortase labeling method placed the Alexa532 (acceptor) at the C terminus. The orthogonal placement of BADAN and Alexa532 on the prothrombin molecule resulted in extensive energy transfer between the two probes. The cleavage of doubly-labeled prothrombin to produce meizothrombin was associated with a change in intramolecular energy transfer. The variation of the energy transfers observed between the zymogen and protease forms can be interpreted as global rearrangement of the prothrombin molecule renders the second cleavage site accessible of active site docking. The FRET studies provide a comprehensive physical explanation for the ordered cleavage of prothrombin by prothrombinase. Initial cleavage occurs at the spatially accessible cleavage site R320. The prothrombin molecule then significantly re-orients itself making the second R271 cleavage site accessible to prothrombinase in order for activation to proceed and produce thrombin. Disclosures:No relevant conflicts of interest to declare.

Mechanism for Calcium Ion Sensing by the C2A Domain of Synaptotagmin I

The C2A domain is one of two calcium ion (Ca2+)- and membrane-binding domains within synaptotagmin I (Syt I), the identified Ca2+ sensor for regulated exocytosis of neurotransmitter. We propose that the mechanistic basis for C2A's response to Ca2+ and cellular function stems from marginal stability and ligand-induced redistributions of protein conformers. To test this hypothesis, we used a combination of calorimetric and fluorescence techniques. We measured free energies of stability by globally fitting differential scanning calorimetry and fluorescence lifetime spectroscopy denaturation data, and found that C2A is weakly stable. Additionally, using partition functions in a fluorescence resonance energy transfer approach, we found that the Ca2+- and membrane-binding sites of C2A exhibit weak cooperative linkage. Lastly, a dye-release assay revealed that the Ca2+- and membrane-bound conformer subset of C2A promote membrane disruption. We discuss how these phenomena may lead to both cooperative and functional responses of Syt I.

Distinct Dendritic Spine and Nuclear Phases of Calcineurin Activation after Exposure to Amyloid-β Revealed by a Novel Fluorescence Resonance Energy Transfer Assay

Calcineurin (CaN) activation is critically involved in the regulation of spine morphology in response to oligomeric amyloid-β (Aβ) as well as in synaptic plasticity in normal memory, but no existing techniques can monitor the spatiotemporal pattern of CaN activity. Here, we use a spectral fluorescence resonance energy transfer approach to monitor CaN activation dynamics in real time with subcellular resolution. When oligomeric Aβ derived from Tg2576 murine transgenic neurons or human AD brains were applied to wild-type murine primary cortical neurons, we observe a dynamic progression of CaN activation within minutes, first in dendritic spines, and then in the cytoplasm and, in hours, in the nucleus. CaN activation in spines leads to rapid but reversible morphological changes in spines and in postsynaptic proteins; longer exposure leads to NFAT (nuclear factor of activated T-cells) translocation to the nucleus and frank spine loss. These results provide a framework for understanding the role of calcineurin in synaptic alterations associated with AD pathogenesis.

Evaluation of Fluorescence Resonance Energy Transfer Approaches as a Tool to Quantify the Stability of Antisense Oligodeoxynucleotides

Antisense oligodeoxynucleotides (AsODN) are rapidly degraded by nucleases in biological fluids which compromises their efficacy as therapeutic agents. This study evaluated two Fluorescence Resonance Energy Transfer (FRET) approaches, Acceptor Photobleaching and Sensitized Emission, in terms of their suitability for quantification of oligonucleotide stability in various colloidal carrier systems in vitro. The influence of the formulations pH and viscosity on the validity of the two approaches was determined and showed that the donor fluorescence intensity was highly susceptible to pH fluctuations of the medium. Moreover, the Acceptor Photobleaching approach proved to be unsuitable for the proposed studies due to Brownian motion of molecules in liquid formulations, suggesting that this method can only be used for immobilized specimens. The stability of a 30-mer AsODN incorporated into various in situ gelling systems was evaluated using the Sensitized Emission approach. This approach appeared to offer a simple tool to evaluate the stability of AsODN and showed stable molecules over a period of one week. However, a number of criteria, such as photobleaching due to repeated exposure, pH of the surrounding medium and sample preparation need to be carefully considered when performing quantitative FRET measurements. Keywords: Acceptor photobleaching, Brownian motion, Fluorescence resonance energy transfer, In situ gelling systems, Stability, pH, Image analysis, Sensitized emission, Viscosity, High performance liquid chromatography, Electrospray ionisation mass spectrometry

