| Biological processes are complex processes, possibly, governed by simple laws. Insights into laws concerning molecular recognition are crucial to understanding cellular processes and communication.; This dissertation deals with developing a more accurate model that best describes the binding process for a simple binding system: recognition between Sem-5 C-SH3 domain and the Sos ligand. Ideas gained from this simple system could, in principle, be extended to more complex processes. Such information can have far-reaching applications in structure-based drug design and molecular simulations.; Existing models are often modified, replaced by newer models, which can simulate the existing experimental data better. For this reason, a broad base knowledge of the behavior of the system is necessary for experimental and theoretical correlations. The first part of this dissertation involves extensive biophysical characterization of the model protein, Sem-5 C-SH3 domain. This includes understanding the structural, dynamic and energetic properties of the protein, as well as, aspects concerning its association with the ligand.; Proteins exist as rapidly interconverting conformations. The probability of the various states is dictated by the boltzmann energy distribution. For this reason, a ‘rigid-body’ binding model might not be adequate to describe the association process. In the second part of this dissertation, an alternative model is proposed and tested. Ala and Gly mutations are introduced into solvent-exposed sites in the Sem-5 C-SH3 domain FIT and distal loop regions. These mutations are demonstrated to have minimal structural perturbation, thereby facilitating the dissection of the different energetic contributions on binding. As interactions are the same in the binding interface for both the Ala and the Gly mutants, the apparent binding free energy difference will be directly related to the energies involved in the conformational redistribution of the binding-incompetent to binding-competent species. |
