Biochemical and biophysical characterization of the manganese transport regulator (MntR) from Bacillus subtilis

Metal ions are employed in biology for several reasons including their ability to participate in redox chemistry, catalysis, and structural stabilization of proteins. However, the properties that make metal ions so widely utilized in biology can be potentially hazardous, particularly if abnormal quantities of these ions are accumulated. This necessitates a mechanism by which the balance between uptake of essential metal ions and efflux of excess essential or toxic metal ions, otherwise referred to as metal homeostasis, can be maintained. Bacteria employ a unique set of metal responsive transcription factors (metalloregulators) to manage this delicate balance. The biochemical and biophysical characterization of MntR, a manganese responsive regulator from the DtxR family is the focus of this thesis. Fluorescence anisotropy was used to probe the DNA-binding of wild type MntR, MntR D8M, and MntR E99C mutants to the cognate DNA recognition sequences mntH and mntA in the presence of various divalent metal ions. Our studies demonstrate the extent to which these metal ions are able to activate MntR to bind DNA. In addition, these studies shed light on the origin of metal specificity between MntR and DtxR and are in agreement with in vivo data reported in the literature. In addition to investigating the DNA- binding abilities of MntR, we also examined the metal binding affinities of this protein in order explain how it fits into the DtxR family and the general field of metalloregulatory proteins. The results demonstrate that MntR metal-binding affinities loosely follow the Irving- Williams series. Interestingly, the protein exhibits the weakest affinity for one of its cognate metal ions. Finally, the metal-mediated mechanism of DNA binding by MntR was studied. Initial investigations using circular dichroism and an environmentally-sensitive dye ANS showed that metal binding stabilizes either tertiary or quaternary structure of MntR. Subsequent studies focused on localizing these structural changes using deuterium exchange mass spectrometry (DXMS) and demonstrated that metal-binding serves to rigidify the pre-organized structure of MntR. Moreover, contrary to typical observation of transcription factors, cofactor (metal) binding does not appear to alter the structure of helix- turn-helix DNA-binding moti

Rational design of FRET sensor proteins based on mutually exclusive domain interactions

Abstract Proteins that switch between distinct conformational states are ideal to monitor and control molecular processes within the complexity of biological systems. Inspired by the modular architecture of natural signalling proteins, our group explores generic design strategies for the construction of FRET-based sensor proteins and other protein switches. In the present article, I show that designing FRET sensors based on mutually exclusive domain interactions provides a robust method to engineer sensors with predictable properties and an inherently large change in emission ratio. The modularity of this approach should make it easily transferable to other applications of protein switches in fields ranging from synthetic biology, optogenetics and molecular diagnostics

Monitoring bile acid transport in single living cells using a genetically encoded Förster resonance energy transfer sensor

Bile acids are pivotal for the absorption of dietary lipids and vitamins and function as important signaling molecules in metabolism. Here, we describe a genetically encoded fluorescent bile acid sensor (BAS) that allows for spatiotemporal monitoring of bile acid transport in single living cells. Changes in concentration of multiple physiological and pathophysiological bile acid species were detected as robust changes in Förster resonance energy transfer (FRET) in a range of cell types. Specific subcellular targeting of the sensor demonstrated rapid influx of bile acids into the cytoplasm and nucleus, but no FRET changes were observed in the peroxisomes. Furthermore, expression of the liver fatty acid binding protein reduced the availability of bile acids in the nucleus. The sensor allows for single cell visualization of uptake and accumulation of conjugated bile acids, mediated by the Na(+)-taurocholate cotransporting protein (NTCP). In addition, cyprinol sulphate uptake, mediated by the putative zebrafish homologue of the apical sodium bile acid transporter, was visualized using a sensor based on the zebrafish farnesoid X receptor. The reversible nature of the sensor also enabled measurements of bile acid efflux in living cells, and expression of the organic solute transporter αβ (OSTαβ) resulted in influx and efflux of conjugated chenodeoxycholic acid. Finally, combined visualization of bile acid uptake and fluorescent labeling of several NTCP variants indicated that the sensor can also be used to study the functional effect of patient mutations in genes affecting bile acid homeostasis. CONCLUSION: A genetically encoded fluorescent BAS was developed that allows intracellular imaging of bile acid homeostasis in single living cells in real tim