Direct Observation of Kinetic Pathways of Biomolecular Recognition

Abstract

The pathways of molecular recognition, which is a central event in all biological processes, belong to the most important subjects of contemporary research in biomolecular science. By using fluorescence spectroscopy in a microfluidics channel, it can be determined that molecular recognition of α-chymotrypsin in hydrous surroundings at two different pH values (3.6 and 6.3) follows two distinctly different pathways. Whereas one corroborates an induced-fit model (pH 3.6), the other one (pH 6.3) is consistent with the selected-fit model of biomolecular recognition. The role of massive structural perturbations of differential recognition pathways could be ruled out by earlier XRD studies, rather was consistent with the femtosecond-resolved observation of dynamic flexibility of the protein at different pH values. At low concentrations of ligands, the selected-fit model dominates, whereas increasing the ligand concentration leads to the induced-fit model. From molecular modelling and experimental results, the timescale associated with the conformational flexibility of the protein plays a key role in the selection of a pathway in biomolecular recognition.

Modulation of Kinetic Pathways of Enzyme–Substrate Interaction in a Microfluidic Channel: Nanoscopic Water Dynamics as a Switch

“…This is consistent with molecular recognition of the enzyme by the substrate through the “conformational selection” fit mechanism . From the magnitude of the pseudo‐first‐order reaction rate constant, the value of the corresponding second‐order rate constant is found to be 1.8×10 6 m −1 s −1 , which agrees well with that (≈10 6 m −1 s −1 ) for the interaction of ANS with CHT at pH 6.3 . The first‐order rate constant, k 2 , for structural reorganization of the enzyme is also of the same order as reported (≈10–100 s −1 ) earlier .…”

“…From the magnitude of the pseudo‐first‐order reaction rate constant, the value of the corresponding second‐order rate constant is found to be 1.8×10 6 m −1 s −1 , which agrees well with that (≈10 6 m −1 s −1 ) for the interaction of ANS with CHT at pH 6.3 . The first‐order rate constant, k 2 , for structural reorganization of the enzyme is also of the same order as reported (≈10–100 s −1 ) earlier . In the case of the anionic reverse micelle (AOT/benzene), the pseudo‐first‐order rate constant, k 1 , is much smaller (Table ) than that for the cationic reverse micelle, and the estimated second‐order rate constant is about 1.1×10 5 m −1 s −1 , which is consistent with a weak binding interaction of AMC with CHT in the first stage .…”

“…The first‐order rate constant, k 2 , for structural reorganization of the enzyme is also of the same order as reported (≈10–100 s −1 ) earlier . In the case of the anionic reverse micelle (AOT/benzene), the pseudo‐first‐order rate constant, k 1 , is much smaller (Table ) than that for the cationic reverse micelle, and the estimated second‐order rate constant is about 1.1×10 5 m −1 s −1 , which is consistent with a weak binding interaction of AMC with CHT in the first stage . In the second step, structural reorganization is associated with a time constant ( k 2 ) nearly three times smaller than that for the cationic reverse micelles.…”

“…In the second step, structural reorganization is associated with a time constant ( k 2 ) nearly three times smaller than that for the cationic reverse micelles. Thus, the molecular recognition pathway in the anionic reverse micelle of AOT involves a much weaker binding interaction between AMC and CHT in the first step than for the cationic reverse micelle, followed by complexation through slow structural reorganization, and is consistent with the “induced fit” mechanism …”

Ultrafast differential flexibility of Cro-protein binding domains of two operator DNAs with different sequences

“…[8][9][10] In a series of studies from our group, we have established that l-repressor and Gal-repressor show altered ultrafast dynamical motions in the C-terminal domain upon operator DNA-binding in the distant N-terminal domain. [15][16][17][18][19] In an earlier study, 20 it was concluded that the flexibility of a target DNA may also be important for the specific DNA binding affinity. [15][16][17][18][19] In an earlier study, 20 it was concluded that the flexibility of a target DNA may also be important for the specific DNA binding affinity.…”