By: Christopher P. Toseland1 2 
, Martin R. Webb1Affiliation(s): (1) MRC National Institute for Medical Research, London, UK
(2) Institut für Zellul?re Physiologie, Physiologisches Institut, Ludwig Maximilians Universit?t, Munich, Germany
Book Title: Single Molecule Enzymology : Methods and ProtocolsSeries: Methods in Molecular Biology | Volume: 778 | Pub. Date: Aug-31-2011 | Page Range: 161-174 | DOI: 10.1007/978-1-61779-261-8_11Subject: Biochemistry The interconversion of nucleoside triphosphate (NTP) and diphosphate occurs in some of the most -important cellular reactions. It is catalyzed by diverse classes of enzymes, such as nucleoside triphosphatases, kinases, and ATP synthases. Triphosphatases include helicases, myosins, and G-proteins, as well as many other energy-transducing enzymes. The transfer of phosphate by kinases is involved in many metabolic pathways and in control of enzyme activity through protein phosphorylation. To understand the processes catalyzed by these enzymes, it is important to measure the kinetics of individual elementary steps and conformation changes. Fluorescent nucleotides can directly report on the binding and release steps, and conformational changes associated with these processes. In single-molecule studies, fluorescent nucleotides can allow their role to be explored by following precisely the temporal and spatial changes in the bound nucleotide. Here, the selection of fluorophores and nucleotide modifications are discussed and methods are described to prepare ATP analogs with examples of two alternate fluorophores, diethylaminocoumarin and Cy3.Key Words: Fluorescent nucleotides - ATP - GTP - Motor proteins - TIRF microscopy
1 Introduction
The conversions of ATP to ADP and GTP to GDP are mediated by a wide range of enzymes. These include motor proteins such as myosins, helicases, and kinesins, along with proteins from signaling pathways, such as kinases and G-proteins. With respect to many motor proteins, the energy from the ATP hydrolysis is coupled to changes in protein conformation, and/or protein–track interactions, enabling functions such as muscle contraction, DNA unwinding, and modulation of protein–protein interactions.
Fluorescence nucleotides are widely applied to investigate solution kinetics of such triphosphatases and kinases. For example, they are used to measure the kinetics of individual steps in the enzymic reaction (binding, hydrolysis, product release, and associated -structural changes) and to understand fully how such activities are coupled to the protein function. In such measurements, a change in the fluorescence properties is required to give a signal associated with the process of interest. Most often, this change is in intensity, but other properties such as anisotropy are also used. Importantly, significant fluorescence changes are more important in this type of use than overall fluorophore brightness. In addition, fluorophore photobleaching is usually not a major problem, as light sources can be of lower intensity than those for single-molecule visualization, described below.
The use of fluorescent nucleotides in single-molecule assays has increased over the past 15 years, and this has been especially driven by the study of motor proteins, in which there is a precise relationship between movement and nucleotide hydrolysis. Total internal reflection fluorescence microscopy (TIRFM) is readily used to visualize individual fluorescent ATP and ADP molecules allowing the measurements of ATP turnovers by single myosin molecules (1–4). For such measurements, a bright, photostable, fluorophore is a major factor: the light sources must be intense to get sufficient photons emitted from single complexes. More recently, single-molecule fluorescence measurements have been combined with translocation measurements to show the coupling between ATPase activity and translocation along actin (4). TIRFM selectively excites molecules within 100–200 nm of the surface, dramatically reducing the background fluorescence from unbound fluorophores in the bulk solution. This improvement in the signal-to-noise ratio allows detection of individual fluorescent ATP molecules, when bound to surface-attached proteins. However, the possibility of further improvement in the signal-to-noise ratio, through a fluorescence intensity increase on protein binding, could improve either the spatial or temporal, resolution of measurements. This chapter considers only the use of fluorescence intensity measurements of a single fluorophore on the nucleotide. However, developments such as spFRET (single-particle F?rster Resonance Energy Transfer) (5) has clear potential to extend the applications of fluorescent nucleotides.
In this chapter, the selection of the fluorophores and types of nucleotide modifications are discussed. Two example syntheses, purifications, and characterizations are described that result in fluorescent adducts, differing both in the type of fluorophore and in the linkage between the fluorophore and nucleotide. The structures of the two adducts are shown in Fig. 1.
Fig. 1. Fluorescent nucleotide analogs. (a) Deac-aminoATP and (b) Cy3-edaATP.