In vivo expression of metalloproteins requires specific metal trafficking and incorporation machinery inside the cell. Synthetic designed metalloproteins are typically purified without the target metal, which is subsequently introduced through in vitro reconstitution. The extra step complicates protein optimization by high-throughput library screening or laboratory evolution. We demonstrate that a designed coiled-coil iron−sulfur protein (CCIS) assembles robustly with [4Fe-4S] clusters in vivo. While in vitro reconstitution produces a mixture of oligomers that depends on solution conditions, in vivo production generates a stable homotrimer coordinating a single, diamagnetic [4Fe-4S] 2+ cluster. The multinuclear cluster of in vivo assembled CCIS is more resistant to degradation by molecular oxygen. Only one of the two metal coordinating half-sites is required in vivo, indicating specificity of molecular recognition in recruitment of the metal cluster. CCIS, unbiased by evolution, is a unique platform to examine iron−sulfur protein biogenesis and develop synthetic multinuclear oxidoreductases.

Signaling through c-Raf downstream pathways is the crucial subject of extensive studies because over expressed or mutated genes in this pathway lead to a variety of human cancers. On the basis of cellular localization, this pathway has been sub-divided into two cascades. The first RAF1-MEK1-ERK2 cascade which remains in the cytosol, whereas the second MEK1-ERK2-RSKs transduces into the nucleus and regulates the transactivation function. But how a few amino acids critically regulate the transcriptional function remains unclear. In this paper, we have performed in silico studies to unravel how atomic complexities at the MEK1-ERK2-RSKs pathways intercedes different functional responses. The secondary structure of the ERK, RSKs have been modeled using Jpred3, PSI-PHRED, protein modeler, and Integrated sequence analyzer from Discovery Studio software. Peptides of RSKs isozymes (RSK1/2/3/4) were built and docked on ERK2 structure using ZDOCK module. The hydropathy index for the RSKs molecules was determined using the KYTE-DOOLITTLE plot. The simulations of complex molecules were carried out using a CHARMM force field. The protein-protein interactions (PPIs) in different cascade of MAP kinase (MAPK) have been shown to be similar to those predicted in vivo. PPIs elucidate that the amino acids located at the conserved domains of MAPK pathways are responsible for transactivation functions.

Ribosomal S6 kinases (RSKs) are the major functional components in mitogen-activated protein kinase (MAPK) pathway, and these are activated by upstream Extracellular signal-regulated kinase. Upon activation, RSKs activate a number of substrate molecules involved in transcription, translation and cell-cycle regulation. But how cellular binding partners are engaged in the MAPK pathways and regulate the molecular mechanisms have not been explored. Considering the importance of protein-protein interactions in cell signalling and folding pattern of native protein, functional C-terminal kinase domain of RSK3 has been characterized using in vitro, in silico and biophysical approaches. RSKs discharge different functions by binding to downstream kinase partners. Hence, depending upon cellular binding partners, RSKs translocate between cytoplasm and nucleus. In our study, it has been observed that the refolded C-terminal Kinase domain (CTKD) of RSK 3 has a compact domain structure which is predominantly α-helical in nature by burying the tryptophans deep into the core, which was confirmed by CD, Fluorescence spectroscopy and limited proteolysis assay. Our study also revealed that RSK 3 CTKD was found to be a homotrimer from DLS experiments. A model was also built for RSK 3 CTKD and was further validated using PROCHECK and ProSA webservers.