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Fluorescent Nucleoside Triphosphates for Single-Molecule Enzymology
Affiliation(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:
Series: Methods in Molecular Biology&&|&&Volume: 778&&|&&Pub. Date: Aug-31-2011&&|&&Page Range: 161-174&&|&&DOI: 10.-_11
Subject: &
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:
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 proteinCtrack interactions, enabling functions such as muscle contraction, DNA unwinding, and modulation of proteinCprotein 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 (C). 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 (). TIRFM selectively excites molecules within 100C200 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) () 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. .
Fig. 1.& Fluorescent nucleotide analogs. (a) Deac-aminoATP and (b) Cy3-edaATP.
Fluorescent adenine and guanine nucleotides have been widely used to report upon binding, protein release and structural changes (C). Fluorophores are sensitive probes, easily used at submicromolar concentrations, and can have properties that report rapidly, even on small perturbations in the region of the fluorophore. Thus, ATP and analogs have been modified with fluorophores at several locations in the molecular structure and the range of modifications has been reviewed (, ). The choice of attachment site is important, both to get a fluorophore in a position to report but also to modify the parent nucleotide without significant perturbation of the biochemical properties. First, the purine base can be made fluorescent either by modification or by using a fluorescent analog of the natural base, as in the case of formycin triphosphate (FTP) (). However, such modifications may disrupt the proteinCnucleotide interactions, as there is often high selectivity in the base binding site of proteins (). The phosphate chain can also be modified, as with γ-AMNS-ATP for investigations of Escherichia coli RNA polymerase (), but these modifications frequently disrupt the cleavage& step by preventing &correct binding of the phosphates or by blocking& access to the γ-phosphate. Ribose modifications are often the most successful, whereby the analog closely mimics the activity of ATP. In many enzymes that bind GTP or ATP, the 2′- and/or 3′-hydroxyl groups of the ribose are partly exposed to the protein surface, while the base and phosphates are well buried. This allows a ribose label to sit at the entrance to the binding site and potentially report on changes in that region, with only a small effect on the binding and catalytic properties.
When fluorescent analogs are to be used for microscopy, the main criteria for choices of fluorophore are high fluorescence intensity (high extinction coefficient and fluorescence quantum yield), excitation and emission maxima best suited to the excitation source, without interference from other components in the system, and stability against photobleaching. With some fluorophores, the extinction coefficient and particularly the quantum yield can change significantly with the chemical environment of the fluorophore. Such changes can be problematic in choosing a suitable fluorophore, but if the intensity changes are monitored, then changes in intensity can be harnessed, as described below for a diethylaminocoumarin. Long-wavelength fluorophores, such as the cyanine dyes (e.g., Cy3), have good properties for fluorescence microscopy and have been used for detecting fluorescence from single molecules. Typically, fluorophores that excite at longer wavelengths are relatively large moieties with multiring structures and may have significantly hydrophobic regions. This may lead to nonspecific binding to proteins and surfaces. There are now many commercially available fluorophore-labeling reagents that give variations on quantum yield, photostability, and wavelengths. A discussion of this variety is outside the scope of this chapter. For additional information and for commercial sources of such labels as well as ATP analogs see: , ,
and . Quantum dots have significant potential for future use, but their large size relative to nucleoside triphosphates is likely to make them difficult to apply generally here.
As described above, labeling at the ribose hydroxyl groups has been useful because this modification may not lead to large perturbations of the biochemical properties. However, such labeling of the hydroxyl groups leads to the formation of a mixture of 2′- and 3′-isomers, whose biochemical and fluorescent properties may differ when bound (). This problem can be circumvented by using a parent nucleotide, in which only one hydroxyl is available for modification, for example the commercially available 2′-deoxyATP, or the synthetic 3′-amino-3′-deoxyATP (). Alternatively, the isomers of some labeled nucleotides interconvert only very slowly and can be successfully separated by chromatography and stored as single isomers (, ).
