Notes on Columbo, Lappalainen, Michelot Nature Communications volume 12, Article number: 548 (2021)


* Fig 1b. Binding ability of different ATTO-488 fluorescent ATP analogs (0.2 µM) to G-actin (2 µM) in NFG + MEI buffer, revealed by measuring changes in steady-state anisotropy values 30 min after the initiation of the experiment. Light (resp. dark) gray are values obtained in the absence (resp. presence) of G-actin. Bar graphs indicate mean values and standard deviations. n = 20 for each condition.

* Only one that works is N6-(6-Amino)hexyl-ATP-ATTO-488 because others' dyes clash w/ binding pocket.
 - i.e. DON'T use γ-[6-Aminohexyl]-ATP-ATTO-488 or 2′(3′)-O-(N-(2-(amino)ethyl)carbamoyl) ATP

* versus εATP (note εATP is intensity not anisotropy)
 - compared the exchange kinetics of εATP and ATP-ATTO-488 with G-actin
 - Fig 2a.  εATP faster (maybe too fast if you want to boost with profilin), also it photobleaches.

* Fig 2, shows binding and dissociation over time by anisotropy, variety of actin and dye conc's
 - looks like 200-500 nM dye is best w/ 2 uM actin in NFG + MEI
 - looks like 1 uM Ca-G-actin (-KCl) is best w/ 200 nM dye

* ATP-ATTO-488 displays similar exchange kinetics on actin monomers compared to ATP. However, its dissociation rate from actin is approximately three times slower, and this may be due to weak interactions of the fluorescent dye with actin
 - this means you can use this for ATP binding assays, but there would be caveats if you want to do dissociation
 - worth noting/citing this in the paper

* does not inhibit the activities of key ABPs (i.e. profilin) on actin

* yeast profilin accelerated exchange of ATP to ATP-ATTO-488 (and inversely of ATP-ATTO-488 to ATP) by 3- to 4-fold on actin monomers (Fig 4a)
 - cited Eads, J. C. et al. Structure determination and characterization of Saccharomyces cerevisiae profilin. Biochemistry 37, 11171–11181 (1998)

* Fig 4a is the profilin stuff
 - 340-8000 nM profilin (or noPro), 0.2 uM dye, 2 uM G-Actin, in NFG + MEI

* assay protocol
 - NFG = nucleotide-free G-buffer; 5 mM Tris pH 8, 0.2 mM CaCl2, 0.5 mM DTT, 0.02% Sodium Azide
 - MEI = 1 mM MgCl2, 1 mM EGTA, 10 mM Imidazole-HCl, pH 7.0

 - actin is stored at 4 °C in G buffer (5 mM Tris pH 8; 0.1 mM CaCl2; 0.2 mM ATP; 0.5 mM DTT; 0.02% Sodium Azide)
 - dilute G-actin to 100 uM in NFG, then to assay conc (e.g. 2 uM) in NFG + MEI
 - final assay conditions have 2-fold excess ATP over G-actin (e.g. 4 uM ATP w/ 2 uM G-actin)
 - reaction initiated by adding ATP-ATTO-488 (e.g. 200 nM)
 - ATTO-488 excitation at 504 nm, emission at 521 nm