................................................................................................................................... 35 3.1 Biological background ....................................................................................................... 36 3.1.1 Mechanism of action of Rapamycin .......................................................................... 38 3.2 Structural details of the complex ....................................................................................... 41 CHAPTER 4STABILIZATION OF FKBP12-FRB COMPLEX BY RAPAMYCIN .......... 43 4.1 Materials and methods ....................................................................................................... 43 4.2 Free energy calculations .................................................................................................... 45 4.3 Computational Alanine Scanning Studies ......................................................................... 48 4.4 Analysis of the Interaction Network .................................................................................. 54 Conclusions ............................................................................................................................. 59 PART II Computational Modelling of the application of mechanical force to the biomolecules CHAPTER 5COMPUTATIONAL METHODS II ............................................................... 62 5.1 Steered Molecular Dynamics ............................................................................................. 62 5.2 Umbrella Sampling and Weighted Histogram Analysis Method ...................................... 68 CHAPTER 6MODELLING THE EFFECT OF OSMOLYTES ON PEPTIDE MECHANICAL UNFOLDING .............................................................................................. 72 Abstract.................................................................................................................................... 72 6.1 Osmolytes and Protein Stability ........................................................................................ 73 6.2 Materials & Methods ......................................................................................................... 77 6.3 Free Energy Calculation of Mechanical Unfolding Of the Peptide ................................... 81 6.4 Structural Changes during Mechanical Unfolding of the Peptide ..................................... 83 Conclusions ............................................................................................................................. 88 CHAPTER 7PROTEIN-PROTEIN INTERACTIONS AMONG TUBULIN HETERODIMERS: AN in-silico STUDY. ............................................................................. 89 Abstract .................................................................................................................................... 89 7.1 Microtubules ...................................................................................................................... 90 7.1.1 Structure and Dynamics of Microtubules ................................................................... 90 7.1.2 Biological role of Microtubules .................................................................................. 95 7.1.3 Microtubules as a drug-target ..................................................................................... 97 7.2 Study of Longitudinal Interactions Among Tubulin Heterodimers ................................. 100 7.2.1 Materials and Methods ............................................................................................. 101 7.2.2 Results and Discussion ............................................................................................. 103 7.3 Study of Lateral Interactions among Tubulin Heterodimers ............................................ 109 7.3.1 Materials and methods .............................................................................................. 110 7.3.2 Results and Discussion ............................................................................................. 111 Conclusions & Future perspectives........................................................................................ 115 _______________________________________________________________________ _______________________________________________________________________ 1 THESIS OVERVIEW Targeting protein-protein interactions is a challenging task in drug discovery process. Despite the challenges, several studies have provided evidences for the development of small molecules modulating protein-protein interactions. In Part I, it is demonstrated that how a small molecule can induce the formation of an otherwise unstable protein-protein complex. A study of the stabilization of a FKBP12-FRB complex by a small molecule rapamycin is presented. The stability of the complex is analyzed and its interactions are characterized at the atomic level by performing free energy calculations and computational alanine scanning. It is shown that rapamycin stabilizes the complex by acting as a bridge between the two proteins; and the complex is stable only in the presence of rapamycin. The reported results and the good performance of standard molecular modeling techniques in describing the model system can be interesting not only in the design and development of improved molecules acting as FKBP12–FRB protein interaction stabilizers, but also in the somehow neglected study of protein-protein interactions stabilizers in general. In Part II, studies regarding computational modeling of the application of mechanical force to biomolecules is presented. This part is further divided into two chapters since the investigations have been performed on two biological systems. In the first chapter of Part II (chapter 6), it is described that how the osmolyte molecules affect the mechanical unfolding of a peptide. The mechanical unfolding of peptide has been performed by using Steered Molecular Dynamics. In this study, the effect of four different osmolytes on the free energy difference between the folded and the denatured state have been calculated. The observed trend mirrors the expected behavior of the studied osmolytes and unfolding pathways analysis allows an insight into the mechanism of action of osmolytes. _______________________________________________________________________ _______________________________________________________________________ 2 After the successful application of Steered molecular dynamics technique on the β-hairpin peptide, the same is applied on tubulin heterodimers for the in-depth study of the lateral and longitudinal interactions which are responsible for the stability and dynamics of the microtubules. In the other chapter of Part II (chapter 7), these interactions are studied with the help of mechanical dissociation of the tubulin heterodimers. These studies have allowed the identification of the critical interactions responsible for the binding of tubulin heterodimers laterally as well as longitudinally. The observations obtained could be important for the design of compounds that target these interactions and acts as microtubule inhibitors or stabilizers. _______________________________________________________________________ _______________________________________________________________________ 3 _______________________________________________________________________ _______________________________________________________________________ 4 PART I Molecular Insights into the Stabilization of Protein-Protein Interactions with Small Molecule: The FKBP12-Rapamycin-FRB Case Study Chapter 1: Protein-Protein Interactions _______________________________________________________________________ _______________________________________________________________________ 5 CHAPTER 1 PROTEIN-PROTEIN INTERACTIONS 1.