Fluorescence tools to investigate riboswitch structural dynamics

Riboswitches are novel regulatory elements that respond to cellular metabolites to control gene expression. They are constituted of highly conserved domains that have evolved to recognize specific metabolites. Such domains, so-called aptamers, are folded into intricate structures to enable metabolite recognition. Over the years, the development of ensemble and single-molecule fluorescence techniques has allowed to probe most of the mechanistic aspects of aptamer folding and ligand binding. In this review, we summarize the current fluorescence toolkit available to study riboswitch structural dynamics. We fist describe those methods based on fluorescent nucleotide analogues, mostly 2-aminopurine (2AP), to investigate short-range conformational changes, including some key steady-state and time-resolved examples that exemplify the versatility of fluorescent analogues as structural probes. The study of long-range structural changes by Förster resonance energy transfer (FRET) is mostly discussed in the context of single-molecule studies, including some recent developments based on the combination of single-molecule FRET techniques with controlled chemical denaturation methods. This article is part of a Special Issue entitled: Riboswitches.     

Optical investigation of the electron transfer protein azurin-gold nanoparticle system

The hybrid system obtained by conjugating the protein azurin, which is a very stable and well-described protein showing a unique interplay among its electron transfer and optical properties, with 20-nm sized gold nanoparticles has been investigated. Binding of azurin molecules to gold nanoparticle surface results in the red shift of the nanoparticle resonance plasmon band and in the quenching of the azurin single tryptophan fluorescence signal. These findings together with the estimate of the hydrodynamic radius of the composite, obtained by means of Dynamic Light Scattering, are consistent with the formation of a monolayer of protein molecules, with preserved natural folding, on nanoparticle surface. The fluorescence quenching of azurin bound molecules is explained by an energy transfer from protein to metal surface and it is discussed in terms of the involvement of the Az electron transfer route in the interaction of the protein with the nanoparticle.     

Influence of spectral heterogeneity of prodan and laurdan solutions on the transfer of electronic energy to octadecyl rhodamine B

Fluorescence quenching and resonance energy transfer have been studied by steady-state fluorescence spectroscopy. The experimental and theoretical values for the rate constants of the electronic energy transfer (kET) and critical radius (R0) were determined for prodan and laurdan as donors and octadecyl rhodamine B as acceptor. The spectroscopic data show, that prodan and laurdan in solution create an inhomogeneous spectroscopic medium in which multi-channel luminescence phenomena take place. This finding indicated that the modified form of the Stern-Volmer relation should be used for analyzing fluorescence quenching data. Results of performed studies point out, that dipole-dipole interaction is responsible for the resonance energy transfer from prodan and laurdan to octadecyl rhodamine B. The relative quenching efficiencies of both dyes depend on polarity of the medium and are higher for more polar solvent (AcN).     

Tryptophan fluorescence quenching by alkaline pH and ternary complex formation in human beta 1 beta 1 and horse EE alcohol dehydrogenases.

The horse EE and human β₁β₁ alcohol dehydrogenase isoenzymes have almost identical protein backbone folding patterns and contain 2 tryptophans per subunit (Trp-15 and Trp-314). Tyr-286, which had been proposed to quench the fluorescence of Trp-314 by resonance energy transfer at alkaline pH in EE, is substituted by Cys in β₁β₁. The proposed role of Tyr-286 in pH-dependent quenching of EE is confirmed by our observation that tryptophan fluorescence of β₁β₁ is not substantially quenched at alkaline pH. Tyr-286 had also been implicated in the quenching of Trp-314 upon formation of the EE-NAD⁺-trifluoroethanol ternary complex. However, β₁β₁ exhibits the same extent of tryptophan fluorescence quenching as EE upon complexation, which strongly suggests that Tyr-286 is not involved in ternary complex quenching.     

Single-molecule Förster resonance energy transfer studies of RNA structure, dynamics and function

Single-molecule fluorescence microscopy experiments on RNA molecules brought to light the highly complex dynamics of key biological processes, including RNA folding, catalysis of ribozymes, ligand sensing of riboswitches and aptamers, and protein synthesis in the ribosome. By using highly advanced biophysical spectroscopy techniques in combination with sophisticated biochemical synthesis approaches, molecular dynamics of individual RNA molecules can be observed in real time and under physiological conditions in unprecedented detail that cannot be achieved with bulk experiments. Here, we review recent advances in RNA folding and functional studies of RNA and RNA-protein complexes addressed by using single-molecule Förster (fluorescence) resonance energy transfer (smFRET) technique.     

