Synchrotron radiation circular dichroism spectroscopy study of recombinant T$\beta $4 folding

Thymosin beta 4 (T$\beta $4) is a 43-amino acid small peptide, has been demonstrated that it can promote cardiac repair, wound repair, tissue protection, and involve in the proliferation of blood cell precursor stem cells of bone marrow. Moreover, T$\beta $4 has been identified as a multifunction intrinsically disordered protein, which is lacking the stable tertiary structure. Owing to the small size and disordered character, the T$\beta $4 protein degrades rapidly and the storage condition is critical. Therefore, it is not easy to reveal its folding mechanism of native T$\beta $4. However, recombinant T$\beta $4 protein (rT$\beta $4), which fused with a 5-kDa peptide in its amino-terminal, is stable and possesses identical function of T$\beta $4. Therefore, rT$\beta $4 can be used to study its folding mechanism. By using over-critical folding process, stable folding intermediates of rT$\beta $4 can be obtained. Structure analysis of folding intermediates by synchrotron radiation circular dichroism (SRCD) and fluorescence spectroscopies indicate that rT$\beta $4 is a random coli major protein and its hydrophobic region becomes compact gradually. Moreover, the rT$\beta $4 folding is a two state transition. Thermal denaturation analysis indicates that rT$\beta $4 lacks stable tertiary structure. These results indicated that rT$\beta $4, similar to T$\beta $4, is an intrinsically disordered protein.