What is it about?
The binding of a peptide to a protein is a fundamental process in biology. It helps regulate and control systems in the human body. Understanding how a protein molecule bends and contorts itself to bind a target is critical to understanding the structure and function of the protein. Our paper demonstrates how to label a protein with two unique fluorescent dyes to monitor the binding process. Analyzing single molecules one at a time helps us see different binding configurations that might not be visible in a bulk average experiment with millions of molecules.
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Why is it important?
In the past, researchers labeled the target peptide and the receiving protein with a unique dye. This is a simple process where a single dye modification was made to the peptide and the protein. In our work, we label the protein with two unique fluorescent dyes. Whole protein HPLC separates the labeled proteins that might result from the labeling process. Labeling, isolating, and purifying the labeled protein so that only a donor and an acceptor dye are present on the molecule was demonstrated. Immobilization of the protein for study by single molecule spectroscopy was accomplished by binding CaM to maltose binding protein. Agarose gels were used to create microenvironments for each molecule. These pockets in the gel were large enough to accommodate the free movement of one protein, providing it free range of motion inside the pocket. Single-molecule data collection and analysis reveal sub-states of binding hidden in ensemble average measurements made with bulk samples.
Perspectives
This groundbreaking work was the first example of an energy transfer study performed with the donor and acceptor dyes on the same protein. Labeling, isolating, and purifying the properly labeled compound was a chromatography challenge. Furthermore, a unique agarose gel immobilization protocol was developed to "hold" the protein in place while providing access to the freely diffusing peptide target. Future work could be extended to different signaling proteins or any protein-binding process. This unique approach is also the basis of an Analytical Chemistry paper where single molecules are "counted" to determine very low binding constants.
Dr. Michael W Allen
Metrohm AG
Read the Original
This page is a summary of: Single-Molecule Assays of Calmodulin Target Binding Detected with a Calmodulin Energy-Transfer Construct, Analytical Chemistry, May 2004, American Chemical Society (ACS),
DOI: 10.1021/ac0497656.
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