TWO-PHOTON CROSS-CORRELATION ANALYSIS OF INTRACELLULAR REACTIONS WITH VARIABLE STOICHIOMETRY

¹ U. Texas at Houston
² TU Dresden

^* To whom correspondence should be addressed. E-mail: pschwil{at}gwdg.de.

Submitted on November 5, 2004
Revised on December 17, 2004
Accepted on 9 March 2005

	Abstract

We successfully demonstrate the effectiveness of two-photon fluorescence cross-correlation spectroscopy (TPCCS) to study the complex binding stoichiometry of calmodulin (CaM) and Ca2+/CaM-dependent protein kinase II (CaMKII). Practical considerations are made for developing an intracellular cross-correlation assay, including characterization of the fluorescent molecules involved, calibration procedures of the setup, and optimal measurement conditions. Potential pitfalls and artifacts are discussed, and the complex stoichiometry of the molecular system is accounted for by a new ex-perimental and theoretical framework for TPCCS. Our tailored model accommodates up to 12 red-labeled CaMs binding to a single green-labeled dodecameric CaMKII holoen-zyme and accounts for the probability distributions of bound ligand as well as the respec-tive changes in fluorescence emission upon binding. The model was experimentally dem-onstrated both in solution and in living cells by analyzing the binding of Alexa 633(C2)CaM to eGFP-CaMKII under different biochemical conditions known to induce the basal, activated and autophosphorylated forms of the enzyme. Key binding parame-ters, such as binding degree, concentrations of reactants, and binding affinities, were determined under varying conditions with certain assumptions. TPCCS thus offers the unique ability to test our biochemical understanding of protein dynamics in the intracellular milieu.

Key Words: CaMKII, calmodulin, cross-correlation, intracellular FCS, single molecule, two-photon excitation

This article has been cited by other articles:

				A. B. Goins, H. Sanabria, and M. N. Waxham Macromolecular Crowding and Size Effects on Probe Microviscosity Biophys. J., December 1, 2008; 95(11): 5362 - 5373. [Abstract] [Full Text] [PDF]

				S. Zorrilla, A. Ortega, D. Chaix, C. Alfonso, G. Rivas, S. Aymerich, M. P. Lillo, N. Declerck, and C. A. Royer Characterization of the Control Catabolite Protein of Gluconeogenic Genes Repressor by Fluorescence Cross-Correlation Spectroscopy and Other Biophysical Approaches Biophys. J., November 1, 2008; 95(9): 4403 - 4415. [Abstract] [Full Text] [PDF]

				J. W. D. Comeau, S. Costantino, and P. W. Wiseman A Guide to Accurate Fluorescence Microscopy Colocalization Measurements Biophys. J., December 15, 2006; 91(12): 4611 - 4622. [Abstract] [Full Text] [PDF]

				L. N. Hillesheim, Y. Chen, and J. D. Muller Dual-Color Photon Counting Histogram Analysis of mRFP1 and EGFP in Living Cells Biophys. J., December 1, 2006; 91(11): 4273 - 4284. [Abstract] [Full Text] [PDF]

				J. Ries and P. Schwille Studying Slow Membrane Dynamics with Continuous Wave Scanning Fluorescence Correlation Spectroscopy Biophys. J., September 1, 2006; 91(5): 1915 - 1924. [Abstract] [Full Text] [PDF]