We present a comprehensive toolkit for Förster resonance energy transfer (FRET)-restrained modeling of biomolecules and their complexes for quantitative applications in structural biology. A dramatic improvement in the precision of FRET-derived structures is achieved by explicitly considering spatial distributions of dye positions, which greatly reduces uncertainties due to flexible dye linkers. The precision and confidence levels of the models are calculated by rigorous error estimation. The accuracy of this approach is demonstrated by docking a DNA primer-template to HIV-1 reverse transcriptase. The derived model agrees with the known X-ray structure with an r.m.s. deviation of 0.5 Å. Furthermore, we introduce FRET-guided 'screening' of a large structural ensemble created by molecular dynamics simulations. We used this hybrid approach to determine the formerly unknown configuration of the flexible single-strand template overhang.

No abstract

In Förster resonance energy transfer (FRET) experiments, the donor (D) and acceptor (A) fluorophores are usually attached to the macromolecule of interest via long flexible linkers of up to 15 Å in length. This causes significant uncertainties in quantitative distance measurements and prevents experiments with short distances between the attachment points of the dyes due to possible dye-dye interactions. We present two approaches to overcome the above problems as demonstrated by FRET measurements for a series of dsDNA and dsRNA internally labeled with Alexa488 and Cy5 as D and A dye, respectively. First, we characterize the influence of linker length and flexibility on FRET for different dye linker types (long, intermediate, short) by analyzing fluorescence lifetime and anisotropy decays. For long linkers, we describe a straightforward procedure that allows for very high accuracy of FRET-based structure determination through proper consideration of the position distribution of the dye and of linker dynamics. The position distribution can be quickly calculated with geometric accessible volume (AV) simulations, provided that the local structure of RNA or DNA in the proximity of the dye is known and that the dye diffuses freely in the sterically allowed space. The AV approach provides results similar to molecular dynamics simulations (MD) and is fully consistent with experimental FRET data. In a benchmark study for ds A-RNA, an rmsd value of 1.3 Å is achieved. Considering the case of undefined dye environments or very short DA distances, we introduce short linkers with a propargyl or alkenyl unit for internal labeling of nucleic acids to minimize position uncertainties. Studies by ensemble time correlated single photon counting and single-molecule detection show that the nature of the linker strongly affects the radius of the dye's accessible volume (6-16 Å). For short propargyl linkers, heterogeneous dye environments are observed on the millisecond time scale. A detailed analysis of possible orientation effects (κ(2) problem) indicates that, for short linkers and unknown local environments, additional κ(2)-related uncertainties are clearly outweighed by better defined dye positions.

This work is dedicated to Christoph Bräuchle on the occasion of his 65th birthday in honor of his pioneering research in single molecule fluorescence spectroscopy and microscopy.

Two complementary methods in confocal single-molecule fluorescence spectroscopy are presented to analyze conformational dynamics by Forster resonance energy transfer (FRET) measurements considering simulated and experimental data. First, an extension of photon distribution analysis (PDA) is applied to characterize conformational exchange between two or more states via global analysis of the shape of FRET peaks for different time bins. PDA accurately predicts the shape of FRET efficiency histograms in the presence of FRET fluctuations, taking into account shot noise and background contributions. Dynamic-PDA quantitatively recovers FRET efficiencies of the interconverting states and relaxation times of dynamics on the time scale of the diffusion time t(d) (typically milliseconds), with a dynamic range of the method of about +/-1 order of magnitude with respect to t(d). Correction procedures are proposed to consider the factors limiting the accuracy of dynamic-PDA, such as brightness variations, shortening of the observation time due to diffusion, and a contribution of multimolecular events. Second, an analysis procedure for multiparameter fluorescence detection is presented, where intensity-derived FRET efficiency is correlated with the fluorescence lifetime of the donor quenched by FRET. If a maximum likelihood estimator is applied to compute a mean fluorescence lifetime of mixed states, one obtains a fluorescence weighted mean lifetime. Thus a mixed state is detected by a characteristic shift of the fluorescence lifetime, which becomes longer than that expected for a single species with the same intensity-derived FRET efficiency. Analysis tools for direct visual inspection of two-dimensional diagrams of FRET efficiency versus donor lifetime are presented for the cases of static and dynamic FRET. Finally these new techniques are compared with fluorescence correlation spectroscopy.