In the fluorescent spotlight: Global and local conformational changes of small catalytic RNAs

RNA is a ubiquitous biopolymer that performs a multitude of essential cellular functions involving the maintenance, transfer, and processing of genetic information. RNA is unique in that it can carry both genetic information and catalytic function. Its secondary structure domains, which fold stably and independently, assemble hierarchically into modular tertiary structures. Studies of these folding events are key to understanding how catalytic RNAs (ribozymes) are able to position reaction components for site‐specific chemistry. We have made use of fluorescence techniques to monitor the rates and free energies of folding of the small hairpin and hepatitis delta virus (HDV) ribozymes, found in satellite RNAs of plant and the human hepatitis B viruses, respectively. In particular, fluorescence resonance energy transfer (FRET) has been employed to monitor global conformational changes, and 2‐aminopurine fluorescence quenching to probe for local structural rearrangements. In this review we illuminate what we have learned about the reaction pathways of the hairpin and HDV ribozymes, and how our results have complemented other biochemical and biophysical investigations. The structural transitions observed in these two small catalytic RNAs are likely to be found in many other biological RNAs, and the described fluorescence techniques promise to be broadly applicable. © 2002 Wiley Periodicals, Inc. Biopoly (Nucleic Acid Sci) 61: 224–241, 2002     

Partitioning of 2,6-Bis(1H-Benzimidazol-2-yl)pyridine fluorophore into a phospholipid bilayer: complementary use of fluorescence quenching studies and molecular dynamics simulations

Successful use of fluorescence sensing in elucidating the biophysical properties of lipid membranes requires knowledge of the distribution and location of an emitting molecule in the bilayer. We report here that 2,6-bis(1H-benzimidazol-2-yl)pyridine (BBP), which is almost non-fluorescent in aqueous solutions, reveals a strong emission enhancement in a hydrophobic environment of a phospholipid bilayer, making it interesting for fluorescence probing of water content in a lipid membrane. Comparing the fluorescence behavior of BBP in a wide variety of solvents with those in phospholipid vesicles, we suggest that the hydrogen bonding interactions between a BBP fluorophore and water molecules play a crucial role in the observed “light switch effect”. Therefore, the loss of water-induced fluorescence quenching inside a membrane are thought to be due to deep penetration of BBP into the hydrophobic, water-free region of a bilayer. Characterized by strong quenching by transition metal ions in solution, BBP also demonstrated significant shielding from the action of the quencher in the presence of phospholipid vesicles. We used the increase in fluorescence intensity, measured upon titration of probe molecules with lipid vesicles, to estimate the partition constant and the Gibbs free energy (ΔG) of transfer of BBP from aqueous buffer into a membrane. Partitioning BBP revealed strongly favorable ΔG, which depends only slightly on the lipid composition of a bilayer, varying in a range from − 6.5 to − 7.0 kcal/mol. To elucidate the binding interactions of the probe with a membrane on the molecular level, a distribution and favorable location of BBP in a POPC bilayer were modeled via atomistic molecular dynamics (MD) simulations using two different approaches: (i) free, diffusion-driven partitioning of the probe molecules into a bilayer and (ii) constrained umbrella sampling of a penetration profile of the dye molecule across a bilayer. Both of these MD approaches agreed with regard to the preferred location of a BBP fluorophore within the interfacial region of a bilayer, located between the hydrocarbon acyl tails and the initial portion of the lipid headgroups. MD simulations also revealed restricted permeability of water molecules into this region of a POPC bilayer, determining the strong fluorescence enhancement observed experimentally for the membrane-partitioned form of BBP.     

