Single-molecule Forster resonance energy transfer study of protein dynamics under denaturing conditions

Proteins are highly complex systems, exhibiting a substantial degree of structural variability in their folded state. In the presence of denaturants, the heterogeneity is greatly enhanced, and fluctuations among vast numbers of folded and unfolded conformations occur via many different pathways. Here, we have studied the structure and dynamics of the small enzyme ribonuclease HI (RNase H) in the presence of the chemical denaturant guanidinium chloride (GdmCl) using single-molecule fluorescence microscopy, with a particular focus on the characterization of the unfolded-state ensemble. A dye pair was specifically attached to the enzyme to measure structural changes through F?rster resonance energy transfer (FRET). Enzyme immobilization on star-polymer surfaces that were specially developed for negligible interaction with folded and unfolded proteins enabled us to monitor conformational changes of individual proteins for several hundred seconds. FRET efficiency histograms were calculated from confocal scan images. They showed an expansion of the unfolded proteins with increasing GdmCl concentration. Cross-correlation analysis of donor and acceptor fluorescence intensity time traces from single molecules revealed reconfiguration of the polypeptide chain on a timescale of approximately equal to 20 micros at 1.7 M GdmCl. Slow conformational dynamics gave rise to characteristic, stepwise FRET efficiency changes. Transitions between folded and unfolded enzyme molecules occurred on the 100-s timescale, in excellent agreement with bulk denaturation experiments. Transitions between unfolded conformations were more frequent, with characteristic times of approximately equal to 2 s. These data were analyzed to obtain information on the free energy landscape of RNase H in the presence of chemical denaturants.     

Watching proteins fold one molecule at a time

Recent theoretical work suggests that protein folding involves an ensemble of pathways on a rugged energy landscape. We provide direct evidence for heterogeneous folding pathways from single-molecule studies, facilitated by a recently developed immobilization technique. Individual fluorophore-labeled molecules of the protein adenylate kinase were trapped within surface-tethered lipid vesicles, thereby allowing spatial restriction without inducing any spurious interactions with the environment, which often occur when using direct surface-linking techniques. The conformational fluctuations of these protein molecules, prepared at the thermodynamic midtransition point, were studied by using fluorescence resonance energy transfer between two specifically attached labels. Folding and unfolding transitions appeared in experimental time traces as correlated steps in donor and acceptor fluorescence intensity. The size of the steps, in fluorescence resonance energy transfer efficiency units, shows a very broad distribution. This distribution peaks at a relatively low value, indicating a preference for small-step motion on the energy landscape. The time scale of the transitions is also distributed, and although many transitions are too fast to be time-resolved here, the slowest ones may take >1 sec to complete. These extremely slow changes during the folding of single molecules highlight the possible importance of correlated, non-Markovian conformational dynamics.     

Mg2+-dependent conformational change of RNA studied by fluorescence correlation and FRET on immobilized single molecules

Fluorescence correlation spectroscopy (FCS) of fluorescence resonant energy transfer (FRET) on immobilized individual fluorophores was used to study the Mg2+-facilitated conformational change of an RNA three-helix junction, a structural element that initiates the folding of the 30S ribosomal subunit. Transitions of the RNA junction between open and folded conformations resulted in fluctuations in fluorescence by FRET. Fluorescence fluctuations occurring between two FRET states on the millisecond time scale were found to be dependent on Mg2+ and Na+ concentrations. Correlation functions of the fluctuations were used to determine transition rates between the two conformations as a function of Mg2+ or Na+ concentration. Both the opening and folding rates were found to vary with changing salt conditions. Assuming specific binding of divalent ions to RNA, the Mg2+ dependence of the observed rates cannot be explained by conformational change induced by Mg2+ binding/unbinding, but is consistent with a model in which the intrinsic conformational change of the RNA junction is altered by uptake of Mg2+ ion(s). This version of FCS/FRET on immobilized single molecules is demonstrated to be a powerful technique in the study of conformational dynamics of biomolecules over time scales ranging from microseconds to seconds.     

