Effect of Molecular Crowding on the Structure and Dynamics of Human Apo and Holo Transferrin using 2D-IR Correlation Spectroscopy
Human serum transferrin is an iron transport glycoprotein that is involved in the regulation and balance of iron content in blood plasma and cells. Little is known about the effect of molecular crowding on the structure, dynamics and aggregation of transferrin. Therefore, we investigated secondary structure, thermal denaturation, aggregation and hydrogen-deuterium (H/2H) exchange of apo and holo transferrin in the presence and absence of the molecular crowding agent dextran using Fourier transform infrared (FTIR) spectroscopy. The data obtained was analysed using second-derivative, deconvolution and two-dimensional correlation infrared (2D-IR) spectroscopy. Protein samples were prepared in phosphate buffer solutions (H2O and 2H2O; pH 7.4) at a concentrations of 20 and 50mg/ml. For the crowded environment condition, the same conditions were used but dextran was also included at a concentration of 200 mg/ml. No molecular crowding-induced changes in the secondary structure of transferrin was detected. However, the H/D exchange of apo and holo transferrin in dextran solution was significantly reduced suggesting a more rigid, solvent inaccessible structure induced by molecular crowding. The thermal study of transferrin in the range 25-95 oC was analysed using the synchronous 2D-IR correlation. The 2D-IR correlation spectra of holo transferrin in presence and absence of dextran revealed different negative peaks. In the absence of dextran, the loss of α-helical structure (1656cm-1) is associated with an increase in the intensity of a band at 1674cm-1. However, in the presence of dextran, the unfolding of the α-helical structure (1656cm-1) is accompanied by the formation of intermolecular β-sheet structure (1616cm-1 and 1690cm-1). Similar crowding effects were also observed for apo transferrin providing direct evidence of the effect of dextran, as molecular crowding agent, on the thermal aggregation characteristics and dynamics of transferrin.
Citation : Abbas, S., Severcan, F. and Haris, P.I. (2015) Effect of Molecular Crowding on the Structure and Dynamics of Human Apo and Holo Transferrin using 2D-IR Correlation Spectroscopy. Biophysical Journal, 108(2), pp.16a.