Molecular Mechanics/ Coarse-Grained hybrid model for investigating protein function

Chapter 1 deals with computational methods used in this thesis. Firstly, the MD simulation method and a short overview of Go model based approach [22] are introduced, then our hybrid model, MM/CG [24], is discussed. Chapter 2 presents the validation of the MM/ CG model [24]. The test systems we chose are two cytoplasmatic Aspartic Protease enzymes: BACE which belongs to pepsin family and HIV-1 PR which belongs to retropepsin family. After a short introduction of the biochemistry of these two proteins of AP class, the validation of our MM/CG model is presented [24]. MM/CG potential is calibrated so as to reproduce the MD data, then we show that our computational approach is able to reproduce both the mesoscopic (i. e. large scale fluctuations) and the local microscopic details (i.e. chemistry in the active site) of HIV-1 PR and BACE, suggesting that MM/CG can be conveniently applied to those systems for which either the size or the necessity of long-time sampling prevents the application of standard MD techniques. Chapter 3 applies the MM/CG method to the membrane protease OmpT [34, 36]. The comparison between MD data [33] with those computed by MM/CG simulations on t he OmpT in its free state suggests that this approach is well suitable to investigate also membrane proteins [34]. From the MM/CG simulation analysis, it seems that OmpT large- scale conformational fluctuations might play a role for its biological function , as for HIV-1 PR [30] and BACE [31]. In order to elucidate the role of large scale fluctuations, we study the dynamics of OmpT in complex with its substrate using the MM/CG approach [36]. We find that large scale motions and fluctuations of the electric field in t he μs time scale may impact on the biological function. Such conclusion can not be drawn within the time scale typical of molecular dynamic simulations and the MM/CG approach is further shown to be a fast and useful tool to provide structure/ function relationships of mutants affecting the enzymatic activity [36]. Two Appendices are present: Appendix A and B. Appendix A is devoted to the investigation of a particular CG model, the ,8Gaussian model (,8GM) [11], which is compared to MM/ CG model in Section 2.3. Appendix B provides further information of our simulation of OmpT in complex with its substrate