Confocal single molecule fluorescence detection - methodical developments and applications to biological specimens

Over the last few decades, single-molecule FRET has become a valuable tool enlightening the fields of molecular conformational dynamics, folding and structure determination of biological macro-molecules. However, quantitative statements about any parameter obtained using FRET are based on aprecise calibration of the acquired data, among others taking into account the properties of the FRET-pair used. In this work, two methods are developed and established that facilitate this calibration by means of confocal microscopy, automatically allowing a sample characterization under application relevant conditions, i.e. close to the single-molecule level. To assess the orientation factor entering the Förster radius calculation, time-resolved anisotropy measurements are performed. In this regard, the depolarization effects induced by the use of a high numerical aperture objective have to be taken into account by two correction factors. These are precisely determined by combining an extended experimental calibration procedure adjusted to the temporal resolution of the setup at hand with theoretical predictions considering the exact measurement conditions. To determine the fluorescence quantum yields of donor and acceptor used in the FRET efficiency calculation, the linear relation between the molecular brightness, made accessible by Fluorescence Correlation Spectroscopy, and the fluorescence quantum yield in the limit of low excitation intensities is exploited. Based on this, the presented quantum yield determination method lowers the needed amount of sample by a factor of around thirty as compared to a commonly applied optical method, but still provides at least the same precision. As compared to fluorescence lifetime based quantum yield determination methods, the novel approach is more comprehensive as it is sensitive not only to dynamic, but also to static fluorescence quenching. Hence, apart from its relevance for smFRET, a reliable characterization of biological samples with limited expression yields is made possible. With these two methodical developments available, smFRET data of structurally rigid, double-stranded DNA oligonucleotides in aqueous buffer and in buffers with specific amounts of glycerol, guanidine hydrochloride and sodium chloride added are analyzed. It is demonstrated that the calculation of inter-dye distances, without taking into account solvent-induced spectral and photo-physical changes of the labels, leads to deviations of up to 4 Å from the real inter-dye distances and furthermore to a misinterpretation of the underlying structural changes. Additionally, it is experimentally shown that electrostatic dye dye repulsions are negligible for the inter-dye distance regime considered here (> 50 Å ). Expanding the given framework of accessible volume calculations by taking into account the electrostatic interaction potential of donor and acceptor in the respective solvent environment, these findings are supported by theoretical predictions. Finally, all methodical approaches and experimental/theoretical findings are combined to validate the further compaction of the already unfolded state of the protein Phosphoglycerate Kinase (PGK) with decreasing concentrations of denaturant, a mechanism known as coil-globule transition

Inter-Dye Distance Distributions Studied by a Combination of Single-Molecule FRET-Filtered Lifetime Measurements and a Weighted Accessible Volume (wAV) Algorithm

Förster resonance energy transfer (FRET) is an important tool for studying the structural and dynamical properties of biomolecules. The fact that both the internal dynamics of the biomolecule and the movements of the biomolecule-attached dyes can occur on similar timescales of nanoseconds is an inherent problem in FRET studies. By performing single-molecule FRET-filtered lifetime measurements, we are able to characterize the amplitude of the motions of fluorescent probes attached to double-stranded DNA standards by means of flexible linkers. With respect to previously proposed experimental approaches, we improved the precision and the accuracy of the inter-dye distance distribution parameters by filtering out the donor-only population with pulsed interleaved excitation. A coarse-grained model is employed to reproduce the experimentally determined inter-dye distance distributions. This approach can easily be extended to intrinsically flexible proteins allowing, under certain conditions, to decouple the macromolecule amplitude of motions from the contribution of the dye linkers

Nanosecond Dynamics of Calmodulin and Ribosome-Bound Nascent Chains Studied by Time-Resolved Fluorescence Anisotropy

We report a time-resolved fluorescence anisotropy study of ribosome-bound nascent chains (RNCs) of calmodulin (CaM), a prototypical member of the EF-hand family of calcium-sensing proteins. As shown in numerous studies, in vitro protein refolding can differ substantially from biosynthetic protein folding, which takes place cotranslationally and depends on the rate of polypeptide chain elongation. A challenge in this respect is to characterize the adopted conformations of nascent chains before their release from the ribosome. CaM RNCs (full-length, half-length, and first EF-hand only) were synthesized in vitro. All constructs contained a tetracysteine motif site-specifically incorporated in the first N-terminal helix; this motif is known to react with FlAsH, a biarsenic fluorescein derivative. As the dye is rotationally locked to this helix, we characterized the structural properties and folding states of polypeptide chains tethered to ribosomes and compared these with released chains. Importantly, we observed decelerated tumbling motions of ribosome-tethered and partially folded nascent chains, compared to released chains. This indicates a pronounced interaction between nascent chains and the ribosome surface, and might reflect chaperone activity of the ribosome

Single-Molecule FRET Measurements in Additive-Enriched Aqueous Solutions

The addition of high amounts of chemical denaturants, salts, viscosity enhancers or macro-molecular crowding agents has an impact on the physical properties of buffer solutions. Among others, the (microscopic) viscosity, the refractive index, the dielectric constant, and the ionic strength can be affected. Here, we systematically evaluate the importance of solvent characteristics with respect to single-molecule FRET (smFRET) data. First, we present a confocal based method for the determination of fluorescence quantum yields to facilitate a fast characterization of smFRET-samples at sub-nM-concentrations. As a case study, we analyze smFRET data of structurally rigid, double-stranded DNA-oligonucleotides in aqueous buffer and in buffers with specific amounts of glycerol, guanidine hydrochloride (GdnHCl), and sodium chloride (NaCl) added. We show that the calculation of interdye distances, without taking into account solvent-induced spectral and photophysical changes of the labels, leads to deviations of up to 4 Å from the real interdye distances. Additionally, we demonstrate that electrostatic dye–dye repulsions are negligible for the interdye distance regime considered here (>50 Å). Finally, we use our approach to validate the further compaction of the already unfolded state of phosphoglycerate kinase (PGK) with decreasing denaturant concentrations, a mechanism known as coil–globule transition

Ungewöhnlich großer orbitaler Fremdkörper - eine diagnostische und therapeutische Herausforderung

Here, we present a comparative method for the accurate determination of fluorescence quantum yields (QYs) by fluorescence correlation spectroscopy. By exploiting the high sensitivity of single-molecule spectroscopy, we obtain the QYs of samples in the microliter range and at (sub)­nanomolar concentrations. Additionally, in combination with fluorescence lifetime measurements, our method allows the quantification of both static and collisional quenching constants. Thus, besides being simple and fast, our method opens up the possibility to photophysically characterize labeled biomolecules under application-relevant conditions and with low sample consumption, which is often important in single-molecule studies