1. Enrico Spiga 1,2 Andrea Scorciapino 1, Arturo Robertazzi 1, 2, Roberto Anedda 2, Mariano Casu 2, Paolo Ruggerone 1 and Matteo Ceccarelli 1 1 University of Cagliari, Department of Physics CNR-SLACS: Sardinian LAboratory for Computational Materials Science 2 University of Cagliari, Department of Chemistry Funded by PRIN n o 2006059902003 from MIUR-Italy Introduction The relatively small size of myoglobin makes it an ideal model system suitable for the investigation of the ligand escape process in respiratory proteins and, in general, for the study of the more general structure-function paradigm. The most known function of myoglobin is the oxygen storage as well as deliverer inside muscles. However, Myoglobin was discovered to be able to bind also other small ligands, such as CO, NO, H 2 O 2, free radicals and so on. While the toxic action of CO is well known for a long time, the role of myoglobin as scavenger of the other ligands is still under debate [1-3]. Molecular Dynamics Simulations of Ligand Binding in Myoglobins www.slacs.it www.unica.it e-mail:matteo.ceccarelli@dsf.unica.it Myoglobins have 4 cavities that can be occupied permanently by Xenon, as observed experimentally (orange in figure). It was recently shown [1] that internal cavities are not only responsible for protein flexibility but define a dynamical pathway for ligands to and from the active site, the Distal pocket. In our work we performed Molecular Dynamics simulations of different myoglobins (sperm-whale and horse) for several nanoseconds using the code Orac with the Amber force field. Then we used the code VOIDOO for searching cavities on the sampled structures. This program provides sizes and positions of cavities. In the figure we show in gray the center of all the cavities during a trajectory of 12 ns (2,400 conformers and approx. 37,000 cavities). Beside Xenon Cavities Due to the fluctuations of the protein, the center of each cavity fluctuates as well. We used the cluster analysis to quantitatively find the most probable position in the protein where cavities can occur. From our analysis the Xenon-cavities and the Distal represent only a small part of all cavities found. In orange are the Xenon-cavities and in yellow we represented the most populated clusters obtained (at least 10% of occurrence) with the cluster analysis. Computationally it is most expensive to find cavities than to perform the simulation itself. Approximately we can analyse 50 conformers in a day on a single opteron 2218. We combined the structural analysis of cavities and metadynamics to understand in more details the role of cavities for the escape of ligands and the more general problem of ligand- protein interactions. As ligand we considered the Xenon, used experimentally as a probe. One of the main question regarding Xenon is whether it perturbs or not the protein ( Anedda et al, submitted JPCB ). We started metadynamics simulations with Xenon inside the Xe1 cavity, and after 120 ns we observed the exit of Xenon. Another simulations has been started with the Xenon in the Xe4 cavity, and after 120 ns the Xenon is still inside the protein. Analysing the path followed by Xenon (green spheres in the figure) we can see that it visits all the Xenon cavities (orange) and some of the secondary cavities (white and yellow, the latter occupied during this trajectory) as obtained from our previous analysis without any ligand inside the protein. Conclusions Our results show that Xenon cavities are only a part of the cavities used by ligands to escape from the binding site to the solvent. A quantitative analysis of the secondary cavities as we performed could be useful for example to search for differences between myoglobins of different species (Horse, pig, sperm-whale, human). REFERENCES: 1. Brunori M. 2001, Trends Biochem. Sci. 26, 21-23. 2. Flogel U. et al 2001, Proc. Natl. Acad.Sci. USA 98, 735-740 3. Witting P.K. et al. 2000, J. Biol. Chem, 10, 1074 Myoglobin-Xenon interactions with metadynamics FF F[s  (r i )] ss KK srisri ss Extra Microscopic V(t) Perturbation H0H0 H=H 0 +V(t) Ligands escape with metadynamics Standard MD simulations are not able to follow activated processes when  F >> k B T. Approximating the process as described by a few reaction coordinates [s  ], we can accelerate the evolution of these variables through the addition of a perturbation V(t) added during the simulation (non-markovian process). The transition state becomes accessible within typical MD time (nanoseconds). The integration of the inverted perturbation provides the approximated free energy surface for the process in the subspace of variables [s  ]. A. Laio and M. Parrinello, PNAS 2002, 20:12562 We developed metadynamics to be able to investigate ligands escape in myioglobin. Using a cartesian coordinate system centered around the Heme group, we set the position of the ligand within this system as meta variable. The perturbation is added on the position visited by the ligand. The bias acts as a penalty function and avoid to revisit the same internal regions. In this case the integration of the bias is simply the probability for the ligand to visit any internal point of the protein (in white). We obtained the escape of CO after 36 ns. Ceccarelli et al. Proteins 2008, 71:1231 From the FES we can obtain minima and transition states, in this case the energy level of each Xenon cavity and barriers.