As mentioned in the above section, fluorophores for fluorescence microscopy are relatively large and so may disrupt the biochemical cycle. To ameliorate this problem, several linkers are available to space the fluorophores further away from the catalytic site. Essentially, a zero-length linker is achieved by direct labeling of the amine group on the ribose ring of 3′-amino-3′-deoxyATP. Longer chemical linkers can use different lengths of diamino-n-alkanes, such as 2′(3′)-O-[N-(2-aminoethyl)-carbamoyl]ATP (edaATP ()) and 2′(3′)-O-[N-(3-aminopropyl)carbamoyl]ATP (pdaATP ()). In some cases, including Cy3, the commercially available labels include a spacer chain between the fluorophore and the reactive group used for attachment. In these cases, the fluorophore will be positioned further away from the protein and, therefore, should not interfere significantly with the nucleotide association and catalysis. However, moving the fluorophore further away from the protein may reduce any changes to fluorescent properties on binding. All modifications may be deleterious to the enzymic activity, and therefore, it is important to assess the impact of these changes. Methods to assess these effects are described later.
The requirements for the synthesis of ATP analogs vary widely. Those described here are relatively simple labeling reactions, performed under aqueous conditions, so potentially high yields can be obtained using conditions and equipment available in most laboratories. The success of the labeling depends both on the chemical reactions per se and on the properties of the fluorophore. For example, very hydrophobic groups may impair the success of a reaction that occurs perfectly well with simpler labels. The purification of the product also may depend on the physical properties of the fluorescent label. Two specific examples are described for the labeling nucleotides at the ribose ring with Cy3 and diethylaminocoumarin (Fig. ).
The visualization of the binding of fluorescent nucleotides to proteins by light microscope has been limited by technical problems such as the nonspecific binding of the fluorescent nucleotide to the coverslip. This has limited the maximum nucleotide concentration that could be used with analogs such as 2′(3′)-(Cy3-O-[N-(2-aminoethyl)carbamoyl])ATP (Cy3-edaATP, Fig. b ()) to &100 nM. Fluorescent groups may also bind to macromolecules such as proteins, independently of the nucleotide and its binding site, and particularly if used at high concentration. A control, such as displacing the labeled with unlabeled nucleotide, will test if such nonspecific binding occurs.
The fluorescent ATP analog, (3′-(7-diethylaminocoumarin-3-carbonylamino)-3′-deoxyadenosine-5′-triphosphate (deac-aminoATP)) (Fig. a) has a low quantum yield when in solution, but this increases dramatically when bound to some proteins. This generates large fluorescence changes, such as the 20-fold increase when bound to myosin Va (). This enables a distinction between coverslip bound “background” molecules and those bound by proteins (), which may compensate for the relatively low optimal excitation wavelength and photostability.
2′,3′-O-(2-Aminoethyl-carbamoyl)-adenosine-5′-triphosphate (edaATP) (Jena Biosciences) (see Note ).
3′-Amino-3′-deoxyATP triethylammonium salt (aminoATP) ().
Cy3 N-hydroxysuccinimide ester (NHS-ester) (GE Healthcare).
7-Diethylaminocoumarin-3-carboxylic acid (Invitrogen).
20 mM Sodium bicarbonate, pH 8.4.
Dimethylformamide (DMF).
Tributylamine.
Isobutyl chloroformate.
HPLC system, preferably with both absorbance and fluorescence detectors.
Strong anion exchange (SAX) Partisphere column (0.4  ×  10 cm) (Whatman).
0.4 M (NH4)2HPO4 adjusted to pH 4.0 with concentrated HCl.
HPLC-grade Methanol.
HPLC-grade Acetonitrile.
DEAE cellulose column (2  ×  30 cm).
Triethylamine (technical grade).
Glass distillation apparatus suitable for up to 500 ml and &having ground glass joint.
Boiling chips.
2-L Buchner flask, with bung and plastic tubing on the side arm, connected to a glass-scinter gas bubbler.
Chromatography system (fraction collector, gradient maker, pump, etc.) at 4°C with absorbance and fluorescence detector, if possible.
Rotary evaporator, equipped with a cold finger condenser and high-vacuum oil pump.
Methanol (highest grade available).
