Investigation of protein-metal ion and protein-protein interactions using mass spectrometry and nuclear magnetic spectroscopy
Protein-protein interaction networks provide a global picture of cellular function and biological processes. Some proteins act as hub proteins, highly connected to others, whereas some others have few interactions. The dysfunction of a single highly connected interactor can cause widespread disruption of cellular processors including diseases and cancer. Therefore, detailed study of the interactions made by cancer-related proteins will help explain their role in the interaction networks. The investigation of proteins by mass spectrometry (MS) has provided unique opportunities to gain insight into the dynamics of these proteins at the molecular level. MS uses mass analysis for protein characterization, and is currently the most comprehensive and versatile tool in proteomics. MS can provide confirmation of protein samples of interest, accurate molecular mass measurements of proteins, purity of protein samples, detection of posttranslational modifications, and more recently, interactions between two or more proteins. The conventional way of investigating the structure of proteins involves nuclear magnetic resonance (NMR) or X-ray crystallography. Compared to MS these methods are time consuming methods and, furthermore, require a considerable amount of protein. MS has proved to be useful in this regard as it provides insights into the structural arrangement of proteins, and/or their interacting partners, without the need for crystalliastion or the tedious process of backbone assignment before structural and functional annotations can be attributed to the protein of interest. However, in many cases, conventional methods are used parallel to MS to serve as validation of the MS data. The broad objective of this MSc study was to provide structural and functional insights into the function of Retinoblastoma Binding Protein-6 (RBBP6), using a MS approach. The aims were twofold: 1) to investigate metal ion binding by RING (Really Interesting New Gene) finger domains from RBBP6, and 2) to investigate the in vitro interaction between RBBP6 and Hsp 70(Heat Shock Protein 70), and between RBBP6 and Murine Double Minute-2 (Mdm2).