Pseudoknot Formation Seeds the Twister Ribozyme Cleavage Reaction Coordinate

The twister RNA is a recently discovered nucleolytic ribozyme that is present in both bacteria and eukarya. While its biological role remains unclear, crystal structure analyses and biochemical approaches have revealed critical features of its catalytic mechanism. Here, we set out to explore dynamic aspects of twister RNA folding along the cleavage reaction coordinate. To do so, we have employed both bulk and single-molecule fluorescence resonance energy transfer (FRET) methods to investigate a set of twister RNAs with labels strategically positioned at communicating segments. The data reveal that folding of the central pseudoknot (T1), the most crucial structural determinant to promote cleavage, exhibits reversible opening and closing dynamics at physiological Mg²⁺ concentration. Uncoupled folding, in which T1 formation precedes structuring for closing of stem P1, was confirmed using pre-steady-state three-color smFRET experiments initiated by Mg²⁺ injection. This finding suggests that the folding path of twister RNA requires proper orientation of the substrate prior to T1 closure such that the U5-A6 cleavage site becomes embraced to achieve its cleavage competent conformation. We also find that the cleaved 3′-fragment retains its compacted pseudoknot fold, despite the absence of the phylogenetically conserved stem P1, rationalizing the poor turnover efficiency of the twister ribozyme

On the Mechanisms of Cyanine Fluorophore Photostabilization

Cyanine fluorophores exhibit greatly improved photostability when covalently linked to stabilizers, such as cyclooctatetraene (COT), nitrobenzyl alcohol (NBA), or Trolox. However, the mechanism by which photostabilization is mediated has yet to be determined. Here, we present spectroscopic evidence that COT, when covalently linked to Cy5, substantially reduces the lifetime of the Cy5 triplet state and that the degree of triplet-state quenching correlates with enhancements in photostability observed in single-molecule fluorescence measurements. By contrast, NBA and Trolox did not quench the Cy5 triplet state under our conditions, suggesting that their mechanism of photostabilization is different from that of COT and does not target the fluorophore triplet state directly. These findings provide insights into the mechanisms of fluorophore photostabilization that may lead to improved fluorophore designs for biological imaging applications

Engineering a Prototypic P‑type ATPase Listeria monocytogenes Ca²⁺-ATPase 1 for Single-Molecule FRET Studies

Approximately 30% of the ATP generated in the living cell is utilized by P-type ATPase primary active transporters to generate and maintain electrochemical gradients across biological membranes. P-type ATPases undergo large conformational changes during their functional cycle to couple ATP hydrolysis in the cytoplasmic domains to ion transport across the membrane. The Ca²⁺-ATPase from Listeria monocytogenes, LMCA1, was found to be a suitable model of P-type ATPases and was engineered to facilitate single-molecule FRET studies of transport-related structural changes. Mutational analyses of the endogenous cysteine residues in LMCA1 were performed to reduce background labeling without compromising activity. Pairs of cysteines were introduced into the optimized low-reactivity background, and labeled with maleimide derivatives of Cy3 and Cy5 resulting in site-specifically double-labeled protein with moderate activity. Ensemble and confocal single-molecule FRET studies revealed changes in FRET distribution related to structural changes during the transport cycle, consistent with those observed by X-ray crystallography for the sarco/endoplasmic reticulum Ca²⁺ ATPase (SERCA). Notably, the cytosolic headpiece of LMCA1 was found to be distinctly more compact in the E1 state than in the E2 state. Thus, the established experimental system should allow future real-time FRET studies of the structural dynamics of LMCA1 as a representative P-type ATPase