Single-molecule fluorescence spectroscopy of enzyme conformational dynamics and cleavage mechanism

Fluorescence resonance energy transfer and fluorescence polarization anisotropy are used to investigate single molecules of the enzyme staphylococcal nuclease. Intramolecular fluorescence resonance energy transfer and fluorescence polarization anisotropy measurements of fluorescently labeled staphylococcal nuclease molecules reveal distinct patterns of fluctuations that may be attributed to protein conformational dynamics on the millisecond time scale. Intermolecular fluorescence resonance energy transfer measurements provide information about the dynamic interactions of staphylococcal nuclease with single substrate molecules. The experimental methods demonstrated here should prove generally useful in studies of protein folding and enzyme catalysis at single-molecule resolution.     

Evaluation of the distribution of distances between energy donors and acceptors by fluorescence decay

When transfer of electronic excitation energy occurs between a donor-acceptor pair by the Förster mechanism, the decay of fluorescence of the donor follows first-order kinetics, with a rate constant that depends on the distance from donor to acceptor. In a system that contains donor-acceptor pairs of different separations, the fluorescence decay of the donors will not be exponential, but will depend on the distribution function of donor-acceptor distances, f(r). Various approaches are outlined for the extraction of information about f(r) from the decay curve of donor fluorescence. Specifically, if a plausible expression with adjustable parameters is assumed for f(r), numerical methods can be used to evaluate the parameters that yield the closest fit between the observed decay curve and that calculated from the assumed f(r). The technique of fluorescence decay may prove to be useful for determination of distribution functions of end-to-end distances of polymers to the edges of which suitable donor-acceptor chromophore pairs have been attached.     

Protein dynamics and electron transfer: electronic decoherence and non-Condon effects

We compute the autocorrelation function of the donor-acceptor tunneling matrix element for six Ru-azurin derivatives. Comparison of this decay time to the decay time of the time-dependent Franck-Condon factor {computed by Rossky and coworkers [Lockwood, D. M., Cheng, Y.-K. & Rossky, P. J. (2001) Chem. Phys. Lett. 345, 159-165]} reveals the extent to which non-Condon effects influence the electron-transfer rate. is studied as a function of donor-acceptor distance, tunneling pathway structure, tunneling energy, and temperature to explore the structural and dynamical origins of non-Condon effects. For azurin, the correlation function is remarkably insensitive to tunneling pathway structure. The decay time is only slightly shorter than it is for solvent-mediated electron transfer in small organic molecules and originates, largely, from fluctuations of valence angles rather than bond lengths.     

Complementarity of ensemble and single-molecule measures of protein motion: a relaxation dispersion NMR study of an enzyme complex

Single-molecule fluorescence experiments have shown that the conformation of the complex between Escherichia coli general NAD(P)H:flavin oxidoreductase (FRE) and flavin adenine dinucleotide (FAD) fluctuates over a range of timescales between 10(-4) and 1 s. Here we use (15)N and (13)C relaxation dispersion NMR methods to study millisecond-timescale dynamics in the complex. In this time regime, the protein is extremely flexible, with residues that undergo conformational exchange located throughout the molecule. Three distinct regions of dynamics are quantified, with two of them involving residues making contact to the donor (Tyr-35) and acceptor (FAD) sites that participate in the electron transfer reaction monitored in single-molecule experiments. Modulation of the donor-acceptor distance through these conformational exchange processes, occurring with rates of approximately 400 and 1,200 s(-1) (22 degrees C), affects the rate of electron transfer and partially accounts for the range of the observed dynamics monitored in the fluorescence experiments.     

