Use of CD and FT-IR to determine the secondary structure of purified proteins in the low-microgram range

The spectroscopic characterization of protein secondary structure is often partially unreliable when samples are not extremely pure and abundant. This problem may be overcome by the combination of circular dichroism (CD) and Fourier transform infrared spectroscopy (FT-IR). We used these methods to c haracterize the secondary structure of two proteins of neurobiological inte rest, calexcitin (CE) and the cellular isoform of prion protein (PrPC). Bot h proteins were purified with multiple chromatographic steps and were obtai ned in buffer with high purity (> 95%) and in low amount (similar to 2 mu g ). The samples were analyzed by circular dichroism (down to 184 or 182 nm), recovered, and deposited on films for infrared analysis. The spectral deco nvolution from the two methods yielded secondary structures in good agreeme nt with each other as well as with theoretical predictions based on amino a cid sequence. The conformation of CE was found to be dependent on its conce ntration and on calcium binding. The secondary structure of cellular native PrP varied dramatically with the detergent used. In conclusion, the combin ation of CD and FT-IR analysis is suitable for the characterization of the conformational changes induced by ligand binding and/or by different solven t conditions when the protein of interest is only scarcely available. The m ethods used here provide valuable insights into the putative correlation be tween protein structure and activity.