A Man-Made ATP-Binding Protein Evolved Independent of Nature Causes Abnormal Growth in Bacterial Cells

Recent advances in de novo protein evolution have made it possible to create synthetic proteins from unbiased libraries that fold into stable tertiary structures with predefined functions. However, it is not known whether such proteins will be functional when expressed inside living cells or how a host organism would respond to an encounter with a non-biological protein. Here, we examine the physiology and morphology of Escherichia coli cells engineered to express a synthetic ATP-binding protein evolved entirely from non-biological origins. We show that this man-made protein disrupts the normal energetic balance of the cell by altering the levels of intracellular ATP. This disruption cascades into a series of events that ultimately limit reproductive competency by inhibiting cell division. We now describe a detailed investigation into the synthetic biology of this man-made protein in a living bacterial organism, and the effect that this protein has on normal cell physiology

Activation of orphan receptor-mediated transcription by Ca(2+)/calmodulin-dependent protein kinase IV

Retinoid-related receptor α (RORα) is an orphan nuclear receptor that constitutively activates transcription from its cognate response element. We show that RORα is Ca(2+)responsive, and a Ca(2+)/calmodulin-independent form of Ca(2+)/calmodulin-dependent protein kinase IV (CaMKIV) potentiates RORα-dependent transcription 20- to 30-fold. Other orphan receptors including RORα2, RORγ and COUP-TFI are also potentiated by CaMKIV. Transcriptional activation by CaMKIV is orphan receptor selective and does not occur with either the thyroid hormone or estrogen receptor. CaMKIV does not phosphorylate RORα or its ligand-binding domain (LBD) in vitro, although the LBD is essential for transactivation. Therefore, the RORα LBD was used in the mammalian two-hybrid assay to identify a single class of small peptide molecules containing LXXLL motifs that interacted with greater affinity in the presence of CaMKIV. This class of peptides antagonized activation of orphan receptor-mediated transcription by CaMKIV. These studies demonstrate a pivotal role for CaMKIV in the regulation of orphan receptor-mediated transcription