Single-pair fluorescence resonance energy transfer on freely diffusing molecules: observation of Förster distance dependence and subpopulations

Photon bursts from single diffusing donor-acceptor labeled macromolecules were used to measure intramolecular distances and identify subpopulations of freely diffusing macromolecules in a heterogeneous ensemble. By using DNA as a rigid spacer, a series of constructs with varying intramolecular donor-acceptor spacings were used to measure the mean and distribution width of fluorescence resonance energy transfer (FRET) efficiencies as a function of distance. The mean single-pair FRET efficiencies qualitatively follow the distance dependence predicted by F?rster theory. Possible contributions to the widths of the FRET efficiency distributions are discussed, and potential applications in the study of biopolymer conformational dynamics are suggested. The ability to measure intramolecular (and intermolecular) distances for single molecules implies the ability to distinguish and monitor subpopulations of molecules in a mixture with different distances or conformational states. This is demonstrated by monitoring substrate and product subpopulations before and after a restriction endonuclease cleavage reaction. Distance measurements at single-molecule resolution also should facilitate the study of complex reactions such as biopolymer folding. To this end, the denaturation of a DNA hairpin was examined by using single-pair FRET.     

Nonexponential fluorescence decay of aqueous tryptophan and two related peptides by picosecond spectroscopy

Time-resolved fluorescence spectroscopy of tryptophan and two related dipeptides, tryptophylalanine and alanyltryptophan, has been carried out on the subnanosecond time scale by using picosecond exciting pulses at a wavelength of 264 nm. Detection was with an ultrafast streak camera coupled to an optical multichannel analyzer. The zwitterions of these molecules show a definite nonexponential fluorescence decay which can be analyzed in terms of two exponentials. The two decay rates increase strongly with increasing temperature, as does the weight of the faster component. In tryptophan at pH 11, where the amino group is deprotonated, there remains only a single temperature-dependent exponential. The results are interpreted in terms of two kinds of trapped conformers in the excited state that interconvert no quicker than the time scale of the fluorescence. A model is suggested in which the nonradiative processes in one conformer approximate those in the bare indole moiety. The nonradiative decay rate of the other conformer is substantially faster. It is believed that the process responsible for this fast decay is intramolecular electron transfer from the indole to the amino acid side chain. The predilection for this electron transfer depends on steric relationships as well as on the electron-attracting power of the carbonyl group. This picture is consistent with earlier fluorescence quantum yield results. In fact, a self-consistent picture emerges from the temporal and yield data that quantitatively explains most important facets of tryptophan photochemistry in aqueous solution.     

Ligand-induced conformational changes observed in single RNA molecules

We present the first demonstration that fluorescence resonance energy transfer can be used to track the motion of a single molecule undergoing conformational changes. As a model system, the conformational changes of individual three-helix junction RNA molecules induced by the binding of ribosomal protein S15 or Mg(2+) ions were studied by changes in single-molecule fluorescence. The transition from an open to a folded configuration was monitored by the change of fluorescence resonance energy transfer between two different dye molecules attached to the ends of two helices in the RNA junction. Averaged behavior of RNA molecules closely resembles that of unlabeled molecules in solution determined by other bulk assays, proving that this approach is viable and suggesting new opportunities for studying protein-nucleic acids interactions. Surprisingly, we observed an anomalously broad distribution of RNA conformations at intermediate ion concentrations that may be attributed to foldability differences among RNA molecules. In addition, an experimental scheme was developed where the real-time response of single molecules can be followed under changing environments. As a demonstration, we repeatedly changed Mg(2+) concentration in the buffer while monitoring single RNA molecules and showed that individual RNA molecules can measure the instantaneous Mg(2+) concentration with 20-ms time resolution, making it the world's smallest Mg(2+) meter.     

