As the DNA switching process is extremely sensitive to changes in the hydrodynamic drag, minor changes in the molecular structure of proteins and other macromolecules can be detected as changes in the switching dynamics. A typical application of such an experiment, is the detection of ligand-induced conformational changes, for example upon association of a small molecule to a protein.For the successful detection of a conformational change, please consider the following aspects:
- The molecule of interest can be captured via hybridization after covalent coupling, or via a tag system
(His6-Tag, Strep-Tag, biotin-streptavidin interaction, etc.) - Significant conformational changes can be resolved as changes of a protein’s hydrodynamic diameter, which is calculated using the Lollipop model. Please note that this kind of analysis is only available when using appropriate buffer conditions.
Also see “What is the Lollipop Model?” - Please analyze changes in the hydrodynamic diameter using the “Sizing” evaluation tool in switchANALYSIS.
- Often, conformational changes affect only minor portions of the protein molecule, which might not be resolvable by use of the Lollipop model.
- Nevertheless, these changes usually still change the hydrodynamic properties of the protein, resulting in changes in the switching speed.
- These changes can be evaluated as relative Dynamic Response change in comparison to the bare DNA reference measurement.
- For such analyses, please use the “Conformational Change” evaluation tool in switchANALYSIS.
Category:
Specific Experimental Considerations