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Transient Incomplete Separation Facilitates Finding Accurate Equilibrium Dissociation Constant of Protein–Small Molecule Complex
Author(s) -
Sisavath Nicolas,
Rukundo JeanLuc,
Le Blanc J. C. Yves,
Galievsky Victor A.,
Bao Jiayin,
Kochmann Sven,
Stasheuski Alexander S.,
Krylov Sergey N.
Publication year - 2019
Publication title -
angewandte chemie international edition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.831
H-Index - 550
eISSN - 1521-3773
pISSN - 1433-7851
DOI - 10.1002/anie.201901345
Subject(s) - dissociation (chemistry) , superposition principle , small molecule , molecule , chemistry , transient (computer programming) , constant (computer programming) , kinetic energy , dissociation constant , equilibrium constant , capillary action , thermodynamics , chemical physics , computational chemistry , physics , computer science , classical mechanics , organic chemistry , biochemistry , receptor , quantum mechanics , programming language , operating system
Current practical methods for finding the equilibrium dissociation constant, K d , of protein–small molecule complexes have inherent sources of inaccuracy. Introduced here is “accurate constant via transient incomplete separation” (ACTIS), which appears to be free of inherent sources of inaccuracy. Conceptually, a short plug of the pre‐equilibrated protein–small molecule mixture is pressure‐propagated in a capillary, causing fast transient incomplete separation of the complex from the unbound small molecule. A superposition of signals from these two components is measured near the capillary exit and used to calculate a fraction of unbound small molecule, which, in turn, is used to calculate K d . Herein the validity of ACTIS is proven theoretically, its accuracy is verified by computer simulation, and its practical use is demonstrated. ACTIS has the potential to become a reference‐standard method for determining K d values of protein–small molecule complexes.