Reversible and irreversible intermediates during photoinhibition of photosystem II: stable reduced QA species promote chlorophyll triplet formation

Photoinhibition of photosynthesis was studied in isolated photosystem II membranes by using chlorophyll fluorescence and electron paramagnetic resonance (EPR) spectroscopy combined with protein analysis. Under anaerobic conditions four sequentially intermediate steps in the photoinhibitory process were identified and characterized. These intermediates show high dark chlorophyll fluorescence (Foi) with typical decay kinetics (fast, semistable, stable, and nondecaying). The fast-decaying state has no bound QB but possesses a single reduced QA species with a 30-s decay half-time in the dark (QB, second quinone acceptor; QA, first quinone acceptor). In the semistable state, Q-A is stabilized for 2-3 min, most likely by protonation, and gives rise to the Q-A Fe2+ EPR signal in the dark. In the stable state, QA has become double reduced and is stabilized for 0.5-2 hr by protonation and a protein conformational change. The final, nondecaying state is likely to represent centers where QA H2 has left its binding site. The first three photoinhibitory states are reversible in the dark through reestablishment of QA to QB electron transfer. Significantly, illumination at 4 K of anaerobically photoinhibited centers trapped in all but the fast state gives rise to a spinpolarized triplet EPR signal from chlorophyll P680 (primary electron donor). When oxygen is introduced during anaerobic illumination, the light-inducible chlorophyll triplet is lost concomitant with induction of D1 protein degradation. The results are integrated into a model for the photoinhibitory process involving initial loss of bound QB followed by stable reduction and subsequent loss of QA facilitating chlorophyll P680 triplet formation. This in turn mediates light-induced formation of highly reactive and damaging singlet oxygen.     

Fast, long-range, reversible conformational fluctuations in nucleosomes revealed by single-pair fluorescence resonance energy transfer

The nucleosome core particle, the basic repeated structure in chromatin fibers, consists of an octamer of eight core histone molecules, organized as dimers (H2A/H2B) and tetramers [(H3/H4)2] around which DNA wraps tightly in almost two left-handed turns. The nucleosome has to undergo certain conformational changes to allow processes that need access to the DNA template to occur. By single-pair fluorescence resonance energy transfer, we demonstrate fast, long-range, reversible conformational fluctuations in nucleosomes between two states: fully folded (closed), with the DNA wrapped around the histone core, or open, with the DNA significantly unraveled from the histone octamer. The brief excursions into an extended open state may create windows of opportunity for protein factors involved in DNA transactions to bind to or translocate along the DNA.     

Direct single-molecule observation of a protein living in two opposed native structures

Biological activity in proteins requires them to share the energy landscape for folding and global conformational motions, 2 key determinants of function. Although most structural studies to date have focused on fluctuations around a single structural basin, we directly observe the coexistence of 2 symmetrically opposed conformations for a mutant of the Rop-homodimer (Repressor of Primer) in single-molecule fluorescence resonance energy transfer (smFRET) measurements. We find that mild denaturing conditions can affect the sensitive balance between the conformations, generating an equilibrium ensemble consisting of 2 equally occupied structural basins. Despite the need for large-scale conformational rearrangement, both native structures are dynamically and reversibly adopted for the same paired molecules without separation of the constituent monomers. Such an ability of some proteins or protein complexes to switch between conformations by thermal fluctuations and/or minor environmental changes could be central to their ability to control biological function.     

Differential flexibilities in three branches of an N-linked triantennary glycopeptide.

The solution conformation behavior of complex oligosaccharides was studied by resonance energy transfer, as measured by the time-resolved fluorescence method, to determine the conformational heterogeneity of a triantennary glycopeptide at various temperatures. Groups that acted as a fluorescence donor (naphthyl-2-acetyl, Nap) or acceptor (dansylethylenediamine, Dan) were selectively attached to the N terminus of the peptide and a Gal residue [either 6' (shown below), 6, or 8] of the oligosaccharide, respectively. [formula: see text] Time-resolved fluorescence energy-transfer measurements revealed two populations of conformers when Dan was attached to either Gal-6' or Gal-6. One conformer contained the antenna folded back toward the core region, and a second was in an extended conformation. The two conformations differed in donor-acceptor distance by about 10 A. Systematically increasing the temperature from 0 degrees C to 40 degrees C increased the ratio of extended to folded forms 2-fold for the Gal-6 isomer and 4-fold for the Gal-6' isomer, whereas the Gal-8 isomer showed only a single distance population throughout this temperature range. From these data, delta H and delta S for the reversible conformational change were calculated to be 3.1 kcal/mol and 10.8 cal/(mol.K) for the Gal-6 isomer and 7.1 kcal/mol and 25.8 cal/(mol.K) for the Gal-6' isomer. In addition to the structural microheterogeneity commonly associated with glycoproteins, the differential flexibilities of the different branches in the oligosaccharides contribute conformational heterogeneity and should be considered in conformational analysis. The data are discussed in terms of the most probable linkages that contribute to the observed flexibility of the individual triantennary branches, and the biological significance of flexible linkages in complex carbohydrates is considered.     

