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Designed phosphoprotein recognition in Escherichia coli.

ABSTRACT: Protein phosphorylation is a central biological mechanism for cellular adaptation to environmental changes. Dysregulation of phosphorylation signaling is implicated in a wide variety of diseases. Thus, the ability to detect and quantify protein phosphorylation is highly desirable for both diagnostic and research applications. Here we present a general strategy for detecting phosphopeptide-protein interactions in Escherichia coli. We first redesign a model tetratricopeptide repeat (TPR) protein to recognize phosphoserine in a sequence-specific fashion and characterize the interaction with its target phosphopeptide in vitro. We then combine in vivo site-specific incorporation of phosphoserine with split mCherry assembly to observe the designed phosphopeptide-protein interaction specificity in E. coli. This in vivo strategy for detecting and characterizing phosphopeptide-protein interactions has numerous potential applications for the study of natural interactions and the design of novel ones.

SUBMITTER: Sawyer N

PROVIDER: S-EPMC4245168 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Designed phosphoprotein recognition in Escherichia coli.

Sawyer Nicholas N Gassaway Brandon M BM Haimovich Adrian D AD Isaacs Farren J FJ Rinehart Jesse J Regan Lynne L

ACS chemical biology 20141006 11

Protein phosphorylation is a central biological mechanism for cellular adaptation to environmental changes. Dysregulation of phosphorylation signaling is implicated in a wide variety of diseases. Thus, the ability to detect and quantify protein phosphorylation is highly desirable for both diagnostic and research applications. Here we present a general strategy for detecting phosphopeptide-protein interactions in Escherichia coli. We first redesign a model tetratricopeptide repeat (TPR) protein t ...[more]

PMID: 25272187

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Project description:Correct targeting of membrane proteins is essential for membrane integrity, cell physiology, and viability. Cotranslational targeting depends on the universally conserved signal recognition particle (SRP), which is a ribonucleoprotein complex comprised of the protein component Ffh and the 4.5S RNA in Escherichia coli About 25?years ago it was reported that Ffh is an unstable protein, but the underlying mechanism has never been explored. Here, we show that Lon is the primary protease responsible for adjusting the cellular Ffh level. When overproduced, Ffh is particularly prone to degradation during transition from exponential to stationary growth and the cellular Ffh amount is lowest in stationary phase. The Ffh protein consists of two domains, the NG domain, responsible for GTP hydrolysis and docking to the membrane receptor FtsY, and the RNA-binding M domain. We find that the NG domain alone is stable, whereas the isolated M domain is degraded. Consistent with the importance of Lon in this process, the M domain confers synthetic lethality to the lon mutant. The Ffh homolog from the model plant Arabidopsis thaliana, which forms a protein-protein complex rather than a protein-RNA complex, is stable, suggesting that the RNA-binding ability residing in the M domain of E. coli Ffh is important for proteolysis. Our results support a model in which excess Ffh not bound to 4.5S RNA is subjected to proteolysis until an appropriate Ffh concentration is reached. The differential proteolysis adjusts Ffh levels to the cellular demand and maintains cotranslational protein transport and membrane integrity.IMPORTANCE Since one-third of all bacterial proteins reside outside the cytoplasm, protein targeting to the appropriate address is an essential process. Cotranslational targeting to the membrane relies on the signal recognition particle (SRP), which is a protein-RNA complex in bacteria. We report that the protein component Ffh is a substrate of the Lon protease. Regulated proteolysis of Ffh provides a simple mechanism to adjust the concentration of the essential protein to the cellular demand. This is important because elevated or depleted SRP levels negatively impact protein targeting and bacterial fitness.

Dataset Information

Designed phosphoprotein recognition in Escherichia coli.

Publications

Designed phosphoprotein recognition in Escherichia coli.

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