Automated Circular Dichroism Spectroscopy for Medium-Throughput Quantification of Protein Conformation

Abstract

Circular dichroism (CD) spectroscopy is a powerful method for monitoring conformational changes of biomolecules. For proteins, it is highly sensitive to changes in secondary structure, which, in turn, are influenced by amino acid composition, posttranslational modifications, solution conditions (e.g., temperature, pH, salts, detergents, denaturants, excipients, etc.), and ligand binding. The CD signal is directly linked to protein structure, the analyte is in solution and label-free, 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 that is equipped with a flow-through cell and coupled to a 3-axis robotic liquid handler. This enables completely 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 demonstrate that the accuracy, precision, and data quality of the automated CD spectrometer are as good as those of a conventional, manually operated instrument and exemplify how the advantages offered by automated CD spectroscopy can be exploited in protein-unfolding experiments using chemical denaturants.