Interaction of cromolyn sodium with human serum albumin: a fluorescence quenching study

The interaction between cromolyn sodium (CS) and human serum albumin (HSA) was investigated using tryptophan fluorescence quenching. In the discussion of the mechanism, it was proved that the fluorescence quenching of HSA by CS is a result of the formation of a CS–HSA complex. Quenching constants were determined using the Sterns–Volmer equation to provide a measure of the binding affinity between CS and HSA. The thermodynamic parameters ΔG, ΔH, and ΔS at different temperatures were calculated. The distance r between donor (Trp²¹⁴) and acceptor (CS) was obtained according to fluorescence resonance energy transfer (FRET). Furthermore, synchronous fluorescence spectroscopy data and UV–vis absorbance spectra have suggested that the association between CS and HSA changed the molecular conformation of HSA and the electrostatic interactions play a major role in CS–HSA association.     

Calmodulin, conformational states, and calcium signaling. A single-molecule perspective

Single-molecule fluorescence measurements can provide a new perspective on the conformations, dynamics, and interactions of proteins. Recent examples are described illustrating the application of single-molecule fluorescence spectroscopy to calcium signaling proteins with an emphasis on the new information available in single-molecule fluorescence burst measurements, resonance energy transfer, and polarization modulation methods. Calcium signaling pathways are crucial in many cellular processes. The calcium binding protein calmodulin (CaM) serves as a molecular switch to regulate a network of calcium signaling pathways. Single-molecule spectroscopic methods can yield insights into conformations and dynamics of CaM and CaM-regulated proteins. Examples include studies of the conformations and dynamics of CaM, binding of target peptides, and interaction with the plasma-membrane Ca2+ pump. Single-molecule resonance energy transfer measurements revealed conformational substates of CaM, and single-molecule polarization modulation spectroscopy was used to probe interactions between CaM and the plasma-membrane Ca2+-ATPase.     

From Ca2+ in muscle contraction to IP3 receptor/Ca2+ signaling In memory of Setsuro Ebashi

The red fluorescent protein, DsRed, and a few of its mutants have been shown to bind copper ions resulting in quenching of its fluorescence. The response to Cu²⁺ is rapid, selective, and reversible upon addition of a copper chelator. DsRed has been employed as an in vitro probe for Cu²⁺ determination by us and other groups. It is also envisioned that DsRed can serve as an intracellular genetically encoded indicator of Cu²⁺ concentration, and can be targeted to desired subcellular locations for Cu²⁺ determination. However, no information has been reported yet regarding the mechanism of the fluorescence quenching of DsRed in the presence of Cu²⁺. In this work, we have performed spectroscopic investigations to determine the mechanism of quenching of DsRed fluorescence in the presence of Cu²⁺. We have studied the effect of Cu²⁺ addition on two representative mutants of DsRed, specifically, DsRed-Monomer and DsRed-Express. Both proteins bind Cu²⁺ with micromolar affinities. Stern-Volmer plots generated at different temperatures indicate a static quenching process in the case of both proteins in the presence of Cu²⁺. This mechanism was further studied using absorption spectroscopy. Stern-Volmer constants and quenching rate constants support the observation of static quenching in DsRed in the presence of Cu²⁺. Circular dichroism (CD)-spectroscopic studies revealed no effect of Cu²⁺-binding on the secondary structure or conformation of the protein. The effect of pH changes on the quenching of DsRed fluorescence in the presence of copper resulted in pK_a values indicative of histidine and cysteine residue involvement in Cu²⁺-binding.     

The localization of the local anesthetic tetracaine in phospholipid vesicles: A fluorescence quenching and resonance energy transfer study

Fluorescence quenching and resonance energy transfer have been used to determine the localization of the local anesthetic tetracaine in vesicles composed of 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) as a function of both temperature and ionic strength. The fluorescence behaviour of tetracaine in vesicles can be attributed to its different partition coefficients in acid and basic solution, in gel phase and fluid phase vesicles, respectively. Using both steady-state and time-resolved fluorescence measurements we show that a saturable binding rather than a partitioning model holds for the interaction of tetracaine with gel phase bilayers. The relative quenching efficiencies of the series of n-AS dyes depend on the phase state of the bilayer and suggest a deeper incorporation of tetracaine in fluid phase than in gel phase membranes. Resonance energy transfer measurements support the view that tetracaine is incorporated predominantly in the region of the 9-AS chromophore in DMPC-bilayers.     

