Insight into signaling linked to catalytic turnover (GSAM) and understanding of protein stability and storage properties (RHCC)

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The primary goal of the proposed research is to understand in detail the structure-function relationship of proteins as dynamic systems with either enzymatic activity or storage and delivery functions. The research will be focused (i) on the vitamin B6-dependent key enzyme in chlorophyll biosynthesis, glutamate-1-semialdehyde aminomutase (GSAM) and (ii) the Staphylothermus marinus right-handed coiled coil (RHCC). The purpose is to obtain mechanistic and chemical information about the two systems to shed light on the intersubunit signaling in the negative cooperativity of GSAM and to investigate the extreme stability and storage properties of the archaeal protein RHCC. Research will address atomic level mechanistic questions that have arisen or have not yet  been answered in the on-going structure-function studies. Not only will the research enhance our knowledge of the allosteric communication in GSAM linked to catalytic turnover, but insights will be gained into structure-based design of highly selective herbicides. Because no counterpart of this enzyme exists in animals, it is a promising target for safe, selective herbicides and the rational design of such compounds. Understanding the intricate mechanisms of thermostability at extreme low pH, together with the complex formation of RHCC with a protease of the subtilisin family, will provide further information about the nature of protein stability under extreme conditions. The unusual capability of RHCC to incorporate cluster-like arranged water molecules makes it suitable for a new direction of research, the storage function of coiled coils. All these studies involve a combination of structural (X-ray crystallography and solution NMR) and spectroscopic (UV-Vis, CD, DSC and FRET) techniques complemented by in-vitro functional assays.