Amino Acid Specific Effects on RNA Tertiary Interactions: Single-Molecule Kinetic and Thermodynamic Studies

In light of the current models for an early RNA-based universe, the potential influence of simple amino acids on tertiary folding of ribozymal RNA into biochemically competent structures is speculated to be of significant evolutionary importance. In the present work, the folding–unfolding kinetics of a ubiquitous tertiary interaction motif, the GAAA tetraloop–tetraloop receptor (TL–TLR), is investigated by single-molecule fluorescence resonance energy transfer spectroscopy in the presence of natural amino acids both <i>with</i> (e.g., lysine, arginine) and <i>without</i> (e.g., glycine) protonated side chain residues. By way of control, we also investigate the effects of a special amino acid (e.g., proline) and amino acid mimetic (e.g., betaine) that contain secondary or quaternary amine groups rather than a primary amine group. This combination permits systematic study of amino acid induced (or amino acid like) RNA folding dynamics as a function of side chain complexity, p<i>K</i>_a, charge state, and amine group content. Most importantly, each of the naturally occurring amino acids is found to <i>destabilize</i> the TL–TLR tertiary folding equilibrium, the kinetic origin of which is dominated by a <i>decrease</i> in the folding rate constant (<i>k</i>_dock), also affected by a strongly amino acid selective <i>increase</i> in the unfolding rate constant (<i>k</i>_undock). To further elucidate the underlying thermodynamics, single-molecule equilibrium constants (<i>K</i>_eq) for TL–TLR folding have been probed as a function of temperature, which reveal an amino acid dependent decrease in both overall exothermicity (ΔΔ<i>H</i>° > 0) and entropic cost (−<i>T</i>ΔΔ<i>S</i>° < 0) for the overall folding process. Temperature-dependent studies on the folding/unfolding kinetic rate constants reveal analogous amino acid specific changes in both enthalpy (ΔΔ<i>H</i>^⧧) and entropy (ΔΔ<i>S</i>^⧧) for accessing the transition state barrier. The maximum destabilization of the TL–TLR tertiary interaction is observed for arginine, which is consistent with early studies of arginine and guanidine ion-inhibited self-splicing kinetics for the full <i>Tetrahymena</i> ribozyme [Yarus, M.; Christian, E. L. Nature 1989, 342, 349−350; Yarus, M. Science 1988, 240, 1751–1758]

Excited State Proton Transfer Dynamics of Topotecan Inside Biomimicking Nanocavity

The excited state proton transfer (ESPT) dynamics of a potentially important anticancer drug, Topotecan (TPT), has been explored in aqueous reverse micelle (RM) using steady-state and time-resolved fluorescence measurements. Both the time-resolved emission spectrum and time-resolved area normalized emission spectrum infer the generation of excited state zwitterionic form of TPT from the excited state cationic form of TPT, as a result of ESPT process from the −OH group of TPT to the nearby water molecule. The ESPT dynamics were found to be severely retarded inside the nanocavities of RMs, yielding time constants of 250 ps to 1.0 ns, which is significantly slower than the dynamics obtained in bulk water (32 ps). The observed slow ESPT dynamics in RM compared to bulk water is mainly attributed to the sluggish hydrogen-bonded network dynamics of water molecules inside the nanocavity of RM and the screening of the sodium ions present at the interface

Prototropical and Photophysical Properties of Ellipticine inside the Nanocavities of Molecular Containers

Host–guest interactions between an anticancer drug, ellipticine (EPT), and molecular containers (cucurbitruils (CB<i>n</i>) and cyclodextrins (CD)) are investigated with the help of steady state and time-resolved fluorescence measurements. Our experimental results confirm the formation of 1:1 inclusion complexes with CB7 and CB8. The protonated form of EPT predominantly prevails in the inclusion complexes due to the stabilization achieved through ion–dipole interaction between host and positively charged drug. Drug does not form an inclusion complex with CB6, which is smaller in cavity size compared to either CB7 or CB8. In the case of cyclodextrins, α-CD does not form an inclusion complex, whereas β-CD forms a 1:1 inclusion complex with the protonated form of the drug, and the binding affinity of EPT with β-CD is less compared to CB7/CB8. Interestingly, in the case of γ-CD, drug exists in different forms depending on the concentration of the host. At lower concentration of γ-CD, 1:1 inclusion complex formation takes place and EPT exists in protonated form due to accessibility of water by the drug in the inclusion complex, whereas, at higher concentration, a 2:1 inclusion complex (γ-CD:EPT) is observed, in which EPT is completely buried inside the hydrophobic cavity of the capsule formed by two γ-CD molecules, and we believe the hydrophobic environment inside the capsule stabilizes the neutral form of the drug in the 2:1 inclusion complex. Deep insight into the molecular picture of these host–guest interactions has been provided by the docking studies followed by quantum chemical calculations

Urea Induced Unfolding Dynamics of Flavin Adenine Dinucleotide (FAD): Spectroscopic and Molecular Dynamics Simulation Studies from Femto-Second to Nanosecond Regime

Here, we investigate the effect of urea in the unfolding dynamics of flavin adenine dinucleotide (FAD), an important enzymatic cofactor, through steady state, time-resolved fluorescence spectroscopic and molecular dynamics (MD) simulation studies. Steady state results indicate the possibility of urea induced unfolding of FAD, inferred from increasing emission intensity of FAD with urea. The TCSPC and up-conversion results suggest that the stack–unstack dynamics of FAD severely gets affected in the presence of urea and leads to an increase in the unstack conformation population from 15% in pure water to 40% in 12 M urea. Molecular dynamics simulation was employed to understand the nature of the interaction between FAD and urea at the molecular level. Results depict that urea molecules replace many of the water molecules around adenine and isoalloxazine rings of FAD. However, the major driving force for the stability of this unstack conformations arises from the favorable stacking interaction of a significant fraction of the urea molecules with adenine and isoalloxazine rings of FAD, which overcomes the intramolecular stacking interaction between themselves observed in pure water