David P. Millar

Stability of hairpin ribozyme tertiary structure is governed by the interdomain junction

The equilibrium distributions of hairpin ribozyme conformational isomers have been examined by time-resolved fluorescence resonance energy transfer. Ribozymes partition between active (docked) and inactive (extended) conformers, characterized by unique interdomain distance distributions, which define differences in folding free energy. The active tertiary structure is stabilized both by specific interactions between the catalytic and the substrate-binding domains and by the structure of the intervening helical junction. Under physiological conditions, the docking equilibrium of the natural four-way junction dramatically favors the active conformer, while those of a three-way and the two-way junction used in gene therapy applications favor the inactive conformer.

Torsion and bending of nucleic acids studied by subnanosecond time‐resolved fluorescence depolarization of intercalated dyes

Subnanosecond time‐resolved fluorescence depolarization has been used to monitor the reorientation of ethidium bromide intercalated in native DNA, synthetic polynucleotide complexes, and in supercoiled plasmid DNA. The fluorescence polarization anisotropy was successfully analyzed with an elastic model of DNA dynamics, including both torsion and bending, which yielded an accurate value for the torsional rigidity of the different DNA samples. The dependence of the torsional rigidity on the base sequence, helical structure, and tertiary structure was experimentally observed. The magnitude of the polyelectrolyte contribution to the torsional rigidity of DNA was measured over a wide range of ionic strength, and compared with polyelectrolyte theories for the persistence length. We also observed a rapid initial reorientation of the intercalated ethidium which had a much smaller amplitude in RNA than in DNA.

Time-resolved fluorescence spectroscopy

Time-resolved fluorescence spectroscopy is used to monitor molecular interactions and motions that occur in the picosecond-nanosecond time range, and is especially useful in the analysis of biomolecular structure and dynamics. Recent advances in the application of time-resolved fluorescence spectroscopy to biological systems have led to a better understanding of the origin of nonexponential fluorescence decay in proteins, the use of tryptophan analogs as unique spectroscopic probes of protein-protein interactions, the detailed characterization of protein-folding processes and intermediates, and the development of new approaches to the study of DNA-protein interactions.

Fluorescence studies of DNA and RNA structure and dynamics

Fluorescence spectroscopy is increasingly being used as a technique for probing the structure and dynamics of nucleic acids. Recently, fluorescence methods have been used to elucidate the three-dimensional arrangement of complex DNA and RNA structures, characterize structural perturbations resulting from base bulges in helices and junctions, determine helical handedness in solution, and analyze the protein-induced melting of DNA.

Picosecond dynamics of electronic energy transfer in condensed phases

Energy transfer between donor and acceptor molecules randomly distributed in condensed phases is investigated by time-resolved spectroscopy on the picosecond and nanosecond time scales. The effects of translational diffusion and excitation transfer among the donors is experimentally observed and used to test theoretical models based on a diffusion equation for the donor excitation. The time-resolved data demonstrate that the Forster dipole–dipole model is valid in the cresyl violet (donor):azulene (acceptor) system from 1 ps to at least 10 ns after excitation, and over a 1000-fold range of acceptor concentration. The critical transfer distance obtained from the transient experiments (26.6 A) is in excellent agreement with the value obtained from the spectral overlap (27.8 A) at all acceptor concentrations. In fluid solutions the donor decay agrees very well with the approximate solution of the diffusion equation including a sink term for energy transfer. The deviations observed at high donor concentrations suggest that donor–donor excitation transfer is nondiffusive on the picosecond time scale.

HIV-1 Rev protein assembles on viral RNA one molecule at a time

Oligomerization of the HIV-1 protein Rev on the Rev Response Element (RRE) regulates nuclear export of genomic viral RNA and partially spliced viral mRNAs encoding for structural proteins. Single-molecule fluorescence spectroscopy has been used to dissect the multistep assembly pathway of this essential ribonucleoprotein, revealing dynamic intermediates and the mechanism of assembly. Assembly is initiated by binding of Rev to a high-affinity site in stem-loop IIB of the RRE and proceeds rapidly by addition of single Rev monomers, facilitated by cooperative Rev-Rev interactions on the RRE. Dwell-time analysis of fluorescence trajectories recorded during individual Rev-RRE assembly reactions has revealed the microscopic rate constants for several of the Rev monomer binding and dissociation steps. The high-affinity binding of multiple Rev monomers to the RRE is achieved on a much faster timescale than reported in previous bulk kinetic studies of Rev-RRE association, indicating that oligomerization is an early step in complex assembly.

Picosecond dynamics of barrier crossing in solution: A study of the conformational change of excited state 1,1’‐binaphthyl

The rates of optically induced conformational changes of excited state 1,1’‐binaphthyl in a series of alcohol solvents were measured by a picosecond laser technique. Excellent agreement with experiment is found for the Kramers model for both the intermediate and high friction regimes using a frequency independent friction coefficient evaluated from classical hydrodynamics. This is the first observation of Kramers behavior in the intermediate friction regime and indicates that deviations from Kramers behavior is not a universal phenomenon in the conformational dynamics of flexible molecules in solution. It also emphasizes the possibility that the observed deviations from the Kramers model can be due to effects other than non‐Markovian friction.

Picosecond saturation spectroscopy of cresyl violet: rotational diffusion by a “sticking” boundary condition in the liquid phase

Picosecond pulses (0.6–2.5 ps duration) from a passively mode-locked and cavity-dumped dye laser have been used to study the molecular reorientation of cresyl violet in liquids. The dipole correlation function is obtained from the decay of the induced dichroism. The results when compared with hydrodynamics give good agreement with the stick boundary condition, and predict effective shape of cresyl violet in the liquid.

Deeply Inverted Electron-Hole Recombination in a Luminescent Antibody-Stilbene Complex

The blue-emissive antibody EP2-19G2 that has been elicited against trans-stilbene has unprecedented ability to produce bright luminescence and has been used as a biosensor in various applications. We show that the prolonged luminescence is not stilbene fluorescence. Instead, the emissive species is a charge-transfer excited complex of an anionic stilbene and a cationic, parallel π-stacked tryptophan. Upon charge recombination, this complex generates exceptionally bright blue light. Complex formation is enabled by a deeply penetrating ligand-binding pocket, which in turn results from a noncanonical interface between the two variable domains of the antibody.

Frictional effects on barrier crossing in solution: Comparison with the Kramers’ equation

In our efforts to examine the validity of the Kramers’ equation, the rate constants of the excited state isomerization of 1,1’‐binaphthyl in n‐alkane solvents were measured at room temperature using picosecond spectroscopy. These data, and data measured previously in n‐alcohols, were compared with Kramers’ model using two forms for the friction. When a hydrodynamic model for the friction was used, good agreement was found for the alcohol data only. When the isomerization friction is assumed to scale linearly with the friction for overall reorientational motion, we find excellent agreement for both the alcohol and alkane solvents. In addition, the friction in alkanes is found to be considerably larger than that of alcohols of comparable viscosity. This provides a direct indication that the molecular aspects of the solute–solvent interaction play a role in the barrier crossing process.