Influence of α-Helical Content on the Thermodiffusion of Apomyoglobin

Apo-myoglobin (Apo-Mb) is an extensively studied model system for investigating protein folding due to its distinct stable native, partially folded molten globule (MG), and unfolded states at acidic pH. This study examines the impact of structural conformational changes on the thermodiffusive behavior of Apo-Mb using the infrared thermal diffusion forced Rayleigh scattering (TDFRS) technique. The conformational states were modulated by varying pH and buffer conditions, with their structural changes confirmed via circular dichroism (CD) spectroscopy. The α-helical content decreased with decreasing pH. The thermodiffusion parameter Δ<i>S</i>_T(Δ<i>T</i>), a measure of the temperature sensitivity of the Soret coefficient <i>S</i>_T, also showed a decrease, which is typically related to a decreasing hydrophilicity of the solute. Additionally, the buffer composition significantly influenced the thermodiffusive behavior: phosphate buffer promoted Apo-Mb aggregation through electrostatic screening, whereas acetate buffer favored Apo-Mb solubilization. Microsecond-long discrete protonation state constant pH molecular dynamics (CpHMD) simulations support the experimentally observed, pH- and buffer-dependent changes in α-helical content and highlight the differences in protein-buffer interactions for phosphate buffer versus acetate buffer. In conclusion, a strong correlation was observed between the thermodiffusion parameter Δ<i>S</i>_T(Δ<i>T</i>) and the α-helical content, with Δ<i>S</i>_T(Δ<i>T</i>) increasing alongside hydrophilicity and α-helical content. These findings highlight the role of structural conformation and buffer environment in modulating the thermodiffusive properties of proteins.