Development of a high-throughput recombinant protein expression pipeline and methods to analyze and validate physiologically assembled complexes are essential for the success of the HARC Center structural efforts. As a starting point, it is important to carefully re-examine the physiological relevance of a reported interaction, as many have been initially identified using over-expression systems such as yeast two-hybrid assays, that can result in unnatural, 'nonspecific'binding at high concentration. The 'completeness1 of any given complex also is an issue, particularly for structural studies where each macromolecular interface may contribute to complex stability and surfaces may be remodeled by the interactions. Tandem affinity purification strategies coupled with mass spectrometry have been used successfully on many proteins in multiple organisms. Much of these data have been generated using S. cerevisiae where mid-scale proteomics studies have defined machinery involved in various cellular processes, including kinetochore function (353, 354), mRNA splicing (355, 356), and transcriptional regulation (357). Krogan has used this approach extensively to identify novel complexes and new components of previously characterized complexes involved in transcriptional regulation, rRNA processing, mRNA splicing, protein degradation, DNA repair, and protein trafficking (358-378) and has developed cutting-edge experimental and computational technology.
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