Enhanced Detection of Lipid Transfer Protein Activity by Resonance Energy Transfer with Tetramethyl-Bodipy Labeled Lipids

Real-time monitoring of lipid transfer between membranes can be achieved with fluorescence-based approaches. Ideally, the fluorophore probe should have a high extinction coefficient, quantum yield, and photostability. We previously showed the usefulness of 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-yl (Me4-BODIPY-8) fluorophore as a lipid-probe for overcoming deficiencies of other probes [1,2]. Here, we have expanded our evaluation of Me4-BODIPY-8 sphingolipids and glycerolipids by using rhodamine 101-diglyceride (Rh101-DG), rhodamine B-diglyceride (RhB-DG), and C18-tetramethylindocarbocyanine-5,5’-disulfonic acid (C18-diI) as energy acceptors during resonance energy transfer (RET). Each RET system was assessed for sensitivity and accuracy of time-dependent, glycolipid intervesicular transfer by human glycolipid transfer protein (GLTP). Me4-BODIPY-8-PC served as negative control for GLTP specificity. A long linker chain for attaching Me4-BODIPY-8 was essential for maintaining low spontaneous transfer of Me4-BODIPY-8 lipids. Use of Rh-B-DG, Rh-101-DG and C18-DiI also minimized spontaneous transfer compared to RhB-PE and avoided the self-quenching observed when DPH-PC was used as energy donor in the RET system. Our results show that tetramethyl-BODIPY-based energy transfer approaches provide increased sensitivity over previous lipid transfer protein assay systems ([2] and references therein) and show promise for direct monitoring of lipid protein transfer activities within the cellular milieu in the visible light range. [Support: NIH/NIGMS GM45928, Hormel Foundation; Russian Foundation for Basic Research 09-04-00313] [1] Boldyrev, I.A., X. Zhai, M.M. Momsen, H.L. Brockman, R.E. Brown, and J.G. Molotkovsky. 2007. New BODIPY lipid probes for fluorescence studies of membranes. J. Lipid Res. 48: 1518-1532. [2] Mattjus, P., J. G. Molotkovsky, J.M. Smaby, R.E. Brown. 1999. A fluorescence resonance energy transfer approach for monitoring protein-mediated glycolipid transfer between vesicle membranes. Anal. Biochem. 268: 297-304.

Computer Aided Distance Reconstruction in Single Molecule FRET Experiments through Combination with Dye Dynamics from Molecular Dynamics Simulations

Fluorescence resonance energy transfer (FRET) experiments probe molecular distances via the efficiency of energy transfer from an excited donor dye to its acceptor counterpart. To study flexibilities and structural changes in biomolecules, single molecule FRET settings allow probing distance distributions and fluctuations.However, the measured energy transfer efficiency depends not only on the distance between the two dyes, but also on their mutual orientation which is typically inaccessible to experiments. Thus, assumptions on the orientation distributions have to be made such as an isotropic orientation distribution. These approximations severely limit the accuracy of the distance distributions extracted from FRET experiments alone.Here we show that estimates of the distance distributions can be improved considerably by combining measured efficiency distributions with dye orientation statistics obtained from molecular dynamics (MD) simulations. Quantitative agreement of calculated efficiency distributions from simulations with the experimental efficiency distributions was obtained for poly-proline chains with Alexa488 and Alexa594 dyes attached. The experimentally observed heterogeneity was related to the presence of cis-isomers and previously undetected distinct dye conformers.This agreement of our in silico FRET approach with experiments provides a solid basis to use the obtained dye orientation dynamics from MD simulations in the distance reconstruction. We propose a transfer-function based approach and show that the poly-proline all-trans peak location in the reconstructed distances deviates by only 0.35A from the true peak location in contrast to a 6.2A deviation when employing the isotropic approximation of the dye orientation.