Ultrafast real-time visualization of active site flexibility of flavoenzyme thymidylate synthase ThyX

In many bacteria the flavoenzyme thymidylate synthase ThyX produces the DNA nucleotide deoxythymidine monophosphate from dUMP, using methylenetetrahydrofolate as carbon donor and NADPH as hydride donor. Because all three substrates bind in close proximity to the catalytic flavin adenine dinucleotide group, substantial flexibility of the ThyX active site has been hypothesized. Using femtosecond time-resolved fluorescence spectroscopy, we have studied the conformational heterogeneity and the conformational interconversion dynamics in real time in ThyX from the hyperthermophilic bacterium Thermotoga maritima. The dynamics of electron transfer to excited flavin adenine dinucleotide from a neighboring tyrosine residue are used as a sensitive probe of the functional dynamics of the active site. The fluorescence decay spanned a full three orders of magnitude, demonstrating a very wide range of conformations. In particular, at physiological temperatures, multiple angstrom cofactor-residue displacements occur on the picoseconds timescale. These experimental findings are supported by molecular dynamics simulations. Binding of the dUMP substrate abolishes this flexibility and stabilizes the active site in a configuration where dUMP closely interacts with the flavin cofactor and very efficiently quenches fluorescence itself. Our results indicate a dynamic selected-fit mechanism where binding of the first substrate dUMP at high temperature stabilizes the enzyme in a configuration favorable for interaction with the second substrate NADPH, and more generally have important implications for the role of active site flexibility in enzymes interacting with multiple poly-atom substrates and products. Moreover, our data provide the basis for exploring the effect of inhibitor molecules on the active site dynamics of ThyX and other multisubstrate flavoenzymes.     

Probing the interaction between two single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor.

We extend the sensitivity of fluorescence resonance energy transfer (FRET) to the single molecule level by measuring energy transfer between a single donor fluorophore and a single acceptor fluorophore. Near-field scanning optical microscopy (NSOM) is used to obtain simultaneous dual color images and emission spectra from donor and acceptor fluorophores linked by a short DNA molecule. Photodestruction dynamics of the donor or acceptor are used to determine the presence and efficiency of energy transfer. The classical equations used to measure energy transfer on ensembles of fluorophores are modified for single-molecule measurements. In contrast to ensemble measurements, dynamic events on a molecular scale are observable in single pair FRET measurements because they are not canceled out by random averaging. Monitoring conformational changes, such as rotations and distance changes on a nanometer scale, within single biological macromolecules, may be possible with single pair FRET.     

Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy

We have investigated the pH dependence of the dynamics of conformational fluctuations of green fluorescent protein mutants EGFP (F64L/S65T) and GFP-S65T in small ensembles of molecules in solution by using fluorescence correlation spectroscopy (FCS). FCS utilizes time-resolved measurements of fluctuations in the molecular fluorescence emission for determination of the intrinsic dynamics and thermodynamics of all processes that affect the fluorescence. Fluorescence excitation of a bulk solution of EGFP decreases to zero at low pH (pK_a = 5.8) paralleled by a decrease of the absorption at 488 nm and an increase at 400 nm. Protonation of the hydroxyl group of Tyr-66, which is part of the chromophore, induces these changes. When FCS is used the fluctuations in the protonation state of the chromophore are time resolved. The autocorrelation function of fluorescence emission shows contributions from two chemical relaxation processes as well as diffusional concentration fluctuations. The time constant of the fast, pH-dependent chemical process decreases with pH from 300 μs at pH 7 to 45 μs at pH 5, while the time-average fraction of molecules in a nonfluorescent state increases to 80% in the same range. A second, pH-independent, process with a time constant of 340 μs and an associated fraction of 13% nonfluorescent molecules is observed between pH 8 and 11, possibly representing an internal proton transfer process and associated conformational rearrangements. The FCS data provide direct measures of the dynamics and the equilibrium properties of the protonation processes. Thus FCS is a convenient, intrinsically calibrated method for pH measurements in subfemtoliter volumes with nanomolar concentrations of EGFP.     

Conformational fluctuations in single DNA molecules

Measurement of fluorescent lifetimes of dye-tagged DNA molecules reveal the existence of different conformations. Conformational fluctuations observed by fluorescence correlation spectroscopy give rise to a relaxation behavior that is described by “stretched” exponentials and indicates the presence of a distribution of transition rates between two conformations. Whether this is an inhomogeneous distribution, where each molecule contributes with its own reaction rate to the overall distribution, or a homogeneous distribution, where the reaction rate of each molecule is time-dependent, is not yet known. We used a tetramethylrhodamine-linked 217-bp DNA oligonucleotide as a probe for conformational fluctuations. Fluorescence fluctuations from single DNA molecules attached to a streptavidin-coated surface directly show the transitions between two conformational states. The conformational fluctuations typical for single molecules are similar to those seen in single ion channels in cell membranes.     

Conformational dependence of electron transfer across de novo designed metalloproteins.

Flash photolysis and pulse radiolysis measurements demonstrate a conformational dependence of electron transfer rates across a 16-mer helical bundle (three-helix metalloprotein) modified with a capping CoIII(bipyridine)3 electron acceptor at the N terminus and a 1-ethyl-1'-ethyl-4,4'- bipyridinium donor at the C terminus. For the CoIII(peptide)3-1-ethyl-1'-ethyl-4,4'-bipyridinium maquettes, the observed transfer is a first order, intramolecular process, independent of peptide concentration or laser pulse energy. In the presence of 6 M urea, the random coil bundle (approximately 0% helicity) has an observed electron transfer rate constant of kobs = 900 +/- 100 s-1. In the presence of 25% trifluoroethanol (TFE), the helicity of the peptide is 80% and the kobs increases to 2000 +/- 200 s-1. Moreover, the increase in the rate constant in TFE is consistent with the observed decrease in donor-acceptor distance in this solvent. Such bifunctional systems provide a class of molecules for testing the effects of conformation on electron transfer in proteins and peptides.     

Polyproline and the "spectroscopic ruler" revisited with single-molecule fluorescence

To determine whether Forster resonance energy transfer (FRET) measurements can provide quantitative distance information in single-molecule fluorescence experiments on polypeptides, we measured FRET efficiency distributions for donor and acceptor dyes attached to the ends of freely diffusing polyproline molecules of various lengths. The observed mean FRET efficiencies agree with those determined from ensemble lifetime measurements but differ considerably from the values expected from Forster theory, with polyproline treated as a rigid rod. At donor-acceptor distances much less than the Forster radius R(0), the observed efficiencies are lower than predicted, whereas at distances comparable to and greater than R(0), they are much higher. Two possible contributions to the former are incomplete orientational averaging during the donor lifetime and, because of the large size of the dyes, breakdown of the point-dipole approximation assumed in Forster theory. End-to-end distance distributions and correlation times obtained from Langevin molecular dynamics simulations suggest that the differences for the longer polyproline peptides can be explained by chain bending, which considerably shortens the donor-acceptor distances.