Abstract
Understanding biological function, such as the fascinating rate acceleration of enzymes, specificity of protein/protein interactions or the delicately controlled action of signaling has been a long-standing challenge. Despite remarkable information generated using chemical tools in the last 100 years, complemented more recently with structural and computational approaches, we cannot yet identify with a complete energy inventory how ANY protein works. The secret of enzymes lies in their ability to partition energetic contributions among many atoms in a well-coordinated style. To unravel these secrets, proteins in action are spied on at atomic resolution to provide a comprehensive description of enzyme catalysis in the form of an energy landscape. Since the rate of catalysis is determined by the climb over a sequence of energy barriers, we focus here on the critical question of transition pathways with the highest energy state being the transition state.
I will discuss our exploration of the full energy landscape of enzyme catalysis through a combination of time-resolved NMR including high-pressure NMR, crystallography, single-molecule FRET and MD simulation. Allosteric is in play for 3 very different systems: an enzyme, a phoshorylation-mediated signaling protein and the inhibition of rhodopsin kinase via protein/protein interactions. For the latter example, binding by conformational selection and not via an induced fit is directly demonstrated by flux measurements, the only rigorous test for the two opposing mechanisms.
The presented data stress the point that highly choreographed chemical integrity AND optimized conformational sampling is a prerequisite for efficient enzyme catalysis. The power of an intimate marriage between NMR and other biophysical methods and MD simulations including a variety of novel pathway algorithms will be illustrated.
