Jasco J-815 Circular Dichroism (CD) Spectropolarimeter

Circular dichroism (CD) spectroscopy measures difference in the absorption of left-handed polarized light versus right-handed polarized light that arises due to structural asymmetry. The absence of regular structure results in zero CD intensity, while an ordered structure results in a spectrum that can contain both positive and negative signals. CD can be used for: Determination if a protein is folded Characterization of secondary structure (α-helix, β-sheet) Detection of changes in structure upon mutagenesis Studying conformational stability of proteins by varying: pH denaturant concentration (urea, guanidinium chloride ) temperature buffers components stabilizers Detection of Changes in the conformation of a protein upon protein:protein interaction Characterization of secondary structure is probably the most used CD spectroscopy application. Secondary structure can be identified in the "far-UV" spectral region (190-250 nm). Thepeptide bond is the chromophore, and it is possible to detect a signal if the protein is in a specific secondary structural conformation (α-helix, β-sheet). Alpha-helix, beta-sheet, and random coil structures each give rise to a characteristic shape and magnitude of the CD spectrum. Like all spectroscopic techniques, the CD signal reflects an average of the entire molecular population. CD Spectroscopy requirements: Far-UV CD spectra require between 100 µl- 700 µl of ~ 3-10 µM protein solution, in any buffer which does not have high absorbance in this region of the spectrum. Substances not optimal for CD: DTT (high concentrations only), imidazole, TritonX-100. Accessories: Pelitier temperature control. Automated Titration Systems allow chemical denaturation, and ligand binding studies in a completely unattended mode. Bio-Logic: SFM-20; two-channel stop-flow setup for kinetics measurements.