RNA conformation and folding studied with fluorescence resonance energy transfer

Fluorescence resonance energy transfer (FRET) results from nonradiative coupling of two fluorophores and reports on distances in the range 10-100 A. It is therefore a suitable probe to determine distances in RNA molecules and define their global structure, to follow kinetics of RNA conformational changes during folding in real time, to monitor ion binding, or to analyze conformational equilibria and assess the thermodynamic stability of tertiary structure conformers. Along with the basic principles of steady-state and time-resolved fluorescence resonance energy transfer measurements, approaches to investigate RNA conformational transitions and folding are described and illustrated with selected examples. The versatility of FRET-based techniques has recently been demonstrated by implementations of FRET in high-throughput screening of potential drugs as well as studies of energy transfer that monitor RNA conformational changes on the single-molecule level.     

Sensitive detection of the redox state of copper proteins using fluorescence

The blue copper protein azurin from Pseudomonas aeruginosa has been covalently labelled with the fluorescing dye Cy5. The optical spectrum of the azurin changes markedly with its redox state. These changes are reflected in the fluorescence intensity of the dye through fluorescence resonance energy transfer (FRET). This provides a sensitive way to monitor biological redox events. The method shown to work in the nanomolar range of protein concentrations, can be easily extended into the sub-nanomolar regime and holds promise for single-molecule detection.     

Active Cage Mechanism of Chaperonin-Assisted Protein Folding Demonstrated at Single-Molecule Level

The cylindrical chaperonin GroEL and its lid-shaped cofactor GroES of Escherichia coli have an essential role in assisting protein folding by transiently encapsulating non-native substrate in an ATP-regulated mechanism. It remains controversial whether the chaperonin system functions solely as an infinite dilution chamber, preventing off-pathway aggregation, or actively enhances folding kinetics by modulating the folding energy landscape. Here we developed single-molecule approaches to distinguish between passive and active chaperonin mechanisms. Using low protein concentrations (100 pM) to exclude aggregation, we measured the spontaneous and GroEL/ES-assisted folding of double-mutant maltose binding protein (DM-MBP) by single-pair fluorescence resonance energy transfer and fluorescence correlation spectroscopy. We find that GroEL/ES accelerates folding of DM-MBP up to 8-fold over the spontaneous folding rate. Accelerated folding is achieved by encapsulation of folding intermediate in the GroEL/ES cage, independent of repetitive cycles of protein binding and release from GroEL. Moreover, photoinduced electron transfer experiments provided direct physical evidence that the confining environment of the chaperonin restricts polypeptide chain dynamics. This effect is mediated by the net-negatively charged wall of the GroEL/ES cavity, as shown using the GroEL mutant EL(KKK2) in which the net-negative charge is removed. EL(KKK2)/ES functions as a passive cage in which folding occurs at the slow spontaneous rate. Taken together our findings suggest that protein encapsulation can accelerate folding by entropically destabilizing folding intermediates, in strong support of an active chaperonin mechanism in the folding of some proteins. Accelerated folding is biologically significant as it adjusts folding rates relative to the speed of protein synthesis.     

Tertiary structure of human alpha1-acid glycoprotein (orosomucoid). Straightforward fluorescence experiments revealing the presence of a binding pocket

Binding of hemin to alpha1-acid glycoprotein has been investigated. Hemin binds to the hydrophobic pocket of hemoproteins. The fluorescent probe 2-(p-toluidino)-6-naphthalenesulfonate (TNS) binds to a hydrophobic domain in alpha1-acid glycoprotein with a dissociation constant equal to 60 microM. Addition of hemin to an alpha1-acid glycoprotein-TNS complex induces the displacement of TNS from its binding site. At saturation (1 hemin for 1 protein) all the TNS has been displaced from its binding site. The dissociation constant of hemin-alpha1-acid glycoprotein was found equal to 2 microM. Thus, TNS and hemin bind to the same hydrophobic site: the pocket of alpha1-acid glycoprotein. Energy-transfer studies performed between the Trp residues of alpha1-acid glycoprotein and hemin indicated that efficiency (E) of Trp fluorescence quenching was equal to 80% and the F?rster distance, R0 at which the efficiency of energy transfer is 50% was calculated to be 26 A, revealing a very high energy transfer.     

