Parole chiave: Circular Dichroism Spectroscopy, Prep1, Protein Domain Design, Protein Purification, Protein-Protein Interaction, Thermal Stability,
*** IBB - CNR *** Institute of Biostructure and Bioimaging - National Research Council and Interuniversity Research Centre on Bioactive Peptides, Naples, Italy., Department of Environmental Biology, Biological and Pharmaceutical Sciences and Technologies, Second University of Naples, Caserta, Italy., Department of Pharmacy, University of Naples "Federico II", Naples, Italy., Institute of Biostructure and Bioimaging - National Research Council and Interuniversity Research Centre on Bioactive Peptides, Naples, Italy. firstname.lastname@example.org.,
PREP1/p160 is a protein complex with relevant physiopathological roles in vivo. p160 regulates PREP1 transcriptional activity by preventing the formation of other PREP1-containing complexes, whereas PREP1 regulates p160 activity by increasing its stability. This induces the repression of the insulin-regulated glucose transporter GLUT4 dampening insulin sensitivity. In spite of the considerable amount of functional studies performed on the PREP1/p160 complex in vivo, a biochemical and structural characterization of this complex has not been so far undertaken, given the poor stability of the recombinant full-length proteins. Here, we report the design and preparation of PREP1 and p160 domains together with preliminary structural and binding studies. PREP1, residues 45-155, and p160, residues 20-160, have been expressed and purified as folded, monomeric domains. The two domains show both all-alpha secondary structures, as demonstrated by CD studies and are endowed with unusually high thermal stabilities. We have also estimated for the first time the PREP1-p160 interaction strength finding that the two recombinant domains interact with a KD ranging between about 0.3 and 1 muM. Altogether, data suggest that the selected PREP1 and p160 domains are structurally independent and that their structure is underlined by high stability and a prevailing alpha-helical organization.
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