The nucleosome has a central role in the compaction of genomic DNA and the control of DNA accessibility for transcription and replication. To help understanding the mechanism of nucleosome opening and closing in these processes, we studied the disassembly of mononucleosomes by quantitative single-molecule FRET with high spatial resolution, using the SELEX-generated ''Widom 601'' positioning sequence labeled with donor and acceptor fluorophores. Reversible dissociation was induced by increasing NaCl concentration. At least 3 species with different FRET were identified and assigned to structures: (i) the most stable high-FRET species corresponding to the intact nucleosome, (ii) a less stable mid-FRET species that we attribute to a first intermediate with a partially unwrapped DNA and less histones, and (iii) a low-FRET species characterized by a very broad FRET distribution, representing highly unwrapped structures and free DNA formed at the expense of the other 2 species. Selective FCS analysis indicates that even in the low-FRET state, some histones are still bound to the DNA. The interdye distance of 54.0 Å measured for the high-FRET species corresponds to a compact conformation close to the known crystallographic structure. The coexistence and interconversion of these species is first demonstrated under non-invasive conditions. A geometric model of the DNA unwinding predicts the presence of the observed FRET species. The different structures of these species in the disassembly pathway map the energy landscape indicating major barriers for 10-bp and minor ones for 5-bp DNA unwinding steps.chromatin ͉ mononucleosomes ͉ structure T he nucleosome, the basic unit of genome compaction (1), consists of Ϸ2 turns of double-stranded DNA wound around a histone protein octamer. Its structure is known to a resolution of 1.9 Å (2). DNA sequence, chemical modifications and histone composition modulate gene activity through this structure.Central to nucleosomal function is its restructuring during processes that act on DNA, e.g., transcription or replication. Biochemical data indicate that nucleosome dissociation is temporary. Mechanisms for nucleosome unfolding, unwrapping or repositioning have been proposed (3), but so far none has been proven by direct physical evidence (e.g., detection of intermediate states). Here, we use single molecule Förster resonance energy transfer (FRET) to obtain quantitative structural information for elucidating such mechanisms.Bulk solution FRET is a proven tool for measuring average distances; nucleosome dynamics have been quantified by FRET between dyes attached to DNA and/or histone proteins (4-6). Analyzing FRET from single molecules (7, 8) can give detailed information on structural diversity; e.g., FRET on surfacetethered nucleosomes demonstrated spontaneous structure fluctuations (9, 10), whereas confocal spFRET experiments (11, 12) on freely diffusing single nucleosomes detected structural subpopulations under various conditions.The transit of a single molecule through the laser focus ...

Heat shock proteins 70 (Hsp70) represent a ubiquitous and conserved family of molecular chaperones involved in a plethora of cellular processes. The dynamics of their ATP hydrolysis-driven and cochaperone-regulated conformational cycle are poorly understood. We used fluorescence spectroscopy to analyze, in real time and at single-molecule resolution, the effects of nucleotides and cochaperones on the conformation of Ssc1, a mitochondrial member of the family. We report that the conformation of its ADP state is unexpectedly heterogeneous, in contrast to a uniform ATP state. Substrates are actively involved in determining the conformation of Ssc1. The J protein Mdj1 does not interact transiently with the chaperone, as generally believed, but rather is released slowly upon ATP hydrolysis. Analysis of the major bacterial Hsp70 revealed important differences between highly homologous members of the family, possibly explaining tuning of Hsp70 chaperones to meet specific functions in different organisms and cellular compartments.

Single-molecule FRET experiments on freely diffusing rigid molecules frequently show FRET efficiency (E) distributions broader than those defined by photon statistics. It is often unclear whether the observed extra broadening can be attributed to a physical donor-acceptor distance (R(DA)) distribution. Using double-stranded DNA (dsDNA) labeled with Alexa488 and Cy5 (or Alexa647) as a test system, we investigate various possible contributions to the E distribution width. On the basis of simultaneous analysis of donor and acceptor intensities and donor lifetimes, we conclude that dsDNA chain dynamics can be ruled out as a possible reason for the observed E distribution broadening. We applied a set of tools to demonstrate that complex acceptor dye photophysics can represent a major contribution to the E distribution width. Quantitative analysis of the correlation between FRET efficiency and donor fluorescence lifetime in 2D multiparameter histograms allows one to distinguish between broadening due to distinct FRET or dye species. Moreover, we derived a simple theory, which predicts that the apparent distance width due to acceptor fluorescence quantum yield variations increases linearly with physical donor-acceptor distance. This theory nicely explains the experimentally observed FRET broadening of a series of freely diffusing labeled dsDNA and dsRNA molecules. Accounting for multiple acceptor states allowed the fitting of experimental E distributions, assuming a single fixed donor-acceptor distance.