Caged FEDA-ATP: a new tool in the measurement of ATP turnover during the in vitro motility assay

Abstract

In an attempt to distinguish between the various models of muscle contraction, attention has been focused upon calculation of the myosin crossbridge step-size (the distance moved during active interaction of a myosin head with acM per ATP molecule hydrolysed) using in virro motility assays. We have previously used a fluorescein derivative of ATP, FEDA-ATP as a potential probe for nucleotide turnover under in virro assay conditions [l].Although an array of myosin heads can be visualised in the presence of FEDA-ATP by fluorescence microscopy, only low concentrations (50 nM) of the latter can be used, so as to minimise the background signal from the free fluorophore. Total internal reflectance fluorescence microscopy offers one possibility around this problem [2]. An alternative strategy is to cage the fluorescein [ 31 to make a non-fluorescent derivative which can be photoactivated in localised regions containing the array of myosin heads. Any free fluorophores activated in this process are expected to diffuse from the site of interest within several milliseconds.We have prepared a non-fluorescent biscaged derivative of FEDA-ATP by reacting the succinimidyl ester of carboxyfluorescein-bis-(4,5dimethoxy-2-ni~benzyl) ether (Molecular Probes Inc., Oregon, U.S.A.)) with the 2', (3') ethylenediamine derivative of ATP (Fig. 1). The latter (EDA-ATP) was prepared as described previously [ 11. Such a caged FEDA-ATP is a substrate for rabbit skeletal myosin subfragment 1. Caged FEDA-ATP quenches tryptophan fluorescence allowing the binding and turnover steps to be monitored by stopped-flow methods (Fig. 2). As expected, fluorescence emission at the fluorescein wavelengths is close to background levels. Irradiation of caged FEDA-ATP with 360 nm light releases a product with comparable properties to FEDA-ATP (note because of the use of a P 0. 0. 0. "f-; o . -i -o -i -o~~-o -a c 6 6 3 p -. J oac Fig. 1 The structure of caged FEDA-ATP succinimidyl ester rather than isothiocyanate to link to the amine of EDA-ATP, a ureido rather that thioureido group is present in the photoactivated product). The irradiated preparation shows a quench in fluorescein emission on binding to myosin subfragment 1, followed by a recovery corresponding to the product release phase. This is similar to FEDA-ATP itself [l].As a preliminary to the photoactivation of caged FEDA-ATP when bound to myosin molecular arrays, it is desirable to produce a long-lived myosin products complex to allow optimisation of the microbeam optics. We have therefore explored the formation of the M.FEDA-ADP.AlF4-complex using AlF4-as a phosphate analogue [4]. Addition of 0.1 mM AIC13 and 5 mM NaF to stoichiometric amounts of rabbit myosin subfragment 1 and FEDA-ADP produced a 60 8 quench in fluorescein fluorescence, Biochemical Society Transactions ( 1 995) 23 1 0 I 1 I I I I I I 1 1 401 S -1 0 0 1 0 20 30 40 50 6 0 7 0 80 90 100 Time (secs) Fig. 2. Stopped-flow record showing the turnover of 1 ph'l caged FEDA-ATP by 0.5 ph'l myosin subfragment 1 as monitored by a quench in txyp...

Kinetic and Spectroscopic Characterization of Fluorescent Ribose-Modified ATP Analogs upon Interaction with Skeletal Muscle Myosin Subfragment 1

“…Although we have carried out fewer measurements with other fluorophores, we have shown that the overall kinetics of interaction are similar for both REDA-ATP and Cy3-EDA-ATP. Also, we have introduced photoactivatable caged fluorescein in this manner and shown that the caged FEDA-ATP is a substrate for myosin (Jeffreys et al, 1995).…”

Measurement of nucleotide exchange kinetics with isolated synthetic myosin filaments using flash photolysis

“…Conibeap; C.R. Bagshaw/FEBS Letters 380 (1996) [13][14][15][16] otides is likely to effect only the amplitude rather than the kinetics of observed intensity changes. The REDA-ADP-dissociation kinetics provide a test of the time resolution of the apparatus.…”

“…As a consequence, we can now monitor displacement from a l-/~m segment of a synthetic myosin filament estimated to contain a few hundred sites with a time resolution of 0.1 s. Furthermore, the technique has made records much more reproducible owing to the lack of mechanical artefacts inherent to flow methods. The finding that ATP can be released photolytically without damage to the catalytic activity of myosin is a necessary prerequisite for the general use of flash photolysis and bodes well for studies using caged fluorescent ATP analogs [14]. We have initiated the sliding of actin in in vitro motility assays with this apparatus [15], but this process is more susceptible to damage from the free radicals released during photolysis and inclusion of thiol reagents is essential.…”