A review of TNP-ATP in protein binding studies: benefits and pitfalls

Structure of VanS from Vancomycin-Resistant Enterococci: A Sensor Kinase with Weak ATP Binding

“…Our affinity estimates are robust, as two orthogonal methods yield K D estimates in agreement with each other (Table 2). It is worth noting that our affinity estimate for TNP-ATP binding to EnvZ is 20-fold weaker than previously reported (27); this may reflect the fact that the earlier work titrated TNP-ATP into a fixed concentration of protein, whereas we performed the opposite titration, in order to avoid complications from the inner-filter effect (25). Despite this difference, however, our estimates for EnvZ's ATP-binding affinity agree well with published values.…”

“…We first used the fluorescent ATP analog TNP-ATP. This molecule’s emission profile is highly sensitive to the fluorophore’s environment, and thus it functions as a useful probe for protein binding (25,26). As a positive control, we used the CA domain from E. coli EnvZ, for which the TNP-ATP and ATP binding affinities have previously been reported (27).…”

Identification of novel functional mini-receptors by combinatorial screening of split-WW domains

“…This peptide competes very well with other engineered systems 43 and natural ATP-binding proteins, the latter having K d values from the high nanomolar to the millimolar regime. 44 Sequence optimization of WW-2-10 could improve ATP binding even further. Only a single artificial ATP-binding protein of 80 amino acids, identified by in vitro selection of a messenger RNA displayed protein library with 6 × 10 12 members, showed a K d value of 0.1 μM.…”

“…By comparison, natural ATPbinding proteins bind their ligand in the high nM to mM range. 44 To further investigate the relationship between sequence and activity, we also relied on computational methods. A structural model of WW-2-10 was constructed with the Rosetta Relax application 41 based on an available NMR structure of hPin1 WW (pdb code 1i6c), in which the corresponding amino acid residues were mutated.…”

Biophysical Reports: Open for infinite possibilities

“…Shimon Weiss and colleagues report on the recording of membrane potentials by using fluorescent nanodiscs, a technique that could have broad applications in studying the biophysics of neurons ( 6 ). And in the journal’s first review paper, Dixon J. Woodbury and colleagues provide a comprehensive overview of the use of 2,4,6-trinitrophenol-adenosine triphosphate for protein binding studies, discussing the potential of this important method as well as its challenges and problems ( 7 ).…”