Structure-based calculation of multi-donor multi-acceptor fluorescence resonance energy transfer in the 4×6-mer tarantula hemocyanin

Hemocyanins are oxygen carriers of arthropods and molluscs. The oxygen is bound between two copper ions, forming a Cu(II)-O₂ ^2–-Cu(II) complex. The oxygenated active sites create two spectroscopic signals indicating the oxygen load of the hemocyanins: first, an absorption band at 340 nm which is due to a ligand-to-metal charge transfer complex, and second, a strong quenching of the intrinsic tryptophan fluorescence, the cause of which has not been definitively identified. We showed for the 4×6-mer hemocyanin of the tarantula Eurypelma californicum that the fluorescence quenching of oxygenated hemocyanin is caused exclusively by fluorescence resonance energy transfer (FRET). The tarantula hemocyanin consists of 24 subunits containing 148 tryptophans acting as donors and 24 active sites as acceptors. The donor–acceptor distances are determined on the basis of a closely related crystal structure of the horseshoe crab Limulus polyphemus hemocyanin subunit II (68–79% homology). Calculation of the expected fluorescence quenching and the measured transfer efficiency coincided extraordinary well, so that the fluorescence quenching of oxygenated tarantula hemocyanin can be completely explained by Förster transfer. This results explain for the first time, on a molecular basis, why fluorescence quantum yield can be used as an intrinsic signal for oxygen load of at least one arthropod hemocyanin, in particular that from the tarantula.     

Spectral diagnostics of the interaction between pyridoxine hydrochloride and bovine serum albumin in vitro

The interaction between pyridoxine hydrochloride (VB6) and bovine serum albumin (BSA) were studied by spectroscopic methods including fluorescence spectroscopy and UV-visible absorption spectra. The quenching mechanism of fluorescence of BSA by VB6 was discussed. The number of binding sites n and observed binding constant K(b) was measured by fluorescence quenching method. The thermodynamic parameters DeltaH(theta), DeltaG(theta), DeltaS(theta) at different temperatures were calculated and the results indicate the binding reaction is mainly entropy-driven and hydrophobic interaction played major role in the reaction. The distance r between donor (BSA) and acceptor (VB6) was obtained according to FOrster theory of non-radiation energy transfer. Synchronous fluorescence and three-dimensional fluorescence spectra were used to investigate the structural change of BSA molecules with addition of VB6, the result indicates that the secondary structure of BSA molecules is changed in the presence of VB6.     

Transition Path Times Measured by Single-Molecule Spectroscopy

The transition path is a tiny fraction of a molecular trajectory during which the free-energy barrier is crossed. It is a single-molecule property and contains all mechanistic information of folding processes of biomolecules such as proteins and nucleic acids. However, the transition path has been difficult to probe because it is short and rarely visited when transitions actually occur. Recent technical advances in single-molecule spectroscopy have made it possible to directly probe transition paths, which has opened up new theoretical and experimental approaches to investigating folding mechanisms. This article reviews recent single-molecule fluorescence and force spectroscopic measurements of transition path times and their connection to both theory and simulations.     

Probing conformational changes of gramicidin ion channels by single-molecule patch-clamp fluorescence microscopy

Complex conformational changes influence and regulate the dynamics of ion channels. Such conformational changes are stochastic and often inhomogeneous, which makes it extremely difficult, if not impossible, to characterize them by ensemble-averaged experiments or by single-channel recordings of the electric current that report the open-closed events but do not specifically probe the associated conformational changes. Here, we report our studies on ion channel conformational changes using a new approach, patch-clamp fluorescence microscopy, which simultaneously combines single-molecule fluorescence spectroscopy and single-channel current recordings to probe the open-closed transitions and the conformational dynamics of individual ion channels. We demonstrate patch-clamp fluorescence microscopy by measuring gramicidin ion channel conformational changes in a lipid bilayer formed at a patch-clamp micropipette tip under a buffer solution. By measuring single-pair fluorescence resonance energy transfer and fluorescence self-quenching from dye-labeled gramicidin channels, we observed that the efficiency of single-pair fluorescence resonance energy transfer and self-quenching is widely distributed, which reflects a broad distribution of conformations. Our results strongly suggest a hitherto undetectable correlation between the multiple conformational states of the gramicidin channel and its closed and open states in a lipid bilayer.