Determination of the Solution Structure of Antifreeze Glycoproteins Using Two-Dimensional Infrared Spectroscopy.

We study the solution structure of antifreeze glycoproteins (AFGPs) with linear and two-dimensional infrared spectroscopy (2D-IR). With 2D-IR, we study the coupling between the amide I and amide II vibrations of AFGPs. The measured nonlinear spectral response constitutes a much more clearly resolved amide I spectrum than the linear absorption spectrum of the amide I vibrations and allows us to identify the different structural elements of AFGPs in solution. We find clear evidence for the presence of polyproline II (PPII) helical structures already at room temperature, and we find that the fraction of PPII structures increases when the temperature is decreased to the biological working temperature of AFGP. We observe that inhibition of the antifreeze activity of AFGP using borate buffer or enhancing the antifreeze activity using sulfate buffer does not lead to significant changes in the protein conformation. This finding indicates that AFGPs bind to ice with their sugar side chains.