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

DNA origami-based single-molecule forcespectroscopy elucidates RNA Polymerase IIIpre-initiation complex stability

The TATA-binding protein (TBP) and a transcription factor (TF) IIB-like factor are important constituents of all eukaryotic initiation complexes. The reason for the emergence and strict requirement of the additional initiation factor Bdp1 in the RNA polymerase (RNAP) III system, however, remained elusive. A poorly studied aspect in this context is the effect of DNA strain arising from DNA compaction and transcriptional activity on initiation complex formation. We made use of a DNA origami-based force clamp to follow the assembly of human initiation complexes in the RNAP II and RNAP III systems at the single-molecule level under piconewton forces. We demonstrate that TBP-DNA complexes are force-sensitive and TFIIB is sufficient to stabilise TBP on a strained promoter. In contrast, Bdp1 is the pivotal component that ensures stable anchoring of initiation factors, and thus the polymerase itself, in the RNAP III system. Thereby, we offer an explanation for the crucial role of Bdp1 for the high transcriptional output of RNAP III

Design considerations for haptic-enabled virtual reality simulation for interactive learning of nanoscale science in schools

This paper reports on a study which investigated whether the addition of haptics (virtual touch) to a 3D virtual reality (VR) simulation promotes understanding of key nanoscale concepts in membrane systems for students aged 12 to 13. We developed a virtual model of a section of the cell membrane and a haptic enabled interface that enables students to interact with the model and to manipulate objects in the model. Students, in two schools in England, worked collaboratively in pairs on activities designed to develop their understanding of key concepts of cell membrane function. Results of pre-and post-tests of conceptual knowledge and understanding showed significant knowledge gains but there were no significant differences between the haptic and non-haptic condition. However, findings from observation of the activities and student interviews revealed that students were very positive about using the system and believed that being able to feel structures and manipulate objects within the model assisted their learning. We examine some of the design challenges and issues affecting the perception of haptic feedback

Thermoswitchable Nanoparticles Based on Elastin-like Polypeptides

The design of biocompatible particles with defined size on the nanometer scale has proven to be a challenging task in current biomedical research. Here we present an approach toward temperature-responsive nanoparticles by covalently cross-linking micelles based on trimeric constructs of elastin-like polypeptides. These trimers can be triggered to assemble into micelles by heating the solution above a specific transition temperature (<i>T</i>_t) which was shown in previous studies. Here we show that the disassembly of the micelles below the <i>T</i>_t can be prevented by the incorporation of covalent cross-links in the core of the micelles. This facilitates a temperature-triggered swelling and collapsing by around 35% in diameter, as determined by dynamic light scattering. Size distribution was confirmed by fluorescence correlation spectroscopy, atomic force microscopy, and transmission electron microscopy. We show switchable nanoparticles with reversible volume changes in the temperature region between 30 and 40 °C, making these particles promising candidates for switchable drug delivery carriers

Dynamics of heat shock protein 90 C-terminal dimerization is an important part of its conformational cycle

The molecular chaperone heat shock protein 90 (Hsp90) is an important and abundant protein in eukaryotic cells, essential for the activation of a large set of signal transduction and regulatory proteins. During the functional cycle, the Hsp90 dimer performs large conformational rearrangements. The transient N-terminal dimerization of Hsp90 has been extensively investigated, under the assumption that the C-terminal interface is stably dimerized. Using a fluorescence-based single molecule assay and Hsp90 dimers caged in lipid vesicles, we were able to separately observe and kinetically analyze N- and C-terminal dimerizations. Surprisingly, the C-terminal dimer opens and closes with fast kinetics. The occupancy of the unexpected C-terminal open conformation can be modulated by nucleotides bound to the N-terminal domain and by N-terminal deletion mutations, clearly showing a communication between the two terminal domains. Moreover our findings suggest that the C- and N-terminal dimerizations are anticorrelated. This changes our view on the conformational cycle of Hsp90 and shows the interaction of two dimerization domains

A nucleotide-switch mechanism mediates opposing catalytic activities of Rel enzymes

Bifunctional Rel stringent factors, the most abundant class of RelA/SpoT homologs, are ribosome-associated enzymes that transfer a pyrophosphate from ATP onto the 3 ' of guanosine tri-/diphosphate (GTP/GDP) to synthesize the bacterial alarmone (p)ppGpp, and also catalyze the 3 ' pyrophosphate hydrolysis to degrade it. The regulation of the opposing activities of Rel enzymes is a complex allosteric mechanism that remains an active research topic despite decades of research. We show that a guanine-nucleotide-switch mechanism controls catalysis by Thermus thermophilus Rel (Rel(Tt)). The binding of GDP/ATP opens the N-terminal catalytic domains (NTD) of Rel(Tt) (Rel(Tt)(NTD)) by stretching apart the two catalytic domains. This activates the synthetase domain and allosterically blocks hydrolysis. Conversely, binding of ppGpp to the hydrolase domain closes the NTD, burying the synthetase active site and precluding the binding of synthesis precursors. This allosteric mechanism is an activity switch that safeguards against futile cycles of alarmone synthesis and degradation