Conformational dynamics of Calcium Green 1 by fluorescence correlation spectroscopy

Abstract

Fluorescence correlation spectroscopy was used to characterise the conformational equilibrium in “Calcium Green 1’', a fluorescent indicator of calcium. In the calcium bound form, a triplet bunching time of 6.7 µs is seen. We show that, in the absence of calcium, 10% of the indicator is in a fluorescent conformation that inter-converts to a non-fluorescent form in 10 µs. This confirms the probing mechanism based on a conformation controlled PET proposed for Calcium Green 1.

Introduction

We have used fluorescence correlation spectroscopy (FCS) to provide direct evidence of the existence of a conformational equilibrium in a fluorescent indicator. Fluorescence correlation spectroscop has already been used to study conformational dynamics of oligonucleotide or with fluorescent indicators of ions, where the exchange rate of the ion with the probe was measured Here we measure a more intimate process: the conformational equilibrium inside the fluorescent indicator. Calcium Green 1 is composed of a fluorochrom and a chelating group of calcium: the BAPTA (1,2-bis(o-aminophenoxy)ethane N,N,N′,N′-tetraacetic acid). When this fluorescent indicator is not complexing calcium, the fluorescence decay is composed of two components: a short-lived component with a 0.5 ns lifetime and a long-lived component with a 3.5 ns lifetime. The long-lived component has been neglected for a long time and attributed to impurities or to residual calcium in the mixture We have proposed that the long-lived component be due to an open conformer of the molecule where the quenching of the fluorescence by through space electron transfer from the BAPTA does not occur. Indeed the balance between the short and long lifetime components depends on the dielectric constant as expected for charged species of different volumes

We have used FCS to provide direct evidence of the conformational equilibrium since an impurity will give a very different signal from that of a quickly inter-converting conformer.

Materials and methods