In vitro evolution of a new family of metal-sensors by phage display

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Project objective

In recent years, a new family of metal-responsive transcription factors has been identified, two of which, SmtB and a novel sequence designated as PacR, are present in cyanobacteria (Prof. Robinson's lab). Most of these metal-responsive repressors control the expression of proteins involved in metal ion homeostasis or detoxification, and are characterized by their ability to sense specific metal ions. The existence of a family of related regulatory proteins which differ in the spectrum of metal ions that they sense provides a unique opportunity to identify the determinants of metal specificity. The objective of this program is to identify the metal-binding sites in these repressors and to establish what structures determine the different preferences within the spectrum of metal-ions sensed by these repressors. The relationship between the structure and the function of the metal-binding site of several members of this repressor family, in particular of SmtB, the repressor of the first (characterized) prokaryotic metallothionein gene, smtA, and PacR, the putative repressor of a gene encoding a predicted copper ATPase, will be analyzed in detail to understand how these metals are perceived by cyanobacteria. We will use the strategy of in vitro evolution by phage display technology to modify ligand affinity and specificity of SmtB and PacR. This approach will reveal which amino acids form the metal-binding site in these repressors and also lead to the engineering of novel metal-sensor proteins. There are potential long-term applications for the development of metal-specific biosensors and for engineering metal-resistance/-accumulation.

Original text from CORDIS.

Participants

• Aristotle University of Thessaloniki · ThessalonikiCoordinatorGreece

Links

• View on CORDIS