Structural Changes of Yellow Cameleon Domains Observed by Quantitative FRET Analysis and Polarized Fluorescence Correlation Spectroscopy

Förster resonance energy transfer (FRET) is a widely used method for monitoring interactions between or within biological macromolecules conjugated with suitable donor-acceptor pairs. Donor fluorescence lifetimes in absence and presence of acceptor molecules are often measured for the observation of FRET. However, these lifetimes may originate from interacting and noninteracting molecules, which hampers quantitative interpretation of FRET data. We describe a methodology for the detection of FRET that monitors the rise time of acceptor fluorescence on donor excitation thereby detecting only those molecules undergoing FRET. The large advantage of this method, as compared to donor fluorescence quenching method used more commonly, is that the transfer rate of FRET can be determined accurately even in cases where the FRET efficiencies approach 100% yielding highly quenched donor fluorescence. Subsequently, the relative orientation between donor and acceptor chromophores is obtained from time-dependent fluorescence anisotropy measurements carried out under identical conditions of donor excitation and acceptor detection. The FRET based calcium sensor Yellow Cameleon 3.60 (YC3.60) was used because it changes its conformation on calcium binding, thereby increasing the FRET efficiency. After mapping distances and orientation angles between the FRET moieties in YC3.60, cartoon models of this FRET sensor with and without calcium could be created. Independent support for these representations came from experiments where the hydrodynamic properties of YC3.60 under ensemble and single-molecule conditions on selective excitation of the acceptor were determined. From rotational diffusion times as found by fluorescence correlation spectroscopy and consistently by fluorescence anisotropy decay analysis it could be concluded that the open structure (without calcium) is flexible as opposed to the rather rigid closed conformation. The combination of two independent methods gives consistent results and presents a rapid and specific methodology to analyze structural and dynamical changes in a protein on ligand bindin

Precision and accuracy of single-molecule FRET measurements - a multi-laboratory benchmark study

Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods

Oxide-supported Rh particle structure probed with carbon monoxide

Rh deposits in a wide range of metal exposures have been grown on a thin, well-ordered alumina film under ultra-high vacuum conditions. Their morphologies range from individual Rh particles with sizes below ten atoms to closed metal films, as determined by scanning tunneling microscopy (STM). CO chemisorption on these deposits has been studied by infrared reflection absorption spectroscopy (IRAS) and X-ray photoelectron spectroscopy (XPS). At low metal coverages, the formation of isolated Rh carbonyl species associated with surface defects is observed. Changes in the infrared spectra of CO adsorbed on annealed Rh aggregates are attributed to a thermally induced ordering of the Rh particle surface on a local scale, leading to a reduction in the number of low coordinated metal atoms. This is accompanied by a marked decrease of the CO chemisorption capacity. Adsorbed CO reduces the extent of this ordering process, while the adlayer itself reorganizes

Diffusion of macromolecules in a polymer hydrogel: from microscopic to macroscopic scales

To gain insight into the fundamental processes determining the motion of macromolecules in polymeric matrices{,} the dynamical hindrance of polymeric dextran molecules diffusing as probe through a polyacrylamide hydrogel is systematically explored. Three complementary experimental methods combined with Brownian dynamics simulations are used to study a broad range of dextran molecular weights and salt concentrations. While multi-parameter fluorescence image spectroscopy (MFIS) is applied to investigate the local diffusion of single molecules on a microscopic length scale inside the hydrogel{,} a macroscopic transmission imaging (MTI) fluorescence technique and nuclear magnetic resonance (NMR) are used to study the collective motion of dextrans on the macroscopic scale. These fundamentally different experimental methods{,} probing different length scales of the system{,} yield long-time diffusion coefficients for the dextran molecules which agree quantitatively. The measured diffusion coefficients decay markedly with increasing molecular weight of the dextran and fall onto a master curve. The observed trends of the hindrance factors are consistent with Brownian dynamics simulations. The simulations also allow us to estimate the mean pore size for the herein investigated experimental conditions. In addition to the diffusing molecules{,} MFIS detects temporarily trapped molecules inside the matrix with diffusion times above 10 ms{,} which is also confirmed by anisotropy analysis. The fraction of bound molecules depends on the ionic strength of the solution and the charge of the dye. Using fluorescence intensity analysis{,} also MTI confirms the observation of the interaction of dextrans with the hydrogel. Moreover{,} pixelwise analysis permits to show significant heterogeneity of the gel on the microscopic scale

Resolving dynamics and function of transient states in single enzyme molecules

We use a hybrid fluorescence spectroscopic toolkit to monitor T4 Lysozyme (T4L) in action by unraveling the kinetic and dynamic interplay of the conformational states. In particular, by combining single-molecule and ensemble multiparameter fluorescence detection, EPR spectroscopy, mutagenesis, and FRET-positioning and screening, and other biochemical and biophysical tools, we characterize three short-lived conformational states over the ns-ms timescale. The use of 33 FRET-derived distance sets, to screen available T4L structures, reveal that T4L in solution mainly adopts the known open and closed states in exchange at 4 µs. A newly found minor state, undisclosed by, at present, more than 500 crystal structures of T4L and sampled at 230 µs, may be actively involved in the product release step in catalysis. The presented fluorescence spectroscopic toolkit will likely accelerate the development of dynamic structural biology by identifying transient conformational states that are highly abundant in biology and critical in enzymatic reactions