Event Information
Characterizing the Thermodynamic Stability of Multi-Domain Proteins by Using an H/D Exchange- and Mass Spectrometry-Based Technique
- Abstract:
- The investigation of the thermodynamic stability of proteins and protein-ligand complexes has been of great interest to biochemists and biologists because it provides important biological information for understanding the functions and structures of the proteins. Conventional techniques to characterize the thermodynamic properties of proteins have typically relied on the use of calorimetric methods or the use of optical spectroscopies, such as circular dichroism (CD), fluorescence and UV absorption. Although these techniques have been used over the years, they have experimental limitations. For example, pure and large amount of samples are required, and the experiments are rather time-consuming, hence, low throughput. In contrast, an H/D exchange and mass spectrometry - based technique, termed SUPREX (Stability of Unpurified Proteins by Rates of H/D Exchange) has no such limitations. The most important of this technique is that it allows the analysis of unpurified sample or complex biological mixtures.
To date, the thermodynamic stability measurements of non-two-state proteins have been a challenge because distinct optical probes are required for the characterization of individual domains. However, such probes are usually not available. To solve this problem, SUPREX was employed in combination with protease digestion to characterize the thermodynamic stability of a model non-two-state, multi-domain protein, calmodulin. Calmodulin is a regulatory protein in eukaryotic cells, regulating the cellular functions. This protein contains a total of 148 amino acids that fold into two domains, each with two calcium binding sites. A central linker between the two domains can be cleaved by trypsin to generate fragments that represent the C-terminal half and the N-terminal half.
A new protocol was developed for using SUPREX to characterize the thermodynamic stability of this two-domain protein at the domain level. In this protocol, H/D exchange of intact calmodulin was performed followed by a trypsin digestion. Ultimately, the exchange properties of the two domains were defined. The folding stability and Ca2+ binding affinity of the two domains in the intact calmodulin were successfully analyzed by SUPREX using the new protocol. The N-terminal domain was observed to be more stable than the C-terminal domain, while its binding affinity to Ca2+ was lower than that of the C-terminal domain. Our results are qualitatively in good agreement with published results obtained by using conventional techniques.
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