Single-molecule assays of calmodulin target binding detected with a calmodulin energy-transfer construct

Analytical Chemistry
Michael W AllenCarey K Johnson

Abstract

We have detected single-molecule binding interactions of a target peptide with the calcium-signaling protein calmodulin (CaM) immobilized in an agarose gel, and we have demonstrated the application of a single-molecule binding assay to measure the binding strength of CaM with the CaM-binding domain of calmodulin-dependent protein kinase II (CaMKII). The results demonstrate the potential for ultrasensitive assays of CaM-target interactions and the measurement of a picomolar dissociation constant. To detect single-molecule protein interactions, single-molecule assays require that the analyte molecule be confined to the focal spot of the objective for the time scale of the measurement. We demonstrate the deleterious effect of surface immobilization on CaM. As an alternative to surface immobilization, we have constructed a CaM/maltose binding protein fusion protein, which renders CaM translationally immobile in a low weight percent agarose gel. The target binding functionality of CaM assayed in agarose gels is in good agreement with solution assays. The utility of the construct for detecting interactions with CaM targets was demonstrated in a single-molecule assay of binding interactions of MBP-CaM with the CaMKII CaM-binding domai...Continue Reading

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Citations

Oct 5, 2007·Langmuir : the ACS Journal of Surfaces and Colloids·Jian ZhangJoseph R Lakowicz
May 4, 2011·World Journal of Biological Chemistry·Asma Zaidi
Apr 19, 2011·The Journal of Chemical Physics·Mihailo BackovićJohn P Ralston
Jan 4, 2012·Microscopy Research and Technique·Peter AdanyRongqing Hui
Jan 18, 2005·Physiology·Carey K JohnsonBrian D Slaughter
Jul 21, 2006·The Journal of Physical Chemistry. B·Brian D SlaughterCarey K Johnson
Aug 25, 2005·Journal of the American Chemical Society·Brian D SlaughterCarey K Johnson

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