Isopropanol (technical grade).
Spectrophotometer.
HPLC system, preferably with both absorbance and fluorescence detectors.
Strong anion exchange (SAX) Partisphere column (0.4  ×  10 cm) (Whatman).
0.4 M (NH4)2HPO4, pH 4 with concentrated HCl.
HPLC-grade methanol.
HPLC-grade acetonitrile.
Fluorescence spectrophotometer.
MDCC-PBP (). Phosphate binding protein (A197C) from E. coli, labeled with (N-[2-(1-maleimidyl)ethyl]-7-diethylaminocoumarin-3-carboxamide) (Invitrogen) (see Note ).
Rhodamine-PBP (). Phosphate binding protein (A17C, A197C) from E. coli, labeled with 6-iodoacetamidotetramethylrhodamine (see Note ).
Fluorescence spectrophotometer.
Inorganic phosphate standard solution.
This method is based on that described by Oiwa et al. () and gives mixed (2′,3′) isomers (Fig. b).
3.1.1 & Labeling
Mix 4 μmol Cy3 NHS-ester with 20 μmol edaATP in 20 mM sodium bicarbonate, pH 8.4, for 1 h at room temperature (see Note ).
Analyze the reaction mixture using HPLC to confirm the formation of Cy3-edaATP. Equilibrate a Partisphere SAX column with 0.4 M (NH4)2HPO4 with 20% (v/v) methanol: flow rate of 1 ml/min at room temperature (see Notes 4 and 5).
Add an aliquot of the reaction mixture (1C10 nmol) to 100 μL of the running buffer.
Inject the solution onto the column.
Follow the absorbance at 254 nm and fluorescence with excitation of 550 nm and emission of 570 nm. The chromatogram will show the elution of Cy3 NHS-ester, edaATP, and Cy3-edaATP, respectively (see Note ).
Inject known standards of Cy3 NHS-ester and edaATP at the same concentration as the reaction mixture to identify peaks.
3.1.2 & Preparation of Triethylammonium Bicarbonate Solution
Distil triethylamine (500 ml), discarding the first and last 10% of the distillate. Use the middle 80% of the distillate (see Note ).
Add cold (4°C), distilled, deionized water to 139.4 ml distilled triethylamine to give 1 L of a 1 M solution (see Note ).
In a fume hood, put dry ice in the Buchner flask, and with the solution in ice, bubble CO2 through the solution until the pH is 7.5C7.6 (approximately 2 h) using the scintered glass bubbler. Keep the Buchner flask, containing the dry ice, raised above the solution to reduce the risk of sucking back.
Store triethylammonium bicarbonate (TEAB) at 4°C in a well-stoppered container. It lasts approximately 1C2 months, but the pH gradually rises with time. In this case, rebubble CO2 through it.
3.1.3 & Purification of Nucleotide
Preequilibrate the DEAE-cellulose column with 10 mM TEAB, pH 7.6 at 1 ml/min at 4°C.
Alter the pH of the reaction mixture to 7.6 using acid or base, reduce the conductivity by dilution in water so it is close to that of 10 mM TEAB and load onto the column.
Wash the column with 10 mM TEAB, pH 7.6 at a flow rate of 1 ml/min until no more pink material is eluted.
Elute the nucleotide with a linear gradient of 10C800 mM TEAB (total volume 600 ml). Follow the absorbance at 254 nm. Unreacted edaATP is eluted first followed by Cy3-edaATP (see Note ).
Identify the fractions containing Cy3-edaATP by measuring the absorbance at 550 nm and 260 nm.
3.1.4 & Concentration
Pool fractions containing Cy3-edaATP and remove TEAB by rotary evaporation. Use a flask with a capacity at least four times the volume of the solution.
Fill the condenser with dry iceCisopropanol.
Add the pooled fractions to the flask, rotate, and slowly apply the vacuum to begin evaporation. Warm the flask in a water bath at 30°C. Reduce the volume to ∼5 ml. When the solution &volume is reduced to 10C20%, frothing may begin (see Note ).
