An RNA toolbox for single-molecule force spectroscopy studies

Precise, controllable single-molecule force spectroscopy studies of RNA and RNA-dependent processes have recently shed new light on the dynamics and pathways of RNA folding and RNA-enzyme interactions. A crucial component of this research is the design and assembly of an appropriate RNA construct. Such a construct is typically subject to several criteria. First, single-molecule force spectroscopy techniques often require an RNA construct that is longer than the RNA molecules used for bulk biochemical studies. Next, the incorporation of modified nucleotides into the RNA construct is required for its surface immobilization. In addition, RNA constructs for single-molecule studies are commonly assembled from different single-stranded RNA molecules, demanding good control of hybridization or ligation. Finally, precautions to prevent RNase- and divalent cation-dependent RNA digestion must be taken. The rather limited selection of molecular biology tools adapted to the manipulation of RNA molecules, as well as the sensitivity of RNA to degradation, make RNA construct preparation a challenging task. We briefly illustrate the types of single-molecule force spectroscopy experiments that can be performed on RNA, and then present an overview of the toolkit of molecular biology techniques at one's disposal for the assembly of such RNA constructs. Within this context, we evaluate the molecular biology protocols in terms of their effectiveness in producing long and stable RNA constructs.     

Comparative single-molecule and ensemble myosin enzymology: sulfoindocyanine ATP and ADP derivatives

Single-molecule and macroscopic reactions of fluorescent nucleotides with myosin have been compared. The single-molecule studies serve as paradigms for enzyme-catalyzed reactions and ligand-receptor interactions analyzed as individual stochastic processes. Fluorescent nucleotides, called Cy3-EDA-ATP and Cy5-EDA-ATP, were derived by coupling the dyes Cy3.29.OH and Cy5.29.OH (compounds XI and XIV, respectively, in, Bioconjug. Chem. 4:105-111)) with 2'(3')-O-[N-(2-aminoethyl)carbamoyl]ATP (EDA-ATP). The ATP(ADP) analogs were separated into their respective 2'- and 3'-O-isomers, the interconversion rate of which was 30[OH(-)] s(-1) (0.016 h(-1) at pH 7.1) at 22 degrees C. Macroscopic studies showed that 2'(3')-O-substituted nucleotides had properties similar to those of ATP and ADP in their interactions with myosin, actomyosin, and muscle fibers, although the ATP analogs did not relax muscle as well as ATP did. Significant differences in the fluorescence intensity of Cy3-nucleotide 2'- and 3'-O-isomers in free solution and when they interacted with myosin were evident. Single-molecule studies using total internal reflection fluorescence microscopy showed that reciprocal mean lifetimes of the nucleotide analogs interacting with myosin filaments were one- to severalfold greater than predicted from macroscopic data. Kinetic and equilibrium data of nucleotide-(acto)myosin interactions derived from single-molecule microscopy now have a biochemical and physiological framework. This is important for single-molecule mechanical studies of motor proteins.     

Energy transfer in the peridinin-chlorophyll protein complex reconstituted with mixed chlorophyll sites

We use femtosecond transient absorption spectroscopy to study chlorophyll (Chl)-Chl energy transfer in the peridinin-chlorophyll protein (PCP) reconstituted with mixtures of either chlorophyll b (Chlb) and Chld or Chla and bacteriochlorophyll a (BChla). Analysis of absorption and transient absorption spectra demonstrated that reconstitution with chlorophyll mixtures produces a significant fraction of PCP complexes that contains a different Chl in each domain of the PCP monomer. The data also suggest that binding affinity of Chla is less than that of the other three Chl species. By exciting the Chl species lying at higher energy, we obtained energy transfer times of 40 ± 5 ps (Chlb-Chld) and 59 ± 3 ps (Chla-BChla). The experimental values match those obtained from the Förster equation, 36 and 50 ps, respectively, showing that energy transfer proceeds via the Förster mechanism. Excitation of peridinin in the PCP complex reconstituted with Chla/BChla mixture provided time constants of 2.6 and 0.4 ps for the peridinin-Chla and peridinin-BChla energy transfer, matching those obtained from studies of PCP complexes reconstituted with single chlorophyll species.     

