Session A9 - Proteins.
ORAL session, Monday morning, March 22
510B, Palais des Congres
Enzymes are complex molecules evolving on a very convoluted and restricted free energy surface. It is expected that, upon closer examination, their dynamics and kinetics will exhibit complicated behavior. We use single molecule fluorescence spectroscopy to study these spatial and temporal heterogeneities. Adenylate Kinase (AK) is an important enzyme in the regulation of the cellular energy balance. It catalyzes the interconversion of ATP, ADP, and AMP. Because of its critical roles in cellular function, any spatial or temporal heterogeneities in the activity of AK may have important consequences on the cellular level. We have labeled a mutant of E. Coli AK with a pair of fluorescent substituents to monitor its dynamics using single-molecule Förster Resonance Energy Transfer. To accurately measure the distance between these two residues in real time, we have developed a new non-parametric method of analysis that delivers time and distance resolution approaching the theoretical limit. Our maximum information algorithm analyzes the trajectory on a photon-by-photon basis, utilizing the information carried by each photon to achieve sufficient time resolution to observe the reactive complex in detail. These measurements allow us to calculate the static conformational distributions of individual molecules. The time dependent observations also allow us to measure temporal heterogeneity in the reactivity of AK. The measured reactive trajectories indicate a correlated structural flexibility and reaction dynamics. This newly developed methodology may allow us to begin to study the ways in which reactive heterogeneity affects its cellular function.