Add methanol (∼10% of initial solution volume) and repeat the evaporation.
Repeat methanol additions and evaporation three times: &during this it should be possible to remove essentially all the solvent before adding more methanol. At the final stage, evaporate all of the methanol. The Cy3-edaATP will remain as a gum.
Dissolve in &3 ml methanol and transfer to a pear flask (10 ml) and reconcentrate, with very careful application of the vacuum to avoid frothing. Finally, dissolve in water or buffer and adjust to pH  ∼ &# before storing at −80°C (see Note ).
3.1.5 & Characterization
Measure the absorbance spectra of Cy3-edaATP in 50 mM TrisCHCl, pH 7.5 between 220 and 700 nm. Taking the extinction coefficient for the Cy3 to be 150,000 M−1 cm−1 at 552 nm () and the extinction coefficient for adenosine to be 15,200 M−1 cm−1 at 260 nm, calculate the concentration of the nucleotide (see Note ).
Characterize Cy3-edaATP by HPLC using the same method as above. The major peak should be Cy3-edaATP. Check for the presence of Cy3-edaADP, edaATP, and edaADP. Determine the purity by integrating the Cy3-edaATP peak with any other peaks (see Note ).
Measure the fluorescence excitation and emission spectrum of Cy3-edaATP in 50 mM TrisCHCl (pH 7.5). Typically, 1 μM in a solution of 60 μl will be used. Use the peak wavelength from the absorbance measurement as the excitation wavelength to measure the emission. Then, use the peak in the emission spectrum for the excitation spectrum. Add an excess of the protein of interest to the sample (e.g., 10-fold) and repeat the measurement (see Notes 14 and 15). Compare the two spectra to determine the change in fluorescence when bound to protein.
3.1.6 & Generating Cy3-edaADP
Cy3-edaADP can be obtained by hydrolysis of Cy3-edaATP. Add the desired concentration of Cy3-edaATP (e.g., 100 μM) to rabbit skeletal muscle myosin (1 mg/ml) in 1 mM MgCl2, 0.2 mM dithiothreitol (DTT), and 10 mM TrisCHCl, pH 7.0. 4°C for 2 h (see Note ).
Centrifuge the sample at 235,000  &  g at 4°C to remove the myosin.
Analyze the product using HPLC, as described above.
Store the supernatant at −80°C.
This method is based on that described by Webb et al. () and gives a single product as reaction occurs only at the 3′-amine (Fig. a).
3.2.1 & Labeling
This method requires the starting material 3′-amino-3′-deoxy&&ATP ().
Activate 7-diethylaminocoumarin-3-carboxylic acid (16.4 mg, 62.8 μmol) by dissolving in dry DMF (1 ml), cooling it on ice, and adding tributylamine (25 μl, 103 μmol) and isobutyl chloroformate (10 μl, 77 μmol).
Leave the reaction mixture on ice for 50 min.
Add 3′-amino-3′-deoxyATP (40 μmol, triethylammonium salt) in water (300 μl) to the activated coumarin and stir at room temperature for 2 h.
Analyze the reaction mixture using HPLC to confirm the formation of deac-aminoATP. Equilibrate a Partisphere SAX column with 0.4 M (NH4)2HPO4 with 5% (v/v) acetonitrile at a flow rate of 1.5 ml/min at room temperature.
Add an aliquot of the reaction mixture (1C10 nmol) to 100 μl of the running buffer.
Inject the solution onto the column.
Follow the absorbance at 254 nm and fluorescence with excitation 435 nm and emission 465 nm. Elution times are approximately 1.6 min for 7-diethylaminocoumarin-3-carboxylic acid, 3.5 min for 3′-amino-3′-deoxyATP, and 13 min for deac-&aminoATP (see Note ).
3.2.2 & Purification
The reaction mixture was purified on a DEAE-cellulose &column. Equilibrate the column with 10 mM TEAB, pH 7.6 at 1 ml/min at 4°C.
Alter the pH of the reaction mixture to 7.6 using acid or base, reduce the conductivity by dilution in water so it is close to that of 10 mM TEAB and load on to the column.
