An optofluidic antenna for enhancing the sensitivity of single-emitter measurements.

Many single-molecule investigations are performed in fluidic environments, for example, to avoid unwanted consequences of contact with surfaces. Diffusion of molecules in this arrangement limits the observation time and the number of collected photons, thus, compromising studies of processes with fast or slow dynamics. Here, we introduce a planar optofluidic antenna (OFA), which enhances the fluorescence signal from molecules by about 5 times per passage, leads to about 7-fold more frequent returns to the observation volume, and significantly lengthens the diffusion time within one passage.

Integrative dynamic structural biology unveils conformers essential for the oligomerization of a large GTPase.

Guanylate binding proteins (GBPs) are soluble dynamin-like proteins that undergo a conformational transition for GTP-controlled oligomerization and disrupt membranes of intracellular parasites to exert their function as part of the innate immune system of mammalian cells. We apply neutron spin echo, X-ray scattering, fluorescence, and EPR spectroscopy as techniques for integrative dynamic structural biology to study the structural basis and mechanism of conformational transitions in the human GBP1 (hGBP1). We mapped hGBP1's essential dynamics from nanoseconds to milliseconds by motional spectra of sub-domains.

Reliability and accuracy of single-molecule FRET studies for characterization of structural dynamics and distances in proteins.

Single-molecule Förster-resonance energy transfer (smFRET) experiments allow the study of biomolecular structure and dynamics in vitro and in vivo. We performed an international blind study involving 19 laboratories to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems with distinct conformational changes and dynamics, we obtained an uncertainty of the FRET efficiency ≤0.