Two-photon cross-correlation analysis of intracellular reactions with variable stoichiometry.

Abstract:

:We successfully demonstrate the effectiveness of two-photon fluorescence cross-correlation spectroscopy (TPCCS) to study the complex binding stoichiometry of calmodulin (CaM) and Ca(2+)/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 experimental and theoretical framework for TPCCS. Our tailored model accommodates up to 12 red-labeled CaMs binding to a single green-labeled dodecameric CaMKII holoenzyme and accounts for the probability distributions of bound ligand as well as the respective changes in fluorescence emission upon binding. The model was experimentally demonstrated 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 parameters, 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.

journal_name

Biophys J

journal_title

Biophysical journal

authors

Kim SA,Heinze KG,Bacia K,Waxham MN,Schwille P

doi

10.1529/biophysj.104.055319

subject

Has Abstract

pub_date

2005-06-01 00:00:00

pages

4319-36

issue

6

eissn

0006-3495

issn

1542-0086

pii

S0006-3495(05)73481-0

journal_volume

88

pub_type

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