Single-Molecule Fluorescence Meets DNA Origami

Recent developments in DNA nanotechnology facilitate a new degree of control to place arbitrary objects. Therefore, DNA is folded into arbitrary 2- and 3-dimensional structures on a nanometer to micrometer scale. In this so-called origami technique, introduced by Paul Rothemund in 2006, one ∼7.3 kbases long single-stranded DNA is hybridized with ∼200 short synthetic DNA “staple” strands to build a desired structure by self-assembly. Objects of interest, e.g. single fluorophores, are attached to individual incorporated DNA strands at specific positions within the structure. Several applications of this approach are shown using single-molecule fluorescence techniques.Revisiting the distance dependence of fluorescence resonance energy transfer (FRET), we used the DNA origami technique to build a spectroscopic ruler. In contrast to double stranded DNA, a commonly used spacer molecule, this technique offers distinct advantages. We designed a rigid DNA origami block, which has a higher persistence length and additionally allows placing the dye molecules all oriented in the same direction on the top surface, limiting static effects of the linker lengths. In contrast to dsDNA, for the origami block the Forster Radius R0 could directly be obtained from the distance dependence of energy transfer based on single-molecule FRET measurements.Guided by the idea to build complex spectroscopic networks by self-assembly, we used rectangular DNA origami as a molecular breadboard to precisely position individual fluorophores. In this artificial system the path of energy transfer can be manipulated on the nanoscale. Fluorophores were incorporated such that the light from the “blue” input dye could either be guided to the “red” or “IR” output dye, by a “green” dye that was placed at two alternative positions. We used a single-molecule four-color FRET approach with alternating laser excitation for analysis of the different energy transfer paths.

Fluorescence Detection of Potassium Ion Using the G-Quadruplex Structure

Oligonucleotides with sequences of human telomere DNA or thrombin binding aptamer (TBA) are known to form tetraplex structures upon binding the K(+) ion. Structural changes associated with the formation of tetraplex assemblies led to the development of potassium-sensing oligonucleotide (PSO) probes, in which two fluorescent dyes were attached to both termini of particular oligonucleotide. The combination of dyes included fluorescence resonance energy transfer (FRET) and excimer emission approaches, and the structural changes upon binding K(+) ion could be monitored by a fluorescence technique. These systems showed a very high preference for K(+) over Na(+) ion, which was suitable for fluorescence imaging of the potassium concentration gradient in a living cell. In the case of human telomere DNA, it was also possible to follow the polymorphism of its tetraplex structures.

Quantification of protein interaction in living cells by two‐photon spectral imaging with fluorescent protein fluorescence resonance energy transfer pair devoid of acceptor bleed‐through

Fluorescence resonance energy transfer (FRET) between fluorescent proteins (FPs) is a powerful method to visualize and quantify protein-protein interaction in living cells. Unfortunately, the emission bleed-through of FPs limits the usage of this complex technique. To circumvent undesirable excitation of the acceptor fluorophore, using two-photon excitation, we searched for FRET pairs that show selective excitation of the donor but not of the acceptor fluorescent molecule. We found this property in the fluorescent cyan fluorescent protein (CFP)/yellow fluorescent protein (YFP) and YFP/mCherry FRET pairs and performed two-photon excited FRET spectral imaging to quantify protein interactions on the later pair that shows better spectral discrimination. Applying non-negative matrix factorization to unmix two-photon excited spectral imaging data, we were able to eliminate the donor bleed-through as well as the autofluorescence. As a result, we achieved FRET quantification by means of a single spectral acquisition, making the FRET approach not only easy and straightforward but also less prone to calculation artifacts. As an application of our approach, the intermolecular interaction of amyloid precursor protein and the adaptor protein Fe65 associated with Alzheimer's disease was quantified. We believe that the FRET approach using two-photon and fluorescent YFP/mCherry pair is a promising method to monitor protein interaction in living cells.