Wash the column with 10 mM TEAB, pH 7.6 at a flow rate of 1 ml/min for two column volumes.
Elute the nucleotide with a linear gradient of 10C600 mM TEAB (total volume 1 L). Follow the absorbance at 254 nm (see Note ). Unreacted aminoATP is eluted first followed by deac-aminoATP.
3.2.3 & Concentration
The product deac-aminoATP is concentrated as described for Cy3-edaATP and stored at −80°C.
3.2.4 & Characterization
Measure the absorbance spectra of deac-aminoATP in 50 mM TrisCHCl, pH 7.5 between 220 and 700 nm. Taking the extinction coefficient for the coumarin to be 46,800 M−1 cm−1 at 429 nm and for adenosine to be 15,200 M−1 cm−1 at 260 nm, calculate the concentrations of the nucleotide (see Note ).
Characterize deac-aminoATP by HPLC using the same method as above. The major peak should be deac-aminoATP. Determine the purity by integrating the deac-aminoATP peak with any other peaks.
Measure the fluorescence spectra as described for Cy3-edaATP, but using the corresponding excitation and emission peaks for the coumarin (Fig. ).
Fig. 2.& Fluorescence change upon binding of deac-aminoADP to myosin S1. Excess &myosin S1 was added to bind all nucleotides. Deac-aminoADP (0.3 μM) was excited at 435 nm and 10 μM S1 was added. Insert shows the titration of myosin S1 into a solution of deac-aminoADP. This highlights the need to saturate the nucleotide to determine the maximum fluorescence change. Myosin S1 was added to a solution of 0.1 μM nucleotide, and the fluorescence was monitored at 480 nm, with excitation at 435 nm.
Follow the same procedure described for the Cy3-edaATP to generate the diphosphate.
The method describes here is for an ATPase or GTPase. This specific example uses a DNA helicase Bacillus stearothermophilus PcrA. The easiest method to provide an overall assessment of the effect of an ATP modification is to measure a steady-state ATPase assay. Should there be a change in the steady-state parameters (greater than 20%), then the individual steps of the ATP cycle could be investigated. It is common for the diphosphate affinity to increase with modifications to the ribose ring (, , , ).
It is also highly recommended that a functional activity assay is performed, such as measuring DNA unwinding by a DNA helicase or an in vitro motility assay with myosin. This is an alternate assessment of the modification effect: the label may interfere with one criterion which may not be noticeable in the other.
Prepare a mixture (60 μl) of 2 nM PcrA helicase, 500 nM dT20 oligonucleotide and 10 μM MDCC-PBP (or 6IATR-PBP) in a buffer containing 50 mM TrisCHCl, pH 7.5, 3 mM MgCl2 and 150 mM NaCl.
Record the fluorescence by exciting at 436 nm and emission at 465 nm for MDCC-PBP, or excitation 555 nm and emission 575 nm for 6IATR-PBP.
Add ATP at various concentrations (1 mM to 0.5 μM) (see Note ).
Repeat the measurements at the same concentrations of deac-aminoATP or Cy3-edaATP (see Note ).
Perform a calibration of the fluorescence signal using known concentrations of inorganic phosphate.
Compare the V max and K m values for the native and modified nucleotides.
It is also possible to synthesize edaATP (, , ).
MDCC-PBP is available commercially from Invitrogen, but cannot be used with diethylaminocoumarin-labeled nucleotides because the fluorophores are the same. Similarly, 6IATR-PBP cannot be used if a fluorophore with similar wavelengths is present, such as Cy3 or other rhodamine.
Use an excess of nucleotide over Cy3 NHS-ester due to the expense of the fluorophore.
Filter and degas the (NH4)2HPO4 and then add the HPLC grade methanol.
Alternatively, the reaction can be followed by thin-layer chromatography on silica plates ().
Using the fluorescence detection is approximately 100-fold more sensitive than absorbance. If necessary, absorbance peaks can be collected, their fluorescence measured in a fluorimeter, and their full absorbance spectrum measured in a spectrometer.