Ternary SNARE complexes in parallel versus anti-parallel orientation: Examination of their disassembly using single-molecule force spectroscopy

Interactions between the proteins of the ternary soluble N-ethyl maleimide-sensitive fusion protein attachment protein receptor (SNARE) complex, synaptobrevin 2 (Sb2), syntaxin 1A (Sx1A) and synaptosome-associated protein of 25kDa (SNAP25) can be readily assessed using force spectroscopy single-molecule measurements. We studied interactions during the disassembly of the ternary SNARE complex pre-formed by binding Sb2 in parallel or anti-parallel orientations to the binary Sx1A-SNAP25B acceptor complex. We determined the spontaneous dissociation lifetimes and found that the stability of the anti-parallel ternary SNARE complex is ～1/3 less than that of the parallel complex. While the free energies were very similar, within 0.5 k(B)T, for both orientations, the enthalpy changes (42.1 k(B)T and 39.8 k(B)T, for parallel and anti-parallel orientations, respectively) indicate that the parallel ternary complex is energetically advantageous by 2.3 k(B)T. Indeed, both ternary SNARE complex orientations were much more stable (by ～4-13 times) and energetically favorable (by ～9-13 k(B)T) than selected binary complexes, constituents of the ternary complex, in both orientations. We propose a model which considers the geometry for the vesicle approach to the plasma membrane with favorable energies and stability as the basis for preferential usage of the parallel ternary SNARE complex in exocytosis.     

Monitoring fluorescence of individual chromophores in peridinin-chlorophyll-protein complex using single molecule spectroscopy

Single molecule spectroscopy experiments are reported for native peridinin-chlorophyll a-protein (PCP) complexes, and three reconstituted light-harvesting systems, where an N-terminal construct of native PCP from Amphidinium carterae has been reconstituted with chlorophyll (Chl) mixtures: with Chl a, with Chl b and with both Chl a and Chl b. Using laser excitation into peridinin (Per) absorption band we take advantage of sub-picosecond energy transfer from Per to Chl that is order of magnitude faster than the Förster energy transfer between the Chl molecules to independently populate each Chl in the complex. The results indicate that reconstituted PCP complexes contain only two Chl molecules, so that they are spectroscopically equivalent to monomers of native-trimeric-PCP and do not aggregate further. Through removal of ensemble averaging we are able to observe for single reconstituted PCP complexes two clear steps in fluorescence intensity timetraces attributed to subsequent bleaching of the two Chl molecules. Importantly, the bleaching of the first Chl affects neither the energy nor the intensity of the emission of the second one. Since in strongly interacting systems Chl is a very efficient quencher of the fluorescence, this behavior implies that the two fluorescing Chls within a PCP monomer interact very weakly with each other which makes it possible to independently monitor the fluorescence of each individual chromophore in the complex. We apply this property, which distinguishes PCP from other light-harvesting systems, to measure the distribution of the energy splitting between two chemically identical Chl a molecules contained in the PCP monomer that reaches 280 cm^− 1. In agreement with this interpretation, stepwise bleaching of fluorescence is also observed for native PCP complexes, which contain six Chls. Most PCP complexes reconstituted with both Chl a and Chl b show two emission lines, whose wavelengths correspond to the fluorescence of Chl a and Chl b. This is a clear proof that these two different chromophores are present in a single PCP monomer. Single molecule fluorescence studies of PCP complexes, both native and artificially reconstituted with chlorophyll mixtures, provide new and detailed information necessary to fully understand the energy transfer in this unique light-harvesting system.     