Single-molecule FRET study of SNARE-mediated membrane fusion.

Membrane fusion is one of the most important cellular processes by which two initially distinct lipid bilayers merge their hydrophobic cores, resulting in one interconnected structure. Proteins, called SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor), play a central role in the fusion process that is also regulated by several accessory proteins. In order to study the SNARE-mediated membrane fusion, the in vitro protein reconstitution assay involving ensemble FRET (fluorescence resonance energy transfer) has been used over a decade. In this mini-review, we describe several single-molecule-based FRET approaches that have been applied to this field to overcome the shortage of the bulk assay in terms of protein and fusion dynamics.

Rice A20/AN1 zinc‐finger containing stress‐associated proteins (SAP1/11) and a receptor‐like cytoplasmic kinase (OsRLCK253) interact via A20 zinc‐finger and confer abiotic stress tolerance in transgenic Arabidopsis plants

• The inbuilt mechanisms of plant survival have been exploited for improving tolerance to abiotic stresses. Stress-associated proteins (SAPs), containing A20/AN1 zinc-finger domains, confer abiotic stress tolerance in different plants, however, their interacting partners and downstream targets remain to be identified. • In this study, we have investigated the subcellular interactions of rice SAPs and their interacting partner using yeast two-hybrid and fluorescence resonance energy transfer (FRET) approaches. Their efficacy in improving abiotic stress tolerance was analysed in transgenic Arabidopsis plants. Regulation of gene expression by genome-wide microarray in transgenics was used to identify downstream targets. • It was found that the A20 domain mediates the interaction of OsSAP1 with self, its close homolog OsSAP11 and a rice receptor-like cytoplasmic kinase, OsRLCK253. Such interactions between OsSAP1/11 and with OsRLCK253 occur at nuclear membrane, plasma membrane and in nucleus. Functionally, both OsSAP11 and OsRLCK253 could improve the water-deficit and salt stress tolerance in transgenic Arabidopsis plants via a signaling pathway affecting the expression of several common endogenous genes. • Components of a novel stress-responsive pathway have been identified. Their stress-inducible expression provided the protection against yield loss in transgenic plants, indicating the agronomic relevance of OsSAP11 and OsRLCK253 in conferring abiotic stress tolerance.

Unravelling the Symmetry Mismatch of the Two Coupled Rotary Motors of a Single FOF1-ATP Synthase by Three Color Fret

FoF1-ATP synthase is the enzyme that provides the ‘chemical energy currency’ adenosine triphosphate, ATP, for living cells. The enzyme consists of the membrane-embedded Fo part and the F1 part with three catalytic nucleotide binding sites. The formation of ATP is accomplished by a stepwise internal rotation of subunits within both parts. During ATP synthesis, proton translocation through the Fo part drives a 10-step rotary motion of the c10 ring with respect to the non-rotating subunits a and b2 [1]. This rotation is transmitted to the γ and ε subunits of the F1 part which perform three 120° steps per full rotation [2]. To localize transient elastic energy storage within the enzyme due to the symmetry mismatch, we monitor subunit rotation by a single-molecule fluorescence resonance energy transfer (FRET) approach using three fluorophores specifically attached to the enzyme. Cy5 is attached to one c subunit and EGFP to the a subunit of the Fo part [3]. The F1 part has a fluorophore bound at the ε subunit. To reduce photophysical artifacts due to spectral fluctuations of the fluorophores, a duty cycle-optimized alternating three laser scheme (DCO-ALEX) has been developed. Simultaneous observation of the stepsizes for both motors in a single enzyme revealed elastic deformations in the rotor parts of Fo and F1 during catalysis. [1] M. G. Düser, N. Zarrabi, D. J. Cipriano, S. Ernst, G. D.Glick, S.D. Dunn, M. Börsch (2009) EMBO Journal 28, 2689-2696. [2] B. Zimmermann, M. Diez, N. Zarrabi, P. Gräber, M. Börsch (2005) EMBO Journal 24, 2053-2063. [3] M. G. Düser, Y. Bi, N. Zarrabi, S.D. Dunn, M. Börsch (2008) J. Biol. Chem. 283, 33602-33610.

