XII SCHOOL OF PURE AND APPLIED BIOPHYSICS The ever changing world of (hemo)globins

Bright and short x-ray flashes extracted from a synchrotron allow the study of the structuraldynamics of proteins at medium resolution (~ 10 Ang) with 100ps time resolution. Heme-basedproteins, due to their natural light sensitivity and robustness, are particularly suited for such studies.In this contribution I will discuss the basic components of the experiment apparatus and theinformations contained in a scattering experiment (of water/protein solution).To conclude the structural dynamics accompanying the R-to-T quaternary transition of HumanHemoglobin is presented. The main results are: An unexpected fast time scale (~1.5us) for the transitionAbsence of any (quaternary) intermediatesDemonstration of the structural sensitivity of the current apparatusIf time permits, a short introduction of the next generation x-ray sources and the future plans for thedata analysis will be given. XII SCHOOL OF PURE AND APPLIED BIOPHYSICSAbstracts of lectures 19A Hierarchy of Functionally Important Relaxations in Myoglobin Noam AgmonDepartment of Physical Chemistry and the Fritz Haber Research CenterThe Hebrew University, Jerusalem 91904, Israel Various protein relaxation modes couple to its activity on different timescales leading tonon-exponential kinetics. As a ligand migrates within the protein, these modes can localize atdifferent points along its trajectory. How can one identify these different modes and determine theiractivation energies? In order to answer this question, we have used a unique combination ofsolvents, mutations and theoretical approach. We have studied (in collaboration with Joel M.Friedman) geminate CO rebinding to myoglobin for two viscous solvents, trehalose and sol-gel(bathed in 100% glycerol) at several temperatures. Mutations in key distal hemepocket residueswere used to eliminate or enhance specific relaxation modes. The time-resolved data were thenanalyzed with a modified Agmon-Hopfield model providing excellent fits in cases where a singlerelaxation mode is dominant. Using this approach we determine the relaxation rate constants ofspecific functionally important modes and their Arrhenius activation energies. We have found a hierarchy of distal pocket modes controlling the rebinding kinetics. The``heme access mode'' (HAM) is responsible for the major slow-down in rebinding. It is a solvent-coupled cooperative mode which restricts ligand return from the xenon cavities. Bulky side-chains,like those His64 and Trp29 (in the L29W mutant), operate like overdamped pendulums which moveover the binding site. They may be either unslaved (His64) or moderately slaved (Trp29) to thesolvent. Small side-chain relaxations, most notably of leucines, are revealed in some mutants(V68L, V68A). They are conjectured to facilitate inter-cavity ligand motion. When all relaxationsare arrested (H64L in trehalose) we observe pure inhomogeneous kinetics with no temperaturedependence, suggesting that proximal relaxation is not a factor on the investigated timescale. The first lecture will provide a detailed discussion of the physical observations, whereas thesecond talk will demonstrate how to use our SSDP Windows application (E.B. Krissinel`) to solvethe underlying diffusion equation and fit the time dependence of the protein survival probability toobtain its relaxation rate parameter.