Forschungsschwerpunkt BioComp

BioComp-Projekt #12

High-throughput quantification of protein conformation by automated circular dichroism spectroscopy

 

Christoph Garth - Sandro Keller

Circular dichroism (CD) spectroscopy is a powerful method for monitoring conformational changes of proteins caused by alterations in solution conditions, amino acid composition, posttranslational 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 established data-analysis approaches are straightforward. However, CD spectroscopy has remained a low-throughput method until the world’s first academic automated CD spectrometer recently became available at the TU Kaiserslautern. This instrument enables unattended measurements on up to 384 samples and thus novel experimental protocols that yield new insights into the conformational stability of proteins as a function of solution conditions.

In order to fully exploit the potential of automated CD spectroscopy, this tremendous increase in experimental throughput needs to be complemented with new data-analysis approaches that make use of the high information content now available (i.e., several hundred spectra per experiment, each spectrum consisting of a matrix of average intensities with associated standard deviations). Moreover, conventional analysis approaches are of limited use in extracting the peak shapes of CD spectra, and need to be adjusted to the specific conditions and properties of protein conformation. To aid an experimenter in interpreting the output of such sophisticated analysis methods, domain-specific visualisation techniques are required that facilitate intuitive and interactive visual analysis of conformation states. Tightly coupled, mathematical analysis and interactive visualisation will facilitate detailed, quantitative insights into changes in protein conformation not accessible through conventional statistical analysis. This approach is especially needed to interpret the large basis of data available for the first time through automated CD spectrometry.

In this project, the combination of automated CD spectroscopy on the experimental side as well as mathematical analysis and visualisation on the computational side will be applied to the quantification of protein stability and the reconstitution of membrane proteins, both of which are of great interest to fundamental and applied research, including protein biotechnology and drug development.

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