Contrary to the classical view, according to which all proteins adopt a specific folded conformation necessary for their function, intrinsically unstructured proteins (IUPs) display random-coil-like conformation under physiological conditions, although they have specific biological function. Upon ligand binding, however, many of them may adopt a well-defined three-dimensional structure.Titin is a giant protein responsible for striated-muscle elasticity. It contains a series of ordered domains and a large disordered segment called the PEVK domain. It acts as an entropic spring and is thought to be responsible for the generation of passive contractile force in muscle. The ordered domains belong to the immunoglobulin (Ig) type C2 and fibronectin (FN) type III superfamilies.We expressed a 171-residue-long fragment of the PEVK domain (polyE) and an Ig domain (I27) in BL21 derivative E.coli Rosetta competent strains. FTIR spectroscopy combined with a diamond anvil cell was used as a non-perturbing method for investigating the secondary structures of these recombinant proteins. Fluorescence spectra of I27 were also recorded.PolyE preserves its disordered characteristics across a wide range of pressure (0-16 kbar), temperature (0-100 °C), pD (3-10.5) and in presence of several cosolvents. Upon pressure treatment, titin I27 unfolds at 10.7 kbar at 30 °C. As the function of temperature we observed two transitions. At 50 °C the secondary structure is loosened, and the protein transforms into a molten-globule state. At 70 °C the protein completely unfolds. Unfolding is followed by aggregation at ambient pressure. Moderate pressures (>2 kbar), however, can prevent the protein from aggregation. We determined the detailed temperature-pressure phase diagram of titin I27, which contains metastable regions as well.