Monitoring fluorescence of individual chromophores in peridinin-chlorophyll-protein complex using single molecule spectroscopy

Single molecule spectroscopy experiments are reported for native peridinin-chlorophyll a-protein (PCP) complexes, and three reconstituted light-harvesting systems, where an N-terminal construct of native PCP from Amphidinium carterae has been reconstituted with chlorophyll (Chl) mixtures: with Chl a, with Chl b and with both Chl a and Chl b. Using laser excitation into peridinin (Per) absorption band we take advantage of sub-picosecond energy transfer from Per to Chl that is order of magnitude faster than the F?rster energy transfer between the Chl molecules to independently populate each Chl in the complex. The results indicate that reconstituted PCP complexes contain only two Chl molecules, so that they are spectroscopically equivalent to monomers of native-trimeric-PCP and do not aggregate further. Through removal of ensemble averaging we are able to observe for single reconstituted PCP complexes two clear steps in fluorescence intensity timetraces attributed to subsequent bleaching of the two Chl molecules. Importantly, the bleaching of the first Chl affects neither the energy nor the intensity of the emission of the second one. Since in strongly interacting systems Chl is a very efficient quencher of the fluorescence, this behavior implies that the two fluorescing Chls within a PCP monomer interact very weakly with each other which makes it possible to independently monitor the fluorescence of each individual chromophore in the complex. We apply this property, which distinguishes PCP from other light-harvesting systems, to measure the distribution of the energy splitting between two chemically identical Chl a molecules contained in the PCP monomer that reaches 280 cm(-1). In agreement with this interpretation, stepwise bleaching of fluorescence is also observed for native PCP complexes, which contain six Chls. Most PCP complexes reconstituted with both Chl a and Chl b show two emission lines, whose wavelengths correspond to the fluorescence of Chl a and Chl b. This is a clear proof that these two different chromophores are present in a single PCP monomer. Single molecule fluorescence studies of PCP complexes, both native and artificially reconstituted with chlorophyll mixtures, provide new and detailed information necessary to fully understand the energy transfer in this unique light-harvesting system.     

Thermal stability studies of photosystem II complexes reconstituted into phosphatidylcholine liposomes

Light-harvesting II complexes (LHCII) and photosystem II core complexes (PSIICC) were isolated from spinach (Spinacia oleracea L.) and reconstituted into phosphatidylcholine liposomes and, under heat stress, PSIICC-LHCII proteoliposomes were found to exhibit significantly higher oxygen evolution activity than PSIICC proteoliposomes lacking LHCII. In the presence of LHCII, the temperature of a 10-min heat stress that caused semi-inactivation of oxygen-evolving activity in these liposomes increased from 34 to ～37°C and the total inactivation temperature increased from ～50 to ～60°C. Moreover, with heat stress, decreases in the absorbance and fluorescence spectra of PSIICC-LHCII proteoliposomes were smaller than in LHCII-lacking PSIICC proteoliposomes. These results demonstrated that reconstitution of PSII into liposomes with LHCII increased the antenna size and light harvesting cross-section of PSII and thus, under heat stress, enhanced PSII photochemical activity and thermal stability.     

Fluorescence resonance energy transfer (FRET) and competing processes in donor-acceptor substituted DNA strands: a comparative study of ensemble and single-molecule data

We studied the fluorescence resonance energy transfer (FRET) efficiency of different donor-acceptor labeled model DNA systems in aqueous solution from ensemble measurements and at the single molecule level. The donor dyes: tetramethylrhodamine (TMR); rhodamine 6G (R6G); and a carbocyanine dye (Cy3) were covalently attached to the 5'-end of a 40-mer model oligonucleotide. The acceptor dyes, a carbocyanine dye (Cy5), and a rhodamine derivative (JA133) were attached at modified thymidine bases in the complementary DNA strand with donor-acceptor distances of 5, 15, 25 and 35 DNA-bases, respectively. Anisotropy measurements demonstrate that none of the dyes can be observed as a free rotor; especially in the 5-bp constructs the dyes exhibit relatively high anisotropy values. Nevertheless, the dyes change their conformation with respect to the oligonucleotide on a slower time scale in the millisecond range. This results in a dynamic inhomogeneous distribution of donor/acceptor (D/A) distances and orientations. FRET efficiencies have been calculated from donor and acceptor fluorescence intensity as well as from time-resolved fluorescence measurements of the donor fluorescence decay. Dependent on the D/A pair and distance, additional strong fluorescence quenching of the donor is observed, which simulates lower FRET efficiencies at short distances and higher efficiencies at longer distances. On the other hand, spFRET measurements revealed subpopulations that exhibit the expected FRET efficiency, even at short D/A distances. In addition, the measured acceptor fluorescence intensities and lifetimes also partly show fluorescence quenching effects independent of the excitation wavelength, i.e. either directly excited or via FRET. These effects strongly depend on the D/A distance and the dyes used, respectively. The obtained data demonstrate that besides dimerization at short D/A distances, an electron transfer process between the acceptor Cy5 and rhodamine donors has to be taken into account. To explain deviations from FRET theory even at larger D/A distances, we suggest that the pi-stack of the DNA double helix mediates electron transfer from the donor to the acceptor, even over distances as long as 35 base pairs. Our data show that FRET experiments at the single molecule level are rather suited to resolve fluorescent subpopulations in heterogeneous mixture, information about strongly quenched subpopulations gets lost.     