Do not store distilled triethylamine for later use without redistillation: it decomposes on storage. It may be possible to use high-purity triethylamine without distillation, but triethylamine does form impurities on storage. The distillation ensures that only volatile components end up in the TEAB solution.
Triethylamine itself is only partially miscible with water: there will be two layers initially, which becomes a single solution after some CO2 has been absorbed.
Alternatively, follow the fluorescence of Cy3 using an excitation of 550 nm and emission of 570 nm.
Apply and remove the vacuum slowly to prevent the solution splashing and frothing, and so passing into the condenser.
Aliquot the ATP into small amounts before freezing to avoid freeze-thaw cycles. It is advisable to check the purity of the nucleotide by HPLC periodically during long-term storage.
The ratio between the molar amount of the fluorophore and adenosine should be ∼1. If not, there is likely to be contaminating fluorophore in the preparation.
Determine the limit of sensitivity for the instrument by injecting known amounts. Typically, 1% contaminatio for example, if 10 nmol is injected, it should be possible to detect 0.1 nmol. It is possible that a greater amount of nucleotide will have to be injected.
Ideally, the addition of excess protein to the nucleotide sample would lead to the maximum potential signal change. However, this is dependent on the affinity between nucleotide and protein.
Unless the protein has a low hydrolysis rate constant, or the protein requires an activator, it is likely that any fluorescence change occurs due to the formation of diphosphate. The diphosphate fluorescence change should be measured independently.
It is also possible to begin the labeling with edaADP and repeat the same protocol as describe above to produce the fluorescent diphosphate. In addition, it is possible to use other ATPases or commercially available glycerol kinase with d-glyceraldehyde (albeit that ribose-modified nucleotides are poor substrates for this kinase) to achieve the hydrolysis of the triphosphate (). The resulting ADP analog is purified by a desalting column (PD10) or repeating the ion-exchange chromatography.
Alternatively, follow the fluorescence of the diethylaminocoumarin using an excitation of 430 nm and emission of 465 nm.
Avoid diluting the ATP samples to low concentrations. Use 60× concentrated stocks. By adding 1 μl of the ATP to the 60 μl reaction mixture, the correct ATP concentration is achieved.
We would like to thank the various coworkers, who have been involved in synthesis and use of fluorescent nucleotides and are coauthors of publications cited here. We thank the Medical Research Council, UK (C.P.T. and M.R.W.) and European Molecular Biology Organization (C.P.T) for financial support.
References
Funatsu, T., Harada, Y., Tokunaga, M., Saito, K., and Yanagida, T. (1995) Imaging of single fluorescent molecules and individual ATP turnovers by single myosin molecules in aqueous solution. Nature 374, 555C559.
Ishijima, A., Kojima, H., Funatsu, T., Tokunaga, M., Higuchi, H., Tanaka, H., and Yanagida, T. (1998) Simultaneous observation of individual ATPase and mechanical events by a single myosin molecule during interaction with actin. Cell 92, 161C171.
Oiwa, K., Eccleston, J. F., Anson, M., Kikumoto, M., Davis, C. T., Reid, G. P., Ferenczi, M. A., Corrie, J. E., Yamada, A., Nakayama, H., and Trentham, D. R. (2000) Comparative single-molecule and ensemble myosin enzymology: sulfoindocyanine ATP and ADP derivatives. Biophys. J. 78, .
Sakamoto, T., Webb, M. R., Forgacs, E., White, H. D., and Sellers, J. R. (2008) Direct observation of the mechanochemical coupling in myosin Va during processive movement. Nature 455, 128C132.
Ha, T. (2001) Single-molecule fluorescence resonance energy transfer. Methods 25, 78C86.
Henn, A., Cao, W., Hackney, D. D., and De La Cruz, E. M. (2008) The ATPase cycle mechanism of the DEAD-box rRNA helicase, DbpA. J. Mol. Biol. 377, 193C205.
Moore, K. J., and Lohman, T. M. (1994) Kinetic mechanism of adenine nucleotide binding to and hydrolysis by the Escherichia coli Rep &monomer. 2. Application of a kinetic competition approach. Biochemistry 33, .
