Dynamics of ligand substitution in labile cobalt complexes resolved by ultrafast T-jump.

Ligand exchange of hydrated metal complexes is common in chemical and biological systems. Using the ultrafast T-jump, we examined this process, specifically the transformation of aqua cobalt (II) complexes to their fully halogenated species. The results reveal a stepwise mechanism with time scales varying from hundreds of picoseconds to nanoseconds. The dynamics are significantly faster when the structure is retained but becomes rate-limited when the octahedral-to-tetrahedral structural change bottlenecks the transformation. Evidence is presented, from bimolecular kinetics and energetics (enthalpic and entropic), for a reaction in which the ligand assists the displacement of water molecules, with the retention of the entering ligand in the activated state. The reaction time scale deviates by one to two orders of magnitude from that of ionic diffusion, suggesting the involvement of a collisional barrier between the ion and the much larger complex.

DNA folding and melting observed in real time redefine the energy landscape.

We report real-time observations of the folding and melting of DNA by probing two active sites of a hairpin structure, the bases and the stem end, and using an ultrafast T-jump. Studies at different initial temperatures (before, during, and after melting) provide the time scale of water heating (<20 ps), single-strand destacking (700 ps to 2 ns), and hairpin destacking (microseconds and longer) in solutions of various ionic strengths and pH values. The behavior of transient changes gives direct evidence to the existence of intermediate collapsed structures, labile in destacking but compact in nature, and indicates that melting is not a two-state process. We propose a landscape that is defined by these nucleation structures and destacking for efficient folding and melting.

Ultrafast T-jump in water: studies of conformation and reaction dynamics at the thermal limit.

We report the development of ultrafast T-jump with time resolution reaching the fundamental time scale of water thermalization time ( approximately 5 ps). The T-jump heats the bulk water up to 20 degrees C via the overtone absorption of the OH- stretch at 1.5 mum. The example given here shows the application of the methodology, for the first time, and the results demonstrate distinct time scales for solvation, conformational change, and thermal reaction.

RNA-protein recognition: single-residue ultrafast dynamical control of structural specificity and function.

The transcription antiterminator N protein from bacteriophage lambda uses its arginine-rich motif to specifically bind a stem-loop RNA hairpin (boxB) as a bent alpha-helix. A single stacking interaction between a tryptophan (Trp-18) and an adenosine (A7) in the RNA loop is robust and necessary for antitermination activity in vivo. Previously, femtosecond fluorescence up-conversion experiments from this laboratory indicated that the N/boxB complex exists in a dynamical two-state equilibrium between stacked and unstacked conformations and that the extent of stacking depends on the identity of peptide residues 14 and 15. In the present work, we have combined transient absorption and fluorescence up-conversion to determine the nature of interactions responsible for this sequence-dependent behavior. Analysis of mutant complexes supports the idea that the beta-carbon of residue 14 enforces the stacked geometry by hydrophobic interaction with the ribose of A7, whereas a positive charge at this residue plays only a secondary role. A positive charge at position 15 substantially disfavors the stacked state but retains much of the binding energy. Remarkably, in vivo antitermination experiments show strong correlation with our femtosecond dynamics, demonstrating how conformational interplay can control the activity of a macromolecular machine.

The RNA-protein complex: direct probing of the interfacial recognition dynamics and its correlation with biological functions.

The N protein from bacteriophage lambda is a key regulator of transcription antitermination. It specifically recognizes a nascent mRNA stem loop termed boxB, enabling RNA polymerase to read through downstream terminators processively. The stacking interaction between Trp-18 of WT N protein and A7 of boxB RNA is crucial for efficient antitermination. Here, we report on the direct probing of the dynamics for this interfacial binding and the correlation of the dynamics with biological functions. Specifically, we examined the influence of structural changes in four peptides on the femtosecond dynamics of boxB RNA (2-aminopurine labeled in different positions), through mutations of critical residues of N peptide (residues 1-22). We then compare their in vivo (Escherichia coli) transcription antitermination activities with the dynamics. The results demonstrate that the RNA-peptide complexes adopt essentially two dynamical conformations with the time scale for interfacial interaction in the two structures being vastly different, 1 ps for the stacked structure and nanosecond for the unstacked one; only the weighted average of the two is detected in NMR by nuclear Overhauser effect experiments. Strikingly, the amplitude of the observed ultrafast dynamics depends on the identity of the amino acid residues that are one helical turn away from Trp-18 in the peptides and is correlated with the level of biological function of their respective full-length proteins.

Femtosecond charge transfer dynamics of a modified DNA base: 2-aminopurine in complexes with nucleotides.

As a fluorescent isomer of adenine, 2-aminopurine (Ap) is a powerful probe of DNA dynamics and DNA-mediated charge transfer processes. Here, we report studies with femtosecond resolution of the excited-state dynamics of Ap in various solvents and in bimolecular complexes with nucleotides. Using time-resolved transient absorption and fluorescence up-conversion methods we identify charge transfer as the origin for the quenching of the Ap fluorescence by all four DNA nucleotides. The direction of the redox process is, however, dependent on the base, and from the rates we deduce the nature of the transfer, hole versus electron transfer. The pH and the kinetic isotope effects of these charge transfer reactions revealed no evidence for proton transfer involvement in the rate-determining step. From the measured rates and using electron transfer theory we estimate the driving force for charge transfer between all four nucleobases and Ap. The results are important for the studies of dynamics using Ap in DNA assemblies.