Energy transfer method in membrane studies: some theoretical and practical aspects

Some applications of resonance energy transfer (RET) method to distance estimation in membrane systems are considered. The model of energy transfer between donors and acceptors randomly distributed over parallel planes localized at the outer and inner membrane leaflets is presented. It is demonstrated that RET method can provide evidence for specific orientation of the fluorophore relative to the lipid-water interface. An approach to estimating the depth of the protein penetration in lipid bilayer is suggested.     

DNA origami as biocompatible surface to match single-molecule and ensemble experiments

Single-molecule experiments on immobilized molecules allow unique insights into the dynamics of molecular machines and enzymes as well as their interactions. The immobilization, however, can invoke perturbation to the activity of biomolecules causing incongruities between single molecule and ensemble measurements. Here we introduce the recently developed DNA origami as a platform to transfer ensemble assays to the immobilized single molecule level without changing the nano-environment of the biomolecules. The idea is a stepwise transfer of common functional assays first to the surface of a DNA origami, which can be checked at the ensemble level, and then to the microscope glass slide for single-molecule inquiry using the DNA origami as a transfer platform. We studied the structural flexibility of a DNA Holliday junction and the TATA-binding protein (TBP)-induced bending of DNA both on freely diffusing molecules and attached to the origami structure by fluorescence resonance energy transfer. This resulted in highly congruent data sets demonstrating that the DNA origami does not influence the functionality of the biomolecule. Single-molecule data collected from surface-immobilized biomolecule-loaded DNA origami are in very good agreement with data from solution measurements supporting the fact that the DNA origami can be used as biocompatible surface in many fluorescence-based measurements.     

Bridging conformational dynamics and function using single-molecule spectroscopy

In a typical structure-function relation study, the primary structure of proteins or nucleic acids is changed by mutagenesis and its functional effect is measured via biochemical means. Single-molecule spectroscopy has begun to give a whole new meaning to the "structure-function relation" by measuring the real-time conformational changes of individual biological macromolecules while they are functioning. This review discusses a few recent examples: untangling internal chemistry and conformational dynamics of a ribozyme, branch migration landscape of a Holliday junction at a single-step resolution, tRNA selection and dynamics in a ribosome, repetitive shuttling and snapback of a helicase, and discrete rotation of an ATP synthase.     

Protein-protein unbinding induced by force: single-molecule studies

Experiments in which two specifically interacting protein molecules are dissociated by external force have yielded new insights into mechanisms involved in cell adhesion, leukocyte rolling, regulation of integrin activity, antigen-antibody interactions and other protein-mediated reactions contingent upon molecular recognition. Another important aspect of force-induced protein-protein unbinding studies is the new information being gleaned about the thermodynamics and kinetics of bond rupture.     

