As part of the MAGGIE group we are focused on developing methodologies and testing model systems to facilitate the larger goal of proteome mapping and proteome-wide technology development central to DOE missions. In Component 2, we aim to exploit advantages unique to the hyperthermophilic Archaea Sulfolobus solfataricus to isolate, identify, and characterize recombinant multiprotein complexes and modified proteins from the native organism. We are advancing Sulfolobus molecular biology and protein isolation technologies to isolate transient and unstable complexes. Taking advantage of the hyperthermophilic nature of S. solfataricus (80°C), we are isolating complexes at lower temperatures (4°C), thereby providing significantly increased complex stability. We will compare and contrast our data across species with datasets from both Pyrococcus furiosus and Halobacterium to identify orthologous complexes among MAGGIE organisms as well as complexes unique to each of these organisms.
Immediate Goals:
- To develop molecular biology tools to affinity tag S. solfataricus genes and reintroduce them into the native organism in a high-throughput manner.
- To isolate tagged protein complexes and modified proteins from S. solfataricus.
- To characterize protein complex components and stoichiometry by 1D/2D gel separation, mass spectrometry, and small angle X-ray scattering (SAXS).
- To assess protein complex conservation between S. solfolobus, P. furiosus and Halobacterium using protein tagging and isolation in S. solfataricus.
- To isolate tagged-protein complexes under prototypical stress conditions (oxygen stress, desiccation, ionizing radiation, UV radiation, and heavy metals).
- To evaluate subunit changes in protein complexes in response to prototypical stresses using developing protein microarray technologies.
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