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The use of a quartz crystal microbalance with dissipation for the measurement of protein-protein interactions : a qualitative and quantitative analysis of the interactions between molecular chaperones


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Biotechnology research and innovation depends on the ability to understand the molecular mechanisms of biological processes such as protein-protein and protein-ligand interactions. Surface plasmon resonance (SPR) spectroscopy is now well established as a quantitative technique for monitoring biomolecular interactions. In this study, we examined the recently developed quartz crystal microbalance with dissipation (QCM-D) method as an alternative to SPR spectroscopy to investigate protein-protein interactions, in particular, for chaperone-co-chaperone interactions. In mammalian cells, the Hsp70 / Hsp90 organizing protein (Hop) is a co-chaperone required for the association of the molecular chaperones, heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90). The objective of this research was to characterize qualitatively and quantitatively the interaction of Hsp70 with Hop. A truncated version of Hop consisting of only the C-terminal region and lacking the Hsp70-binding domain (GST-C-Hop) was used as a non-Hsp70- binding control. Immobilized GST-Hop was found to bind Hsp70 successfully, displaying a QCM-D response consistent with formation of a complex that became slightly more flexible as the concentration of bound Hsp70 increased. GST-C-Hop did not bind to Hsp70, thereby validating the specificity of the GST-Hop interaction with Hsp70. The kinetics of the interaction was followed at different concentrations of Hsp70, and an apparent thermodynamic dissociation constant (KD value) in the micromolar range was determined that correlated well with the value derived previously using SPR. This study represents a proof-of-principle that QCM-D can be applied to the analysis of chaperone-co-chaperone interactions. The economic and technical accessibility of QCM-D makes it a valuable tool for analyses of chaperone interactions, and protein- protein interactions in general.
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Document Type: Research Article

Publication date: November 1, 2004

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