Rossomando, E. F., Jahngen, J. H., and Eccleston, J. F. (1981) Formycin 5′-triphosphate, a fluorescent analog of ATP, as a substrate for adenylate cyclase. Proc. Natl. Acad. Sci. USA 78, .
Toseland, C. P., Martinez-Senac, M. M., Slatter, A. F., and Webb, M. R. (2009) The ATPase Cycle of PcrA Helicase and Its Coupling to Translocation on DNA. J. Mol. Biol. 392, .
Woodward, S. K., Eccleston, J. F., and Geeves, M. A. (1991) Kinetics of the interaction of 2′(3′)-O-(N-methylanthraniloyl)-ATP with myosin subfragment 1 and actomyosin subfragment 1: characterization of two acto-S1-ADP complexes. Biochemistry 30, 422C430.
Phillips, R. A., Hunter, J. L., Eccleston, J. F., and Webb, M. R. (2003) The mechanism of Ras GTPase activation by neurofibromin. Biochemistry 42, .
Cremo, C. R. (2003) Fluorescent nucleotides: synthesis and characterization. Methods Enzymol. 360, 128C177.
Jameson, D. M., and Eccleston, J. F. (1997) Fluorescent nucleotide analogs: synthesis and applications. Methods Enzymol. 278, 363C390.
Yarbrough, L. R., Schlageck, J. G., and Baughman, M. (1979) Synthesis and properties of fluorescent nucleotide substrates for &DNA-dependent RNA polymerases. J. Biol. Chem. 254, .
Webb, M. R., Reid, G. P., Munasinghe, V. R., and Corrie, J. E. (2004) A series of related nucleotide analogues that aids optimization of fluorescence signals in probing the mechanism of P-loop ATPases, such as actomyosin. Biochemistry 43, .
Webb, M. R., and Corrie, J. E. (2001) Fluorescent coumarin-labeled nucleotides to measure ADP release from actomyosin. Biophys. J. 81, .
Cremo, C. R., Neuron, J. M., and Yount, R. G. (1990) Interaction of myosin subfragment 1 with fluorescent ribose-modified nucleotides. A comparison of vanadate trapping and SH1-SH2 cross-linking. Biochemistry 29, .
Forgacs, E., Cartwright, S., Kovacs, M., Sakamoto, T., Sellers, J. R., Corrie, J. E., Webb, M. R., and White, H. D. (2006) Kinetic &mechanism of myosinV-S1 using a new &fluorescent ATP analogue. Biochemistry 45, .
Brune, M., Hunter, J. L., Howell, S. A., Martin, S. R., Hazlett, T. L., Corrie, J. E., and Webb, M. R. (1998) Mechanism of inorganic phosphate interaction with phosphate binding &protein from Escherichia coli. Biochemistry 37, .
Okoh, M. P., Hunter, J. L., Corrie, J. E., and Webb, M. R. (2006) A biosensor for inorganic phosphate using a rhodamine-labeled phosphate binding protein. Biochemistry 45, .
Mujumdar, R. B., Ernst, L. A., Mujumdar, S. R., Lewis, C. J., and Waggoner, A. S. (1993) Cyanine dye labeling reagents: sulfoindocyanine succinimidyl esters. Bioconjug. Chem. 4, 105C111.
Kurzawa-Goertz, S. E., Perreault-Micale, C. L., Trybus, K. M., Szent-Gyorgyi, A. G., and Geeves, M. A. (1998) Loop I can modulate ADP affinity, ATPase activity, and motility of different scallop myosins. Transient kinetic analysis of S1 isoforms. Biochemistry 37, .
Talavera, M. A., and De La Cruz, E. M. (2005) Equilibrium and kinetic analysis of nucleotide binding to the DEAD-box RNA helicase DbpA. Biochemistry 44, 959C970.
Webb, M. R. (1980) A method for determining the positional isotope exchange in a nucleoside triphosphate: cyclization of nucleoside triphosphate by dicyclohexylcarbodiimide. Biochemistry 19, .
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