title: Automated circular dichroism spectroscopy for medium-throughput analysis of protein conformation.
abstract: Circular dichroism (CD) spectroscopy is a powerful method for monitoring conformational changes of biomolecules. For peptides and proteins, it is highly sensitive to changes in secondary structure, which may be caused by alterations in amino acid composition or solution conditions (e.g., temperature, pH, salts, detergents, denaturants, and excipients), post-translational modifications, self-association, or ligand binding. The assets of CD spectroscopy are that the signal is directly linked to structure, the analyte is measured without labels and in solution, the technique requires low sample amounts, and data analysis is straightforward. However, CD spectroscopy has remained a low-throughput method because it imposes high requirements on the optical quality of sample cells and thus cannot be performed in microplate-reader format. Here, we introduce an automated CD spectrometer equipped with a low-birefringence flow-through cell that is coupled to a three-axis robotic liquid-handling system. This enables unattended CD measurements on up to 384 samples, including sample transfer from 96-well plates into the flow-through cell, data acquisition, and cell cleaning. We show that the accuracy, precision, and reproducibility afforded by the new instrument are excellent and exemplify how the advantages offered by automated CD spectroscopy can be exploited to quantify protein stability by titration with chemical denaturants.
authors.affiliation: Molecular Biophysics, University of Kaiserslautern, Erwin-Schrödinger-Str. 13, 67663 Kaiserslautern, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Fiedler
authors.forename: Lindsay
authors.initials: L
authors.lastname: Cole
authors.forename: Sandro
authors.initials: S
authors.lastname: Keller
mesh_terms: D057205:Automation, Laboratory / Q000295:instrumentation / Q000379:methods*; D002942:Circular Dichroism / Q000295:instrumentation / Q000379:methods*; D011487:Protein Conformation