Comparative Analysis of RNA/Protein Dynamics for the Arginine-Rich-Binding Motif and Zinc-Finger-Binding Motif Proteins Encoded by HIV-1

We report a comparative study in which a single-molecule fluorescence resonance energy transfer approach was used to examine how the binding of two families of HIV-1 viral proteins to viral RNA hairpins locally changes the RNA secondary structures. The single-molecule fluorescence resonance energy transfer results indicate that the zinc finger protein (nucleocapsid) locally melts the TAR RNA and RRE-IIB RNA hairpins, whereas arginine-rich motif proteins (Tat and Rev) may strengthen the hairpin structures through specific binding interactions. Competition experiments show that Tat and Rev can effectively inhibit the nucleocapsid-chaperoned annealing of complementary DNA oligonucleotides to the TAR and RRE-IIB RNA hairpins, respectively. The competition binding data presented here suggest that the specific nucleic acid binding interactions of Tat and Rev can effectively compete with the general nucleic acid binding/chaperone functions of the nucleocapsid protein, and thus may in principle help regulate critical events during the HIV life cycle.

Long-range tertiary interactions in single hammerhead ribozymes bias motional sampling toward catalytically active conformations

Enzymes generally are thought to derive their functional activity from conformational motions. The limited chemical variation in RNA suggests that such structural dynamics may play a particularly important role in RNA function. Minimal hammerhead ribozymes are known to cleave efficiently only in ∼ 10-fold higher than physiologic concentrations of Mg(2+) ions. Extended versions containing native loop-loop interactions, however, show greatly enhanced catalytic activity at physiologically relevant Mg(2+) concentrations, for reasons that are still ill-understood. Here, we use Mg(2+) titrations, activity assays, ensemble, and single molecule fluorescence resonance energy transfer (FRET) approaches, combined with molecular dynamics (MD) simulations, to ask what influence the spatially distant tertiary loop-loop interactions of an extended hammerhead ribozyme have on its structural dynamics. By comparing hammerhead variants with wild-type, partially disrupted, and fully disrupted loop-loop interaction sequences we find that the tertiary interactions lead to a dynamic motional sampling that increasingly populates catalytically active conformations. At the global level the wild-type tertiary interactions lead to more frequent, if transient, encounters of the loop-carrying stems, whereas at the local level they lead to an enrichment in favorable in-line attack angles at the cleavage site. These results invoke a linkage between RNA structural dynamics and function and suggest that loop-loop interactions in extended hammerhead ribozymes-and Mg(2+) ions that bind to minimal ribozymes-may generally allow more frequent access to a catalytically relevant conformation(s), rather than simply locking the ribozyme into a single active state.

Monitoring multiple distances within a single molecule using switchable FRET

The analysis of structure and dynamics of biomolecules is important for understanding their function. Toward this aim, we introduce a method called 'switchable FRET', which combines single-molecule fluorescence resonance energy transfer (FRET) with reversible photoswitching of fluorophores. Typically, single-molecule FRET is measured within a single donor-acceptor pair and reports on only one distance. Although multipair FRET approaches that monitor multiple distances have been developed, they are technically challenging and difficult to extend, mainly because of their reliance on spectrally distinct acceptors. In contrast, switchable FRET sequentially probes FRET between a single donor and spectrally identical photoswitchable acceptors, dramatically reducing the experimental and analytical complexity and enabling direct monitoring of multiple distances. Our experiments on DNA molecules, a protein-DNA complex and dynamic Holliday junctions demonstrate the potential of switchable FRET for studying dynamic, multicomponent biomolecules.

