Conformations of a Metastable SH3 Domain Characterized by smFRET and an Excluded-Volume Polymer Model.

Conformational states of the metastable drkN SH3 domain were characterized using single-molecule fluorescence techniques. Under nondenaturing conditions, two Förster resonance energy transfer (FRET) populations were observed that corresponded to a folded and an unfolded state. FRET-estimated radii of gyration and hydrodynamic radii estimated by fluorescence correlation spectroscopy of the two coexisting conformations are in agreement with previous ensemble x-ray scattering and NMR measurements. Surprisingly, when exposed to high concentrations of urea and GdmCl denaturants, the protein still exhibits two distinct FRET populations. The dominant conformation is expanded, showing a low FRET efficiency, consistent with the expected behavior of a random chain with excluded volume. However, approximately one-third of the drkN SH3 conformations showed high, nearly 100%, FRET efficiency, which is shown to correspond to denaturation-induced looped conformations that remain stable on a timescale of at least 100 µs. These loops may contain interconverting conformations that are more globally collapsed, hairpin-like, or circular, giving rise to the observed heterogeneous broadening of this population. Although the underlying mechanism of chain looping remains elusive, FRET experiments in formamide and dimethyl sulfoxide suggest that interactions between hydrophobic groups in the distal regions may play a significant role in the formation of the looped state.

Sub-diffusion decays in fluorescence correlation spectroscopy: dye photophysics or protein dynamics?

Transitions between bright and dark fluorescent states of several rhodamine dyes were investigated by fluorescence correlation spectroscopy. We resolved two sub-diffusion exponential decays for free rhodamines in aqueous solutions, of which the slower component scales linearly with the viscosity of the solution. Correlation data for proteins and DNA labeled with tetramethylrhodamine were fitted with three to four exponential decays describing flickering dynamics on a time scale between 0.5 and 100 µs. We investigated the nature of these processes by performing experiments under different experimental conditions and for different samples. On the basis of how their population and lifetime change with viscosity, the oxygen content of the solution, the laser irradiance, and the detection geometry, we assigned these states, in the order of increasing lifetimes, to a triplet state, a hybrid between twisted-intramolecular-charge-transfer state and a ground state lactonic state, a lactonic state, and a photoionized state, respectively. Our data suggests that none of the observed sub-diffusion correlation decays can be directly assigned to the intramolecular dynamics of the labeled biomolecules. However, we found evidence that the intrinsic conformational dynamics of the biomolecule appears in the correlation curves as a modulation of the photophysics of the dye label. This shows the importance of accurate control measurements and appropriate modeling of the dye photophysics in fluorescence correlation studies, and it cautions against direct assignments of dark-state relaxation times to folding kinetics in proteins and nucleic acids.

On the performance of bioanalytical fluorescence correlation spectroscopy measurements in a multiparameter photon-counting microscope.

Fluorescence correlation spectroscopy (FCS) data acquisition and analysis routines were developed and implemented in a home-built, multiparameter photon-counting microscope. Laser excitation conditions were investigated for two representative fluorescent probes, Rhodamine110 and enhanced green fluorescent protein (EGFP). Reliable local concentrations and diffusion constants were obtained by fitting measured FCS curves, provided that the excitation intensity did not exceed 20% of the saturation level for each fluorophore. Accurate results were obtained from FCS measurements for sample concentrations varying from pM to µM range, as well as for conditions of high background signals. These experimental constraints were found to be determined by characteristics of the detection system and by the saturation behavior of the fluorescent probes. These factors actually limit the average number of photons that can be collected from a single fluorophore passing through the detection volume. The versatility of our setup and the data analysis capabilities were tested by measuring the mobility of EGFP in the nucleus of Drosophila cells under conditions of high concentration and molecular crowding. As a bioanalytical application, we studied by FCS the binding affinity of a novel peptide-based drug to the cancer-regulating STAT3 protein and corroborated the results with fluorescence polarization analysis derived from the same photon data.