Vectorial charge transfer reactions on the donor side of manganese-depleted and reconstituted photosystem 2 core complexes

The light-induced functioning of photosystem 2 (PS 2) is directly linked to the translocation of both electrons and protons across the membrane, which results in the formation of transmembrane electric potential difference (ΔΨ). Generation of ΔΨ due to S-state transitions of the water oxidation complex was demonstrated for the first time in Mn-depleted and reconstituted PS 2 core complexes incorporated into liposomes. The kinetics and relative amplitudes of the electrogenic reactions in dark-adapted samples during S₁→S₂, S₂→S₃, and S₄→S₀ transitions in response to the first, second and third laser flashes were comparable to those obtained in the intact PS 2 core particles. These results expand current understanding of the nature and mechanisms of electrogenic (vectorial) reactions due to a charge transfer on the donor side of PS 2.     

Methods and estimations of uncertainties in single-molecule dynamic force spectroscopy

In dynamic force spectroscopy, access to the characteristic parameters of single molecular bonds requires nontrivial measurements and data processing as the rupture forces are found not only to be distributed over a wide range, but are also dependent on the loading rate. The choice of measurement procedure and data processing methods has a considerable impact on the accuracy and precision of the final results. We analyze, by means of numerical simulations, methods to minimize and assess the magnitude of the expected errors for different combinations of experimental and evaluation methods. It was found that the choice of fitting function is crucial to extract correct parameter values. Applying a Gaussian function, which is a common practice, is equivalent to introducing a systematic error, and leads to a consequent overestimation of the thermal off-rate by more than 30%. We found that the precision of the bond length and the thermal off-rate, in presence of unbiased noise, were improved by reducing the number of loading rates for a given number of measurements. Finally, the results suggest that the minimum number of measurements needed to obtain the bond strength, with acceptable precision, exceeds the common number of ~100 reported in literature.     

Reference-free alignment and sorting of single-molecule force spectroscopy data

Single-molecule force spectroscopy has become a versatile tool for investigating the (un)folding of proteins and other polymeric molecules. Like other single-molecule techniques, single-molecule force spectroscopy requires recording and analysis of large data sets to extract statistically meaningful conclusions. Here, we present a data analysis tool that provides efficient filtering of heterogeneous data sets, brings spectra into register based on a reference-free alignment algorithm, and determines automatically the location of unfolding barriers. Furthermore, it groups spectra according to the number of unfolding events, subclassifies the spectra using cross correlation-based sorting, and extracts unfolding pathways by principal component analysis and clustering methods to extracted peak positions. Our approach has been tested on a data set obtained through mechanical unfolding of bacteriorhodopsin (bR), which contained a significant number of spectra that did not show the well-known bR fingerprint. In addition, we have tested the performance of the data analysis tool on unfolding data of the soluble multidomain (Ig27)(8) protein.     

Energy transfer in spectrally inhomogeneous light-harvesting pigment-protein complexes of purple bacteria.

Energy transfer within the peripheral light-harvesting antenna of the purple bacteria Rhodobacter sphaeroides and Rhodopseudomonas palustris was studied by one- and two-color pump-probe absorption spectroscopy with approximately 100-fs tunable pulses at room temperature and at 77 K. The energy transfer from B800 to B850 occurs with a time constant of 0.7 +/- 0.05 ps at room temperature and 1.8 +/- 0.2 ps at 77 K and is similar in both species. Anisotropy measurements suggest a limited but fast B800 <--> B800 transfer time (tau approximately 0.3 ps). This is analyzed as incoherent hopping of the excitation in a system of spectrally inhomogeneous antenna pigment-protein complexes, by a master equation approach. The simulations show that the measured B800 dynamics is well described as energy transfer with a characteristic average nearest-neighbor pairwise transfer time of 0.35 ps among approximately 10 Bchl molecules in a circular arrangement, in good agreement with the recent high-resolution structure of LH2. The possible presence of fast intramolecular relaxation processes within the Bchl a molecule was investigated by measurement of time-resolved difference absorption spectra and kinetics of Bchl a in solution and in low-temperature glasses. From these measurements it is concluded that fast transients observed at room temperature are due mainly to solvation processes, whereas at 77 K predominantly slower (> 10-ps) relaxation occurs.