Effect of Single-Strand Break on Branch Migration and Folding Dynamics of Holliday Junctions

The Holliday junction (HJ), or four-way junction, is a central intermediate state of DNA for homologous genetic recombination and other genetic processes such as replication and repair. Branch migration is the process by which the exchange of homologous DNA regions occurs, and it can be spontaneous or driven by proteins. Unfolding of the HJ is required for branch migration. Our previous single-molecule fluorescence studies led to a model according to which branch migration is a stepwise process consisting of consecutive migration and folding steps. Folding of the HJ in one of the folded conformations terminates the branch migration phase. At the same time, in the unfolded state HJ rapidly migrates over entire homology region of the HJ in one hop. This process can be affected by irregularities in the DNA double helical structure, so mismatches almost terminate a spontaneous branch migration. Single-stranded breaks or nicks are the most ubiquitous defects in the DNA helix; however, to date, their effect on the HJ branch migration has not been studied. In addition, although nicked HJs are specific substrates for a number of enzymes involved in DNA recombination and repair, the role of this substrate specificity remains unclear. Our main goal in this work was to study the effect of nicks on the efficiency of HJ branch migration and the dynamics of the HJ. To accomplish this goal, we applied two single-molecule methods: atomic force microscopy and fluorescence resonance energy transfer. The atomic force microscopy data show that the nick does not prevent branch migration, but it does decrease the probability that the HJ will pass the DNA lesion. The single-molecule fluorescence resonance energy transfer approaches were instrumental in detailing the effects of nicks. These studies reveal a dramatic change of the HJ dynamics. The nick changes the structure and conformational dynamics of the junctions, leading to conformations with geometries that are different from those for the intact HJ. On the basis of these data, we propose a model of branch migration in which the propensity of the junction to unfold decreases the lifetimes of folded states, thereby increasing the frequency of junction fluctuations between the folded states.

Heparin Induces Rat Aorta Relaxation via Integrin-Dependent Activation of Muscarinic M 3 Receptors

Previous reports have shown that heparin may promote human hypotension and vascular relaxation by elevation of NO levels through unclear mechanisms. We hypothesized that endothelial muscarinic M(3) receptor activation mediates the heparin-induced vasodilation of rat aortic rings. The experiments were carried out using unfractionated heparin extracted from bovine intestinal mucosa, which elicited an endothelium and NO-dependent relaxation of aortic segments with maximal potency and efficacy (EC(50): 100±10 μmol/L; E(max): 41±3%). Atropine and 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide inhibitors reduced the heparin-dependent relaxation, indicating that M(3) muscarinic receptor is involved in this phenomenon. However, no direct binding of heparin to muscarinic receptors was observed. More importantly, studies performed using the arginine-glycine-aspartic acid peptide and 1-(1,1-dimethylethyl)-3-(1-naphthalenyl)-1H-pyrazolo[3,4-day]pyrimidin-4-amine, an Src family inhibitor, reduced by 51% and 73% the heparin-dependent relaxation, respectively, suggesting the coupling of heparin and M(3) receptor through extracellular matrix molecules and integrin. Furthermore, unfractionated heparin induced activation of focal adhesion protein kinase, Src, and paxillin. Finally, fluorescence resonance energy transfer approach confirmed the interaction of the M(3) receptor to integrin. Taken together, these data demonstrate the participation of M(3) receptor and integrin in heparin-dependent relaxation of vascular smooth muscle. These results provide new insights into the molecular mechanism and potential pharmacological action of heparin in vascular physiology.

A novel FRET approach for in situ investigation of cellulase–cellulose interaction

A novel real-time in situ detection method for the investigation of cellulase-cellulose interactions based on fluorescence resonance energy transfer (FRET) has been developed. FRET has been widely used in biological and biophysical fields for studies related to proteins, nucleic acids, and small biological molecules. Here, we report the efficient labeling of carboxymethyl cellulose (CMC) with donor dye 5-(aminomethyl)fluorescein and its use as a donor in a FRET assay together with an Alexa Fluor 594 (AF594, acceptor)-cellulase conjugate as acceptor. This methodology was successfully employed to investigate the temperature dependency of cellulase binding to cellulose at a molecular level by monitoring the fluorescence emission change of donor (or acceptor) in a homogeneous liquid environment. It also provides a sound base for ongoing cellulase-cellulose study using cellulosic fiber.