The Hydrophobic Core of Twin-Arginine Signal Sequences Orchestrates Specific Binding to Tat-Pathway Related Chaperones

Redox enzyme maturation proteins (REMPs) bind pre-proteins destined for translocation across the bacterial cytoplasmic membrane via the twin-arginine translocation system and enable the enzymatic incorporation of complex cofactors. Most REMPs recognize one specific pre-protein. The recognition site usually resides in the N-terminal signal sequence. REMP binding protects signal peptides against degradation by proteases. REMPs are also believed to prevent binding of immature pre-proteins to the translocon. The main aim of this work was to better understand the interaction between REMPs and substrate signal sequences. Two REMPs were investigated: DmsD (specific for dimethylsulfoxide reductase, DmsA) and TorD (specific for trimethylamine N-oxide reductase, TorA). Green fluorescent protein (GFP) was genetically fused behind the signal sequences of TorA and DmsA. This ensures native behavior of the respective signal sequence and excludes any effects mediated by the mature domain of the pre-protein. Surface plasmon resonance analysis revealed that these chimeric pre-proteins specifically bind to the cognate REMP. Furthermore, the region of the signal sequence that is responsible for specific binding to the corresponding REMP was identified by creating region-swapped chimeric signal sequences, containing parts of both the TorA and DmsA signal sequences. Surprisingly, specificity is not encoded in the highly variable positively charged N-terminal region of the signal sequence, but in the more similar hydrophobic C-terminal parts. Interestingly, binding of DmsD to its model substrate reduced membrane binding of the pre-protein. This property could link REMP-signal peptide binding to its reported proofreading function

Visualizing Interactions along the Escherichia coli Twin-Arginine Translocation Pathway Using Protein Fragment Complementation

The twin-arginine translocation (Tat) pathway is well known for its ability to export fully folded substrate proteins out of the cytoplasm of Gram-negative and Gram-positive bacteria. Studies of this mechanism in Escherichia coli have identified numerous transient protein-protein interactions that guide export-competent proteins through the Tat pathway. To visualize these interactions, we have adapted bimolecular fluorescence complementation (BiFC) to detect protein-protein interactions along the Tat pathway of living cells. Fragments of the yellow fluorescent protein (YFP) were fused to soluble and transmembrane factors that participate in the translocation process including Tat substrates, Tat-specific proofreading chaperones and the integral membrane proteins TatABC that form the translocase. Fluorescence analysis of these YFP chimeras revealed a wide range of interactions such as the one between the Tat substrate dimethyl sulfoxide reductase (DmsA) and its dedicated proofreading chaperone DmsD. In addition, BiFC analysis illuminated homo- and hetero-oligomeric complexes of the TatA, TatB and TatC integral membrane proteins that were consistent with the current model of translocase assembly. In the case of TatBC assemblies, we provide the first evidence that these complexes are co-localized at the cell poles. Finally, we used this BiFC approach to capture interactions between the putative Tat receptor complex formed by TatBC and the DmsA substrate or its dedicated chaperone DmsD. Our results demonstrate that BiFC is a powerful approach for studying cytoplasmic and inner membrane interactions underlying bacterial secretory pathways

Dynamic light scattering study of calmodulin-target peptide complexes.

Dynamic light scattering (DLS) has been used to assess the influence of eleven different synthetic peptides, comprising the calmodulin (CaM)-binding domains of various CaM-binding proteins, on the structure of apo-CaM (calcium-free) and Ca(2+)-CaM. Peptides that bind CaM in a 1:1 and 2:1 peptide-to-protein ratio were studied, as were solutions of CaM bound simultaneously to two different peptides. DLS was also used to investigate the effect of Ca(2+) on the N- and C-terminal CaM fragments TR1C and TR2C, and to determine whether the two lobes of CaM interact in solution. The results obtained in this study were comparable to similar solution studies performed for some of these peptides using small-angle x-ray scattering. The addition of Ca(2+) to apo-CaM increased the hydrodynamic radius from 2.5 to 3.0 nm. The peptides studied induced a collapse of the elongated Ca(2+)-CaM structure to a more globular form, decreasing its hydrodynamic radius by an average of 25%. None of the peptides had an effect on the conformation of apo-CaM, indicating that either most of the peptides did not interact with apo-CaM, or if bound, they did not cause a large conformational change. The hydrodynamic radii of TR1C and TR2C CaM fragments were not significantly affected by the addition of Ca(2+). The addition of a target peptide and Ca(2+) to the two fragments of CaM, suggest that a globular complex is forming, as has been seen in nuclear magnetic resonance solution studies. This work demonstrates that dynamic light scattering is an inexpensive and efficient technique for assessing large-scale conformational changes that take place in calmodulin and related proteins upon binding of Ca(2+) ions and peptides, and provides a qualitative picture of how this occurs. This work also illustrates that DLS provides a rapid screening method for identifying new CaM targets