DNA double-strand breaks (DSBs) 1 arise in cells during physiological processes such as DNA recombination and meiosis (1, 2). Immunological diversity is generated by V(D)J recombination that produces DSBs during the process of rearrangement of genes encoding B cell immunoglobulins and T cell receptors (3, 4). They are also generated by both exogenously and endogenously generated DNA-damaging agents, including ionizing radiation and reactive oxygen species that arise as by-products of DNA metabolism. If left unrepaired, they lead to broken chromosomes and cell death. On the other hand, if DNA DSBs are repaired incorrectly, they may lead to chromosome translocation and cancer. Cells have evolved two different pathways for repairing DSBs, namely homologous recombination and non-homologous end-joining DNA (NHEJ). NHEJ is the dominant pathway in cells of multicellular eukaryotes, while homologous recombination prevails in diploid Saccharomyces cerevisiae (5). Mammalian cells utilize the same reaction to repair both radiation-induced DSBs and breaks induced during V(D)J recombination (6).Five proteins that function in NHEJ in mammalian cells have been identified to date, namely Ku70, Ku80 DNA-PKcs, Xrcc4, and ligase IV (7). Three of these (Ku70, Ku80, and DNA-PKcs) constitute a complex termed the DNA-dependent protein kinase (DNA-PK). DNA-PKcs is a large protein of 469 kDa and a member of a sub-group of phosphatidylinositol 3-kinases, called phosphatidylinositol 3-kinase-related kinases (8). Ku70 and Ku83 are subunits of the heterodimeric protein Ku and require heterodimerization for stability and function (9). Ku has double-stranded (ds) DNA end binding activity (10) and once bound can slide along the DNA in an energy-independent manner (11). Recently, the crystal structure of the Ku heterodimer, both in the presence and absence of DNA, has been determined (12). The structure shows that Ku has the shape of a ring with a large base and a narrow "handle." When bound to DNA, the conformation of Ku does not change, and a dsDNA duplex fits precisely inside the ring. One face of the duplex DNA remains relatively accessible to the solvent, because it is only partially covered by the narrow handle of the Ku molecule. In this way, the processing enzymes may have easy access to this side of the DNA duplex to remove damaged nucleotides and fill gaps prior to ligation. Although the structural studies are very useful in providing a structure of how Ku binds to DNA, they do not provide information on the dynamics of the interaction with Ku. Here, we exploit biophysical studies to evaluate further the information gained from the structural studies. In addition, the structure was determined for a Ku variant that lacked the C terminus of Ku83, a region that does not seem to be involved in the binding of Ku to DNA but is required for the interaction with DNA-PKcs (13). Several laboratories have investigated the binding of Ku to DNA and have shown that Ku cannot bind any DNA substrate shorter than 14 bp (14). It has been also shown t...

Recombinant protein expression has become an invaluable tool for academic and biotechnological projects. With the use of high-throughput screening technologies for soluble protein production, uncountable target proteins have been produced in a soluble and homogeneous state enabling the realization of further studies. Evaluation of hundreds conditions requires the use of high-throughput cloning and screening methods. Here we describe a new versatile vector suite dedicated to the expression improvement of recombinant proteins (RP) with solubility problems. This vector suite allows the parallel cloning of the same PCR product into the 12 different expression vectors evaluating protein expression under different promoter strength, different fusion tags as well as different solubility enhancer proteins. Additionally, we propose the use of a new fusion protein which appears to be a useful solubility enhancer. Above all we propose in this work an economic and useful vector suite to fast track the solubility of different RP. We also propose a new solubility enhancer protein that can be included in the evaluation of the expression of RP that are insoluble in classical expression conditions.

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Recombinant protein expression has become an invaluable tool in basic and applied research. The accumulated knowledge in this field allowed the expression of thousands of protein targets in a soluble, pure, and homogeneous state, essential for biochemical and structural analyses. A lot of progress has been achieved in the last decades, where challenging proteins were expressed in a soluble manner after evaluating different parameters such as host, strain, and fusion partner or promoter strength, among others. In this regard, we have previously developed a vector suite that allows the evaluation of different promoters and solubility enhancer-proteins, through an easy and efficient cloning strategy. Nonetheless, the proper expression of many targets remains elusive, requiring, for example, the addition of complex post-translation modifications and/or passage through specialized compartments. In order to overcome the limitations found when working with a single subcellular localization and a single host type, we herein expanded our previously developed vector suite to include the evaluation of recombinant protein expression in different cell compartments and cell hosts. In addition, these vectors also allow the assessment of alternative purification strategies for the improvement of target protein yields.

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The detection of small molecules in living cells using genetically encoded FRET sensors has revolutionized our understanding of signaling pathways at the sub-cellular level. However, engineering fluorescent proteins and specific binding domains to create new sensors remains challenging because of the difficulties associated with the large size of the polypeptides involved, and their intrinsically huge conformational variability. Indeed, FRET sensors’ design still relies on vague structural notions, and trial and error combinations of linkers and protein modules. We recently designed a FRET sensor for the second messenger cAMP named CUTie (Cyclic nucleotide Universal Tag for imaging experiments), which granted sub-micrometer resolution in living cells. Here we apply a combination of sequence/structure analysis to produce a new-generation FRET sensor for the second messenger cGMP based on Protein kinase G I (PKGI), which we named CUTie2. Coarse-grained molecular dynamics simulations achieved an exhaustive sampling of the relevant spatio-temporal coordinates providing a quasi-quantitative prediction of the FRET efficiency, as confirmed by in vitro experiments. Moreover, biochemical characterization showed that the cGMP binding module maintains virtually the same affinity and selectivity for its ligand thant the full-length protein. The computational approach proposed here is easily generalizable to other allosteric protein modules, providing a cost effective-strategy for the custom design of FRET sensors.

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